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

Abstract

The present invention relates to multivalent immunogenic compositions comprising one or more Streptococcus pneumoniae polysaccharide protein conjugates wherein each of the conjugates comprises a polysaccharide from a Streptococcus pneumoniae serotype conjugated to a carrier protein wherein the serotype of the Streptococcus pneumoniae is as defined herein. In some embodiments, at least one of the polysaccharide protein conjugates is formed from a conjugation reaction comprising an aprotic solvent. In further embodiments, each of the polysaccharide protein conjugates is formed from a conjugation reaction comprising an aprotic solvent. Also provided are methods for inducing a protective immune response in a human patient comprising administering to the patient the multivalent immunogenic composition of the invention. The multivalent immunogenic composition is used to provide protection against Streptococcus pneumoniae infection and / or pneumococcal disease caused by Streptococcus pneumoniae. The compositions of the invention are also useful as part of a therapeutic regimen that provides supplemental protection for patients who have been vaccinated with a multivalent vaccine for the prevention of pneumococcal disease.

Description

[0001] This application is a divisional application of a patent application with an application date of December 17, 2019, application number 201980092227.4, and name “Compositions containing pneumococcal polysaccharide-protein conjugates and methods of use thereof”. Technical Field

[0002] The present invention provides a multivalent immunogenic composition with different polysaccharide-protein conjugates. Each conjugate consists of a capsular polysaccharide prepared from different serotypes of Streptococcus pneumoniae conjugated to a carrier protein (preferably CRM197). The immunogenic composition provides a wide range of coverage against pneumococcal diseases.

[0003] This application contains a sequence listing, which has been submitted electronically in ASCII format, the entire contents of which are incorporated herein by reference. The ASCII copy was created on December 3, 2019, named 24683WOPCT-SEQTXT-03DEC2019, and is 6 kilobytes in size. Background Art

[0004] Streptococcus pneumoniae is a Gram-positive bacterium that is the most common cause of invasive bacterial diseases in infants and young children, such as pneumonia, bacteremia, meningitis, and otitis media. Pneumococci are coated with chemically linked polysaccharides that confer serotype specificity. There are more than 90 known serotypes of pneumococci, and the capsule is the main virulence determinant of pneumococci, because the capsule not only protects the inner surface of the bacteria from complement, but is also poorly immunogenic itself. Polysaccharides are T-cell-dependent antigens and, in most cases, cannot be processed or presented on MHC molecules to interact with T cells. However, they are able to stimulate the immune system through another mechanism that involves cross-linking of surface receptors on B cells.

[0005] The multivalent pneumococcal polysaccharide vaccine, which has been licensed for many years, has proven to be of great value in preventing pneumococcal disease in adults, especially the elderly and high-risk groups. However, infants and young children do not respond well to unconjugated pneumococcal polysaccharides. Pneumococcal conjugate vaccines Containing the seven most commonly isolated serotypes (4, 6B, 9V, 14, 18C, 19F, and 23F) that cause invasive pneumococcal disease in young children and infants at the time, the vaccine was first licensed in the United States in February 2000. In common use in the United States, due to The presence of serotypes in the diet has led to a significant reduction in invasive pneumococcal disease in children. See Centers for Disease Control and Prevention, MMWR Morb Mortal Wkly Rep 2005, 54(36):893-7. However, in some parts of the world, There are limitations in the serotype coverage of the current guidelines, and some evidence that some emerging serotypes (e.g., 19A and others) have emerged 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.

[0006] US Patent Application Publication No. US2006 / 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. CN101590224A describes a 14-valent pneumococcal polysaccharide-protein conjugate vaccine comprising serotypes 1, 2, 4, 5, 6A, 6B, 7F, 9N, 9V, 14, 18C, 19A, 19F and 23F.

[0007] Other PCVs cover serotypes 7, 10, 11, or 13, which are included in PCV-15 (U.S. Publication No. 2011 / 0195086), but immune interference with some serotypes has been observed (e.g., reduced protection against serotype 3 in GSK's PCV-11) and reduced protection against Pfizer's PCV-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.

[0008] Current multivalent pneumococcal vaccines have been effective in reducing the incidence of pneumococcal disease associated with those serotypes present in the vaccine. However, the prevalence of pneumococci expressing serotypes not present in currently available vaccines has been increasing. Therefore, there is a need for additional pneumococcal vaccine compositions that can provide protection against pneumococcal serotypes not present in currently available vaccines. Summary of the invention

[0009] The present invention provides a multivalent immunogenic composition comprising a Streptococcus pneumoniae (S. pneumoniae) polysaccharide protein conjugate, wherein each of the conjugates comprises a polysaccharide from a S. pneumoniae serotype conjugated to a carrier protein, and wherein the polysaccharide protein conjugate comprises a polysaccharide from a group of S. pneumoniae serotypes selected from the group consisting of:

[0010] a) 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 12F, 14, 15A, DeOAc15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;

[0011] b) 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;

[0012] c) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, DeOAc15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;

[0013] d) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;

[0014] e) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;

[0015] f) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, DeOAc15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;

[0016] g) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;

[0017] h) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;

[0018] i) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;

[0019] j) 1, 3, 4, 5, 6A, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;

[0020] k) 1, 3, 4, 5, 6B, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;

[0021] l) 1, 3, 4, 5, 6C, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;

[0022] m) 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;

[0023] n) 1, 3, 4, 5, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;

[0024] o) 1, 3, 4, 5, 6C, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;

[0025] p) 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;

[0026] q)1, 3, 4, 5, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;

[0027] r) 1, 3, 4, 5, 6C, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;

[0028] s)1, 3, 4, 5, 6A, 7F, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;

[0029] t) 1, 3, 4, 5, 6B, 7F, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;

[0030] u) 1, 3, 4, 5, 6C, 7F, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;

[0031] v) 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;

[0032] w) 1, 3, 4, 5, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;

[0033] x) 1, 3, 4, 5, 6C, 7F, 8, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;

[0034] y) 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;

[0035] z) 1, 3, 4, 5, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; and

[0036] aa) 1, 3, 4, 5, 6C, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B.

[0037] The present invention provides a multivalent immunogenic composition comprising 22 different polysaccharide protein conjugates, wherein each of the conjugates comprises a capsular polysaccharide from a serotype of Streptococcus pneumoniae conjugated to a carrier protein, wherein the polysaccharide is prepared from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B of Streptococcus pneumoniae.

[0038] The present invention provides a multivalent immunogenic composition comprising 22 different polysaccharide-protein conjugates, wherein each of the conjugates comprises a polysaccharide from a serotype of Streptococcus pneumoniae conjugated to a carrier protein, and wherein the polysaccharide-protein conjugate comprises a polysaccharide from a group of Streptococcus pneumoniae serotypes selected from the group consisting of: 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B.

[0039] The present invention provides a multivalent immunogenic composition comprising 23 different polysaccharide protein conjugates, wherein each of the conjugates comprises a capsular polysaccharide from a serotype of Streptococcus pneumoniae conjugated to a carrier protein, wherein the polysaccharide is prepared from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B of Streptococcus pneumoniae.

[0040] The present invention provides a multivalent immunogenic composition comprising 23 different pneumococcal polysaccharide-protein conjugates, wherein each of the conjugates comprises a capsular polysaccharide from a pneumococcal serotype conjugated to a carrier protein, wherein each different polysaccharide-protein conjugate comprises a polysaccharide from pneumococcal serotype 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B, respectively, and wherein the carrier protein is CRM197.

[0041] The present invention provides a multivalent immunogenic composition comprising 24 different polysaccharide protein conjugates, wherein each of the conjugates comprises a capsular polysaccharide from a serotype of Streptococcus pneumoniae conjugated to a carrier protein, wherein the polysaccharide is prepared from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B of Streptococcus pneumoniae.

[0042] The present invention provides a multivalent immunogenic composition comprising 24 different pneumococcal polysaccharide-protein conjugates, wherein each of the conjugates comprises a capsular polysaccharide from a pneumococcal serotype conjugated to a carrier protein, wherein each different polysaccharide-protein conjugate comprises a polysaccharide from pneumococcal serotype 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B, respectively, and wherein the carrier protein is CRM197.

[0043] The present invention provides a multivalent immunogenic composition comprising 24 different polysaccharide protein conjugates, wherein each of the conjugates comprises a capsular polysaccharide from a serotype of Streptococcus pneumoniae conjugated to a carrier protein, wherein the polysaccharide is prepared from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B of Streptococcus pneumoniae.

[0044] The present invention provides a multivalent immunogenic composition comprising 24 different pneumococcal polysaccharide-protein conjugates, wherein each of the conjugates comprises a capsular polysaccharide from a pneumococcal serotype conjugated to a carrier protein, wherein each different polysaccharide-protein conjugate comprises a polysaccharide from pneumococcal serotype 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B, respectively, and wherein the carrier protein is CRM197.

[0045] The present invention provides a multivalent immunogenic composition comprising up to 33 different polysaccharide protein conjugates, wherein each of the conjugates comprises a polysaccharide from a serotype of Streptococcus pneumoniae conjugated to a carrier protein, and wherein the polysaccharide protein conjugate comprises a polysaccharide from a group of Streptococcus pneumoniae serotypes selected from the following: 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B, and further comprises one, two, three, four, five, six, seven, eight or nine additional Streptococcus pneumoniae serotypes selected from the following: 7C, 9N, 16F, 21, 23A, 31, 34, 35F and 38.

[0046] The present invention provides a multivalent immunogenic composition comprising up to 30 different polysaccharide-protein conjugates, wherein each of the conjugates comprises a polysaccharide from a serotype of Streptococcus pneumoniae conjugated to a carrier protein, and wherein the polysaccharide-protein conjugate comprises a polysaccharide from a group of Streptococcus pneumoniae serotypes selected from the following: 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B, and further comprises one, two, three, four, five or six additional Streptococcus pneumoniae serotypes selected from the following: 7C, 9N, 16F, 23A, 35F and 38.

[0047] The present invention provides a multivalent immunogenic composition comprising up to 33 different polysaccharide-protein conjugates, wherein each of the conjugates comprises a polysaccharide from a serotype of Streptococcus pneumoniae conjugated to a carrier protein, and wherein the polysaccharide-protein conjugate comprises a polysaccharide from a group of Streptococcus pneumoniae serotypes selected from the following: 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B, and further comprises one, two, three, four, five, six, seven, eight or nine additional Streptococcus pneumoniae serotypes selected from the following: 7C, 9N, 16F, 21, 23A, 31, 34, 35F and 38.

[0048] The present invention provides a multivalent immunogenic composition comprising up to 30 different polysaccharide-protein conjugates, wherein each of the conjugates comprises a polysaccharide from a serotype of Streptococcus pneumoniae conjugated to a carrier protein, and wherein the polysaccharide-protein conjugate comprises a polysaccharide from a group of Streptococcus pneumoniae serotypes selected from the following: 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B, and further comprises one, two, three, four, five or six additional Streptococcus pneumoniae serotypes selected from the following: 7C, 9N, 16F, 23A, 35F and 38.

[0049] In some embodiments, at least one of the polysaccharide protein conjugates is formed by a conjugation reaction, and the conjugation reaction comprises an aprotic solvent, such as dimethyl sulfoxide (DMSO). In a specific embodiment, each of the polysaccharide protein conjugates is formed by a conjugation reaction, and the conjugation reaction comprises an aprotic solvent, such as (DMSO).

[0050] Also provided is a method for inducing a protective immune response in a human patient comprising administering to the patient a multivalent immunogenic composition of the invention. In some embodiments of the methods of the invention, the patient has previously been treated with a multivalent pneumococcal vaccine.

[0051] The multivalent immunogenic composition of the present invention can be used as part of a treatment regimen together with different complementary pneumococcal vaccines. Therefore, the present invention provides a method of inducing a protective immune response in a human patient, which comprises administering to the patient a multivalent immunogenic composition of the present invention, and further comprises administering to the patient a multivalent pneumococcal vaccine in any order. In a specific embodiment, the multivalent pneumococcal vaccine comprises a plurality of Streptococcus pneumoniae polysaccharide-protein conjugates, wherein each of the conjugates comprises a polysaccharide from a serotype of Streptococcus pneumoniae conjugated to a carrier protein. In other embodiments, the multivalent pneumococcal vaccine comprises unconjugated capsular polysaccharides.

[0052] The present invention also provides a multivalent immunogenic composition comprising a Streptococcus pneumoniae polysaccharide protein conjugate, wherein each of said conjugates comprises a polysaccharide from a Streptococcus pneumoniae serotype conjugated to a carrier protein, wherein the selected Streptococcus pneumoniae serotype provides cross-reactivity with other selected serotypes. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 : IgG antibody dilution titers before immunization (Pre), after dose 1 (PD1), after dose 2 (PD2), and after dose 3 (PD3) of mice immunized with PCV22 unadjuvanted (PCV22 unadjuvanted) or aluminum phosphate adjuvanted (PCV22 / APA) formulations as determined by ECL. Reading from left to right; Pre PCV22 unadjuvanted, Pre PCV22 / APA, PD1 PCV22 unadjuvanted, PD1 PCV22 / APA, PD2 PCV22 unadjuvanted, PD2 PCV22 / APA, PD3 PCV22 unadjuvanted, and PD3 PCV22 / APA.

[0054] Figure 2 : ECL dilution titer ratio of PCV22 / APA compared to PCV22 no adjuvant (PCV22 no adjuvant) on PD3.

[0055] Figure 3 : Serotype-specific OPA dilution titers (pre-immunization, PD1, PD2, PD3) in mice immunized with PCV22 unadjuvanted (PCV22 unadjuvanted) or APA (PCV22 / APA) formulations. Read from left to right; Pre PCV22 unadjuvanted, PrePCV22 / APA, PD1 PCV22 unadjuvanted, PD1 PCV22 / APA, PD2 PCV22 unadjuvanted, PD2PCV22 / APA, PD3PCV22 unadjuvanted, and PD3 PCV22 / APA.

[0056] Figure 4: OPA dilution titer ratio of PCV22 / APA compared to PCV22 no adjuvant (PCV22 no adjuvant) on PD3.

[0057] Figure 5 : PCV22-immunized mice were protected against intratracheal challenge with S. pneumoniae 24F.

[0058] Figure 6 : Pre-immunization (Pre), PD1 and PD2 IgG antibody dilution titers of rabbits immunized with PCV22 unadjuvanted or PCV22 / APA as determined by ECL. Error bars represent 95% confidence intervals (CI) of the geometric mean titers (GMT). Reading from left to right; Pre PCV22 unadjuvanted, Pre PCV22 / APA, PD1 PCV22 unadjuvanted, PD1 PCV22 / APA, PD2 PCV22 unadjuvanted, and PD2 PCV22 / APA.

[0059] Figure 7 : ECL GMT ratios of PCV22 / APA compared to PCV22 unadjuvanted on PD2. Error bars represent 95% confidence intervals (CI).

[0060] Figure 8 : Serotype-specific OPA dilution titers (pre-immunization "Pre" and PD2) of rabbits immunized with PCV22 without adjuvant or PCV22 / APA. Error bars indicate the variation of functional antibody titers of five rabbits.

[0061] Figure 9: Pre-immunization (Pre), PD1, PD2, and PD3 IgG antibody dilution titers of IRMs vaccinated with A) PCV23 without adjuvant, B) PCV23 (DMSO) / APA, C) PCV23 (DMSO+Aq) / APA, and D) PCV15 / APA+PCV8 / APA as determined by ECL. Error bars represent 95% confidence intervals (CI) of the geometric mean titers (GMT).

[0062] Figure 10: Comparison of ECL antibody responses in IRM (8-9 per group) after vaccination with PCV23 with or without APA. Symbols represent ratios at A) PD1, B) PD2, or C) PD3. GMT ratios, error bars represent 95% CI.

[0063] Figure 11: Comparison of ECL antibody responses in IRMs (9 per group) following vaccination with PCV23(DMSO+Aq) / APA or co-administered PCV15 / APA+PCV8 / APA. Symbols represent ratios at A) PD1, B) PD2, or C) PD3. GMT ratios, error bars represent 95% CI.

[0064] Figure 12: Comparison of ECL antibody responses elicited in IRM (8-9 per group) after immunization with PCV23 unadjuvanted, PCV23 (DMSO) / APA, PCV23 (DMSO + Aq) / APA or PCV15 / APA + PCV8 / APA. A) PD1 / Pre, B) PD2 / Pre, C) PD3 / Pre, D) PD2 / PD1 and E) PD3 / PD2. Symbols are GMT ratios and error bars represent 95% CI.

[0065] Figure 13: (A) IgG antibody dilution titers before immunization (Pre), after dose 1 (PD1), and after dose 2 (PD2) of New Zealand White rabbits immunized with PCV24 using an aluminum phosphate adjuvant formulation (PCV24 / APA) as determined by ECL. Error bars represent 95% confidence intervals (CI) of the geometric mean titer (GMT). (B) Serotype-specific OPA dilution titers (pre-immunization and PD2) of NZWRs immunized with PCV24 / APA. Error bars represent the variation in functional antibody titers of eight NZWRs.

[0066] Figure 14: (A) IgG antibody dilution titers before immunization (Pre), after dose 1 (PD1), after dose 2 (PD2), and after dose 3 (PD3) of infant rhesus macaques (IRM) immunized with PCV24 using an aluminum phosphate adjuvant formulation (PCV24 / APA) as determined by ECL. Error bars represent 95% confidence intervals (CI) of the geometric mean titer (GMT). (B) Serotype-specific OPA dilution titers (pre-immunization and PD3) of IRM immunized with PCV24 / APA. Error bars represent the variation in functional antibody titers of five IRM. DETAILED DESCRIPTION

[0067] The present invention provides a multivalent immunogenic composition comprising pneumococcal polysaccharide-protein conjugates, wherein each of said conjugates comprises a polysaccharide from a serotype of Streptococcus pneumoniae conjugated to a carrier protein, wherein said serotype of Streptococcus pneumoniae is as defined herein.

[0068] In some embodiments, the present invention provides a multivalent immunogenic composition comprising a Streptococcus pneumoniae polysaccharide-protein conjugate, wherein each of the conjugates comprises a polysaccharide from a serotype of Streptococcus pneumoniae conjugated to a carrier protein, and wherein the polysaccharide-protein conjugate comprises a polysaccharide from a group of Streptococcus pneumoniae serotypes selected from:

[0069] a) 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 12F, 14, 15A, DeOAc15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, and 35B;

[0070] b) 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, and 35B;

[0071] c) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, DeOAc15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, and 35B;

[0072] d) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, and 35B;

[0073] e) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, and 35B;

[0074] f) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, DeOAc15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, and 35B;

[0075] g) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, and 35B;

[0076] h) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, and 35B;

[0077] i) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, and 35B;

[0078] j) 1, 3, 4, 5, 6A, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;

[0079] k) 1, 3, 4, 5, 6B, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;

[0080] l) 1, 3, 4, 5, 6C, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;

[0081] m) 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;

[0082] n) 1, 3, 4, 5, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;

[0083] o) 1, 3, 4, 5, 6C, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;

[0084] p) 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;

[0085] q)1, 3, 4, 5, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;

[0086] r) 1, 3, 4, 5, 6C, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;

[0087] s)1, 3, 4, 5, 6A, 7F, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;

[0088] t) 1, 3, 4, 5, 6B, 7F, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, and 35B;

[0089] u) 1, 3, 4, 5, 6C, 7F, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, and 35B;

[0090] v) 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, and 35B;

[0091] w) 1, 3, 4, 5, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, and 35B;

[0092] x) 1, 3, 4, 5, 6C, 7F, 8, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, and 35B;

[0093] y) 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, and 35B;

[0094] z) 1, 3, 4, 5, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, and 35B;

[0095] aa) 1, 3, 4, 5, 6C, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, and 35B;

[0096] bb) 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15A, DeOAc 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B, and 39;

[0097] cc) 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;

[0098] dd) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 12F, 14, 15A, DeOAc15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;

[0099] ee) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;

[0100] ff) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;

[0101] gg) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 11A, 12F, 14, 15A, DeOAc15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;

[0102] hh) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;

[0103] ii) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;

[0104] jj) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;

[0105] kk) 1, 3, 4, 5, 6A, 7F, 9V, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;

[0106] ll)1, 3, 4, 5, 6B, 7F, 9V, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;

[0107] mm) 1, 3, 4, 5, 6C, 7F, 9V, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;

[0108] nn) 1, 3, 4, 5, 6A, 7F, 8, 9V, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;

[0109] oo)1, 3, 4, 5, 6B, 7F, 8, 9V, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;

[0110] pp) 1, 3, 4, 5, 6C, 7F, 8, 9V, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;

[0111] qq)1, 3, 4, 5, 6A, 7F, 8, 9V, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;

[0112] rr)1, 3, 4, 5, 6B, 7F, 8, 9V, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;

[0113] ss) 1, 3, 4, 5, 6C, 7F, 8, 9V, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;

[0114] tt)1, 3, 4, 5, 6A, 7F, 9V, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;

[0115] uu)1, 3, 4, 5, 6B, 7F, 9V, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;

[0116] vv) 1, 3, 4, 5, 6C, 7F, 9V, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;

[0117] ww)1, 3, 4, 5, 6A, 7F, 8, 9V, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;

[0118] xx) 1, 3, 4, 5, 6B, 7F, 8, 9V, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;

[0119] yy) 1, 3, 4, 5, 6C, 7F, 8, 9V, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;

[0120] zz)1, 3, 4, 5, 6A, 7F, 8, 9V, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;

[0121] aaa) 1, 3, 4, 5, 6B, 7F, 8, 9V, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B, and 39; and

[0122] bbb)1, 3, 4, 5, 6C, 7F, 8, 9V, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39.

[0123] In some embodiments, the multivalent immunogenic composition comprises a pneumococcal serotype selected from the group consisting of: i) 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, and 35B; or ii) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, and 35B. or iv) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, and 35B. In a specific embodiment, a multivalent immunogenic composition of the invention comprises a plurality of pneumococcal polysaccharide-protein conjugates, wherein each of the conjugates comprises a polysaccharide from a serotype of pneumococcus conjugated to a carrier protein, wherein the serotype of pneumococcus comprises serotypes: i) 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 34F; 5B; or ii) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; or iii) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B. The compositions were found to be immunogenic in mice, rabbits and / or monkeys and produced functional antibodies that killed vaccine-type bacterial strains at all doses tested.

[0124] The multivalent immunogenic compositions of the invention are suitable for immunizing patients against vaccine-type pneumococcal serotypes and / or for use as part of a treatment regimen with different complementary pneumococcal vaccines. Thus, the invention provides 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 composition of the invention is administered to a patient who has previously been immunized with a different multivalent pneumococcal vaccine.

[0125] In an embodiment of the invention, a conjugate from at least one pneumococcal serotype is prepared using reductive amination in an aprotic solvent such as DMSO. In other embodiments, a multivalent immunogenic composition comprises pneumococcal conjugates, each of which is prepared using reductive amination in an aprotic solvent. The use of DMSO solvent enhances the covalent binding of polysaccharides to proteins by directly consuming lysine residues on the surface of the carrier protein. Increased covalent binding has a direct benefit in increasing the stability of the polysaccharide protein conjugate of the multivalent immunogenic composition comprising a polysaccharide antigen conjugated in DMSO.

[0126] I. Definitions and Abbreviations

[0127] As used throughout the specification and the appended claims, the following abbreviations shall apply:

[0128] APA Aluminum Phosphate Adjuvant

[0129] APC Antigen presenting cell

[0130] CI confidence interval

[0131] DMSO Dimethyl sulfoxide

[0132] DS Polysaccharide-Protein API

[0133] GMC Geometric Mean Concentration

[0134] GMT geometric mean titer

[0135] HPSEC High Performance Size Exclusion Chromatography

[0136] IM intramuscular or intramuscular

[0137] IRM Baby Rhesus Monkey

[0138] LOS Lipo-oligosaccharide

[0139] LPS Lipopolysaccharide

[0140] MALS Multi-angle Light Scattering

[0141] MBC monovalent conjugate raw material

[0142] Mn number average molecular weight

[0143] MOPA Multiplex Opsonophagocytosis Assay

[0144] MW Molecular weight

[0145] NMWCO Nominal molecular weight cutoff

[0146] NZWR New Zealand White Rabbit

[0147] OPA opsonophagocytosis assay

[0148] PCV Pneumococcal conjugate vaccine

[0149] PD1 after 1st dose

[0150] PD2 after 2nd dose

[0151] PD3 after 3rd dose

[0152] PnPs Pneumococcal polysaccharide

[0153] Ps polysaccharide

[0154] PS-20 Polysorbate-20

[0155] RI Refractive Index

[0156] UV

[0157] w / v weight / volume

[0158] In order to make the present invention more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise specifically defined herein, all other technical and scientific terms used herein have the meanings commonly understood by ordinary technicians in the field to which the present invention belongs.

[0159] As used throughout the specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0160] Unless the context clearly indicates one of the indicated possibilities, references to "or" indicate one or both possibilities. In some cases, "and / or" is used to emphasize one or both possibilities.

[0161] The term "aqueous solvent" or "aqueous conditions" when used in conjunction with conjugation (such as reductive amination) refers to the use of water as the solvent for the conjugation reaction. In addition to the absence of organic solvents, water may contain buffers and other components.

[0162] The terms "aprotic solvent", "DMSO solvent" or "DMSO conditions" when used in conjunction with a conjugation (such as reductive amination) refer to the use of an aprotic solvent or a combination of aprotic solvents (or DMSO, if applicable) as the solvent for the conjugation reaction. The aprotic solvent may have some water present, for example up to 1%, 2%, 5%, 10% or 20%.

[0163] The term "comprising" when used with the immunogenic compositions of the present invention refers to the inclusion of any other components, such as adjuvants and excipients, or the addition of one or more polysaccharide-protein conjugates not specifically listed. When used with a multivalent polysaccharide-protein conjugate mixture, the term "consisting of" refers to a mixture with those specific pneumococcal polysaccharide-protein conjugates without other pneumococcal polysaccharide-protein conjugates from different serotypes. "Essentially consisting of" and variants such as "essentially consisting of" and "essentially consisting of" mean that any of the listed elements or groups of elements are included, and optionally include other elements with similar or different properties to the listed elements, which do not substantially change the basic or novel properties of the specified dosage regimen, method or composition.

[0164] An "effective amount" of a composition of the invention is a dose that elicits antibodies that significantly reduces the likelihood or severity of infection by a microorganism (eg, S. pneumoniae) during a subsequent challenge.

[0165] As used herein, the phrase "for the prevention of pneumococcal disease" means that the vaccine or immunogenic composition is approved by one or more regulatory agencies (such as the U.S. Food and Drug Administration) for the prevention of one or more diseases caused by any serotype of S. pneumoniae, including but not limited to: general pneumococcal disease, pneumococcal pneumonia, pneumococcal meningitis, pneumococcal bacteremia, invasive disease caused by S. pneumoniae, and otitis media caused by S. pneumoniae.

[0166] A "multivalent pneumococcal vaccine" is a pharmaceutical preparation comprising more than one active agent (eg, pneumococcal capsular polysaccharide or pneumococcal polysaccharide-protein conjugate) that provides active immunity against diseases or pathological conditions caused by more than one serotype of S. pneumoniae.

[0167] The term "polysaccharide" is intended to include any antigenic sugar element (or antigenic unit) commonly used in the fields of immunization and bacterial vaccines, including but not limited to "saccharides", "oligosaccharides", "polysaccharides", "liposaccharides", "lipooligosaccharides (LOS)", "lipopolysaccharides (LPS)", "glycosylation", "glycoconjugates", etc.

[0168] In the context of the vaccines or immunogenic compositions of the invention, the term "unadjuvanted" refers to pneumococcal polysaccharide compositions, including but not limited to PCV8, PCV15, PCV22, PCV23, and PCV24, wherein the composition does not contain an adjuvant.

[0169] "PCV8" refers to an immunogenic composition containing Streptococcus pneumoniae polysaccharide (PnPs) serotypes -8, -10A, -12F, -15A, -15C, -23B, -24F and -35B.

[0170] "PCV15" refers to an immunogenic composition containing Streptococcus pneumoniae polysaccharide (PnPs) serotypes -1, -3, -4, -5, -6A, -6B, -7F, -9V, -14, -18C, -19A, -19F, -22F, -23F and -33F.

[0171] "PCV22" refers to an immunogenic composition containing Streptococcus pneumoniae polysaccharide (PnPs) serotypes -1, -3, -4, -5, -6A, -6B, -7F, -9V, -10A, -12F, -14, -15A, -15C, -18C, -19A, -19F, -22F, -23B, -23F, -24F, -33F and -35B.

[0172] "PCV23" refers to an immunogenic composition containing Streptococcus pneumoniae polysaccharide (PnPs) serotypes -1, -3, -4, -5, -6A, -6B, -7F, -8, -9V, -10A, -12F, -14, -15A, -15C, -18C, -19A, -19F, -22F, -23B, -23F, -24F, -33F and -35B.

[0173] "PCV24" refers to an immunogenic composition containing Streptococcus pneumoniae polysaccharide (PnPs) serotypes -1, -3, -4, -5, -6A, -6B, -7F, -8, -9V, -10A, -11A, -12F, -14, -15A, -15C, -18C, -19A, -19F, -22F, -23B, -23F, -24F, -33F and -35B.

[0174] "CpG-containing nucleotides," "CpG-containing oligonucleotides," "CpG oligonucleotides," and similar terms refer to nucleotide molecules of 6-50 nucleotides in length that contain an unmethylated CpG moiety. See, e.g., Wang et al., 2003, Vaccine 21:4297. CpG-containing oligonucleotides include modified oligonucleotides using any synthetic internucleoside linkage, modified bases, and / or modified sugars.

[0175] As defined herein, an "adjuvant" is a substance used to enhance the immunogenicity of the immunogenic compositions of the invention. Immunological adjuvants can enhance the immune response to antigens that are poorly immunogenic when administered alone, for example, inducing no or weak antibody titers or cell-mediated immune responses, increasing antibody titers to the antigen, and / or reducing the dose of the antigen that is effective in achieving an immune response in an individual. Thus, adjuvants are typically administered to enhance immune responses and are well known to those skilled in the art.

[0176] "Patient" (alternatively referred to herein as "subject") refers to a mammal capable of being infected by S. pneumoniae. In a preferred embodiment, the patient is a human. The patient can be treated prophylactically or therapeutically. Prophylactic treatment can provide sufficient protective immunity to reduce the likelihood or severity of pneumococcal infection or its effects (e.g., pneumococcal pneumonia). Therapeutic treatment can be performed to reduce the severity of S. pneumoniae infection or its clinical effects or prevent its recurrence. As described herein, the multivalent immunogenic compositions of the present invention can be used for prophylactic treatment. The compositions of the present invention can be administered to the general population or to those at increased risk of pneumococcal infection, such as the elderly, or to those who live with or care for the elderly.

[0177] Those “in need of treatment” include those who have been previously exposed to or infected with S. pneumoniae, those who have been previously vaccinated against S. pneumoniae, and anyone who is susceptible to infection or needs to reduce the likelihood of infection, such as immunocompromised individuals, elderly, children, adults, or healthy individuals.

[0178] A "stable" multivalent immunogenic composition is one that does not change significantly when observed at refrigerated temperatures (e.g., 2-8°C or 4°C) for at least 1 month, 2 months, 3 months, 6 months, 12 months, and / or 24 months. In addition, a "stable" composition includes a composition that exhibits desired characteristics for a period of time including 1 month, 3 months, 6 months, 12 months, and / or 24 months at temperatures including 25°C and 37°C. Typical stability qualification criteria are as follows: the variability of one or more of the following indicators does not exceed about 5%, about 10%, about 15%, or about 20%: (a) the number average molecular weight (Mn) of the pneumococcal polysaccharide-protein conjugate in the composition, (b) the weight average molecular weight (Mw) of the pneumococcal polysaccharide-protein conjugate in the composition, (c) the total polysaccharide concentration in the composition, (d) the emission maximum of the composition measured at a specific excitation wavelength (e.g., 280 nanometers) using intrinsic protein fluorescence spectroscopy, and (e) the fluorescence intensity of the composition measured at a specific excitation wavelength using intrinsic protein fluorescence spectroscopy. The term "stable" may also be used to refer to specific pneumococcal conjugates in multivalent immunogenic compositions. In such use, the term refers to conjugates that exhibit desired properties over time at a specified temperature, and these properties do not vary by more than about 5%, about 10%, about 15%, or about 20% over the indicated time and temperature.

[0179] II. Multivalent immunogenic compositions

[0180] The present invention provides a multivalent immunogenic composition comprising a plurality of Streptococcus pneumoniae polysaccharide protein conjugates, wherein each of the conjugates comprises a polysaccharide from a serotype of Streptococcus pneumoniae conjugated to a carrier protein. Various aspects and embodiments of the multivalent immunogenic composition of the present invention are described below.

[0181] In one embodiment (embodiment E1), the invention provides a multivalent immunogenic composition comprising a plurality of pneumococcal polysaccharide-protein conjugates, each comprising a capsular polysaccharide from a pneumococcal serotype conjugated to a carrier protein, wherein the pneumococcal serotype comprises, consists of, or consists essentially of: i) 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, In a subembodiment of embodiment El, the immunogenic composition does not comprise any other pneumococcal polysaccharide-protein conjugate.

[0182] As used herein, de-O-acetylated serotype 15B (DeOAc15B) pneumococcal polysaccharide is equivalent to the pneumococcal polysaccharide of serotype 15C, and has the same NMR spectrum (data not shown). As used herein, de-O-acetylated serotype 15B pneumococcal polysaccharide and serotype 15C pneumococcal polysaccharide have an O-acetyl content in each repeating unit of each 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 do not contain O-acetyl. In the report of Spencer BL et al., 15C can be slightly O-acetylated (Spencer, BL et al., Clin. Vac. Immuno. (2017) 24 (8): 1-13). Thus, in any embodiment of the multivalent immunogenic composition herein, de-O-acetylated serotype 15B (DeOAc15B) can be used instead of serotype 15C. Methods for de-O-acetylation are well known in the art, for example, as described in Rajam et al., Clinical and Vaccine Immunology, 2007, 14(9): 1223–1227.

[0183] In certain embodiments of any of the multivalent immunogenic compositions of the invention, including embodiment El and any subembodiments thereof, the composition further comprises a pharmaceutically acceptable carrier.

[0184] Cross-reactivity

[0185] In one embodiment, the present invention provides a multivalent immunogenic composition comprising a pneumococcal polysaccharide-protein conjugate, wherein each of the conjugates comprises a polysaccharide from a pneumococcal serotype (including serotype 6C) conjugated to a carrier protein, wherein serotype 6C of pneumococcus provides cross-reactivity against serotypes 6A and 6B of pneumococcus.

[0186] In one embodiment, the present invention provides a multivalent immunogenic composition comprising a pneumococcal polysaccharide-protein conjugate, wherein each of the conjugates comprises a polysaccharide from a pneumococcal serotype (including serotype 6A) conjugated to a carrier protein, wherein serotype 6A of pneumococcus provides cross-protection against serotypes 6B and / or 6C of pneumococcus.

[0187] In one embodiment, the present invention provides a multivalent immunogenic composition comprising a pneumococcal polysaccharide-protein conjugate, wherein each of the conjugates comprises a polysaccharide from a pneumococcal serotype (including serotype 6B) conjugated to a carrier protein, wherein serotype 6B of pneumococcus provides cross-protection against serotypes 6A and / or 6C of pneumococcus.

[0188] In one embodiment, the present invention provides a multivalent immunogenic composition comprising a pneumococcal polysaccharide-protein conjugate, wherein each of the conjugates comprises a polysaccharide from a pneumococcal serotype (including serotype 15C) conjugated to a carrier protein, wherein serotype 15C of pneumococcus provides cross-protection against serotype 15B of pneumococcus.

[0189] In one embodiment, the present invention provides a multivalent immunogenic composition comprising a pneumococcal polysaccharide-protein conjugate, wherein each of the conjugates comprises a polysaccharide from a pneumococcal serotype (including serotype 15B) conjugated to a carrier protein, wherein serotype 15B of pneumococcus provides cross-protection against serotype 15C of pneumococcus.

[0190] Carrier Protein

[0191] In a specific embodiment of the 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:

[0192]

[0193] In one embodiment, CRM197 is isolated from a culture of Corynebacterium diphtheria strain C7 (β197) grown in a casamino acid and yeast extract-based medium. In another embodiment, CRM197 is recombinantly prepared according to the method described in U.S. Patent No. 5,614,382. Generally, CRM197 is purified by a combination of ultrafiltration, ammonium sulfate precipitation, and ion exchange chromatography. In some embodiments, CRM197 is purified using Pfenex Expression Technology TM (Pfenex Inc., San Diego, CA) was produced in Pseudomonas fluorescens.

[0194] Other suitable carrier proteins include additional inactivated bacterial toxins such as DT (diphtheria toxoid) or fragment B of DT (DTFB), TT (tetanus toxin) or fragment C of TT, pertussis toxoid, cholera toxoid (e.g., as described in WO 2004 / 083251), E. coli LT, E. coli ST and exotoxin A from Pseudomonas aeruginosa. Bacterial outer membrane proteins such as the outer membrane complex (OMPC), porins, transferrin binding proteins, pneumococcal surface protein A (PspA; see WO 02 / 091998), pneumococcal adhesin protein (PsaA), C5a peptidase from group A or group B streptococci, or Haemophilus influenzae protein D, pneumolysin (Kuo et al., 1995, Infect Immun 63; 2706-13) including ply detoxified in some form, such as dPLY-GMBS (see WO 04 / 081515) or dPLY-formaldehyde, PhtX including PhtA, PhtB, PhtD, PhtE and fusions of Pht proteins, such as PhtDE fusions, PhtBE fusions (see WO 01 / 98334 and WO 03 / 54007) can also be used. Other proteins such as ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or tuberculin purified protein derivative (PPD), PorB (from Neisseria meningitidis), PD (Haemophilus influenzae protein D; see, e.g., EP 0 594 610B) or immunologically functional equivalents thereof, synthetic peptides (see EP 0 378 881 and EP 0 427 347), heat shock proteins (see WO 93 / 17712 and WO 94 / 03208), pertussis proteins (see WO 98 / 58668 and EP 0 471 177), cytokines, lymphokines, growth factors or hormones (see WO 91 / 01146), artificial proteins comprising multiple human CD4+ T cell epitopes from antigens derived from various pathogens (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), toxin A or B of Clostridium difficile (C. difficile) (see WO 00 / 61761), and flagellin (see Ben-Yedidia et al., 1998, Immunol Lett 64:9) can also be used as carrier proteins.

[0195] Other DT mutants can be used as carrier proteins, such as CRM176, CRM228, CRM45 (Uchida et al., 1973, J Biol Chem 218:3838-3844); CRM9, CRM45, CRM102, CRM103 and CRM107, and the one described by Nicholls and Youle in Genetically=Engineered=Toxins , Ed: Frankel, Maecel Dekker Inc, 1992; Glu-148 to Asp, Gln or Ser and / or Ala 158 to Gly deletion or mutation and other mutations disclosed in US 4,709,017 or US 4,950,740; at least one or more residues Lys 516, Lys 526, Phe 530 and / or Lys 534 mutations and other mutations disclosed in US 5,917,017 or US Pat. No. 6,455,673; or fragments disclosed in US 5,843,711. Such DT mutants can also be used to prepare DTFB variants, wherein the variant comprising the B fragment comprises the epitope region.

[0196] 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.

[0197] In some embodiments of the invention, a second carrier can be used for one or more polysaccharide-protein conjugates in a multivalent immunogenic composition. The second carrier protein is preferably a protein that is non-toxic and non-reactive and can be obtained in sufficient quantity and purity. The second carrier protein is also conjugated or combined with Streptococcus pneumoniae polysaccharide to enhance the immunogenicity of the antigen. The carrier protein should comply with standard conjugation procedures. In one embodiment, each capsular polysaccharide that is not conjugated to the first carrier protein is conjugated to the same second carrier protein (e.g., each capsular polysaccharide molecule is conjugated to a single carrier protein). In another embodiment, the capsular polysaccharide that is not conjugated to the first carrier protein is conjugated to two or more carrier proteins (each capsular polysaccharide molecule is conjugated to a single carrier protein). In such embodiments, each capsular polysaccharide of the same serotype is typically conjugated to the same carrier protein.

[0198] In an embodiment of the invention, including embodiment E1 and any sub-embodiment thereof, one or more (including 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more, as applicable) polysaccharide serotypes are conjugated to CRM197. In a further embodiment of the invention, including embodiment E1 and any sub-embodiment thereof, each polysaccharide serotype is conjugated to CRM197.

[0199] The preparation of the polysaccharide-protein conjugates of the present invention can be accomplished using methods recognized in the art. For example, a single pneumococcal conjugate can be prepared using a physiologically acceptable carrier to prepare a composition. Examples of such carriers include, but are not limited to, water, buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), and dextrose solutions.

[0200] In a preferred embodiment, the vaccine composition is formulated in an L-histidine buffer containing sodium chloride.

[0201] In some embodiments of the invention, a multivalent immunogenic composition comprises a plurality of pneumococcal polysaccharide protein conjugates and an adjuvant, wherein the conjugate comprises a capsular polysaccharide from a pneumococcal serotype conjugated to a carrier protein, wherein the pneumococcal serotype is as described herein. Suitable adjuvants that enhance the effectiveness of the composition include, but are not limited to:

[0202] (1) Aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc.;

[0203] (2) Oil-in-water emulsion formulations (with or without other specific immunostimulants, such as muramyl peptides (defined below) bacterial cell wall components), such as, for example, (a) MF59 (International Patent Application Publication No. WO 90 / 14837), containing 5% squalene, 0.5% Tween 80 and 0.5% Span 85 (optionally containing varying amounts of MTP-PE), formulated into submicron particles using a microfluidizer, such as a model 110Y Microfluidizer (Microfluidics, Newton, MA), (b) SAF, containing 10% squalene, 0.4% Tween 80, 5% Pluronic block polymer L121, and thr-MDP, microfluidized into submicron emulsions or vortexed to produce emulsions with larger particle sizes, (c) Ribi TM Adjuvant system (RAS) (Corixa, Hamilton, MT) containing 2% squalene, 0.2% Tween 80 and one or more bacterial cell wall components derived from 3-O-deacylated monophosphoryl lipid A (MPL) as described in U.S. Pat. No. 4,912,094. TM ), trehalose dimycolic acid (TDM) and cell wall scaffold (CWS), preferably MPL + CWS (Detox TM ); and (d) Montanide ISA;

[0204] (3) Saponin adjuvants such as Quil A or STIMULON can be used TMQS-21 (Antigenics, Framingham, MA) (see, e.g., U.S. Pat. No. 5,057,540) or particles generated therefrom, such as ISCOM (immunostimulatory complexes formed by a combination of cholesterol, saponin, phospholipids, and amphiphilic proteins) and (having substantially the same structure as ISCOM but without the protein);

[0205] (4) bacterial lipopolysaccharide, synthetic lipid A analogs, such as aminoalkylglucosamine phosphate compounds (AGP) or derivatives or analogs thereof, which are available from Corixa and described in U.S. Pat. No. 6,113,918; one such AGP is 2-[(R)-3-tetradecanoyloxytetradecanoylamino]ethyl 2-deoxy-4-O-phosphono-3-O-[(R)-3-tetradecanoyloxytetradecanoyl]-2-[(R)-3-tetradecanoyloxytetradecanoylamino]-β-D-pyranoglucoside, also known as 529 (formerly RC529), which is formulated in the form of an aqueous solution or a stable emulsion;

[0206] (5) synthetic polynucleotides, such as oligonucleotides containing one or more CpG motifs (U.S. Pat. No. 6,207,646);

[0207] (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.), interferons (e.g., interferon-γ), granulocyte macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), tumor necrosis factor (TNF), costimulatory molecules B7-1 and B7-2, etc.; and

[0208] (7) Complement, such as a trimer of the complement component C3d.

[0209] In another embodiment, the adjuvant is a mixture of 2, 3 or more of the above adjuvants, such as SBAS2 (an oil-in-water emulsion also containing 3-deacylated monophosphoryl lipid A and QS21).

[0210] Muramyl peptides include, but are not limited to, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-n-muramyl-L-alanine-2-(1'-2'dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE), and the like.

[0211] In certain embodiments, the adjuvant is an aluminum salt. An aluminum salt adjuvant can be a vaccine precipitated with alum or a vaccine adsorbed with alum. Aluminum salt adjuvants are well known in the art and are described, for example, in Harlow, E. and D. Lane (1988; Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory) and Nicklas, W. (1992; Aluminum salts. Research in Immunology 143: 489-493). Aluminum salts include, but are not limited to, hydrated alumina, hydrated alumina, trihydrated alumina (ATH), hydrates of aluminum, trihydrates of aluminum, aluminum gel, Superfos, Amphogel, aluminum hydroxide (III), hydroxyaluminum phosphate sulfate (aluminum phosphate adjuvant (APA)), amorphous alumina, trihydrated alumina or trihydroxyaluminum.

[0212] APA is an aqueous suspension of aluminum hydroxyphosphate. APA is prepared by blending aluminum chloride and sodium phosphate in a 1:1 volume ratio to precipitate aluminum hydroxyphosphate. After the blending process, the material is reduced in size using a high shear mixer to achieve a monodisperse particle size distribution. The product is then diafiltered with saline and steam sterilized. In one embodiment, the dosage of the aluminum salt is 10, 15, 20, 25, 30, 50, 70, 100, 125, 150, 200, 300, 500 or 700 μg, or 1, 1.2, 1.5, 2, 3, 5 mg or more. In another embodiment, the dosage of the aluminum salt described above is per μg of recombinant protein.

[0213] In certain embodiments, commercially available Al(OH)3 (e.g., Denmark / Accurate Chemical and Scientific Co., Westbury, NY's Alhydrogel or Superfos) is used to adsorb protein at a ratio of 50-200 μg protein / mg aluminum hydroxide. In another embodiment, the adsorption of protein depends on the pI (isoelectric point pH) of the protein and the pH of the medium. Proteins with lower pI are more strongly adsorbed to positively charged aluminum ions than proteins with higher pI. Aluminum salts can establish antigen reservoirs that are slowly released over a period of 2-3 weeks, involving nonspecific activation of macrophages and complement activation, and / or stimulating innate immune mechanisms (possibly through stimulation of uric acid). See, e.g., See, eg, Lambrecht et al., 2009, Curr Opin Immunol 21:23.

[0214] Typically, the monovalent aqueous conjugate stocks are blended together and diluted to a target of 4 μg / mL for all serotypes except 6B, which will be diluted to a target of 8 μg / mL. Once diluted, the batch is filter sterilized and an equal volume of aluminum phosphate adjuvant is added to a target final aluminum concentration of 250 μg / mL. The adjuvanted, formulated batch will be filled into single-use 0.5 mL / dose vials.

[0215] In certain embodiments, the adjuvant is a CpG-containing nucleotide sequence, such as a CpG-containing oligonucleotide, particularly a CpG-containing oligodeoxynucleotide (CpG ODN). In another embodiment, the adjuvant is ODN 1826, which can be obtained from Coley Pharmaceutical Group.

[0216] 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.

[0217] In alternative embodiments, the immunogenic composition comprises a plurality of S. pneumoniae polysaccharide-protein conjugates as described herein, for example, in embodiment El or any subembodiment thereof, and does not comprise an adjuvant.

[0218] preparation

[0219] The compositions of the present invention may be formulated as single-dose vials, multi-dose vials or pre-filled glass or plastic syringes.

[0220] In another embodiment, the composition of the present invention is administered orally, and is thus formulated in a form suitable for oral administration, i.e., as a solid or liquid preparation. Solid oral preparations include tablets, capsules, pills, granules, pellets, etc. Liquid oral preparations include solutions, suspensions, dispersions, emulsions, oils, etc.

[0221] Pharmaceutically acceptable carriers for liquid preparations are water 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 carriers include water, alcohol / aqueous solutions, emulsions or suspensions, including saline and buffered media. Examples of oils include those of animal, plant or synthetic origin, such as peanut oil, soybean oil, olive oil, sunflower oil, cod liver oil, other marine oils or fat from milk or eggs.

[0222] The multivalent immunogenic compositions of the present invention may be isotonic, hypotonic or hypertonic. However, when administered, it is generally preferred that the composition for infusion or injection is substantially isotonic. Thus, for storage, the composition may preferably be isotonic or hypertonic. If the composition is hypertonic for storage, it may be diluted to become an isotonic solution prior to administration.

[0223] Isotonic agents can be ionic isotonic agents (such as salts) or non-ionic isotonic agents (such as sugars). Examples of ionic isotonic agents include, but are not limited to, NaCl, CaCl2, KCl, and MgCl2. Examples of non-ionic isotonic agents include, but are not limited to, mannitol, sorbitol, and glycerol.

[0224] It is also preferred that at least one pharmaceutically acceptable additive is a buffer. For certain purposes, for example, when the pharmaceutical composition is intended for infusion or injection, it is generally desirable that the composition comprises a buffer 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.

[0225] The buffer may, for example, be selected from TRIS, acetate, glutamate, lactate, maleate, tartrate, phosphate, citrate, carbonate, glycinate, histidine, glycine, succinate and triethanolamine buffers.

[0226] The buffer may additionally be selected, for example, from a USP compatible buffer for parenteral use, particularly when the pharmaceutical formulation is for parenteral use. For example, the buffer may be selected from monoacids such as acetic acid, benzoic acid, gluconic acid, glyceric acid and lactic acid; diacids such as aconitic acid, adipic acid, ascorbic acid, carbonic acid, glutamic acid, malic acid, succinic acid and tartaric acid, polyacids such as citric acid and phosphoric acid; and bases such as ammonium, diethanolamine, glycine, triethanolamine and TRIS.

[0227] Parenteral vehicles (for subcutaneous, intravenous, intra-arterial or intramuscular injection) include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer and fixed oil. Intravenous vehicles include liquid and nutritional supplements, electrolyte supplements such as those based on Ringer's dextrose, etc. Examples are sterile liquids such as water and oil, with or without surfactants and other pharmaceutically acceptable adjuvants. In general, water, saline, aqueous dextrose and related sugar solutions, glycols such as propylene glycol or polyethylene glycol, polysorbate 80 (PS-80), polysorbate 20 (PS-20) and poloxamer 188 (P188) are preferred liquid carriers, particularly for injectable solutions. The example of oil is those of animal, plant or synthetic origin, such as peanut oil, soybean oil, olive oil, sunflower oil, cod liver oil, other marine oils or lipids from milk or eggs.

[0228] The preparation of the present invention may also contain a surfactant. Preferred surfactants include, but are not limited to: polyoxyethylene sorbitan ester surfactants (commonly known as Tween), especially PS-20 and PS-80; TM Copolymers of ethylene oxide (EO), propylene oxide (PO) and / or butylene oxide (BO), such as linear EO / PO block copolymers, sold under the trade names TETRAPOL®; octoxynol, which may vary in the number of repeating ethoxy (oxy-1,2-ethanediyl) groups, with octoxynol-9 (Triton X-100, or tert-octylphenoxypolyethoxyethanol) being particularly advantageous; (octylphenoxy)polyethoxyethanol (IGEPAL CA-630 / NP-40); phospholipids such as phosphatidylcholine (lecithin); nonylphenol ethoxylates such as Tergitol TM NP series; polyoxyethylene fatty ethers derived from lauryl alcohol, cetyl alcohol, stearyl alcohol and oleyl alcohol (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.

[0229] Mixtures of surfactants may be used, such as a Tween 80 / Span 85 mixture. Combinations of polyoxyethylene sorbitan esters, such as polyoxyethylene sorbitan monolaurate (PS-80), and octoxynol, such as tert-octylphenoxypolyethoxyethanol (Triton X-100), are also suitable. Another useful combination comprises laureth 9 plus polyoxyethylene sorbitan esters and / or octoxynol.

[0230] Preferred amounts of surfactants (in % by weight) are: polyoxyethylene sorbitan esters (e.g. PS-80) 0.01-1%, especially about 0.1%; octyl or nonylphenoxy polyoxyethanol (e.g. Triton X-100, or other detergents in the Triton series) 0.001 to 0.1%, especially 0.005 to 0.02%; polyoxyethylene ethers (e.g. laureth 9) 0.1 to 20%, preferably 0.1 to 10%, and especially 0.1 to 1% or about 0.5%.

[0231] In certain embodiments, the composition consists essentially of histidine (20 mM), saline (150 mM), and 0.2% PS-20 at pH 5.8 and 250 μg / mL of APA (aluminum phosphate adjuvant). The range of PS-20 can be from 0.005% to 0.3% (w / v). In another embodiment, the range of PS-20 can be from 0.025% to 0.8% (w / v). In another embodiment, the range of PS-20 can be from 0.05% to 0.8% (w / v). In another embodiment, the range of PS-20 can be from 0.05% to 0.2% (w / v). The method comprises mixing a mixture of up to 24 serotypes in histidine, saline, and PS-20, and then mixing this mixture with APA and saline, with or without the addition of an antimicrobial preservative.

[0232] In a specific embodiment, the multivalent immunogenic composition comprises a polysaccharide protein conjugate of Streptococcus pneumoniae, wherein each of the conjugates comprises a polysaccharide from a serotype of Streptococcus pneumoniae conjugated to a carrier protein, wherein the serotype of Streptococcus pneumoniae in the polysaccharide protein conjugate comprises any serotype group described herein, and further comprises 20-80 mM histidine (pH 5.8) and 150 mM NaCl. In some embodiments, the multivalent immunogenic composition further comprises 0.2% to 0.8% w / v polysorbate 20.

[0233] The multivalent immunogenic composition PCV24 was conjugated to the CRM197 protein to the pneumococcal polysaccharide (PnPs) serotypes -1, -3, -4, -5, -6A, -6B, -7F, -8, -9V, -10A, -11A, -12F, -14, -15A, -15C, -18C, -19A, -19F, -22F, -23B, -23F, -24F, -33F and -35B, respectively, by reductive amination in an aprotic solvent (also known as DMSO chemistry) and in 20 mM L-histidine pH 5.8, 150 mM NaCl and 0.1% w / v polysorbate-20 (PS-20), with 4 μg / mL or 8 μg / mL of each polysaccharide serotype, and a total polysaccharide concentration of 96 μg / mL or 192 μg / mL, respectively, and referred to as "PCV24 without adjuvant". In another specific embodiment, the multivalent immunogenic composition PCV24 is prepared in 20 mM L-histidine pH 5.8, 150 mM NaCl and 0.2% w / v polysorbate-20 (PS-20), with 4 μg / mL of each polysaccharide serotype, and a total polysaccharide concentration of 96 μg / mL, and also includes 250 μg [Al] / mL in the form of aluminum phosphate adjuvant. It is referred to as "PCV24 / APA".

[0234] The choice of surfactant may need to be optimized for different formulations and APIs. For multivalent vaccines containing 15 or more serotypes, PS-20 and P188 are preferred. The choice of chemical method for preparing the conjugate will also affect the stability of the formulation. In particular, when the conjugation reaction used to prepare different polysaccharide-protein conjugates in the multivalent composition includes both aqueous solvents and DMSO solvents, there are significant differences in the stability of the specific surfactant system. Improved stability of the polysaccharide-protein conjugates can be seen when polysorbate 20 is used alone or when poloxamer 188 is used in combination with polyols.

[0235] The exact mechanism of how a particular detergent protects a biotherapeutic is poorly understood and cannot be predicted a priori. Possible stabilization mechanisms include preferential hydration, preferential exclusion, air / liquid interface competition between the biotherapeutic and the surface, surface tension, and / or direct association of the detergent with the biotherapeutic to mask hydrophobic clumps that serve as seeds for aggregation.

[0236] Poloxamers may also be used in the compositions of the present invention. Poloxamers are nonionic triblock copolymers consisting of a central hydrophobic chain of polyoxypropylene (polypropylene oxide) connected to two hydrophilic chains of polyoxyethylene (polyethylene oxide). Poloxamers are also known by the trade name Because the length of the polymer blocks can be tailored, there are many different poloxamers with slightly different properties. For the generic name "poloxamers", these copolymers are usually named with the letter "P" (Poloxamer) followed by three numbers, the first two numbers x 100 indicating the approximate molecular weight of the polyoxypropylene core, and the last number x10 indicating the percentage of polyoxyethylene content (e.g., P407 = poloxamer with a polyoxypropylene molecular weight of 4,000 g / mol and a polyoxyethylene content of 70%). Trade names for these copolymers begin with a letter that defines their physical form at room temperature (L = liquid, P = paste, F = flakes (solid)), followed by a two- or three-digit number. The first digit in the numerical designation (two of the three digits) multiplied by 300 indicates the approximate molecular weight of the hydrophobe; the last digit x 10 indicates the percentage of polyoxyethylene content (e.g., The molecular weight of polyoxypropylene is 1,800 g / mol and the content of polyoxyethylene is 10%). See U.S. Pat. No. 3,740,421.

[0237] Examples of poloxamers have the following general formula:

[0238] HO(C2H4O) a (C3H6O) b (C2H4O) aH, where the a and b blocks have the following values:

[0239]

[0240] As used herein, molecular weight ranges are in Daltons (Da) or g / mol.

[0241] Preferably, the molecular weight of the poloxamer is generally 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 the poloxamer in the formulation is 0.001% to 5% weight / volume, or 0.025% to 1% weight / volume. In certain aspects, the polyol is propylene glycol, and the final concentration is 1% to 20% weight / volume. In certain aspects, the polyol is polyethylene glycol 400, and the final concentration is 1% to 20% weight / volume.

[0242] Suitable polyols for the formulations of the present invention are polymeric polyols, particularly polyether glycols, including but not limited to propylene glycol and polyethylene glycol, polyethylene glycol monomethyl ether. The monomer molecular weight range of propylene glycol is from 425 to 2,700. Polyethylene glycol and polyethylene glycol monomethyl ether with molecular weights of 200 to 35,000 can also be obtained, including but not limited to PEG200, PEG300, PEG400, PEG1000, PEG MME 550, PEG MME 600, PEG MME 2000, PEG MME 3350 and PEGMME4000. A preferred polyethylene glycol is polyethylene glycol 400. The final concentration of the polyol in the formulations of the present invention can be from 1% to 20% weight / volume or from 6% to 20% weight / volume.

[0243] The preparation also includes a pH buffered saline solution. The buffer can be, for example, selected from Tris, acetate, glutamate, lactate, maleate, tartrate, phosphate, citrate, carbonate, glycinate, L-histidine, glycine, succinate, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), MOPS (3-(N-morpholino) propanesulfonic acid), MES (2-(N-morpholino) ethanesulfonic acid) and triethanolamine buffer. The buffer can buffer the solution to a pH of 4 to 10, 5.2 to 7.5 or 5.8 to 7.0. In some aspects of the present invention, the buffer is selected from phosphate, succinate, L-histidine, MES, MOPS, HEPES, acetate or citrate. The buffer can also be, for example, selected from USP compatible buffers for parenteral use, particularly when the pharmaceutical preparation is used for parenteral use. The concentration range of the buffer will be from 1mM to 100mM. The concentration range of the buffer will be from 10mM to 80mM. The concentration range of the buffer will be from 1mM to 50mM or 5mM to 50mM. In some aspects, the buffer is histidine at a final concentration of 5mM to 50mM, or succinic acid at a final concentration of 1mM to 10mM. In some aspects, the final concentration of histidine is 20mM ± 2mM.

[0244] Although saline (i.e., solutions containing NaCl) are preferred, other suitable salts for formulation include, but are not limited to, CaCl2, KCl, and MgCl2 and combinations thereof. Nonionic isotonic agents, including but not limited to sucrose, trehalose, mannitol, sorbitol, and glycerol, may be used in place of salt. Suitable salt ranges include, but are not limited to, 25 mM to 500 mM or 40 mM to 170 mM. In one aspect, the saline is NaCl, optionally present at a concentration of 20 mM to 170 mM.

[0245] In a preferred embodiment, the formulation comprises L-histidine buffer and sodium chloride.

[0246] In another embodiment, the pharmaceutical composition is delivered in a controlled release system. For example, the agent can be administered using intravenous infusion, transdermal patches, liposomes, or other modes of administration. In another embodiment, a polymeric material is used; for example, in a microsphere or in an implant.

[0247] The amount of conjugate in each vaccine dose is selected to be an amount that induces an immunoprotective response without significant adverse effects. This amount can vary depending on the pneumococcal serotype. In general, for polysaccharide-based conjugates, each dose will contain 0.08 to 100 μg of each polysaccharide. In some embodiments of the invention, the dose of each polysaccharide conjugate is from 0.08 to 10 μg. In further embodiments, the dose is from 1 to 5 μg, from 0.4 to 4 μg, from 0.4 to 3 μg, from 0.4 to 2 μg or from 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 μg.

[0248] In some embodiments of the compositions of the invention, all polysaccharide conjugates are present in the same amount in the composition. In further embodiments, the polysaccharide conjugates are present in different amounts in the composition (i.e., at least one polysaccharide conjugate is present in an amount different from one or more other polysaccharide conjugates in the composition).

[0249] The optimal amounts of ingredients for a particular vaccine can be determined by standard studies involving observation of an appropriate immune response in a subject. For example, in another embodiment, the dosage for human immunization is determined by extrapolation from animal studies to human data. In another embodiment, the dosage is determined empirically.

[0250] The compositions of the 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 Publication Nos. WO 02 / 083855 and WO 02 / 053761.

[0251] In certain embodiments, the compositions of the present invention are administered to a subject by one or more methods well known to those skilled in the art (such as parenteral, transmucosal, transdermal, intramuscular, intravenous, intradermal, intranasal, subcutaneous, intraperitoneal), and are prepared accordingly. In one embodiment, the compositions of the present invention are administered by epidermal injection, intramuscular injection, intravenous, intraarterial, subcutaneous injection or intrarespiratory mucosal injection of a liquid preparation. Liquid preparations for injection include solutions etc.

[0252] III. Preparation method

[0253] Capsular polysaccharides from Streptococcus pneumoniae can be prepared by standard techniques known to those skilled in the art. For example, polysaccharides can be isolated from bacteria, and polysaccharide size can be adjusted to a certain extent by known methods (see, for example, European Patent Nos. EP497524 and EP497525); and preferably by microfluidization using a homogenizer or chemical hydrolysis. In one embodiment, each pneumococcal polysaccharide serotype is grown in a soybean-based culture medium. Various polysaccharides are then purified by standard steps (including centrifugation, precipitation and ultrafiltration). See, for example, U.S. Patent Application Publication No. 2008 / 0286838 and U.S. Patent No. 5,847,112. Polysaccharides can be sized using techniques such as mechanical or chemical size adjustment to reduce viscosity and / or improve the filterability of the conjugated product. Chemical hydrolysis can be performed using acetic acid. Mechanical size adjustment can be performed using high-pressure homogenization shearing.

[0254] The purified polysaccharide can be chemically activated to enable the polysaccharide to react with the carrier protein. The purified polysaccharide can be connected to a linker. Once activated or connected to a linker, each capsular polysaccharide is conjugated to a carrier protein to form a glycoconjugate. The polysaccharide conjugate can be prepared by known coupling techniques.

[0255] The polysaccharide can be coupled to a joint to form a polysaccharide-joint intermediate, wherein the free end of the joint is an ester group. Therefore, a joint is a joint in which at least one end is an ester group. The other end is selected so that it can react with the polysaccharide to form a polysaccharide-joint intermediate.

[0256] The polysaccharide can be coupled to the joint using the primary amine groups in the polysaccharide. In this case, the joint usually has an ester group at both ends. This allows one of the ester groups to react with the primary amine groups in the polysaccharide to be coupled by nucleophilic acyl substitution. The reaction produces a polysaccharide-joint intermediate, in which the polysaccharide is coupled to the joint by an amide bond. Therefore, the joint is a bifunctional joint, which provides a first ester group for reacting with the primary amine groups in the polysaccharide and a second ester group for reacting with the primary amine groups in the carrier molecule. A typical joint is adipic acid N-hydroxysuccinimide diester (SIDEA).

[0257] The coupling may also take place indirectly, ie via an additional linker used to derivatize the polysaccharide prior to coupling to the linker.

[0258] The polysaccharide is coupled to another joint using a carbonyl group at the reducing end of the polysaccharide. The coupling comprises two steps: (a1) reacting the carbonyl group with another joint; and (a2) reacting the free end of another joint with the joint. In these embodiments, the other joint generally has a primary amine group at both ends, thereby allowing step (a1) to react one of the primary amine groups with the carbonyl group in the polysaccharide by reductive amination. A primary amine group that reacts with the carbonyl group in the polysaccharide is used. Hydrazide or hydroxylamino are suitable. The same primary amine group is generally present at both ends of another joint. The reaction produces a polysaccharide-another joint intermediate, in which the polysaccharide is coupled to another joint by a CN bond.

[0259] The polysaccharide can be coupled to another joint using different groups, particularly carboxyl groups, in the polysaccharide. The coupling includes two steps: (a1) reacting the group with another joint; and (a2) reacting the free end of another joint with the joint. In this case, the other joint generally has a primary amine group at both ends, thereby allowing step (a1) to react one of the primary amine groups with the carboxyl group in the polysaccharide by EDAC activation. The primary amine group reacting with the carboxyl group activated by EDAC in the polysaccharide is used. Hydrazide groups are suitable. The same primary amine group is generally present at both ends of another joint. The reaction produces a polysaccharide-other joint intermediate, in which the polysaccharide is coupled to another joint by an amide bond.

[0260] In one embodiment, chemical activation of the polysaccharide and subsequent conjugation to the carrier protein by reductive amination can be achieved by methods described in U.S. Patent Nos. 4,365,170, 4,673,574 and 4,902,506, U.S. Patent Application Publication Nos. 2006 / 0228380, 2007 / 184072, 2007 / 0231340 and 2007 / 0184071, and International Patent Application Publication Nos. WO2006 / 110381, WO2008 / 079653 and WO2008 / 143709. The chemical reaction may include activating the pneumococcal polysaccharide by reacting with any oxidizing agent such as periodate (including sodium periodate, potassium periodate or periodic acid) that oxidizes the terminal hydroxyl groups to aldehydes. The reaction results in random oxidative cleavage of the ortho-hydroxyl groups of the carbohydrate to form reactive aldehyde groups.

[0261] The lysyl group of protein is coupled to the carrier protein by direct amination reduction. For example, the mixture of activated polysaccharide and carrier protein is reacted with a reducing agent such as sodium cyanoborohydride in the presence of nickel for conjugation. The conjugation reaction can be carried out in the presence of an aqueous solution or dimethyl sulfoxide (DMSO). See, for example, US2015 / 0231270, US2011 / 0195086 and EP 0471177B1. The unreacted aldehyde is then capped by adding a strong reducing agent (such as sodium borohydride).

[0262] Reductive amination involves two steps, (1) oxidation of the polysaccharide to form a reactive aldehyde, and (2) reduction of the imine (Schiff base) formed between the activated polysaccharide and the carrier protein to form a stable amine conjugate bond. Prior to oxidation, the polysaccharide is optionally size-reduced. Mechanical methods (e.g., homogenization) or chemical hydrolysis may be employed. Chemical hydrolysis may be performed using acetic acid. The oxidation step may involve reaction with a periodate. For purposes of the present invention, the term "periodate" includes periodate and periodic acid; the term also includes metaperiodate (104 - ) and orthoperiodate (IO6 - ), and includes various salts of periodate (e.g., sodium periodate and potassium periodate). In one embodiment, the capsular polysaccharide is oxidized in the presence of metaperiodate, preferably sodium periodate (NaIO4). In another embodiment, the capsular polysaccharide is oxidized in the presence of orthoperiodate, preferably periodic acid.

[0263] In one embodiment, the oxidant is a stable nitroxyl or nitroxide free radical compound, such as a piperidine-N-oxyl or pyrrolidine-N-oxyl compound, which selectively oxidizes the primary hydroxyl group in the presence of the oxidant (as described in WO2014 / 097099). In the reaction, in the catalytic cycle, the actual oxidant is an N-oxoammonium salt. In one aspect, the stable nitroxyl or nitroxide free radical compound is a piperidine-N-oxyl or pyrrolidine-N-oxyl compound. In one aspect, the stable nitroxyl or nitroxide free radical compound carries a TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxy) or PROXYL (2,2,5,5-tetramethyl-1-pyrrolidinyloxy) moiety. In one aspect, the stable nitroxyl free radical compound is TEMPO or a derivative thereof. In one aspect, the oxidant is a molecule with an N-halogenated moiety. In one aspect, the oxidant is selected from N-chlorosuccinimide, N-bromosuccinimide, N-iodosuccinimide, dichloroisocyanuric acid, 1,3,5-trichloro-1,3,5-triazinane-2,4,6-trione, dibromoisocyanuric acid, 1,3,5-tribromo-1,3,5-triazinane-2,4,6-trione, diiodoisocyanuric acid and 1,3,5-triiodo-1,3,5-triazinane-2,4,6-trione. Preferably, the oxidant is N-chlorosuccinimide.

[0264] In certain aspects, the oxidant is 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) free radical and N-chlorosuccinimide (NCS) as a co-oxidant (as described in WO2014 / 097099). Thus, in one aspect, a saccharide conjugate from Streptococcus pneumoniae can be obtained by a method comprising the following steps: a) reacting a saccharide with 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) and N-chlorosuccinimide (NCS) in an aqueous solvent to produce an activated saccharide; and b) reacting the activated saccharide with a carrier protein comprising one or more amine groups (the method is hereinafter referred to as "TEMPO / NCS-reductive amination").

[0265] Optionally, the oxidation reaction is quenched by adding a quencher. The quencher can be selected from vicinal diols, 1,2-amino alcohols, amino acids, glutathione, sulfites, bisulfates, dithionites, metabisulfites, thiosulfates, phosphites, hypophosphites or phosphorous acids (such as glycerol, ethylene glycol, propane-1,2-diol, butane-1,2-diol or butane-2,3-diol, ascorbic acid).

[0266] In certain embodiments, the present invention provides a method for preparing a serotype 8 pneumococcal polysaccharide-protein conjugate using a conjugation reaction in an aprotic solvent, wherein the conjugation reaction does not use cyanoborohydride. In a further embodiment, the conjugation reaction is a Schiff base reduction or a reductive amination. In a further embodiment, the protein is a tetanus toxoid, a diphtheria toxoid, or CRM197. In yet another further embodiment, the protein is CRM197. In a further embodiment, the conjugation reaction is a reductive amination. In a further embodiment, the reductive amination is performed in dimethyl sulfoxide (DMSO).

[0267] In some embodiments, the molecular weight of the oxidized polysaccharide before conjugation is between 30kDa and 1,000kDa. The molecular weight can be calculated by size exclusion chromatography (SEC) in combination with a multi-angle light scattering detector (MALS) and a refractive index detector (RI). In some embodiments, the molecular weight of the polysaccharide is between 50kDa and 300kDa. In some embodiments, the molecular weight of the polysaccharide is between 50kDa and 1,000kDa. In other embodiments, the molecular weight of the polysaccharide is between 70kDa and 900kDa. In other embodiments, the molecular weight of the polysaccharide is between 100kDa and 800kDa. In other embodiments, the molecular weight of the polysaccharide is between 200kDa and 600kDa. In further embodiments, the molecular weight of the polysaccharide is 100 kDa to 1,000 kDa; 100 kDa to 900 kDa; 100 kDa to 800 kDa; 100 kDa to 700 kDa; 100 kDa to 600 kDa; 100 kDa to 500 kDa; 100 kDa to 400 kDa; 100 kDa to 300 kDa; 150 kDa to 1,000 kDa; 150 kDa to 9 ... 0kDa to 800kDa; 150kDa to 700kDa; 150kDa to 600kDa; 150kDa to 500kDa; 150kDa to 400kDa; 150kDa to 300kDa; 200kDa to 1,000kDa; 200kDa to 900kDa; 200kDa to 800kDa; 200kDa to 700kDa; 200kDa to 600kDa; 200kDa to 500kDa kDa; 200 kDa to 400 kDa; 200 kDa to 300; 250 kDa to 1,000 kDa; 250 kDa to 900 kDa; 250 kDa to 800 kDa; 250 kDa to 700 kDa; 250 kDa to 600 kDa; 250 kDa to 500 kDa; 250 kDa to 400 kDa; 250 kDa to 350 kDa; 300 kDa to 1,000 kDa; 300 kDa to 900 kDa; 300 kDa to 800 kDa; 300 kDa to 700 kDa; 300 kDa to 600 kDa; 300 kDa to 500 kDa; 300 kDa to 400 kDa; 400 kDa to 1,000 kDa; 400 kDa to 900 kDa; 400 kDa to 800 kDa; 400 kDa to 700 kDa; 400 kDa to 600 kDa; or 500 kDa to 600 kDa.

[0268] The second step of the reductive amination conjugation process is to use a reducing agent to reduce the imine (Schiff base) bond between the activated polysaccharide and the carrier protein to form a stable conjugated bond (so-called reductive amination). Suitable reducing agents include cyanoborohydrides (such as sodium cyanoborohydride) or sodium borohydride. In one embodiment, the reducing agent is sodium cyanoborohydride.

[0269] In certain embodiments, the reductive amination reaction is carried out in an aprotic solvent (or a mixture of aprotic solvents). In one embodiment, the reduction reaction is carried out in a DMSO (dimethyl sulfoxide) or DMF (dimethylformamide) solvent. If lyophilized, DMSO or DMF solvent can be used to reconstitute the activated polysaccharide and carrier protein. In one embodiment, the aprotic solvent is DMSO.

[0270] At the end of the reduction reaction, unreacted aldehyde groups may remain in the conjugate, which can be capped using a suitable capping agent. In one embodiment, the capping agent is sodium borohydride (NaBH4). Suitable alternatives include sodium triacetoxyborohydride or sodium borohydride or zinc borohydride in the presence of Bronsted or Lewis acids, amine boranes, such as pyridine borane, 2-pyridoline borane, 2,6-diborane-methanol, dimethylamine-borane, t-BuMe'PrN-BH3, benzylamine-BH3 or 5-ethyl-2-methylpyridine borane (PEMB) or borohydride exchange resin. After conjugation (reduction reaction and optional capping), the glycoconjugate can be purified by a variety of techniques known to those skilled in the art (enriched relative to the amount of polysaccharide-protein conjugate). These techniques include diafiltration, 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 glycoconjugate is purified by diafiltration or ion exchange chromatography or size exclusion chromatography.

[0271] Sugar conjugates prepared using reductive amination in aprotic solvents are commonly used in multivalent pneumococcal conjugate vaccines. Thus, in certain embodiments, for multivalent compositions where not all serotypes are prepared in aprotic solvents, the reduction reaction for the remaining serotypes is carried out in an aqueous solvent (e.g., selected from PBS (phosphate buffered saline), MES (2-(N-morpholino)ethanesulfonic acid), HEPES (4-(2-hydroxyethyl)-1-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)-1-piperazineethanesulfonic acid), -2-aminoethanesulfonic acid), MOPS (3-(N-morpholino)propanesulfonic acid), DIPSO (3-bis(2-hydroxyethyl)amino-2-hydroxypropane-1-sulfonic acid), MOBS (4-(N-morpholino)butanesulfonic acid), HEPPSO (N-(2-hydroxyethyl)piperazine-N-(2-hydroxypropanesulfonic acid)), POPSO (piperazine-1,4-bis(2-hydroxy-3-propanesulfonic acid)), TEA (triethanolamine), EPPS (4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid), N-bicine, pH between 6.0 and 8.5, 7.0 and 8.0 or 7.0 and 7.5.

[0272] Pneumococcal capsular polysaccharide-protein conjugates that can be prepared using reductive amination in an aprotic solvent include, but are not limited to, Pneumococcal serotypes: 1, 3, 4, 5, 6A, 6B, 6C, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, and 35B. The polysaccharide can be used in the form of an oligosaccharide. These oligosaccharides are conveniently formed by fragmenting the purified polysaccharide (e.g., by hydrolysis), which is typically purified into fragments of the desired size.

[0273] In certain embodiments, one or more of the following serotypes of pneumococcal polysaccharide-protein conjugates are prepared using reductive amination in an aprotic solvent: 1, 3, 4, 5, 6A, 6B, 6C, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, and 35B. In certain embodiments, each serotype in a multivalent immunogenic composition is prepared using reductive amination in an aprotic solvent. In certain embodiments, polysaccharides of one or more serotypes in a multivalent composition are conjugated to a carrier protein using reductive amination in an aprotic solvent, and polysaccharides of one or more serotypes are conjugated using reductive amination in an aqueous solvent. In certain embodiments, polysaccharides of two or more serotypes in a multivalent composition of the invention are conjugated to a carrier protein using reductive amination in an aprotic solvent. In other embodiments, polysaccharides of 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, twenty-one or more, twenty-two or more, twenty-three or more, or twenty-four or more serotypes in the multivalent compositions of the invention are conjugated to a carrier protein using reductive amination in an aprotic solvent. In certain embodiments, polysaccharides of one or more serotypes in the multivalent compositions of the invention are conjugated to a carrier protein using other chemistries that can be in an aprotic solvent or an aqueous solvent.

[0274] Therefore, the present invention relates to a multivalent immunogenic composition comprising a plurality of pneumococcal polysaccharide protein conjugates, each conjugate comprising a capsular polysaccharide from a pneumococcal serotype conjugated to a carrier protein, wherein the pneumococcal serotype is as described herein (i.e., in Section II "Multivalent Immunogenic Composition"), wherein the conjugation reaction of the polysaccharide of one or more polysaccharide protein conjugates to the carrier protein is carried out in an aprotic solvent. In certain embodiments, at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the pneumococcal serotypes in the multivalent immunogenic composition are prepared in an aprotic solvent. The remaining serotypes are prepared using other chemical methods and / or in aqueous solvents.

[0275] It has been determined that the use of DMSO as a solvent during the reductive amination of polysaccharide-protein conjugates results in unexpectedly superior stability and enhanced immunogenicity of these serotypes relative to the same conjugates prepared under aqueous conditions (see US Patent Application Serial Nos. 62 / 463,216 and 62 / 555,444).

[0276] In certain embodiments of the invention, the total polysaccharide concentration in the composition is from about 0.02 to about 0.288 mg / mL. In certain embodiments of the invention, the total polysaccharide concentration in the composition is from about 0.03 to about 0.192 mg / mL. In certain embodiments of the invention, the total polysaccharide concentration in the composition is from about 0.04 to about 0.192 mg / mL. In other embodiments, the total polysaccharide concentration in the composition is from about 0.065 to about 0.096 mg / mL, about 0.070 to about 0.080 mg / mL, about 0.065 to about 0.080 mg / mL, about 0.070 to about 0.085 mg / mL, about 0.110 to about 0.128 mg / mL, about 0.110 to about 0.175 ... , about 0.110 to about 0.15 mg / mL, about 0.110 to about 0.125 mg / mL, about 0.150 to about 0.170 mg / mL, about 0.150 to about 0.165 mg / mL, about 0.140 to about 0.170 mg / mL, about 0.130 to about 0.170 mg / mL, about 0.150 to about 0.175 mg / mL, about 0.070 to about 0.170 mg / mL, about 0.065 to about 0.175 mg / mL or about 0.065 to about 0.180 mg / mL.

[0277] In an embodiment of the invention, wherein one or more or all of the polysaccharide-protein conjugates in the multivalent immunogenic composition are prepared in an aprotic solvent, the total polysaccharide concentration in the composition is stable at 37°C for 4 weeks or more, at 25°C for 4 weeks or more, or at 4°C for 12 weeks or more.

[0278] In certain embodiments of the invention, wherein one or more or all of the polysaccharide-protein conjugates in the multivalent immunogenic composition are prepared in an aprotic solvent, the average molecular weight (Mw) of all of the pneumococcal polysaccharide-protein conjugates in the composition (the average of all conjugates in the composition) is from about 2,000 to about 6,500 kDa, from about 2,500 to about 6,000 kDa, from about 3,000 to about 5,500 kDa, from about 3,500 to about 5,000 kDa, from about 3,500 to about 4 ...000 to about 4,000 kDa, from about 3,500 to about 4,000 kDa, from about 3,500 to about 4 kDa, from about 3,500 to about 4,700 kDa, from about 3,500 to about 4,600 kDa, from about 3,500 to about 4,500 kDa, from about 3,500 to about 4,400 kDa, from about 3,500 to about 4,300 kDa, from about 3,500 to about 4,200 kDa, from about 3,600 to about 4,700 kDa, from about 3,600 to about 4,600 kDa, from about 3,600 to about 4,500 kDa, from about 3,600 to about 4,400 kDa. kDa, from about 3,600 to about 4,300 kDa, from about 3,600 to about 4,200 kDa, from about 3,700 to about 4,700 kDa, from about 3,700 to about 4,600 kDa, from about 3,700 to about 4,500 kDa, from about 3,700 to about 4,400 kDa, from about 3,700 to about 4,300 kDa, from about 3,700 to about 4,200 kDa, from about 3,800 to about 4,700 kDa, from about 3,800 to about 4,600 kDa, In some embodiments, the present invention relates to a molecular weight ...

[0279] In certain embodiments of the invention, wherein the polysaccharide-protein conjugates in the multivalent immunogenic composition are prepared in an aprotic solvent, the Mw of each pneumococcal polysaccharide-protein conjugate in the composition (for a single serotype) is from about 1,000 to about 10,000 kDa, from about 1,500 to about 5,500 kDa, from about 1,500 to about 5,600 kDa, from about 1,500 to about 5,700 kDa, from about 1,500 to about 5,800 kDa, from about 1,500 to about 5,900 kDa, from about 1,100 to about 1,200 kDa, from about 1,200 to about 1,300 kDa, from about 1,500 to about 1,400 kDa, from about 1,500 to about 1,500 kDa, from about 1,500 to about 1,600 kDa, from about 1,600 to about 1,700 kDa, from about 1,800 to about 1,900 kDa, from about 1,100 to about 1,200 kDa, from about 1,500 to about 1,500 kDa, from about 1, kDa, from about 1,500 to about 5,800 kDa, from about 1,500 to about 5,900 kDa, from about 1,500 to about 6,000 kDa, from about 1,000 to about 5,500 kDa, from about 1,000 to about 5,000 kDa, from about 1,000 to about 4,000 kDa, from about 1,000 to about 4,500 kDa, from about 1,000 to about 4,000 kDa or from about 1,000 to about 3,500 kDa. In other embodiments, the Mw of the conjugate from a single serotype within the composition is about 1,000 kDa, about 1,100 kDa, about 1,200 kDa, about 1,300 kDa, about 1,400 kDa, about 1,500 kDa, about 1,600 kDa, about 1,700 kDa, about 1,800 kDa, about 1,900 kDa, about 2,000 kDa, about 2,100 kDa, about 2,200 kDa, about 2,300 kDa, about 2,400 kDa, about 2,500 kDa, about 2,600 kDa, about 2,700 kDa, about 2,800 kDa, about 2,900 kDa, about 3,000 kDa, about 3,100 kDa, about kDa, about 3,400 kDa, about 3,500 kDa, about 3,600 kDa, about 3,700 kDa, about 3,800 kDa, about 3,900 kDa, about 4,000 kDa, about 4,100 kDa, about 4,200 kDa, about 4,300 kDa, about 4,400 kDa, about 4,500 kDa, about 4,600 kDa, about 4,700 kDa, about 4,800 kDa, about 4,900 kDa, about 5,000 kDa, about 5,100 kDa, about 5,200 kDa, about 5,300 kDa, about 5,400 kDa or about 5,500 kDa.

[0280] In certain embodiments of the invention, the polysaccharide-protein conjugates in the multivalent immunogenic composition are prepared in an aprotic solvent. In certain embodiments, the percentage of S. pneumoniae serotype-specific conjugates prepared in an aprotic solvent (calculated by dividing the number of polysaccharide serotypes prepared in an aprotic solvent by the total number of polysaccharide serotypes, where the total number includes those prepared in an aprotic or protic solvent) may be greater than 50%, or greater than 60%, or greater than 70%, or greater than 80%, or greater than 90%, or 100%.

[0281] In certain embodiments of the invention, the serotype 3 polysaccharide-protein conjugate in the composition is prepared in an aprotic solvent and the Mw of the conjugate is from about 1,000 to about 5,000 kDa, or from about 1,000 to about 4,000 kDa, or from about 1,000 to about 3,000 kDa, or from about 1,000 to about 2,500 kDa, or from about 1,000 to about 2,000 kDa.

[0282] In certain embodiments of the invention, wherein one or more or all of the polysaccharide-protein conjugates in the multivalent immunogenic composition are prepared in an aprotic solvent, the number average molecular weight (Mn) of the Streptococcus pneumoniae polysaccharide-protein conjugate in the composition (average of all conjugates in the composition) is from about 900 to about 3,000 kDa, from about 1,000 to about 3,000 kDa, from about 1,000 to about 2,500 kDa, from about 1,500 to about 2,500 kDa, from about 1,800 to about 2,500 kDa, from about 1,900 to about 2,500 kDa, or from about 2,000 to about 2,500 kDa.

[0283] In certain embodiments of the invention, wherein one or more or all of the polysaccharide-protein conjugates in the multivalent immunogenic composition are prepared in an aprotic solvent, the Mn of each S. pneumoniae polysaccharide-protein conjugate in the composition (for a single serotype) is from about 700 to about 7,000 kDa, from about 1,000 to about 6,000 kDa, from about 1,000 to about 5,000 kDa, from about 1,000 to about 4,000 kDa, from about 1,000 to about 3,000 kDa, from about 900 to about 5,500 kDa, from about 900 to about 5,000 kDa, from about 900 to about 4,500 kDa, from about 900 to about 4,000 kDa, from about 900 to about 3,500 kDa, or from about 900 to about 3,000 kDa.

[0284] In an embodiment of the invention, the Mw and / or Mn of the Streptococcus pneumoniae polysaccharide-protein conjugate in the composition is stable at 37°C for 4 weeks or more, at 25°C for 4 weeks or more, and / or at 4°C for 12 weeks or more.

[0285] In an embodiment of the present invention, HPSEC UV / MALS / RI is used to determine polysaccharide concentration, Mw and / or Mn.

[0286] In some embodiments of the invention, wherein one or more or all of the polysaccharide-protein conjugates in the multivalent immunogenic composition are prepared in an aprotic solvent, the maximum emission of the composition measured at an excitation wavelength of 280 nanometers (nm) using intrinsic protein fluorescence spectroscopy is from about 335 nm to about 342 nm. In some embodiments, the maximum emission is maintained at about 335 nm to about 342 nm at 37° C. for at least 1 week, and the fluorescence intensity is stable. In some embodiments, the maximum emission is maintained at about 335 nm to about 342 nm at 37° C. for 1 week, and the fluorescence intensity is stable.

[0287] In some embodiments, all pneumococcal polysaccharide conjugates in the multivalent composition are prepared using reductive amination in DMSO. In certain subembodiments, the multivalent composition comprising polysaccharide conjugates all prepared using DMSO does not comprise an adjuvant.

[0288] Without wishing to be bound by any theory, one possible mechanism for the observed enhanced immunogenicity using glycoconjugates prepared in DMSO includes an increase in the number of bonds between the carbohydrate (capsular polysaccharide) and the lysine residues on the surface of the carrier protein, which will result in additional attachment points between the protein and the polysaccharide to confer stability and resist chemical depolymerization or destruction 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, pp. 93-104. Another benefit of the increased polysaccharide-protein bonds produced during the conjugation process in DMSO solvent is that it can provide more opportunities for successful presentation of the peptide-carbohydrate to T cells. Another possible mechanism for the observed enhancement of immunogenicity by conjugation in DMSO solvent may be due to the denaturation of CRM197 in organic solvents, which exposes additional lysines for polysaccharide attachment, resulting in increased opportunities for glycopeptide presentation on the APC surface, thereby generating T cell-dependent responses to different peptide epitopes. See Avci et al., 2011, Nature Medicine 17:1602-1610.

[0289] Another benefit of conjugation in an organic solvent that produces denatured CRM197 in the conjugate is the ability to reduce immune interference from antibodies against native CRM197 epitopes. Another benefit of increased polysaccharide-protein linkages produced during conjugation in a DMSO solvent is the ability to form larger polysaccharide-protein conjugates, thereby enhancing immunogenicity. It is believed that the compositions of the present invention provide significant advantages in stimulating human responses.

[0290] In certain embodiments, the conjugation reaction is carried out by reductive amination, wherein the use of nickel can improve the efficiency of the conjugation reaction and help remove free cyanide. It is known that transition metals can form stable complexes with cyanide, and it is known that they can improve the reductive methylation of protein amino groups and formaldehyde with sodium cyanoborohydride (S Gidley et al., Biochem J. 1982, 203: 331-334, etc., Jentoft et al., Anal Biochem. 1980, 106: 186-190). By complexing the inhibitory cyanide remaining, the addition of nickel will increase the consumption of protein in the conjugation process and lead to the formation of a larger, possibly more immunogenic conjugate.

[0291] Variations in the starting cyanide content among batches of sodium cyanoborohydride reagent can also lead to inconsistent conjugation performance, resulting in variable product attributes (such as conjugate size and ratio of conjugate Ps to CRM197). The addition of nickel reduces conjugation inconsistency by complexing cyanide, eliminating variability between sodium cyanoborohydride batches.

[0292] Suitable alternative chemical methods include activating sugars with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) to form cyanate. Therefore, activated sugars can be coupled directly or via a spacer (joint) group to an amino group on a carrier protein. For example, a spacer can be cystamine or cysteamine to obtain a thiolated polysaccharide, which can be coupled to a carrier by a thioether bond obtained after reacting with a maleimide-activated carrier protein (e.g., using GMBS) or a haloacetylated carrier protein (e.g., using iodoacetimidate (e.g., hydrochloric acid iodoacetamide) or N-succinimidyl bromoacetate or SIAB, or SIA or SBAP)). Preferably, cyanate (optionally prepared by CDAP chemical method) is coupled with hexamethylenediamine or adipic acid dihydrazide (ADH), and amino-derived sugars are conjugated to a carrier protein via a carboxyl group on a protein carrier using a carbodiimide (e.g., EDAC or EDC) chemical method. Such conjugates are described in International Patent Application Publication Nos. WO 93 / 15760, WO 95 / 08348, and WO 96 / 29094; and in Chu et al., 1983, Infect. Immunity 40:245-256.

[0293] Other suitable techniques use carbodiimides, hydrazides, active esters, norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, TSTU. Many are described in International Patent Application Publication No. WO 98 / 42721. Conjugation may involve a carbonyl linker, which may be formed by reacting the free hydroxyl groups of the sugar with CDI (see Bethell et al., 1979, J. Biol. Chem. 254: 2572-4; Hearn et al., 1981, J. Chromatogr. 218: 509-18), and then reacting with the protein to form a carbamate bond. This may involve reducing the end group to a primary hydroxyl group, optionally protecting / deprotecting the primary hydroxyl group, reacting the primary hydroxyl group with CDI to form a CDI carbamate intermediate and coupling the CDI carbamate intermediate to an amino group on the protein.

[0294] After the capsular polysaccharide is conjugated to the carrier protein, the polysaccharide-protein conjugate is purified (enriched relative to the amount of the polysaccharide-protein conjugate) by one or more of a variety of techniques. Examples of these techniques are well known to those skilled in the art and include concentration / diafiltration operations, ultrafiltration, precipitation / elution, column chromatography, and depth filtration. See, for example, U.S. Pat. No. 6,146,902.

[0295] After the individual saccharides have been conjugated and purified, they are complexed to formulate the immunogenic compositions of the invention. These pneumococcal conjugates, prepared by separate processes and starting materials, are formulated into single dose formulations.

[0296] An alternative method of characterizing the glycoconjugates of the invention is by the number of lysine residues in the carrier protein (e.g., CRM197) that become conjugated to the sugar, which can be characterized as a series of conjugated lysines (degree of conjugation). Evidence of lysine modification of the carrier protein due to covalent bonding to the polysaccharide can be obtained by amino acid analysis using conventional methods known to those skilled in the art. Conjugation results in a reduction in the number of lysine residues recovered compared to the carrier protein starting material used to generate the conjugate material. In a preferred embodiment, the degree of conjugation of the glycoconjugates of the invention is between 2 and 15, between 2 and 13, between 2 and 10, between 2 and 8, between 2 and 6, between 2 and 5, between 2 and 4, between 3 and 15, between 3 and 13, between 3 and 10, between 3 and 8, between 3 and 6, between 3 and 5, between 3 and 4, between 5 and 15, between 5 and 10, between 8 and 15, between 8 and 12, between 10 and 15, or between 10 and 12. In one embodiment, the degree of conjugation of the glycoconjugates 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 glycoconjugates of the invention is between 4 and 7. In some such embodiments, the carrier protein is CRM197.

[0297] The glycoconjugates of the compositions of the present invention can also be characterized by the ratio of polysaccharide to carrier protein (Ps:Pr) (weight / weight). In some embodiments, the ratio of polysaccharide to carrier protein (w / w) in the glycoconjugates of the composition is between 0.5 and 3.0 (e.g., about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, or about 3.0). In other embodiments, the ratio of polysaccharide to carrier protein (w / w) is between 0.5 and 2.5, between 0.5 and 1.5, between 0.8 and 2.5, between 0.5 and 1.0, between 1.0 and 1.5, between 1.0 and 2.0, between 0.8 and 2.4, between 0.8 and 2.3, between 0.8 and 2.2, between 0.8 and 2.1, between 0.8 and 2.0, between 0.8 and 1.9, between 0.8 and 1.8, between 0.8 and 1.7, between 0.8 and 1.6, between 0.8 and 1.5, between 0.8 and 1.4, between 0.8 and 1.3, between 0.9 and 2.4, between 0.9 and 2.3, between 0.9 and 2.2 In a further embodiment, the ratio (w / w) of polysaccharide to carrier protein is between 0.8 and 1.2. In some such embodiments, the carrier protein is CRM197. The glycoconjugates and immunogenic compositions of the invention may contain free sugars that are not covalently conjugated to the carrier protein but are still present in the glycoconjugate composition. The free sugars may be non-covalently associated with (i.e., non-covalently bound to, adsorbed or entrapped in or with) the glycoconjugate.

[0298] In specific embodiments, for serotype 15A conjugates, the ratio of saccharide to carrier protein (w / w) is from about 1.0 to about 2.0, from about 1.25 to about 1.75, or from about 1.3 to about 1.7. In other embodiments, for serotype 15A, the ratio of saccharide to carrier protein (w / w) is about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, or 1.8.

[0299] In specific embodiments, for serotype 15C conjugation, the ratio of sugar to carrier protein (w / w) is from about 1.0 to about 2.0, from about 1.25 to about 1.75, or from about 1.3 to about 1.7. In other embodiments, for serotype 15C, the ratio of sugar to carrier protein (w / w) is about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, or 1.8.

[0300] In specific embodiments, for serotype 33F conjugates, the ratio of saccharide to carrier protein (w / w) is from about 1.0 to about 2.0, from about 1.25 to about 1.75, or from about 1.3 to about 1.7. In other embodiments, for serotype 33F, the ratio of saccharide to carrier protein (w / w) is about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7 or 1.8.

[0301] In specific embodiments, for serotype 35B conjugates, the ratio of saccharide to carrier protein (w / w) is from about 1.25 to about 2.25, from about 1.25 to about 2.0, or from about 1.3 to about 1.8. In other embodiments, for serotype 35B, the ratio of saccharide to carrier protein (w / w) is about 1.2, 1.3, 1.3, 1.5, 1.6, 1.7, 1.8, 1.9 or 2.0.

[0302] In specific embodiments, for serotype 24F conjugates, the ratio of saccharide to carrier protein (w / w) is from about 0.5 to about 1.5, from about 0.75 to about 1.25, or from about 0.8 to about 1.0. In other embodiments, for serotype 24F, the ratio of saccharide to carrier protein (w / w) is about 0.5, 0.6, 0.7, 0.8, 0.9 or 1.0.

[0303] In a preferred embodiment, the glycoconjugate comprises less than about 50%, 45%, 40%, 35%, 30%, 25%, 20% or 15% free polysaccharide compared to the total amount of polysaccharide. In a preferred embodiment, the glycoconjugate comprises less than about 25% free polysaccharide compared to the total amount of polysaccharide. In a preferred embodiment, the glycoconjugate comprises less than about 20% free polysaccharide compared to the total amount of polysaccharide. In a preferred embodiment, the glycoconjugate comprises less than about 15% free polysaccharide compared to the total amount of polysaccharide.

[0304] IV. How to use

[0305] Embodiments of the invention also include one or more of the multivalent immunogenic compositions described herein (i) for use, (ii) as a medicament or composition for use, or (iii) for the preparation of a medicament for: (a) therapy (e.g., of a human); (b) a medicament; (c) inhibiting pneumococcal infection; (d) inducing an immune response or protective immune response against pneumococcus; (e) preventing pneumococcal infection; (f) preventing recurrence of pneumococcal infection; (g) reducing the progression, onset or severity of pathological symptoms associated with pneumococcal infection, including preventing associated complications such as brain damage, hearing loss and seizures; (h) reducing the likelihood of pneumococcal infection, or (i) treating, preventing or delaying the onset, severity or progression of one or more pneumococcal diseases, including but not limited to: pneumococcal pneumonia, pneumococcal bacteremia, pneumococcal meningitis, otitis media and sinusitis. In these uses, the multivalent pneumococcal polysaccharide-conjugate compositions of the invention can be optionally used in combination with one or more adjuvants, or without an adjuvant.

[0306] Thus, the present invention provides a method for prophylactically treating (ie, protecting against) S. pneumoniae infection or S. pneumoniae disease comprising administering to a patient in need of treatment one or more multivalent immunogenic pneumococcal polysaccharide-protein conjugate compositions of the invention.

[0307] The compositions and formulations of the invention may be used to protect or treat humans susceptible to infection (eg, pneumococcal infection) by administering such compositions or formulations via systemic or mucosal routes.

[0308] In one embodiment, the present invention provides 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 present invention. In another embodiment, the present invention provides a method of vaccinating a human against pneumococcal infection, comprising the step of administering to a human an immunologically effective amount of a multivalent immunogenic composition of the present invention.

[0309] Thus, in one aspect, the invention provides a method for (1) inducing an immune response in a human patient, (2) inducing a protective immune response in a human patient, (3) immunizing a human patient against a pneumococcal infection, or (4) reducing the likelihood of a pneumococcal infection in a human patient, the method comprising administering to the patient a multivalent immunogenic composition of the invention (i.e., any multivalent immunogenic composition described herein, such as a multivalent immunogenic composition described in Section II entitled "Multivalent Immunogenic Compositions," as described above).

[0310] In one embodiment, the invention provides a method for preventing pneumococcal pneumonia and / or invasive pneumococcal disease in an infant (less than 1 year of age), a toddler (approximately 12 to 24 months), or a young child (approximately 2 to 5 years of age).

[0311] In another embodiment, the invention provides a method for preventing pneumococcal pneumonia and / or invasive pneumococcal disease in a patient aged 6 weeks to 17 years.

[0312] In another embodiment, the invention provides a method for preventing pneumococcal pneumonia and / or invasive pneumococcal disease in a patient aged 6 months to 17 years.

[0313] In another embodiment, the invention provides a method for preventing pneumococcal pneumonia and / or invasive pneumococcal disease in adults 18 years of age and older.

[0314] In another embodiment, the invention provides a method for preventing pneumococcal pneumonia and / or invasive pneumococcal disease in adults 50 years of age and older.

[0315] In another embodiment, the invention provides a method for preventing pneumococcal pneumonia and / or invasive pneumococcal disease in adults 65 years of age and older.

[0316] In another embodiment, the invention provides a method for preventing pneumococcal pneumonia and / or invasive pneumococcal disease caused by one or more of the following strains of S. pneumoniae: 1, 3, 4, 5, 6A, 6B, 6C, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B.

[0317] In one embodiment of the above method, the composition comprises a plurality of S. pneumoniae polysaccharide-protein conjugates, wherein each of the conjugates comprises a polysaccharide from a S. pneumoniae serotype conjugated to a carrier protein, wherein the S. pneumoniae serotype comprises serotype: 1, 3, 4, 5, 6A, 6B, 6C, 7F, 8, 9V, 10A, 12F, 14, 15A, 15B, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B, or serotype: 1, 3, 4, 5, 6A, 6B, 6C, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B. In another embodiment of the above method, the composition comprises a plurality of S. pneumoniae polysaccharide-protein conjugates, wherein each of the conjugates comprises a polysaccharide from a serotype of S. pneumoniae conjugated to a carrier protein, wherein the serotype of S. pneumoniae consists of serotype: 1, 3, 4, 5, 6A, 6B, 6C, 7F, 8, 9V, 10A, 12F, 14, 15A, 15B, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B, or serotype: 1, 3, 4, 5, 6A, 6B, 6C, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B. In one embodiment of the above method, the composition comprises a plurality of S. pneumoniae polysaccharide-protein conjugates, wherein each of the conjugates comprises a polysaccharide from a S. pneumoniae serotype conjugated to a carrier protein, wherein the S. pneumoniae serotype comprises serotype: 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B, or serotype: 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B.In another embodiment of the above method, the composition comprises a plurality of S. pneumoniae polysaccharide-protein conjugates, wherein each of the conjugates comprises a polysaccharide from a serotype of S. pneumoniae conjugated to a carrier protein, wherein the serotype of S. pneumoniae consists of serotypes: 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B, or serotypes: 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B. In one embodiment of the above method, the composition comprises a plurality of Streptococcus pneumoniae polysaccharide-protein conjugates, wherein each of the conjugates comprises a polysaccharide from a serotype of Streptococcus pneumoniae conjugated to a carrier protein, wherein the serotype of Streptococcus pneumoniae comprises serotypes: 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B. In another embodiment of the above method, the composition comprises a plurality of Streptococcus pneumoniae polysaccharide-protein conjugates, wherein each of the conjugates comprises a polysaccharide from a serotype of Streptococcus pneumoniae conjugated to a carrier protein, wherein the serotype of Streptococcus pneumoniae consists of the following, serotype: 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B, or serotype:.

[0318] It has been found that pneumococcal conjugate vaccines comprising serotype 6A can provide some cross protection against serotype 6C (Cooper et al., Vaccine 29 (2011) 7207-7211). Therefore, in some embodiments of the above methods, the present invention also provides the use of a multivalent immunogenic composition that does not comprise serotype 6C, but instead comprises serotype 6A or serotypes 6A and 6B. In other embodiments, the immunogenic composition comprises a pneumococcal conjugate of serotypes 6A, 6B and 6C.

[0319] In a specific embodiment of the above method, the multivalent immunogenic composition comprises a pneumococcal conjugate comprising a polysaccharide of a group of S. pneumoniae serotypes selected from the group consisting of:

[0320] a) 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 12F, 14, 15A, DeOAc15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;

[0321] b) 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;

[0322] c) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, DeOAc15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;

[0323] d) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;

[0324] e) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;

[0325] f) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, DeOAc15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;

[0326] g) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;

[0327] h) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;

[0328] i) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;

[0329] j) 1, 3, 4, 5, 6A, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;

[0330] k) 1, 3, 4, 5, 6B, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;

[0331] l) 1, 3, 4, 5, 6C, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;

[0332] m) 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;

[0333] n) 1, 3, 4, 5, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;

[0334] o) 1, 3, 4, 5, 6C, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;

[0335] p) 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;

[0336] q)1, 3, 4, 5, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;

[0337] r) 1, 3, 4, 5, 6C, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;

[0338] s)1, 3, 4, 5, 6A, 7F, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;

[0339] t) 1, 3, 4, 5, 6B, 7F, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;

[0340] u) 1, 3, 4, 5, 6C, 7F, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;

[0341] v) 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;

[0342] w) 1, 3, 4, 5, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;

[0343] x) 1, 3, 4, 5, 6C, 7F, 8, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;

[0344] y) 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;

[0345] z) 1, 3, 4, 5, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; and

[0346] aa) 1, 3, 4, 5, 6C, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B.

[0347] In other embodiments of the above method, the composition comprises a plurality of S. pneumoniae polysaccharide-protein conjugates, wherein each of the conjugates comprises a polysaccharide from a serotype of S. pneumoniae conjugated to a carrier protein, wherein the serotype of S. pneumoniae comprises serotypes: i) 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, and 35B; or ii) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, and 35B. or iv) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, and 35B.

[0348] It has also been found that pneumococcal conjugate vaccines comprising serotype 10A can provide some cross protection against serotype 39 (see WO 2017 / 085586). Therefore, in some embodiments of the above methods, the present invention also provides the use of a multivalent immunogenic composition that does not comprise serotype 10A, but instead comprises serotype 39. In other embodiments, the immunogenic composition comprises a conjugate of pneumococcal serotypes 10A and 39. In a specific embodiment of the above method, the serotype of Streptococcus pneumoniae comprises a serotype selected from the group consisting of:

[0349] bb)1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15A, DeOAc15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;

[0350] cc)1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;

[0351] dd)1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 12F, 14, 15A, DeOAc15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;

[0352] (ee) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;

[0353] (ff) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;

[0354] (gg) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 11A, 12F, 14, 15A, DeOAc15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;

[0355] (hh) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;

[0356] (ii) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;

[0357] (jj) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;

[0358] (kk) 1, 3, 4, 5, 6A, 7F, 9V, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;

[0359] (ll) 1, 3, 4, 5, 6B, 7F, 9V, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;

[0360] (mm) 1, 3, 4, 5, 6C, 7F, 9V, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;

[0361] nn) 1, 3, 4, 5, 6A, 7F, 8, 9V, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;

[0362] oo)1, 3, 4, 5, 6B, 7F, 8, 9V, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;

[0363] pp) 1, 3, 4, 5, 6C, 7F, 8, 9V, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;

[0364] qq)1, 3, 4, 5, 6A, 7F, 8, 9V, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;

[0365] rr)1, 3, 4, 5, 6B, 7F, 8, 9V, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;

[0366] ss) 1, 3, 4, 5, 6C, 7F, 8, 9V, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;

[0367] tt)1, 3, 4, 5, 6A, 7F, 9V, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;

[0368] uu)1, 3, 4, 5, 6B, 7F, 9V, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;

[0369] vv) 1, 3, 4, 5, 6C, 7F, 9V, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;

[0370] ww)1, 3, 4, 5, 6A, 7F, 8, 9V, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;

[0371] xx) 1, 3, 4, 5, 6B, 7F, 8, 9V, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;

[0372] yy) 1, 3, 4, 5, 6C, 7F, 8, 9V, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;

[0373] zz)1, 3, 4, 5, 6A, 7F, 8, 9V, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;

[0374] aaa) 1, 3, 4, 5, 6B, 7F, 8, 9V, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B, and 39; and

[0375] bbb)1, 3, 4, 5, 6C, 7F, 8, 9V, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39.

[0376] It has also been found that immunogenic conjugates comprising pneumococcal serotype 15B capsular polysaccharides covalently linked to a carrier protein can provide some cross-protection against serotype 15C and / or serotype 15A (see WO 2015 / 110942). Therefore, in some embodiments of the above method, the present invention also provides the use of a multivalent immunogenic composition that does not contain serotype 15C (or de-O-acetylated 15B), but instead contains serotype 15B (i.e., serotype 15B polysaccharides that are substantially not de-O-acetylated). In other embodiments, the immunogenic composition comprises a conjugate of pneumococcal serotypes 15B and 15C (or de-O-acetylated 15B).

[0377] The compositions of the invention can be used in methods of providing supplemental protection against S. pneumoniae in patients who have previously received a multivalent pneumococcal vaccine. In such uses, the compositions of the invention can provide protection against a specific serotype of S. pneumoniae against which the patient has not been previously vaccinated, can provide additional protection against a serotype of S. pneumoniae against which the patient has been previously vaccinated, or can provide protection against both a serotype of S. pneumoniae against which the patient has not been previously vaccinated and a serotype of S. pneumoniae against which the patient has been previously vaccinated.

[0378] Therefore, the present invention provides a method for inducing an immune response, vaccination or inducing a protective immune response against Streptococcus pneumoniae in a patient, the method comprising administering to the patient a multivalent immunogenic composition comprising a multivalent Streptococcus pneumoniae polysaccharide-protein conjugate, wherein the polysaccharide-protein conjugate comprises a capsular polysaccharide from a serotype of Streptococcus pneumoniae conjugated to a carrier protein, wherein the patient has previously been immunized against Streptococcus pneumoniae. In an embodiment of this aspect of the invention, the multivalent immunogenic composition may be any multivalent immunogenic composition described herein. In a specific embodiment of the method of the present invention, a multivalent immunogenic composition is administered to a patient who has previously been treated with a multivalent pneumococcal vaccine. The multivalent immunogenic vaccine may be any vaccine for preventing pneumococcal disease caused by more than one serotype of Streptococcus pneumoniae.

[0379] In a specific embodiment of the above method, the patient has previously been treated with a multivalent pneumococcal vaccine suitable for preventing pneumococcal disease caused by one or more serotypes of S. pneumoniae selected from the group consisting of:

[0380] i.4, 6B, 9V, 14, 18C, 19F and 23F;

[0381] ii.4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 6A, 7F and 19A;

[0382] iii.1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F;

[0383] iv.4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 6A, 7F, 19A, 22F and 33F;

[0384] v.4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 7F, 19A, 22F, 33F, 2, 8, 9N, 10A, 11A, 12F, 15B, 17F, and 20; and

[0385] vi. 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 6A, 7F, 19A, 22F, 33F, 8, 10A, 11A, 12F and 15B.

[0386] In a specific embodiment of the above method, the multivalent pneumococcal vaccine comprises a plurality of polysaccharide-protein conjugates, wherein the polysaccharide-protein conjugate comprises a polysaccharide from a serotype of Streptococcus pneumoniae conjugated to a carrier protein. In other embodiments, the multivalent pneumococcal vaccine comprises a plurality of Streptococcus pneumoniae capsular polysaccharides not conjugated to a carrier protein.

[0387] In other embodiments of the above methods, the patient has previously used 13 (Pneumococcal 13-valent conjugate vaccine [diphtheria CRM197 protein], Pfizer, Inc., Philadelphia, PA, USA) treatment.

[0388] In other embodiments of the above methods, the patient has previously used 23 (polyvalent pneumococcal vaccine, Merck & Co., Inc., Kenilworth, NJ, USA) treatment.

[0389] In a further embodiment of the above method, the patient has previously taken SYNFLORIX TM (Pneumococcal polysaccharide conjugate vaccine (adsorbed), GlaxoSmithKline Biologicals sa, Rixensart, Belgium) treatment.

[0390] In an embodiment of the above method, the multivalent immunogenic composition of the invention is administered to the patient at any time after the patient receives the multivalent pneumococcal vaccine, according to a treatment regimen provided by a medical professional (e.g., a physician). In a specific embodiment, the multivalent immunogenic composition of the invention is administered to the patient from 1 month to 5 years after the patient has received the multivalent pneumococcal vaccine, or from 1 month to 1 year, from 1 month to 2 years, from 1 month to 3 years, from 1 month to 4 years, from 1 month to 6 months, from 2 months 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, from 6 months to 2 years, from 6 months to 1 year, from 1 year to 4 years, from 1 year to 3 years, or from 1 year to 2 years. In a further embodiment, the multivalent immunogenic composition is administered to a patient 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, about 11 months, about 1 year, about 1.25 years, about 1.5 years, about 1.75 years, about 2 years, about 2.25 years, about 2.5 years, about 2.75 years, about 3 years, about 3.25 years, about 3.5 years, about 3.75 years, about 4 years, about 4.25 years, about 4.5 years, about 4.75 years, or about 5 years after the patient has received the multivalent pneumococcal vaccine.

[0391] In other embodiments, the present invention provides a method for (1) inducing an immune response in a human patient, (2) inducing a protective immune response in a human patient, (3) immunizing a human patient against a pneumococcal infection, or (4) reducing the likelihood of a pneumococcal infection in a human patient, the method comprising administering to the patient a multivalent immunogenic composition of the present invention and administering a multivalent pneumococcal vaccine in any order. For example, the multivalent pneumococcal vaccine is administered to the patient first, followed by the multivalent immunogenic composition of the present invention. Alternatively, the multivalent immunogenic composition of the present invention is administered to the patient first, followed by the multivalent pneumococcal vaccine. The multivalent pneumococcal vaccine can be any vaccine for preventing pneumococcal disease caused by more than one serotype of S. pneumoniae.

[0392] In a specific embodiment of the above method, the patient is treated with a multivalent immunogenic composition of the invention and a multivalent pneumococcal vaccine for the prevention of pneumococcal disease caused by one or more serotypes of S. pneumoniae selected from the group consisting of:

[0393] i.4, 6B, 9V, 14, 18C, 19F and 23F;

[0394] ii.4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 6A, 7F and 19A;

[0395] iii.1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F;

[0396] iv.4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 6A, 7F, 19A, 22F and 33F;

[0397] v.4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 7F, 19A, 22F, 33F, 2, 8, 9N, 10A, 11A, 12F, 15B, 17F, and 20; and

[0398] vi. 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 6A, 7F, 19A, 22F, 33F, 8, 10A, 11A, 12F and 15B.

[0399] In a specific embodiment of the above method, the multivalent pneumococcal vaccine comprises capsular polysaccharides of pneumococcal 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.

[0400] In a specific embodiment of the above method, the multivalent pneumococcal vaccine comprises a plurality of polysaccharide-protein conjugates, wherein the polysaccharide-protein conjugate comprises a polysaccharide from a serotype of Streptococcus pneumoniae conjugated to a carrier protein. In other embodiments, the multivalent pneumococcal vaccine comprises a plurality of Streptococcus pneumoniae capsular polysaccharides not conjugated to a carrier protein.

[0401] In other embodiments of the above methods, in any order, the multivalent immunogenic composition of the invention and the 13 (Pneumococcal 13-valent conjugate vaccine [diphtheria CRM197 protein], Pfizer, Inc., Philadelphia, PA, USA) is used to treat the patient. In one embodiment, the patient is first administered 13, and then administering the multivalent immunogenic composition of the present invention to the patient. In an alternative embodiment, the multivalent immunogenic composition of the present invention is first administered to the patient, and then administered 13.

[0402] In other embodiments of the above methods, in any order, the multivalent immunogenic composition of the invention and the 23 (polyvalent pneumococcal vaccine, Merck & Co., Inc., Kenilworth, NJ, USA) is used to treat the patient. In one embodiment, the patient is first administered 23, then administer the multivalent immunogenic composition of the present invention to the patient. In an alternative embodiment, the multivalent immunogenic composition of the present invention is first administered to the patient, and then administered twenty three.

[0403] In a further embodiment of the above method, in any order, the multivalent immunogenic composition of the present invention and the SYNFLORIX TM (Pneumococcal polysaccharide conjugate vaccine (adsorbed), GlaxoSmithKline Biologicals sa, Rixensart, Belgium) is used to treat the patient. In one embodiment, SYNFLORIX is first administered to the patient. TM In an alternative embodiment, the multivalent immunogenic composition of the present invention is first administered to the patient, followed by administration of SYNFLORIX TM .

[0404] In some embodiments of the above methods, the multivalent immunogenic composition and the multivalent pneumococcal vaccine are administered concurrently. As used herein, "concurrent administration" is not limited to the simultaneous administration of the two compositions, but also includes administration one after the other in any order. In some embodiments, the multivalent immunogenic composition and the multivalent pneumococcal vaccine are administered to different anatomical sites (e.g., two different arms) by intramuscular or subcutaneous administration.

[0405] In some embodiments of the above methods, the amount of time between administration of the multivalent immunogenic composition of the invention and the multivalent pneumococcal vaccine is from about 4 weeks to about 1 year. In alternative embodiments, the amount of time is from about 1 month to about 5 years.

[0406] In one embodiment, the multivalent pneumococcal vaccine is administered to the patient first, followed by the multivalent immunogenic composition of the invention. In an alternative embodiment, the multivalent immunogenic composition of the invention is administered to the patient first, followed by the multivalent pneumococcal vaccine.

[0407] Also provided is a method of inducing an immune response, immunizing or inducing a protective immune response against Streptococcus pneumoniae in a patient, comprising:

[0408] (1) administering the multivalent immunogenic composition of the present invention to a patient,

[0409] (2) wait for a predetermined amount of time to pass, and

[0410] (3) Administer a multivalent pneumococcal vaccine to the patient.

[0411] In this method, the multivalent immunogenic composition can comprise any combination of the S. pneumoniae polysaccharide-protein conjugates set forth herein, and the multivalent pneumococcal vaccine can be any vaccine for preventing disease caused by more than one serotype of S. pneumoniae.

[0412] Also provided is a method of inducing an immune response, immunizing or inducing a protective immune response against Streptococcus pneumoniae in a patient, comprising:

[0413] (1) administering a polyvalent pneumococcal vaccine to a patient,

[0414] (2) wait for a predetermined amount of time to pass, and

[0415] (3) Administering the multivalent immunogenic composition of the present invention to a patient.

[0416] In this method, the multivalent immunogenic composition can comprise any combination of S. pneumoniae polysaccharide proteins set forth herein, and the multivalent pneumococcal vaccine can be any vaccine for preventing disease caused by more than one serotype of S. pneumoniae.

[0417] In some embodiments of the above methods, the multivalent pneumococcal vaccine comprises a plurality of pneumococcal polysaccharide-protein conjugates, wherein the polysaccharide-protein conjugate comprises capsular polysaccharides from pneumococcal serotypes conjugated to a carrier protein. In alternative embodiments, the multivalent pneumococcal vaccine comprises pneumococcal capsular polysaccharides not conjugated to a carrier protein.

[0418] In any embodiment of the method of the present invention (i.e., any method described herein), the method may further comprise administering to the patient one or more additional doses of the multivalent immunogenic composition of the present invention. In such a method, the patient may have received a multivalent pneumococcal vaccine (as described above) prior to receiving the first dose of the multivalent immunogenic composition of the present invention, or the patient may not have been immunized against S. pneumoniae prior to receiving the multivalent immunogenic composition of the present invention. Thus, in one embodiment, two or more doses of the multivalent immunogenic composition of the present invention are administered to a patient who has received a multivalent pneumococcal vaccine for the prevention of pneumococcal disease caused by S. pneumoniae. In an alternative embodiment, two or more doses of the multivalent immunogenic composition of the present invention are administered to a patient who has not previously been treated with any vaccine for the prevention of pneumococcal disease.

[0419] In an embodiment of the above method, the two or more doses are the same multivalent immunogenic composition of the invention. In an alternative embodiment, the two or more doses are different multivalent immunogenic compositions of the invention.

[0420] In certain embodiments of any of these methods, two, three, or four doses of a multivalent immunogenic composition of the invention are administered to the patient. In certain embodiments, the patient is immunocompromised (eg, on an immunosuppressive regimen following a stem cell transplant).

[0421] In some embodiments, the amount of time between administrations of each dose of the multivalent immunogenic composition of the invention is from about 4 weeks to about 1 year. In alternative embodiments, the amount of time between administrations of each dose of the multivalent immunogenic composition of the invention is from about 1 month to about 5 years.

[0422] In an embodiment of any method of the present invention, the patient treated with one or more compositions of the present invention is a human. In certain embodiments, the human patient is an infant (about 6 weeks to 12 months). In certain embodiments, the human patient is a toddler (about 12 to 24 months) or a toddler (about 2 to 5 years old). The compositions of the present invention are also suitable for older children, teenagers and adults (e.g., 18 to 45 years old, 18 to 50 years old, 18 to 55 years old, 18 to 60 years old or 18 to 65 years old). In other embodiments of any method of the present invention, the age of the patient is about 2 years old to about 18 years old. In a further embodiment of any method of the present invention, the age of the patient is 18 years old or more.

[0423] In other embodiments of the methods of the present invention, the patient is an infant, and the infant is administered 1, 2, or 3 doses of the multivalent immunogenic composition of the present invention. The amount of time between each dose administration may vary, but one example of a dosing regimen includes administering 1 dose at 2 months of age, then 1 dose at 4 months of age, and finally 1 dose at 6 months of age. Another example of an administration regimen in infants is administering 1 dose at 2 months of age, then 1 dose at 3 months of age. Another example of an administration regimen in infants is administering 1 dose at 2 months of age, then 1 dose at 3 months of age, and finally 1 dose at 6 months of age. In other embodiments, when the infant is a toddler, the infant patient may receive an additional "boost" dose of the multivalent immunogenic composition of the present invention. For example, the infant is administered at 2 months of age, then 1 dose at 4 months of age, and finally 1 dose at 6 months of age, and then, when the infant reaches toddler age, an additional "boost" dose of the multivalent immunogenic composition of the present invention is administered at 11 to 15 months of age.

[0424] In one embodiment, the infant is administered 2 doses of the multivalent immunogenic composition of the invention.

[0425] In one embodiment, the infant is administered 3 doses of the multivalent immunogenic composition of the invention.

[0426] In one embodiment, the patient is administered three doses of a multivalent immunogenic composition of the invention, wherein the first and second doses are administered between 2 and 10 months of age and the third dose is administered between 11 and 15 months of age.

[0427] In one embodiment, the patient is administered 4 doses of a multivalent immunogenic composition of the invention, wherein the first dose is administered at 2 months of age, the second dose is administered at 4 months of age, the third dose is administered at 6 months of age, and the fourth dose is administered between 11 and 15 months of age.

[0428] In other embodiments of the methods of the present invention, the human patient is an elderly person. In some embodiments of any of the methods of the present invention, the patient is 50 years of age or older. In some embodiments of any of the methods of the present invention, the patient is 55 years of age or older. In some embodiments of any of the methods of the present invention, the patient is 60 years of age or older. In yet further embodiments of any of the methods of the present invention, the patient is 65 years of age or older. In other embodiments of any of the methods of the present invention, the patient is 70 years of age or older.

[0429] In some embodiments of any of the methods of the invention, the patient treated with the immunogenic composition of the invention is immunocompromised.

[0430] In some embodiments of any of the methods of the invention, the multivalent immunogenic composition is administered concomitantly with a flu vaccine. In certain embodiments, the flu vaccine is a "premium flu vaccine," which is a high-dose flu vaccine for the elderly (e.g., people aged 65 years or older).

[0431] The present invention provides a method for inducing a protective immune response against pneumococcal infection in a patient, comprising administering to the patient an immunologically effective amount of any of the multivalent immunogenic pneumococcal polysaccharide-protein conjugate compositions described herein. The optimal amounts of components of a particular vaccine (i.e., a multivalent immunogenic composition) can be determined by standard studies, which include observing an appropriate immune response in a subject. For example, in another embodiment, the dose for human immunization is determined by extrapolation from animal studies to human data. In another embodiment, the dose is determined empirically.

[0432] The methods of the invention can be used to prevent and / or reduce primary clinical syndromes caused by microorganisms (eg, S. pneumoniae), including invasive infections (meningitis, pneumonia, and bacteremia) and non-invasive infections (acute otitis media and sinusitis).

[0433] Administration of the compositions of the invention may include one or more of the following: injection by intramuscular, intraperitoneal, intradermal or subcutaneous routes; or administration by mucosal administration to the oral / digestive tract, respiratory tract or urogenital tract. In one embodiment, intranasal administration can be used to treat pneumonia or otitis media (because nasopharyngeal transport of pneumococci can be more effectively prevented, thereby alleviating infection at an early stage). In a specific embodiment, the compositions of the invention are administered to a patient by intramuscular or subcutaneous administration.

[0434] All publications mentioned herein are incorporated herein by reference for the purpose of describing and disclosing methodologies and materials that might be used in connection with the present invention.

[0435] Having described different embodiments of the present invention with reference to the accompanying drawings, it should be understood that the present invention is not limited to those precise embodiments and that various changes and modifications may be made therein by those skilled in the art without departing from the scope or spirit of the invention as defined in the appended claims.

[0436] The following examples illustrate but do not limit the invention.

[0437] Example

[0438] Example 1

[0439] Preparation of Streptococcus pneumoniae capsular polysaccharide

[0440] Methods for culturing pneumococci are well known in the art. See, for example, Chase, 1967, Methods of Immunology and Immunochemistry 1:52. Methods for preparing pneumococcal capsular polysaccharides are also well known in the art. See, for example, European Patent No. EP 0 497 524 B1. The methods described below generally follow the methods described in European Patent No. EP 0 497 524 B1 and are generally applicable to all pneumococcal serotypes.

[0441] Serotype 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23F, 33F and 35B pneumococcal strain isolates are obtained from Merck Culture Collection. Serotype 23B strains are obtained from the Centers for Disease Control and Prevention and the University of Alabama at Birmingham. Serotype 24F strains are obtained from the Merck Culture Collection and the University of Alabama at Birmingham. If necessary, specific antisera can be used to distinguish subtypes according to the Quelling reaction. See, for example, U.S. Patent No. 5,847,112. The isolates obtained by further cloning and separation are continuously plated in two stages on agar plates, and the agar plates are composed of a medium without animal components, and the medium contains soy peptone, yeast extract and glucose without hemin. For serotype 7F, the agar plates used also contained heme. Clonal isolates of each serotype were further expanded in liquid culture using animal component-free medium containing soy peptone, yeast extract, HEPES, sodium chloride, sodium bicarbonate, potassium phosphate, glucose and glycerol to prepare pre-master cell banks.

[0442] The production of pneumococcal polysaccharide of each serotype is composed of cell expansion and batch production fermentation, and then chemical inactivation is carried out before downstream purification. The thawed cell bank vials from various serotypes are expanded using a shake flask or culture bottle equipped with a pre-sterilized animal-free growth medium, and the culture medium contains soy peptone or soy peptone ultrafiltrate, yeast extract or yeast extract ultrafiltrate, HEPES, sodium chloride, sodium bicarbonate, potassium phosphate and glucose. The cell expansion culture is grown in a sealed shake flask or culture bottle to minimize gas exchange under temperature and stirring control. In the cell expansion process of these serotypes, temperature, pH, pressure and stirring are controlled. Airflow coverage is also controlled because bubbling (sparging) is not used. After reaching a specified culture density (measured by the optical density of 600nm), a portion of the cell expansion culture is transferred to a production fermenter containing a pre-sterilized animal-free growth medium, and the culture medium contains soy peptone or soy peptone ultrafiltrate, yeast extract or yeast extract ultrafiltrate, sodium chloride, potassium phosphate and glucose. Temperature, pH, pressure and agitation are controlled. Airflow coverage is also controlled since bubbling is not used.

[0443] When the glucose is almost exhausted, the batch fermentation is terminated by adding the chemical inactivator phenol. Pure phenol is 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 occurs in the fermenter for a specified time, where temperature and agitation are continuously controlled. After primary inactivation, the batch is transferred to another container, where it is maintained at a controlled temperature and agitation for an additional specified time for complete inactivation. This can be confirmed by microbial plating techniques or by verifying the phenol concentration and the specified time. The inactivated broth is then purified.

[0444] Example 2

[0445] Purification of Pneumococcal Polysaccharide

[0446] The purification of pneumococcal polysaccharides consists of multiple centrifugation, depth filtration, concentration / diafiltration operations and precipitation steps. All steps were performed at room temperature unless otherwise stated.

[0447] Use cationic polymers (such as BPA-1000, (Baker Hughes Inc., Houston, TX), Spectrum 8160, poly(ethyleneimine) and Millipore pDADMAC) were used to flocculate the inactivated medium from the fermenter culture of Streptococcus pneumoniae. The cationic polymer binds to the impurity proteins, nucleic acids and cell debris. After the flocculation step and the aging stage, the flocculated solids are removed by centrifugation and multiple depth filtration steps. The clarified medium is concentrated and diafiltered using a 100 kDa to 500 kDa MWCO (molecular weight cutoff) filter. The diafiltration is accomplished using Tris, MgCl2 buffer and sodium phosphate buffer. The diafiltration removes residual nucleic acids and proteins.

[0448] Impurities can be further removed by reprecipitating the polysaccharides in sodium acetate and phenol with denatured alcohol and / or isopropanol. In the phenol precipitation step, sodium acetate and phenol (liquefied phenol or solid phenol) in sodium phosphate saline buffer are loaded into the retentate that has been filtered. Alcohol fractionation of the polysaccharides is then performed in two stages. In the first stage, a low percentage of ethanol is added to the preparation to precipitate cell debris and other unwanted impurities, while the crude polysaccharides remain in solution. Impurities are removed via centrifugation and subsequent depth filtration steps. The polysaccharides are then recovered from the solution by adding additional isopropanol or denatured alcohol to the batch. The precipitated polysaccharide precipitate is recovered by centrifugation, ground and dried into a powder, and stored frozen at -70°C.

[0449] Example 3

[0450] Preparation of serotype 1 conjugates for PCV23 (DMSO) multivalent studies using DMSO conjugation

[0451] The polysaccharide is dissolved, reduced in size to the target molecular weight, chemically activated and buffer exchanged by ultrafiltration. The activated polysaccharide and purified CRM197 are lyophilized separately and then redissolved in dimethyl sulfoxide (DMSO). The redissolved polysaccharide and CRM197 solutions are then combined and conjugated as described below. The resulting conjugate is purified by ultrafiltration before a final 0.2 micron filtration. Multiple process parameters such as pH, temperature, concentration and time are controlled in each step to produce a conjugate with desired properties.

[0452] Polysaccharide size reduction and oxidation

[0453] The purified pneumococcal capsule Ps powder was dissolved in water and filtered at 0.45 micron. 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 to 250 bar / 5 passes.

[0454] The size-reduced polysaccharides were concentrated and diafiltered with water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

[0455] The polysaccharide solution was then adjusted to 22°C and pH 5 with sodium acetate buffer to minimize polysaccharide size reduction due to activation. Polysaccharide activation was initiated by adding 100 mM sodium metaperiodate solution. The oxidation reaction was carried out at 22°C for 15 hours.

[0456] The activated product was diafiltered against 10 mM potassium phosphate (pH 6.4) using a 10 kDa NMWCO tangential flow ultrafiltration membrane, followed by diafiltration against water using a 5 kDa NMWCO tangential flow ultrafiltration membrane. The ultrafiltration was performed at 2-8°C.

[0457] Polysaccharide conjugation to CRM197

[0458] Purified CRM197 obtained by expression in Pseudomonas fluorescens as previously described (WO 2012 / 173876A1) was diafiltered using a 5 kDa NMWCO tangential flow ultrafiltration membrane with 2 mM phosphate (pH 7.2) buffer and filtered at 0.2 micron.

[0459] The activated polysaccharide was prepared at 2.5 mg Ps / mL and 10% w / v sucrose for lyophilization. CRM197 was prepared at 6 mg Pr / mL and 1% w / v sucrose for lyophilization.

[0460] The prepared Ps and CRM197 solutions were lyophilized separately. The lyophilized Ps and CRM197 materials were redissolved in an equal volume of DMSO. The polysaccharide and CRM197 solutions were blended to achieve a polysaccharide concentration of 1.0 g Ps / L and a mass ratio of polysaccharide to CRM197 of 1.5. The mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate. Sodium cyanoborohydride (1 mole per mole of polysaccharide repeating unit) was added and conjugation was performed at 22°C.

[0461] Reduction with sodium borohydride

[0462] After the conjugation reaction, sodium borohydride (2 moles per mole of polysaccharide repeating unit) was added and incubated at 22°C for 1 hour. The batch was diluted into 150 mM sodium chloride containing about 0.025% (w / v) polysorbate 20 at about 4°C. Potassium phosphate buffer was then added to neutralize the pH. The batch was dialyzed against 150 mM sodium chloride, 0.05% (w / v) polysorbate 20 using a 300 kD NMWCO dialysis cassette at about 4°C for 3 days.

[0463] Final filtration and product storage

[0464] The batch was filtered at 0.2 micron (using a 0.5 micron pre-filter), dispensed into aliquots and frozen at ≤ -60°C.

[0465] Example 4

[0466] Preparation of serotype 1 conjugates for PCV23 (DMSO+Aq) multivalent studies using aqueous conjugation

[0467] The polysaccharide is dissolved, reduced in size, chemically activated and buffer exchanged by ultrafiltration. Purified CRM197 is then conjugated to the activated polysaccharide using nickel chloride in the reaction mixture, and the resulting conjugate is purified by ultrafiltration before a final 0.2 micron filtration. Multiple process parameters such as pH, temperature, concentration and time are controlled in each step to produce a conjugate with desired properties.

[0468] Polysaccharide size reduction and oxidation

[0469] The purified pneumococcal capsular polysaccharide powder was dissolved in water and filtered at 0.45 micron. The dissolved polysaccharide was homogenized to reduce the molecular weight. The homogenization pressure and the number of passes through the homogenizer were controlled to 250 bar / pass 5 times to reduce the size to the target molecular weight. Subsequently, the reduced size polysaccharide was concentrated and diafiltered with water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

[0470] The polysaccharide solution was then adjusted to 22°C and pH 5 with sodium acetate buffer to minimize polysaccharide size reduction due to activation. Polysaccharide activation was initiated by adding 100 mM sodium metaperiodate solution. The oxidation reaction was carried out at 22°C for 15 hours.

[0471] The activated product was diafiltered against 10 mM potassium phosphate (pH 6.4) using a 10 kDa NMWCO tangential flow ultrafiltration membrane. The ultrafiltration was performed at 2-8°C.

[0472] Polysaccharide conjugation to CRM197

[0473] The oxidized polysaccharide solution is mixed with water and 1.5M potassium phosphate (pH 7.0). The buffer pH is selected to improve the stability of the activated polysaccharide during the conjugation reaction. The purified CRM197 obtained by expression in Pseudomonas fluorescens as described above (WO 2012 / 173876A1) is combined with a buffered polysaccharide solution with a polysaccharide to CRM197 mass ratio of 0.5. This mass ratio is selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate. The concentrations of polysaccharide and phosphate are 6.9g / L and 100mM, respectively. The polysaccharide concentration is selected to control the size of the resulting conjugate. Nickel chloride is added to about 2mM using a 100mM nickel chloride solution. Sodium cyanoborohydride (2 mol / mol polysaccharide repeating unit) is added. The conjugation is carried out for 120 hours to maximize the consumption of polysaccharides and proteins.

[0474] Reduction with sodium borohydride

[0475] After the conjugation reaction, the batch was diluted to a polysaccharide concentration of about 3.5 g / L, cooled to 2-8° C., and filtered at 1.2 microns. The batch was diafiltered at 2-8° C. with 100 mM potassium phosphate (pH 7.0) using a 100 kDa NMWCO tangential flow ultrafiltration membrane. Subsequently, the batch recovered in the retentate was diluted to about 2.0 g polysaccharide / L and the pH was adjusted by adding 1.2 M sodium bicarbonate (pH 9.4). Sodium borohydride (1 mol / mol polysaccharide repeating unit) was added. Subsequently, 1.5 M potassium phosphate (pH 6.0) was added.

[0476] Final filtration and product storage

[0477] The batch was then concentrated and diafiltered using a 300 kDa NMWCO tangential flow ultrafiltration membrane with 150 mM sodium chloride (pH 7.0) containing 10 mM L-histidine at 4° C. Polysorbate 20 was added to the retentate batch to a concentration of 0.05% (w / v) and the batch was then filtered at 0.2 microns.

[0478] The polysaccharide concentration of the batch was adjusted to 1.0 g / L with additional 10 mM L-histidine in 150 mM sodium chloride (pH 7.0) buffer and 0.015% (w / v) polysorbate 20. The batch was dispensed into aliquots and frozen at ≤ -60°C.

[0479] Example 5

[0480] Preparation of serotype 1 conjugates for PCV22 multivalent studies using aqueous conjugation

[0481] The polysaccharide is dissolved, reduced in size, chemically activated and buffer exchanged by ultrafiltration. Purified CRM197 is then conjugated to the activated polysaccharide using nickel chloride in the reaction mixture, and the resulting conjugate is purified by ultrafiltration before a final 0.2 micron filtration. Multiple process parameters such as pH, temperature, concentration and time are controlled in each step to produce a conjugate with desired properties.

[0482] Polysaccharide size reduction and oxidation

[0483] The purified pneumococcal capsular polysaccharide powder was dissolved in water and filtered at 0.45 micron. The dissolved polysaccharide was homogenized to reduce the molecular weight. The homogenization pressure and the number of passes through the homogenizer were controlled to 250 bar / pass 5 times to reduce the size to the target molecular weight. Subsequently, the reduced size polysaccharide was concentrated and diafiltered with water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

[0484] The polysaccharide solution was then adjusted to 22°C and pH 5 with sodium acetate buffer to minimize polysaccharide size reduction due to activation. Polysaccharide activation was initiated by adding 100 mM sodium metaperiodate solution. The oxidation reaction was carried out at 22°C for 15 hours.

[0485] The activated product was diafiltered against 10 mM potassium phosphate (pH 6.4) using a 10 kDa NMWCO tangential flow ultrafiltration membrane. The ultrafiltration was performed at 2-8°C.

[0486] Polysaccharide conjugation to CRM197

[0487] The oxidized polysaccharide solution is mixed with water and 1.5M potassium phosphate (pH 7.0). The buffer pH is selected to improve the stability of the activated polysaccharide during the conjugation reaction. The purified CRM197 obtained by expression in Pseudomonas fluorescens as described above (WO 2012 / 173876A1) is combined with a buffered polysaccharide solution with a polysaccharide to CRM197 mass ratio of 0.5. This mass ratio is selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate. The concentrations of polysaccharide and phosphate are 6.9g / L and 100mM, respectively. The polysaccharide concentration is selected to control the size of the resulting conjugate. The solution is then filtered at 0.2 microns. Nickel chloride is added to about 2mM using a 100mM nickel chloride solution. Sodium cyanoborohydride (2 moles / mole polysaccharide repeating unit) is added. The conjugation is carried out for 120 hours to maximize the consumption of polysaccharides and proteins.

[0488] Reduction with sodium borohydride

[0489] After the conjugation reaction, the batch was diluted to a polysaccharide concentration of about 3.5 g / L, cooled to 2-8° C., and filtered at 1.2 microns. The batch was diafiltered at 2-8° C. with 100 mM potassium phosphate (pH 7.0) using a 100 kDa NMWCO tangential flow ultrafiltration membrane. Subsequently, the batch recovered in the retentate was diluted to about 2.0 g polysaccharide / L and the pH was adjusted by adding 1.2 M sodium bicarbonate (pH 9.4). Sodium borohydride (1 mol / mol polysaccharide repeating unit) was added. Subsequently, 1.5 M potassium phosphate (pH 6.0) was added.

[0490] Final filtration and product storage

[0491] The batch was then concentrated and diafiltered using a 300 kDa NMWCO tangential flow ultrafiltration membrane with 150 mM sodium chloride (pH 7.0) containing 10 mM L-histidine at 4° C. Polysorbate 20 was added to the retentate batch to a concentration of 0.05% (w / v) and the batch was then filtered at 0.2 microns.

[0492] The polysaccharide concentration of the batch was adjusted to 1.0 g / L with additional 10 mM L-histidine in 150 mM sodium chloride (pH 7.0) buffer and 0.015% (w / v) polysorbate 20. The batch was dispensed into aliquots and frozen at ≤ -60°C.

[0493] Example 6

[0494] Preparation of serotype 3 conjugates for PCV23 (DMSO) multivalent studies using DMSO conjugation

[0495] The polysaccharide is dissolved, reduced in size to the target molecular weight, chemically activated and buffer exchanged by ultrafiltration. The activated polysaccharide and purified CRM197 are lyophilized and redissolved in dimethyl sulfoxide (DMSO) respectively. The redissolved polysaccharide is then combined with the CRM197 solution and conjugated as described below. The resulting conjugate is purified by ultrafiltration before a final 0.2 micron filtration. Multiple process parameters such as pH, temperature, concentration and time are controlled in each step to produce a conjugate with desired properties.

[0496] Polysaccharide size reduction and oxidation

[0497] The purified pneumococcal capsule Ps powder was dissolved in water and filtered at 0.45 micron. 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 to 810 bar / 6 times, followed by 900 bar / 3 times.

[0498] The size-reduced polysaccharides were concentrated and diafiltered against water using a 5 kDa NMWCO tangential flow ultrafiltration membrane.

[0499] The polysaccharide solution was then adjusted to 22°C and pH 5 with sodium acetate buffer to minimize polysaccharide size reduction due to activation. 100 mM sodium metaperiodate solution was added to initiate polysaccharide activation. The oxidation reaction was carried out at 22°C for 12 hours.

[0500] The activated product was diafiltered against 10 mM potassium phosphate (pH 6.4) and then against water using a 5 kDa NMWCO tangential flow ultrafiltration membrane. The ultrafiltration was performed at 2-8°C.

[0501] Polysaccharide conjugation to CRM197

[0502] Purified CRM197 obtained by expression in Pseudomonas fluorescens as previously described (WO 2012 / 173876A1) was diafiltered using a 5 kDa NMWCO tangential flow ultrafiltration membrane with 2 mM phosphate (pH 7.2) buffer and filtered at 0.2 micron.

[0503] The activated polysaccharide was prepared at 2.5 mg Ps / mL and 10% w / v sucrose for lyophilization. CRM197 was prepared at 6 mg Pr / mL and 1% w / v sucrose for lyophilization.

[0504] The prepared Ps and CRM197 solutions were lyophilized separately. The lyophilized Ps and CRM197 materials were redissolved in an equal volume of DMSO. The polysaccharide was blended with the CRM197 solution to obtain a polysaccharide concentration of 2.25 g Ps / L and a polysaccharide to CRM197 mass ratio of 1.35. This mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate. Sodium cyanoborohydride (1 mol / mol polysaccharide repeating unit) was added and conjugation was performed at 22°C.

[0505] Reduction with sodium borohydride

[0506] Sodium borohydride (2 mol / mol polysaccharide repeating unit) was added after the conjugation reaction and incubated for 1 hour at 22° C. The batch was diluted to 150 mM sodium chloride and approximately 0.025% (w / v) polysorbate 20 at approximately 4° C. Potassium phosphate buffer was then added to neutralize the pH.

[0507] Final filtration and product storage

[0508] The batch was then concentrated and diafiltered at 4°C using a 300 kDa NMWCO tangential flow ultrafiltration membrane with 150 mM sodium chloride (pH 7.0) and 0.015% (w / v) polysorbate 20 containing 10 mM histidine.

[0509] The retentate batch was filtered at 0.2 micron (using a 0.5 micron prefilter) and then diluted with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0, and 0.015% (w / v) polysorbate 20, dispensed into aliquots and frozen at ≤ -60°C.

[0510] Example 7

[0511] Preparation of serotype 3 conjugates for PCV22 and PCV23 (DMSO+Aq) multivalent studies using aqueous conjugation

[0512] The polysaccharide is dissolved, reduced in size, chemically activated and buffer exchanged by ultrafiltration. Purified CRM197 is then conjugated to the activated polysaccharide using nickel chloride in the reaction mixture, and the resulting conjugate is purified by ultrafiltration before a final 0.2 micron filtration. Multiple process parameters such as pH, temperature, concentration and time are controlled in each step to produce a conjugate with desired properties.

[0513] Polysaccharide size reduction and oxidation

[0514] Purified pneumococcal capsular polysaccharide powder was dissolved in water and filtered at 0.45 micron. The dissolved polysaccharide was homogenized to reduce the molecular weight. The homogenization pressure and the number of passes through the homogenizer were controlled to 380 bar / 5 times to reduce the size to the target molecular weight. The size-reduced polysaccharide was then concentrated and diafiltered with water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

[0515] The polysaccharide solution was then adjusted to 22°C and pH 5 with sodium acetate buffer to minimize polysaccharide size reduction due to activation. 100 mM sodium metaperiodate solution was added to initiate polysaccharide activation. The oxidation reaction was carried out at 22°C for 12 hours.

[0516] The activated product was diafiltered against 10 mM potassium phosphate (pH 6.4) using a 10 kDa NMWCO tangential flow ultrafiltration membrane. The ultrafiltration was performed at 2-8°C.

[0517] Polysaccharide conjugation to CRM197

[0518] The oxidized polysaccharide solution is mixed with water and 1.5M potassium phosphate (pH 6.0). The buffer pH is selected to improve the stability of the activated polysaccharide during the conjugation reaction. The purified CRM197 obtained by expression in Pseudomonas fluorescens as described above (WO 2012 / 173876A1) is filtered at 0.2 microns and combined with a buffered polysaccharide solution with a polysaccharide to CRM197 mass ratio of 0.6. This mass ratio is selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate. The concentrations of polysaccharide and phosphate are 4.1g / L and 150mM, respectively. The polysaccharide concentration is selected to control the size of the resulting conjugate. The solution is then filtered at 0.2 microns. Nickel chloride is added to about 2mM using a 100mM nickel chloride solution. Sodium cyanoborohydride (2 moles / mole polysaccharide repeating unit) is added. Conjugation is performed at 10°C for 120 hours to maximize the consumption of polysaccharides and proteins.

[0519] Reduction with sodium borohydride

[0520] After the conjugation reaction, the batch was diluted to a polysaccharide concentration of about 3.5 g / L, cooled to 2-8° C., and filtered at 1.2 microns. The batch was diafiltered at 2-8° C. with 100 mM potassium phosphate (pH 7.0) using a 100 kDa NMWCO tangential flow ultrafiltration membrane. Subsequently, the batch recovered in the retentate was diluted to about 2.0 g polysaccharide / L and the pH was adjusted by adding 1.2 M sodium bicarbonate (pH 9.4). Sodium borohydride (1 mol / mol polysaccharide repeating unit) was added. Subsequently, 1.5 M potassium phosphate (pH 6.0) was added.

[0521] Final filtration and product storage

[0522] The batch was then concentrated and diafiltered using a 300 kDa NMWCO tangential flow ultrafiltration membrane with 150 mM sodium chloride, pH 7.0, containing 10 mM L-histidine at 4° C. The batch was filtered at 0.2 microns.

[0523] The polysaccharide concentration of the batch was adjusted to 1.0 g / L with additional 150 mM sodium chloride (pH 7.0) buffer containing 10 mM L-histidine. The batch was divided into aliquots and frozen at ≤ -60°C.

[0524] Example 8

[0525] Preparation of serotype 4 conjugates for PCV23 (DMSO) multivalent studies using DMSO conjugation The polysaccharide was dissolved, reduced in size to the target molecular weight, chemically activated and buffer exchanged by ultrafiltration. The activated polysaccharide and purified CRM197 were lyophilized and redissolved in DMSO separately. The redissolved polysaccharide was then combined with the CRM197 solution and conjugated as described below. The resulting conjugate was purified by diafiltration before final filtration at 0.2 microns. Multiple process parameters such as pH, temperature, concentration and time were controlled in each step to produce a conjugate with desired properties.

[0526] Polysaccharide size reduction and oxidation

[0527] The purified pneumococcal capsule Ps powder was dissolved in water and filtered at 0.45 micron. 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 to 300 bar / 5 times.

[0528] The size-reduced polysaccharides were concentrated and diafiltered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

[0529] Then, the polysaccharide solution was adjusted to 50°C and pH 4.1 was adjusted with sodium acetate buffer to partially deketalize the polysaccharide. Then, the polysaccharide solution was cooled to 22°C and then activated. 100 mM sodium metaperiodate solution was added to initiate polysaccharide activation. The oxidation reaction was carried out at 22°C for 4 hours.

[0530] The activated product was diafiltered against 10 mM potassium phosphate (pH 6.4) using a 10 kDa NMWCO tangential flow ultrafiltration membrane, followed by diafiltration against water using a 5 kDa NMWCO tangential flow ultrafiltration membrane. The ultrafiltration was performed at 2-8°C.

[0531] Polysaccharide conjugation to CRM197

[0532] Purified CRM197 obtained by expression in Pseudomonas fluorescens as previously described (WO 2012 / 173876A1) was diafiltered using a 5 kDa NMWCO tangential flow ultrafiltration membrane with 2 mM phosphate (pH 7.2) buffer and filtered at 0.2 micron.

[0533] The activated polysaccharide was prepared at 6 mg Ps / mL and 5% w / v sucrose for lyophilization. CRM197 was prepared at 6 mg Pr / mL and 1% w / v sucrose for lyophilization.

[0534] The prepared Ps and CRM197 solutions were lyophilized separately. The lyophilized Ps and CRM197 materials were redissolved in an equal volume of DMSO. The polysaccharide was blended with the CRM197 solution to obtain a polysaccharide concentration of 5.0 g Ps / L and a polysaccharide to CRM197 mass ratio of 2.0. This mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate. Sodium cyanoborohydride (1 mol / mol polysaccharide repeating unit) was added and the conjugation was performed at 22°C.

[0535] Reduction with sodium borohydride

[0536] Sodium borohydride (2 mol / mol polysaccharide repeating unit) was added after the conjugation reaction and incubated at 22°C for 1 hour. The batch was diluted to 150 mM sodium chloride and about 0.025% (w / v) polysorbate 20 at about 4°C. Potassium phosphate buffer was then added to neutralize the pH. The batch was dialyzed against 150 mM sodium chloride, 0.05% (w / v) polysorbate 20 at about 4°C for 3 days using a 300 kDa NMWCO dialysis cassette.

[0537] Final filtration and product storage

[0538] The batch was filtered at 0.2 micron (using a 0.5 micron pre-filter), dispensed into aliquots, and frozen at ≤ -60°C.

[0539] Example 9

[0540] Preparation of serotype 4 conjugates for PCV23 (DMSO+Aq) and PCV22 multivalent studies using aqueous conjugation

[0541] The polysaccharide is dissolved, reduced in size, chemically activated and buffer exchanged by ultrafiltration. Purified CRM197 is then conjugated to the activated polysaccharide using nickel chloride in the reaction mixture, and the resulting conjugate is purified by ultrafiltration before a final 0.2 micron filtration. Multiple process parameters such as pH, temperature, concentration and time are controlled in each step to produce a conjugate with desired properties.

[0542] Polysaccharide size reduction and oxidation

[0543] Purified pneumococcal capsular polysaccharide powder was dissolved in water and filtered at 0.45 micron. The dissolved polysaccharide was homogenized to reduce the molecular weight. The homogenization pressure and the number of passes through the homogenizer were controlled to 300 bar / 5 times to reduce the size to the target molecular weight. The size-reduced polysaccharide was then concentrated and diafiltered with water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

[0544] Then, the polysaccharide solution was adjusted to 50°C and pH 4.1 was adjusted with sodium acetate buffer to partially deketalize the polysaccharide. Then, the polysaccharide solution was cooled to 22°C and then activated. 100 mM sodium metaperiodate solution was added to initiate polysaccharide activation. The oxidation reaction was carried out at 22°C for 4 hours.

[0545] The activated product was diafiltered against 10 mM potassium phosphate (pH 6.4) using a 10 kDa NMWCO tangential flow ultrafiltration membrane. The ultrafiltration was performed at 2-8°C.

[0546] Polysaccharide conjugation to CRM197

[0547] The oxidized polysaccharide solution is mixed with water and 1.5M potassium phosphate (pH 7.0). The buffer pH is selected to improve the stability of the activated polysaccharide during the conjugation reaction. The purified CRM197 obtained by expression in Pseudomonas fluorescens as described above (WO 2012 / 173876A1) is filtered at 0.2 microns, and combined with a buffered polysaccharide solution with a polysaccharide to CRM197 mass ratio of 0.5. This mass ratio is selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate. The concentrations of polysaccharide and phosphate are 8.3g / L and 100mM respectively. The polysaccharide concentration is selected to control the size of the resulting conjugate. The solution is then filtered at 0.2 microns. Nickel chloride is added to about 2mM using a 100mM nickel chloride solution. Sodium cyanoborohydride (2 moles / mole polysaccharide repeating unit) is added. Conjugation is carried out for 120 hours to maximize the consumption of polysaccharides and proteins.

[0548] Reduction with sodium borohydride

[0549] After the conjugation reaction, the batch was diluted to a polysaccharide concentration of about 3.5 g / L, cooled to 2-8° C., and filtered at 1.2 microns. The batch was diafiltered at 2-8° C. with 100 mM potassium phosphate (pH 7.0) using a 100 kDa NMWCO tangential flow ultrafiltration membrane. Subsequently, the batch recovered in the retentate was diluted to about 2.0 g polysaccharide / L and the pH was adjusted by adding 1.2 M sodium bicarbonate (pH 9.4). Sodium borohydride (1 mol / mol polysaccharide repeating unit) was added. Subsequently, 1.5 M potassium phosphate (pH 6.0) was added.

[0550] Final filtration and product storage

[0551] The batch was then concentrated and diafiltered using a 300 kDa NMWCO tangential flow ultrafiltration membrane with 150 mM sodium chloride, pH 7.0, containing 10 mM L-histidine at 4° C. The batch was then filtered at 0.2 microns.

[0552] The polysaccharide concentration of the batch was adjusted to 1.0 g / L with additional 150 mM sodium chloride (pH 7.0) buffer containing 10 mM L-histidine. The batch was divided into aliquots and frozen at ≤ -60°C.

[0553] Example 10

[0554] Preparation of serotype 5 conjugates for PCV23 (DMSO) multivalent studies using DMSO conjugation

[0555] The polysaccharide is dissolved, reduced in size to the target molecular weight, chemically activated and buffer exchanged by ultrafiltration. The activated polysaccharide and purified CRM197 are lyophilized and redissolved in DMSO separately. The redissolved polysaccharide is then combined with the CRM197 solution and conjugated as described below. The resulting conjugate is purified by diafiltration before final filtration at 0.2 microns. Multiple process parameters such as pH, temperature, concentration and time are controlled in each step to produce a conjugate with desired properties.

[0556] Polysaccharide size reduction and oxidation

[0557] The purified pneumococcal capsule Ps powder was dissolved in water and filtered at 0.45 micron. 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 to 600 bar / 5 times.

[0558] The size-reduced polysaccharides were concentrated and diafiltered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

[0559] The polysaccharide solution was then adjusted to 4°C and pH 4.1 using sodium acetate buffer to minimize polysaccharide size reduction due to activation. 100 mM sodium metaperiodate solution was added to initiate polysaccharide activation. The oxidation reaction was carried out at 4°C for 4 hours.

[0560] The activated product was diafiltered with 10 mM sodium acetate (pH 4.1) using a 10 kDa NMWCO tangential flow ultrafiltration membrane and then diafiltered with water using a 5 kDa NMWCO tangential flow ultrafiltration membrane. The ultrafiltration was performed at 2-8°C.

[0561] Polysaccharide conjugation to CRM197

[0562] Purified CRM197 obtained by expression in Pseudomonas fluorescens as previously described (WO 2012 / 173876A1) was diafiltered using a 5 kDa NMWCO tangential flow ultrafiltration membrane with 2 mM phosphate (pH 7.2) buffer and filtered at 0.2 micron.

[0563] The activated polysaccharide was prepared at 6 mg Ps / mL and 5% w / v sucrose for lyophilization. CRM197 was prepared at 6 mg Pr / mL and 1% w / v sucrose for lyophilization.

[0564] The prepared Ps and CRM197 solutions were lyophilized separately. The lyophilized Ps and CRM197 substances were redissolved in equal volumes of DMSO respectively. The polysaccharide solution was spiked with sodium chloride to a concentration of 20 mM. The polysaccharide was blended with the CRM197 solution to obtain a polysaccharide concentration of 2.0 g Ps / L and a mass ratio of polysaccharide to CRM197 of 1.5. This mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate. Sodium cyanoborohydride (1 mol / mol polysaccharide repeating unit) was added and conjugation was performed at 22°C.

[0565] Dilution and Neutralization

[0566] The batch was diluted into 150 mM sodium chloride and about 0.025% (w / v) polysorbate 20 at about 4°C. Potassium phosphate buffer was then added to neutralize the pH. The batch was dialyzed against 150 mM sodium chloride, 0.05% (w / v) polysorbate 20 at about 4°C for 3 days using a 300 kDa NMWCO dialysis cassette.

[0567] Final filtration and product storage

[0568] The retentate batch was filtered at 0.2 micron (using a 0.5 micron pre-filter), dispensed into aliquots, and frozen at ≤ -60°C.

[0569] Embodiment 11

[0570] Preparation of serotype 5 conjugates for PCV22 and PCV23 (DMSO+Aq) multivalent studies using aqueous conjugation

[0571] The polysaccharide is dissolved, reduced in size, chemically activated and buffer exchanged by ultrafiltration. Purified CRM197 is then conjugated to the activated polysaccharide using nickel chloride in the reaction mixture, and the resulting conjugate is purified by ultrafiltration before a final 0.2 micron filtration. Multiple process parameters such as pH, temperature, concentration and time are controlled in each step to produce a conjugate with desired properties.

[0572] Polysaccharide size reduction and oxidation

[0573] Purified pneumococcal capsular polysaccharide powder was dissolved in water and filtered at 0.45 micron. The dissolved polysaccharide was homogenized to reduce the molecular weight. The homogenization pressure and the number of passes through the homogenizer were controlled to 600 bar / 5 times to reduce the size to the target molecular weight. The size-reduced polysaccharide was then concentrated and diafiltered with water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

[0574] The polysaccharide solution was then adjusted to 4°C and pH 4.1 using sodium acetate buffer to minimize polysaccharide size reduction due to activation. 100 mM sodium metaperiodate solution was added to initiate polysaccharide activation. The oxidation reaction was carried out at 4°C for 4 hours.

[0575] The activated product was diafiltered against 10 mM sodium acetate (pH 4.1) using a 10 kDa NMWCO tangential flow diafiltration membrane. Ultrafiltration was performed at 2-8°C.

[0576] Polysaccharide conjugation to CRM197

[0577] The oxidized polysaccharide solution is mixed with water and 1.5M potassium phosphate (pH 6.0). The buffer pH is selected to improve the stability of the activated polysaccharide during the conjugation reaction. The purified CRM197 obtained by expression in Pseudomonas fluorescens as described above (WO 2012 / 173876A1) is combined with a buffered polysaccharide solution with a polysaccharide to CRM197 mass ratio of 0.4. This mass ratio is selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate. The concentrations of polysaccharide and phosphate are 3.8g / L and 150mM respectively. The polysaccharide concentration is selected to control the size of the resulting conjugate. The solution is then filtered at 0.2 microns. Nickel chloride is added to about 2mM using a 100mM nickel chloride solution. Sodium cyanoborohydride (2 moles / mole polysaccharide repeating unit) is added. Conjugation is carried out for 96 hours to maximize the consumption of polysaccharides and proteins.

[0578] Purification and neutralization

[0579] After the conjugation reaction, the batch was diluted to a polysaccharide concentration of about 3.5 g / L, cooled to 2-8° C., and filtered at 1.2 microns. The batch was diafiltered using a 100 kDa NMWCO tangential flow ultrafiltration membrane at 2-8° C. using 300 mM sodium bicarbonate (pH 9.3). Subsequently, the batch recovered in the retentate was neutralized with 1.5 M potassium phosphate (pH 6.0).

[0580] Final filtration and product storage

[0581] The batch was then concentrated and diafiltered using a 300 kDa NMWCO tangential flow ultrafiltration membrane with 150 mM sodium chloride, pH 7.0, containing 10 mM L-histidine at 4° C. Polysorbate 20 was added to the retentate batch to a concentration of 0.05% (w / v) and the batch was then filtered at 0.2 microns.

[0582] The polysaccharide concentration of the batch was adjusted to 1.0 g / L with additional 10 mM L-histidine in 150 mM sodium chloride (pH 7.0) buffer and 0.015% (w / v) polysorbate 20. The batch was dispensed into aliquots and frozen at ≤ -60°C.

[0583] Example 12

[0584] Preparation of serotype 6A for PCV23 (DMSO) and PCV23 (DMSO + Aq) and PCV22 multivalent studies using DMSO conjugation

[0585] The polysaccharide is dissolved, chemically activated and buffer exchanged by ultrafiltration. The activated polysaccharide and purified CRM197 are lyophilized and redissolved in DMSO separately. The redissolved polysaccharide is then combined with the CRM197 solution and conjugated as described below. The resulting conjugate is purified by ultrafiltration before a final 0.2 micron filtration. Multiple process parameters within each step, such as pH, temperature, concentration and time, are controlled to obtain a conjugate with the desired properties.

[0586] Polysaccharide size reduction and oxidation

[0587] Purified pneumococcal capsule Ps powder was dissolved in water and filtered at 0.45 micron. 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 to 200 bar / 5 times. The size-reduced polysaccharide was concentrated and diafiltered with water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

[0588] The polysaccharide solution was then adjusted to 22°C and pH 5 with sodium acetate buffer to minimize polysaccharide size reduction due to activation. 100 mM sodium metaperiodate solution was added to initiate polysaccharide activation. The oxidation reaction was carried out at 22°C for 2 hours.

[0589] The activated product was diafiltered against 10 mM potassium phosphate (pH 6.4) and then against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane. The ultrafiltration was performed at 2-8°C.

[0590] Polysaccharide conjugation to CRM197

[0591] Purified CRM197 obtained by expression in Pseudomonas fluorescens as previously described (WO 2012 / 173876A1) was diafiltered using a 5 kDa NMWCO tangential flow ultrafiltration membrane with 2 mM phosphate (pH 7.2) buffer and filtered at 0.2 micron.

[0592] The activated polysaccharide was prepared at 6 mg Ps / mL and 5% w / v sucrose for lyophilization. CRM197 was prepared at 6 mg Pr / mL and 1% w / v sucrose for lyophilization.

[0593] The prepared Ps and CRM197 solutions were lyophilized separately. The lyophilized Ps and CRM197 materials were redissolved in an equal volume of DMSO. The polysaccharide was blended with the CRM197 solution to obtain a polysaccharide concentration of 1.5 g Ps / L and a polysaccharide to CRM197 mass ratio of 1.4. This mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate. Sodium cyanoborohydride (1 mol / mol polysaccharide repeating unit) was added and the conjugation was performed at 22°C.

[0594] Reduction with sodium borohydride

[0595] Sodium borohydride (2 mol / mol polysaccharide repeating unit) was added after the conjugation reaction and incubated at 22°C for 3 hours. The batch was diluted to 150 mM sodium chloride and about 0.025% (w / v) polysorbate 20 at about 4°C. Potassium phosphate buffer was then added to neutralize the pH. The batch was concentrated and diafiltered with 150 mM sodium chloride, 25 mM potassium phosphate (pH 7) using a 30 kDa NMWCO tangential flow ultrafiltration membrane at about 4°C.

[0596] Final filtration and product storage

[0597] The batch was then concentrated and diafiltered at 4°C using a 300 kDa NMWCO tangential flow ultrafiltration membrane with 150 mM sodium chloride (pH 7.0) and 0.015% (w / v) polysorbate 20 containing 10 mM histidine.

[0598] The retentate batch was filtered at 0.2 micron and then diluted with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0, and 0.015% (w / v) polysorbate 20, dispensed into aliquots, and frozen at ≤ -60°C.

[0599] Example 13

[0600] Preparation of serotype 6B conjugates for PCV 23 (DMSO) and PCV 23 (DMSO / Aq) multivalent studies using DMSO conjugation

[0601] The polysaccharide is dissolved, reduced in size to the target molecular weight, chemically activated and buffer exchanged by ultrafiltration. The activated polysaccharide and purified CRM197 are lyophilized and redissolved in DMSO separately. The redissolved polysaccharide is then combined with the CRM197 solution and conjugated as described below. The resulting conjugate is purified by ultrafiltration before a final 0.2 micron filtration. Multiple process parameters such as pH, temperature, concentration and time are controlled in each step to produce a conjugate with desired properties.

[0602] Polysaccharide size reduction and oxidation

[0603] The purified pneumococcal capsule Ps powder was dissolved in water and filtered at 0.45 micron. 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 to 200 bar / 5 times.

[0604] The size-reduced polysaccharides were concentrated and diafiltered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

[0605] The polysaccharide solution was then adjusted to 22°C and pH 5 with sodium acetate buffer to minimize polysaccharide size reduction due to activation. 100 mM sodium metaperiodate solution was added to initiate polysaccharide activation. The oxidation reaction was carried out at 22°C for 2 hours.

[0606] The activated product was diafiltered against 10 mM potassium phosphate (pH 6.4) and then against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane. The ultrafiltration was performed at 2-8°C.

[0607] Polysaccharide conjugation to CRM197

[0608] Purified CRM197 obtained by expression in Pseudomonas fluorescens as previously described (WO 2012 / 173876A1) was diafiltered using a 5 kDa NMWCO tangential flow ultrafiltration membrane with 2 mM phosphate (pH 7.2) buffer and filtered at 0.2 micron.

[0609] The activated polysaccharide was prepared at 6 mg Ps / mL and 5% w / v sucrose for lyophilization. CRM197 was prepared at 6 mg Pr / mL and 1% w / v sucrose for lyophilization.

[0610] The prepared Ps and CRM197 solutions were lyophilized separately. The lyophilized Ps and CRM197 materials were redissolved in an equal volume of DMSO. The polysaccharide was blended with the CRM197 solution to obtain a polysaccharide concentration of 1.85 g Ps / L and a polysaccharide to CRM197 mass ratio of 1.35. This mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate. Sodium cyanoborohydride (1 mol / mol polysaccharide repeating unit) was added and conjugation was performed at 22°C.

[0611] Reduction with sodium borohydride

[0612] Sodium borohydride (2 mol / mol polysaccharide repeating unit) was added after the conjugation reaction and incubated at 22°C for 3 hours. The batch was diluted to 150 mM sodium chloride and about 0.025% (w / v) polysorbate 20 at about 4°C. Potassium phosphate buffer was then added to neutralize the pH. The batch was concentrated and diafiltered with 150 mM sodium chloride, 25 mM potassium phosphate (pH 7) using a 30 kDa NMWCO tangential flow ultrafiltration membrane at about 4°C.

[0613] Final filtration and product storage

[0614] The batch was then concentrated and diafiltered at 4°C using a 300 kDa NMWCO tangential flow ultrafiltration membrane with 150 mM sodium chloride (pH 7.0) and 0.015% (w / v) polysorbate 20 containing 10 mM histidine.

[0615] The retentate batch was filtered at 0.2 micron and then diluted with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0, and 0.015% (w / v) polysorbate 20, dispensed into aliquots, and frozen at ≤ -60°C.

[0616] Embodiment 14

[0617] Preparation of serotype 6B conjugates for PCV22 multivalent studies using DMSO conjugation

[0618] The polysaccharide is dissolved, reduced in size to the target molecular weight, chemically activated and buffer exchanged by ultrafiltration. The activated polysaccharide and purified CRM197 are lyophilized and redissolved in DMSO separately. The redissolved polysaccharide is then combined with the CRM197 solution and conjugated as described below. The resulting conjugate is purified by ultrafiltration before a final 0.2 micron filtration. Multiple process parameters such as pH, temperature, concentration and time are controlled in each step to produce a conjugate with desired properties.

[0619] Polysaccharide size reduction and oxidation

[0620] Purified pneumococcal capsule Ps powder was dissolved in water and filtered at 0.45 micron. 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 to 200 bar / 5 times. The size-reduced polysaccharide was concentrated and diafiltered with water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

[0621] The polysaccharide solution was then adjusted to 22°C and pH 5 with sodium acetate buffer to minimize polysaccharide size reduction due to activation. 100 mM sodium metaperiodate solution was added to initiate polysaccharide activation. The oxidation reaction was carried out at 22°C for 2 hours.

[0622] The activated product was diafiltered against 10 mM potassium phosphate (pH 6.4) and then against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane. The ultrafiltration was performed at 2-8°C.

[0623] Polysaccharide conjugation to CRM197

[0624] Purified CRM197 obtained by expression in Pseudomonas fluorescens as previously described (WO 2012 / 173876A1) was diafiltered using a 5 kDa NMWCO tangential flow ultrafiltration membrane with 2 mM phosphate (pH 7.2) buffer and filtered at 0.2 micron.

[0625] The activated polysaccharide was prepared at 6 mg Ps / mL and 5% w / v sucrose for lyophilization. CRM197 was prepared at 6 mg Pr / mL and 1% w / v sucrose for lyophilization.

[0626] The prepared Ps and CRM197 solutions were lyophilized separately. The lyophilized Ps and CRM197 materials were redissolved in an equal volume of DMSO. The polysaccharide was blended with the CRM197 solution to obtain a polysaccharide concentration of 1.75 g Ps / L and a polysaccharide to CRM197 mass ratio of 1.35. This mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate. Sodium cyanoborohydride (1 mol / mol polysaccharide repeating unit) was added and conjugation was performed at 22°C.

[0627] Reduction with sodium borohydride

[0628] Sodium borohydride (2 mol / mol polysaccharide repeating unit) was added after the conjugation reaction and incubated at 22°C for 3 hours. The batch was diluted to 150 mM sodium chloride and about 0.025% (w / v) polysorbate 20 at about 4°C. Potassium phosphate buffer was then added to neutralize the pH. The batch was concentrated and diafiltered with 150 mM sodium chloride, 25 mM potassium phosphate (pH 7) using a 30 kDa NMWCO tangential flow ultrafiltration membrane at about 4°C.

[0629] Final filtration and product storage

[0630] The batch was then concentrated and diafiltered at 4°C using a 300 kDa NMWCO tangential flow ultrafiltration membrane with 150 mM sodium chloride (pH 7.0) and 0.015% (w / v) polysorbate 20 containing 10 mM histidine.

[0631] The retentate batch was filtered at 0.2 micron and then diluted with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0, and 0.015% (w / v) polysorbate 20, dispensed into aliquots, and frozen at ≤ -60°C.

[0632] Embodiment 15

[0633] Preparation of serotype 7F conjugates for PCV23 (DMSO) and PCV23 (DMSO + Aq) multivalent studies using DMSO conjugation

[0634] The polysaccharide is dissolved, reduced in size to the target molecular weight, chemically activated and buffer exchanged by ultrafiltration. The activated polysaccharide and purified CRM197 are lyophilized and redissolved in DMSO separately. The redissolved polysaccharide is then combined with the CRM197 solution and conjugated as described below. The resulting conjugate is purified by ultrafiltration before a final 0.2 micron filtration. Multiple process parameters such as pH, temperature, concentration and time are controlled in each step to produce a conjugate with desired properties.

[0635] Polysaccharide size reduction and oxidation

[0636] Purified pneumococcal capsule Ps powder was dissolved in water and filtered at 0.45 micron. 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 to 150 bar / 7 times. The size-reduced polysaccharide was concentrated and diafiltered with water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

[0637] The polysaccharide was then adjusted to 4°C and to pH 5 with sodium acetate buffer to minimize polysaccharide size reduction due to activation. 100 mM sodium metaperiodate solution was added to initiate polysaccharide activation. The oxidation reaction was carried out at 4°C for 4 hours.

[0638] The activated product was diafiltered against 10 mM potassium phosphate (pH 6.4) and then against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane. The ultrafiltration was performed at 2-8°C.

[0639] Polysaccharide conjugation to CRM197

[0640] Purified CRM197 obtained by expression in Pseudomonas fluorescens as previously described (WO 2012 / 173876A1) was diafiltered using a 5 kDa NMWCO tangential flow ultrafiltration membrane with 2 mM phosphate (pH 7.2) buffer and filtered at 0.2 micron.

[0641] The activated polysaccharide was prepared at 6 mg Ps / mL and 5% w / v sucrose for lyophilization. CRM197 was prepared at 6 mg Pr / mL and 1% w / v sucrose for lyophilization.

[0642] The prepared Ps and CRM197 solutions were lyophilized separately. The lyophilized Ps and CRM197 materials were redissolved in an equal volume of DMSO. The polysaccharide was blended with the CRM197 solution to obtain a polysaccharide concentration of 2.6 g Ps / L and a polysaccharide to CRM197 mass ratio of 1.5. This mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate. Sodium cyanoborohydride (1 mol / mol polysaccharide repeating unit) was added and conjugation was performed at 22°C.

[0643] Reduction with sodium borohydride

[0644] Sodium borohydride (2 mol / mol polysaccharide repeating unit) was added after the conjugation reaction and incubated at 22°C for 3 hours. The batch was diluted to 150 mM sodium chloride and about 0.025% (w / v) polysorbate 20 at about 4°C. Potassium phosphate buffer was then added to neutralize the pH. The batch was concentrated and diafiltered with 150 mM sodium chloride, 25 mM potassium phosphate (pH 7) using a 30 kDa NMWCO tangential flow ultrafiltration membrane at about 4°C.

[0645] Final filtration and product storage

[0646] The batch was then concentrated and diafiltered at 4°C using a 300 kDa NMWCO tangential flow ultrafiltration membrane with 150 mM sodium chloride (pH 7.0) and 0.015% (w / v) polysorbate 20 containing 10 mM histidine.

[0647] The retentate batch was filtered at 0.2 micron and then diluted with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0, and 0.015% (w / v) polysorbate 20, dispensed into aliquots, and frozen at ≤ -60°C.

[0648] Example 16

[0649] Preparation of serotype 7F conjugates for PCV22 multivalent studies using DMSO conjugation

[0650] The polysaccharide is dissolved, reduced in size to the target molecular weight, chemically activated and buffer exchanged by ultrafiltration. The activated polysaccharide and purified CRM197 are lyophilized and redissolved in DMSO separately. The redissolved polysaccharide is then combined with the CRM197 solution and conjugated as described below. The resulting conjugate is purified by ultrafiltration before a final 0.2 micron filtration. Multiple process parameters such as pH, temperature, concentration and time are controlled in each step to produce a conjugate with desired properties.

[0651] Polysaccharide size reduction and oxidation

[0652] Purified pneumococcal capsule Ps powder was dissolved in water and filtered at 0.45 micron. 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 to 150 bar / 7 times. The size-reduced polysaccharide was concentrated and diafiltered with water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

[0653] The polysaccharide was then adjusted to 4°C and to pH 5 with sodium acetate buffer to minimize polysaccharide size reduction due to activation. 100 mM sodium metaperiodate solution was added to initiate polysaccharide activation. The oxidation reaction was carried out at 4°C for 4 hours.

[0654] The activated product was diafiltered against 10 mM potassium phosphate (pH 6.4) and then against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane. The ultrafiltration was performed at 2-8°C.

[0655] Polysaccharide conjugation to CRM197

[0656] Purified CRM197 obtained by expression in Pseudomonas fluorescens as previously described (WO 2012 / 173876A1) was diafiltered using a 5 kDa NMWCO tangential flow ultrafiltration membrane with 2 mM phosphate (pH 7.2) buffer and filtered at 0.2 micron.

[0657] The activated polysaccharide was prepared at 6 mg Ps / mL and 5% w / v sucrose for lyophilization. CRM197 was prepared at 6 mg Pr / mL and 1% w / v sucrose for lyophilization.

[0658] The prepared Ps and CRM197 solutions were lyophilized separately. The lyophilized Ps and CRM197 materials were redissolved in an equal volume of DMSO. The polysaccharide was blended with the CRM197 solution to obtain a polysaccharide concentration of 2.04 g Ps / L and a polysaccharide to CRM197 mass ratio of 1.5. This mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate. Sodium cyanoborohydride (1 mol / mol polysaccharide repeating unit) was added and conjugation was performed at 22°C.

[0659] Reduction with sodium borohydride

[0660] Sodium borohydride (2 mol / mol polysaccharide repeating unit) was added after the conjugation reaction and incubated at 22°C for 3 hours. The batch was diluted to 150 mM sodium chloride and about 0.025% (w / v) polysorbate 20 at about 4°C. Potassium phosphate buffer was then added to neutralize the pH. The batch was concentrated and diafiltered with 150 mM sodium chloride, 25 mM potassium phosphate (pH 7) using a 30 kDa NMWCO tangential flow ultrafiltration membrane at about 4°C.

[0661] Final filtration and product storage

[0662] The batch was then concentrated and diafiltered at 4°C using a 300 kDa NMWCO tangential flow ultrafiltration membrane with 150 mM sodium chloride (pH 7.0) and 0.015% (w / v) polysorbate 20 containing 10 mM histidine.

[0663] The retentate batch was filtered at 0.2 micron and then diluted with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0, and 0.015% (w / v) polysorbate 20, dispensed into aliquots, and frozen at ≤ -60°C.

[0664] Embodiment 17

[0665] Preparation of serotype 8 conjugates for PCV23 (DMSO) and PCV23 (DMSO + Aq) multivalent studies using DMSO conjugation

[0666] The polysaccharide is dissolved, reduced in size to the target molecular weight, chemically activated and buffer exchanged by ultrafiltration. The activated polysaccharide and purified CRM197 are lyophilized and redissolved in DMSO separately. The redissolved polysaccharide is then combined with the CRM197 solution and conjugated as described below. The resulting conjugate is purified by ultrafiltration before a final 0.2 micron filtration. Multiple process parameters such as pH, temperature, concentration and time are controlled in each step to produce a conjugate with desired properties.

[0667] Polysaccharide size reduction and oxidation

[0668] Purified pneumococcal capsule Ps powder was dissolved in water and filtered at 0.45 micron. 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 to 600 bar / 6 times. The size-reduced polysaccharide was concentrated and diafiltered with water using a 5kDa NMWCO tangential flow ultrafiltration membrane.

[0669] The polysaccharide solution was then adjusted to 22°C and pH 5 with sodium acetate buffer to minimize polysaccharide size reduction due to activation. 100 mM sodium metaperiodate solution was added to initiate polysaccharide activation. The oxidation reaction was carried out at 22°C for 4 hours.

[0670] The activated product was diafiltered against 10 mM potassium phosphate (pH 6.4) and then against water using a 5 kDa NMWCO tangential flow ultrafiltration membrane. The ultrafiltration was performed at 2-8°C.

[0671] Polysaccharide conjugation to CRM197

[0672] Purified CRM197 obtained by expression in Pseudomonas fluorescens as previously described (WO 2012 / 173876A1) was diafiltered using a 5 kDa NMWCO tangential flow ultrafiltration membrane with 2 mM phosphate (pH 7.2) buffer and filtered at 0.2 micron.

[0673] The activated polysaccharide was prepared at 6 mg Ps / mL and 5% w / v sucrose for lyophilization. CRM197 was prepared at 6 mg Pr / mL and 1% w / v sucrose for lyophilization.

[0674] The prepared Ps and CRM197 solutions were lyophilized separately. The lyophilized Ps and CRM197 substances were redissolved in an equal volume of DMSO. The polysaccharide and CRM197 solutions were blended to obtain a polysaccharide concentration of 4.5 g Ps / L and a polysaccharide to CRM197 mass ratio of 1.5. This mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate. After mixing, the conjugation reaction was carried out at 22°C.

[0675] Reduction with sodium borohydride

[0676] Sodium borohydride (2 mol / mol polysaccharide repeating unit) was added after the conjugation reaction and incubated for 1 hour at 22° C. The batch was diluted to 150 mM sodium chloride and approximately 0.025% (w / v) polysorbate 20 at approximately 4° C. Potassium phosphate buffer was then added to neutralize the pH.

[0677] Final filtration and product storage

[0678] The batch was then concentrated and diafiltered at 4°C using a 300 kDa NMWCO tangential flow ultrafiltration membrane with 150 mM sodium chloride (pH 7.0) and 0.015% (w / v) polysorbate 20 containing 10 mM histidine.

[0679] The retentate batch was filtered at 0.2 micron (using a 0.5 micron prefilter) and then diluted with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0, and 0.015% (w / v) polysorbate 20, dispensed into aliquots and frozen at ≤ -60°C.

[0680] Embodiment 18

[0681] Preparation of serotype 9V conjugates for PCV23 (DMSO) multivalent studies using DMSO conjugation

[0682] The polysaccharide is dissolved, reduced in size to the target molecular weight, chemically activated and buffer exchanged by ultrafiltration. The activated polysaccharide and purified CRM197 are lyophilized and redissolved in DMSO separately. The redissolved polysaccharide is then combined with the CRM197 solution and conjugated as described below. The resulting conjugate is purified by ultrafiltration before a final 0.2 micron filtration. Multiple process parameters such as pH, temperature, concentration and time are controlled in each step to produce a conjugate with desired properties.

[0683] Polysaccharide size reduction and oxidation

[0684] Purified pneumococcal capsule Ps powder was dissolved in water and filtered at 0.45 micron. 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 to 230 bar / 5.5 times. The size-reduced polysaccharide was concentrated and diafiltered with water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

[0685] The polysaccharide solution was then adjusted to 22°C and pH 5 with sodium acetate buffer to minimize polysaccharide size reduction due to activation. 100 mM sodium metaperiodate solution was added to initiate polysaccharide activation. The oxidation reaction was carried out at 22°C for 6 hours.

[0686] The activated product was diafiltered against 10 mM potassium phosphate (pH 6.4) and then against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane. The ultrafiltration was performed at 2-8°C.

[0687] Polysaccharide conjugation to CRM197

[0688] Purified CRM197 obtained by expression in Pseudomonas fluorescens as previously described (WO 2012 / 173876A1) was diafiltered using a 5 kDa NMWCO tangential flow ultrafiltration membrane with 2 mM phosphate (pH 7.2) buffer and filtered at 0.2 micron.

[0689] The activated polysaccharide was prepared at 6 mg Ps / mL and 5% w / v sucrose for lyophilization. CRM197 was prepared at 6 mg Pr / mL and 1% w / v sucrose for lyophilization.

[0690] The prepared Ps and CRM197 solutions were lyophilized separately. The lyophilized Ps and CRM197 materials were redissolved in an equal volume of DMSO. The polysaccharide was blended with the CRM197 solution to obtain a polysaccharide concentration of 3.0 g Ps / L and a polysaccharide to CRM197 mass ratio of 1.3. This mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate. Sodium cyanoborohydride (1 mol / mol polysaccharide repeating unit) was added and conjugation was performed at 22°C.

[0691] Reduction with sodium borohydride

[0692] Sodium borohydride (2 mol / mol polysaccharide repeating unit) was added after the conjugation reaction and incubated at 22°C for 3 hours. The batch was diluted to 150 mM sodium chloride and about 0.025% (w / v) polysorbate 20 at about 4°C. Potassium phosphate buffer was then added to neutralize the pH. The batch was concentrated and diafiltered with 150 mM sodium chloride, 25 mM potassium phosphate (pH 7) using a 30 kDa NMWCO tangential flow ultrafiltration membrane at about 4°C.

[0693] Final filtration and product storage

[0694] The batch was then concentrated and diafiltered at 4°C using a 300 kDa NMWCO tangential flow ultrafiltration membrane with 150 mM sodium chloride (pH 7.0) and 0.015% (w / v) polysorbate 20 containing 10 mM histidine.

[0695] The retentate batch was filtered at 0.2 micron (using a 0.5 micron prefilter) and then diluted with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0, and 0.015% (w / v) polysorbate 20, dispensed into aliquots, and frozen at ≤ -60°C.

[0696] Embodiment 19

[0697] Preparation of serotype 9V conjugates for PCV22 multivalent studies using aqueous conjugation

[0698] The polysaccharide is dissolved, reduced in size, chemically activated and buffer exchanged by ultrafiltration. Purified CRM197 is then conjugated to the activated polysaccharide using nickel chloride in the reaction mixture, and the resulting conjugate is purified by ultrafiltration before a final 0.2 micron filtration. Multiple process parameters such as pH, temperature, concentration and time are controlled in each step to produce a conjugate with desired properties.

[0699] Polysaccharide size reduction and oxidation

[0700] Purified pneumococcal capsular polysaccharide powder was dissolved in water and filtered at 0.45 micron. The dissolved polysaccharide was homogenized to reduce the molecular weight. The homogenization pressure and the number of passes through the homogenizer were controlled to 100 bar / 5 times to reduce the size to the target molecular weight. The size-reduced polysaccharide was then concentrated and diafiltered with water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

[0701] The polysaccharide solution was then adjusted to 22°C and pH 5 with sodium acetate buffer to minimize polysaccharide size reduction due to activation. 100 mM sodium metaperiodate solution was added to initiate polysaccharide activation. The oxidation reaction was carried out at 22°C for 6 hours.

[0702] The activated product was diafiltered against 10 mM potassium phosphate (pH 6.4) using a 10 kDa NMWCO tangential flow ultrafiltration membrane. The ultrafiltration was performed at 2-8°C.

[0703] Polysaccharide conjugation to CRM197

[0704] The oxidized polysaccharide solution is mixed with water and 1.5M potassium phosphate (pH 7.0). The buffer pH is selected to improve the stability of the activated polysaccharide during the conjugation reaction. The purified CRM197 obtained by expression in Pseudomonas fluorescens as described above (WO 2012 / 173876A1) is filtered at 0.2 microns and combined with a buffered polysaccharide solution with a polysaccharide to CRM197 mass ratio of 0.7. This mass ratio is selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate. The concentrations of polysaccharide and phosphate are 10.0g / L and 100mM, respectively. The polysaccharide concentration is selected to control the size of the resulting conjugate. The solution is then filtered at 0.2 microns. Nickel chloride is added to about 2mM using a 100mM nickel chloride solution. Sodium cyanoborohydride (2 moles / mole polysaccharide repeating unit) is added. Conjugation is carried out for 120 hours to maximize the consumption of polysaccharides and proteins.

[0705] Reduction with sodium borohydride

[0706] After the conjugation reaction, the batch was diluted to a polysaccharide concentration of about 3.5 g / L, cooled to 2-8° C., and filtered at 1.2 microns. The batch was diafiltered at 2-8° C. with 100 mM potassium phosphate (pH 7.0) using a 100 kDa NMWCO tangential flow ultrafiltration membrane. Subsequently, the batch recovered in the retentate was diluted to about 2.0 g polysaccharide / L and the pH was adjusted by adding 1.2 M sodium bicarbonate (pH 9.4). Sodium borohydride (1 mol / mol polysaccharide repeating unit) was added. Subsequently, 1.5 M potassium phosphate (pH 6.0) was added.

[0707] Final filtration and product storage

[0708] The batch was then concentrated and diafiltered using a 300 kDa NMWCO tangential flow ultrafiltration membrane with 150 mM sodium chloride (pH 7.0) containing 10 mM L-histidine at 4° C. Polysorbate 20 was added to the retentate batch to a concentration of 0.05% (w / v) and the batch was then filtered at 0.2 microns.

[0709] The polysaccharide concentration of the batch was adjusted to 1.0 g / L with additional 10 mM L-histidine in 150 mM sodium chloride (pH 7.0) buffer and 0.015% (w / v) polysorbate 20. The batch was dispensed into aliquots and frozen at ≤ -60°C.

[0710] Embodiment 20

[0711] Preparation of serotype 9V conjugates for PCV23 (DMSO+Aq) multivalent studies using aqueous conjugation

[0712] The polysaccharide is dissolved, reduced in size, chemically activated and buffer exchanged by ultrafiltration. Purified CRM197 is then conjugated to the activated polysaccharide using nickel chloride in the reaction mixture, and the resulting conjugate is purified by ultrafiltration before a final 0.2 micron filtration. Multiple process parameters such as pH, temperature, concentration and time are controlled in each step to produce a conjugate with desired properties.

[0713] Polysaccharide size reduction and oxidation

[0714] Purified pneumococcal capsular polysaccharide powder was dissolved in water and filtered at 0.45 micron. The dissolved polysaccharide was homogenized to reduce the molecular weight. The homogenization pressure and the number of passes through the homogenizer were controlled to 100 bar / 5 times to reduce the size to the target molecular weight. The size-reduced polysaccharide was then concentrated and diafiltered with water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

[0715] The polysaccharide solution was then adjusted to 22°C and pH 5 with sodium acetate buffer to minimize polysaccharide size reduction due to activation. 100 mM sodium metaperiodate solution was added to initiate polysaccharide activation. The oxidation reaction was carried out at 22°C for 6 hours.

[0716] The activated product was diafiltered against 10 mM potassium phosphate (pH 6.4) using a 10 kDa NMWCO tangential flow ultrafiltration membrane. The ultrafiltration was performed at 2-8°C.

[0717] Polysaccharide conjugation to CRM197

[0718] The oxidized polysaccharide solution is mixed with water and 1.5M potassium phosphate (pH 7.0). The buffer pH is selected to improve the stability of the activated polysaccharide during the conjugation reaction. The purified CRM197 obtained by expression in Pseudomonas fluorescens as described above (WO 2012 / 173876A1) is filtered at 0.2 microns and combined with a buffered polysaccharide solution with a polysaccharide to CRM197 mass ratio of 0.7. This mass ratio is selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate. The concentrations of polysaccharide and phosphate are 10.0g / L and 100mM, respectively. The polysaccharide concentration is selected to control the size of the resulting conjugate. The solution is then filtered at 0.2 microns. Nickel chloride is added to about 2mM using a 100mM nickel chloride solution. Sodium cyanoborohydride (2 moles / mole polysaccharide repeating unit) is added. Conjugation is carried out for 120 hours to maximize the consumption of polysaccharides and proteins.

[0719] Reduction with sodium borohydride

[0720] After the conjugation reaction, the batch was diluted to a polysaccharide concentration of about 3.5 g / L, cooled to 2-8° C., and filtered at 1.2 microns. The batch was diafiltered at 2-8° C. with 100 mM potassium phosphate (pH 7.0) using a 100 kDa NMWCO tangential flow ultrafiltration membrane. Subsequently, the batch recovered in the retentate was diluted to about 2.0 g polysaccharide / L and the pH was adjusted by adding 1.2 M sodium bicarbonate (pH 9.4). Sodium borohydride (1 mol / mol polysaccharide repeating unit) was added. Subsequently, 1.5 M potassium phosphate (pH 6.0) was added.

[0721] Final filtration and product storage

[0722] The batch was then concentrated and diafiltered using a 300 kDa NMWCO tangential flow ultrafiltration membrane with 150 mM sodium chloride (pH 7.0) containing 10 mM L-histidine at 4° C. Polysorbate 20 was added to the retentate batch to a concentration of 0.05% (w / v) and the batch was then filtered at 0.2 microns.

[0723] The polysaccharide concentration of the batch was adjusted to 1.0 g / L with additional 10 mM L-histidine in 150 mM sodium chloride (pH 7.0) buffer and 0.015% (w / v) polysorbate 20. The batch was dispensed into aliquots and frozen at ≤ -60°C.

[0724] Embodiment 21

[0725] Preparation of serotype 10A conjugates for PCV23 multivalent studies using DMSO conjugation

[0726] The polysaccharide is dissolved, reduced in size to the target molecular weight, chemically activated and buffer exchanged by ultrafiltration. The activated polysaccharide and purified CRM197 are lyophilized and redissolved in DMSO separately. The redissolved polysaccharide is then combined with the CRM197 solution and conjugated as described below. The resulting conjugate is purified by ultrafiltration before a final 0.2 micron filtration. Multiple process parameters such as pH, temperature, concentration and time are controlled in each step to produce a conjugate with desired properties.

[0727] Polysaccharide size reduction and oxidation

[0728] The purified pneumococcal capsule Ps powder was dissolved in water and filtered at 0.45 micron. 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 to 615 bar / 5 times.

[0729] The size-reduced polysaccharides were concentrated and diafiltered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

[0730] The polysaccharide solution was then adjusted to 22°C and pH 5 with sodium acetate buffer to minimize polysaccharide size reduction due to activation. 100 mM sodium metaperiodate solution was added to initiate polysaccharide activation. The oxidation reaction was carried out at 22°C for 2 hours.

[0731] The activated product was diafiltered against 10 mM potassium phosphate (pH 6.4) and then against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane. The ultrafiltration was performed at 2-8°C.

[0732] Polysaccharide conjugation to CRM197

[0733] Purified CRM197 obtained by expression in Pseudomonas fluorescens as previously described (WO 2012 / 173876A1) was diafiltered using a 5 kDa NMWCO tangential flow ultrafiltration membrane with 2 mM phosphate (pH 7.2) buffer and filtered at 0.2 micron.

[0734] The activated polysaccharide was prepared at 6 mg Ps / mL and 5% w / v sucrose for lyophilization. CRM197 was prepared at 6 mg Pr / mL and 1% w / v sucrose for lyophilization.

[0735] The prepared Ps and CRM197 solutions were lyophilized separately. The lyophilized Ps and CRM197 materials were redissolved in an equal volume of DMSO. The polysaccharide was blended with the CRM197 solution to obtain a polysaccharide concentration of 3.5 g Ps / L and a polysaccharide to CRM197 mass ratio of 1.6. This mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate. Sodium cyanoborohydride (1 mol / mol polysaccharide repeating unit) was added and conjugation was performed at 22°C.

[0736] Reduction with sodium borohydride

[0737] Sodium borohydride (2 mol / mol polysaccharide repeating unit) was added after the conjugation reaction and incubated for 1 hour at 22° C. The batch was diluted to 150 mM sodium chloride and approximately 0.025% (w / v) polysorbate 20 at approximately 4° C. Potassium phosphate buffer was then added to neutralize the pH.

[0738] Final filtration and product storage

[0739] The batch was concentrated and diafiltered at 4°C using a 300 kDa NMWCO tangential flow ultrafiltration membrane with 150 mM sodium chloride, pH 7.0, and 0.015% (w / v) polysorbate 20 containing 10 mM histidine.

[0740] The retentate batch was filtered at 0.2 micron (using a 0.5 micron prefilter) and then diluted with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0, and 0.015% (w / v) polysorbate 20, dispensed into aliquots and frozen at ≤ -60°C.

[0741] Embodiment 22

[0742] Preparation of serotype 10A conjugates using DMSO conjugates for PCV23 (DMSO+Aq) multivalent studies

[0743] The polysaccharide is dissolved, reduced in size to the target molecular weight, chemically activated and buffer exchanged by ultrafiltration. The activated polysaccharide and purified CRM197 are lyophilized and redissolved in DMSO separately. The redissolved polysaccharide is then combined with the CRM197 solution and conjugated as described below. The resulting conjugate is purified by ultrafiltration before a final 0.2 micron filtration. Multiple process parameters such as pH, temperature, concentration and time are controlled in each step to produce a conjugate with desired properties.

[0744] Polysaccharide size reduction and oxidation

[0745] Purified pneumococcal capsule Ps powder was dissolved in water and filtered at 0.45 micron. 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 to 600 bar / 5 times. The size-reduced polysaccharide was concentrated and diafiltered with water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

[0746] The polysaccharide solution was then adjusted to 22°C and pH 5 with sodium acetate buffer to minimize polysaccharide size reduction due to activation. 100 mM sodium metaperiodate solution was added to initiate polysaccharide activation. The oxidation reaction was carried out at 22°C for 2 hours.

[0747] The activated product was diafiltered against 10 mM potassium phosphate (pH 6.4) and then against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane. The ultrafiltration was performed at 2-8°C.

[0748] Polysaccharide conjugation to CRM197

[0749] Purified CRM197 obtained by expression in Pseudomonas fluorescens as previously described (WO 2012 / 173876A1) was diafiltered using a 5 kDa NMWCO tangential flow ultrafiltration membrane with 2 mM phosphate (pH 7.2) buffer and filtered at 0.2 micron.

[0750] The activated polysaccharide was prepared at 6 mg Ps / mL and 5% w / v sucrose for lyophilization. CRM197 was prepared at 6 mg Pr / mL and 1% w / v sucrose for lyophilization.

[0751] The prepared Ps and CRM197 solutions were lyophilized separately. The lyophilized Ps and CRM197 materials were redissolved in an equal volume of DMSO. The polysaccharide was blended with the CRM197 solution to obtain a polysaccharide concentration of 3.4 g Ps / L and a polysaccharide to CRM197 mass ratio of 1.6. This mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate. Sodium cyanoborohydride (1 mol / mol polysaccharide repeating unit) was added and conjugation was performed at 22°C.

[0752] Reduction with sodium borohydride

[0753] Sodium borohydride (2 mol / mol polysaccharide repeating unit) was added after the conjugation reaction and incubated for 1 hour at 22° C. The batch was diluted to 150 mM sodium chloride and approximately 0.025% (w / v) polysorbate 20 at approximately 4° C. Potassium phosphate buffer was then added to neutralize the pH.

[0754] Final filtration and product storage

[0755] The batch was then concentrated and diafiltered at 4°C using a 300 kDa NMWCO tangential flow ultrafiltration membrane with 150 mM sodium chloride, 25 mM potassium phosphate, pH 7, followed by 150 mM sodium chloride, pH 7.0, and 0.015% (w / v) polysorbate 20 containing 10 mM histidine.

[0756] The retentate batch was filtered at 0.2 micron (using a 0.5 micron prefilter) and then diluted with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0, and 0.015% (w / v) polysorbate 20, dispensed into aliquots and frozen at ≤ -60°C.

[0757] Embodiment 23

[0758] Preparation of serotype 10A conjugates for PCV22 multivalent studies using DMSO conjugation

[0759] The polysaccharide is dissolved, reduced in size to the target molecular weight, chemically activated and buffer exchanged by ultrafiltration. The activated polysaccharide and purified CRM197 are lyophilized and redissolved in DMSO separately. The redissolved polysaccharide is then combined with the CRM197 solution and conjugated as described below. The resulting conjugate is purified by ultrafiltration before a final 0.2 micron filtration. Multiple process parameters such as pH, temperature, concentration and time are controlled in each step to produce a conjugate with desired properties.

[0760] Polysaccharide size reduction and oxidation

[0761] Purified pneumococcal capsule Ps powder was dissolved in water and filtered at 0.45 micron. 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 to 600 bar / 5 times. The size-reduced polysaccharide was concentrated and diafiltered with water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

[0762] The polysaccharide solution was then adjusted to 22°C and pH 5 with sodium acetate buffer to minimize polysaccharide size reduction due to activation. 100 mM sodium metaperiodate solution was added to initiate polysaccharide activation. The oxidation reaction was carried out at 22°C for 2 hours.

[0763] The activated product was diafiltered against 10 mM potassium phosphate (pH 6.4) and then against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane. The ultrafiltration was performed at 2-8°C.

[0764] Polysaccharide conjugation to CRM197

[0765] Purified CRM197 obtained by expression in Pseudomonas fluorescens as previously described (WO 2012 / 173876A1) was diafiltered using a 5 kDa NMWCO tangential flow ultrafiltration membrane with 2 mM phosphate (pH 7.2) buffer and filtered at 0.2 micron.

[0766] The activated polysaccharide was prepared at 6 mg Ps / mL and 5% w / v sucrose for lyophilization. CRM197 was prepared at 6 mg Pr / mL and 1% w / v sucrose for lyophilization.

[0767] The prepared Ps and CRM197 solutions were lyophilized separately. The lyophilized Ps and CRM197 materials were redissolved in an equal volume of DMSO. The polysaccharide was blended with the CRM197 solution to obtain a polysaccharide concentration of 3.8 g Ps / L and a polysaccharide to CRM197 mass ratio of 1.75. This mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate. Sodium cyanoborohydride (1 mol / mol polysaccharide repeating unit) was added and conjugation was performed at 22°C.

[0768] Reduction with sodium borohydride

[0769] Sodium borohydride (2 mol / mol polysaccharide repeating unit) was added after the conjugation reaction and incubated at 22°C for 1 hour. The batch was diluted to 150 mM sodium chloride and about 0.025% (w / v) polysorbate 20 at about 4°C. Potassium phosphate buffer was then added to neutralize the pH. The batch was concentrated and diafiltered with 150 mM sodium chloride, 25 mM potassium phosphate (pH 7) using a 30 kDa NMWCO tangential flow ultrafiltration membrane at about 4°C.

[0770] Final filtration and product storage

[0771] The batch was then concentrated and diafiltered at 4°C using a 300 kDa NMWCO tangential flow ultrafiltration membrane with 150 mM sodium chloride (pH 7.0) and 0.015% (w / v) polysorbate 20 containing 10 mM histidine.

[0772] The retentate batch was filtered at 0.2 micron (using a 0.5 micron prefilter) and then diluted with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0, and 0.015% (w / v) polysorbate 20, dispensed into aliquots and frozen at ≤ -60°C.

[0773] Example X

[0774] Preparation of serotype 11A conjugates for PCV24 multivalent studies using DMSO conjugation

[0775] The polysaccharide is dissolved, reduced in size to the target molecular weight, chemically activated and buffer exchanged by ultrafiltration. The activated polysaccharide and purified CRM197 are lyophilized and redissolved in DMSO separately. The redissolved polysaccharide is then combined with the CRM197 solution and conjugated as described below. The resulting conjugate is purified by ultrafiltration before a final 0.2 micron filtration. Multiple process parameters such as pH, temperature, concentration and time are controlled in each step to produce a conjugate with desired properties.

[0776] Polysaccharide size reduction and oxidation

[0777] Purified pneumococcal capsule Ps powder was dissolved in water and filtered at 0.45 micron. 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 to 800 bar / 8 times. The size-reduced polysaccharide was concentrated and diafiltered with water using a 5kDa NMWCO tangential flow ultrafiltration membrane.

[0778] The polysaccharide solution was then adjusted to 22°C and pH 5 with sodium acetate buffer to minimize polysaccharide size reduction due to activation. 100 mM sodium metaperiodate solution was added to initiate polysaccharide activation. The oxidation reaction was carried out at 22°C for 2 hours.

[0779] The activated product was diafiltered against 10 mM potassium phosphate (pH 6.4) and then against water using a 5 kDa NMWCO tangential flow ultrafiltration membrane. The ultrafiltration was performed at 2-8°C.

[0780] Polysaccharide conjugate CRM197

[0781] Purified CRM197 obtained by expression in Pseudomonas fluorescens as previously described (WO 2012 / 173876A1) was diafiltered with 2 mM phosphate (pH 7.2) buffer using a 5 kDa NMWCO tangential flow ultrafiltration membrane and filtered at 0.2 microns.

[0782] The activated polysaccharide was prepared at 6 mg Ps / mL and 5% w / v sucrose for lyophilization. CRM197 was prepared at 6 mg Ps / mL and 1% w / v sucrose for lyophilization.

[0783] The prepared Ps and CRM197 solutions were lyophilized separately. The lyophilized Ps and CRM197 materials were redissolved in an equal volume of DMSO. The polysaccharide was blended with the CRM197 solution to obtain a polysaccharide concentration of 2.3 g Ps / L and a polysaccharide to CRM197 mass ratio of 1.5. This mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate. Sodium cyanoborohydride (1 mol / mol polysaccharide repeating unit) was added and conjugation was performed at 22°C.

[0784] Reduction with sodium borohydride

[0785] Sodium borohydride (2 mol / mol polysaccharide repeating unit) was added after the conjugation reaction and incubated for 1 hour at 22° C. The batch was diluted to 150 mM sodium chloride and approximately 0.025% (w / v) polysorbate 20 at approximately 4° C. Potassium phosphate buffer was then added to neutralize the pH.

[0786] Final filtration and product storage

[0787] The batch was then concentrated and diafiltered at 4°C using a 300 kDa NMWCO tangential flow ultrafiltration membrane with 150 mM sodium chloride (pH 7.0) and 0.015% (w / v) polysorbate 20 containing 10 mM histidine.

[0788] The retentate batch was filtered at 0.2 micron (using a 0.5 micron prefilter) and then diluted with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0, and 0.015% (w / v) polysorbate 20, dispensed into aliquots and frozen at ≤ -60°C.

[0789] Embodiment 24

[0790] Preparation of serotype 12F conjugates for PCV23 (DMSO) and PCV23 (DMSO + Aq) multivalent studies using DMSO conjugation

[0791] The polysaccharide is dissolved, reduced in size to the target molecular weight, chemically activated and buffer exchanged by ultrafiltration. The activated polysaccharide and purified CRM197 are lyophilized and redissolved in DMSO separately. The redissolved polysaccharide is then combined with the CRM197 solution and conjugated as described below. The resulting conjugate is purified by ultrafiltration before a final 0.2 micron filtration. Multiple process parameters such as pH, temperature, concentration and time are controlled in each step to produce a conjugate with desired properties.

[0792] Polysaccharide size reduction and oxidation

[0793] Purified pneumococcal capsule Ps powder was dissolved in water and filtered at 0.45 micron. The dissolved polysaccharide size was reduced by acid hydrolysis (by adding acetic acid to 200 mM, incubating at 80°C for 155 minutes, then neutralizing by adding cold potassium phosphate (pH 7) buffer to 400 mM).

[0794] The size-reduced polysaccharides were concentrated and diafiltered against water using a 5 kDa NMWCO tangential flow ultrafiltration membrane.

[0795] The polysaccharide solution was then adjusted to 22°C and pH 5 with sodium acetate buffer to minimize polysaccharide size reduction due to activation. 100 mM sodium metaperiodate solution was added to initiate polysaccharide activation. The oxidation reaction was carried out at 22°C for 2 hours.

[0796] The activated product was diafiltered against 10 mM potassium phosphate (pH 6.4) and then against water using a 5 kDa NMWCO tangential flow ultrafiltration membrane. The ultrafiltration was performed at 2-8°C.

[0797] Polysaccharide conjugation to CRM197

[0798] Purified CRM197 obtained by expression in Pseudomonas fluorescens as previously described (WO 2012 / 173876A1) was diafiltered using a 5 kDa NMWCO tangential flow ultrafiltration membrane with 2 mM phosphate (pH 7.2) buffer and filtered at 0.2 micron.

[0799] The activated polysaccharide was prepared at 6 mg Ps / mL and 5% w / v sucrose for lyophilization. CRM197 was prepared at 6 mg Pr / mL and 1% w / v sucrose for lyophilization.

[0800] The prepared Ps and CRM197 solutions were lyophilized separately. The lyophilized Ps and CRM197 materials were redissolved in an equal volume of DMSO. The polysaccharide was blended with the CRM197 solution to obtain a polysaccharide concentration of 2.7 g Ps / L and a polysaccharide to CRM197 mass ratio of 1.8. This mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate. Sodium cyanoborohydride (1 mol / mol polysaccharide repeating unit) was added and the conjugation was performed at 22°C.

[0801] Reduction with sodium borohydride

[0802] Sodium borohydride (2 mol / mol polysaccharide repeating unit) was added after the conjugation reaction and incubated for 1 hour at 22° C. The batch was diluted to 150 mM sodium chloride and approximately 0.025% (w / v) polysorbate 20 at approximately 4° C. Potassium phosphate buffer was then added to neutralize the pH.

[0803] Final filtration and product storage

[0804] The batch was then concentrated and diafiltered at 4°C using a 300 kDa NMWCO tangential flow ultrafiltration membrane with 150 mM sodium chloride (pH 7.0) and 0.015% (w / v) polysorbate 20 containing 10 mM histidine.

[0805] The retentate batch was filtered at 0.2 micron (using a 0.5 micron prefilter) and then diluted with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0, and 0.015% (w / v) polysorbate 20, dispensed into aliquots and frozen at ≤ -60°C.

[0806] Embodiment 25

[0807] Preparation of serotype 12F conjugates for PCV23 (DMSO) and PCV23 (DMSO + Aq) multivalent studies using DMSO conjugation

[0808] The polysaccharide is dissolved, reduced in size to the target molecular weight, chemically activated and buffer exchanged by ultrafiltration. The activated polysaccharide and purified CRM197 are lyophilized and redissolved in DMSO separately. The redissolved polysaccharide is then combined with the CRM197 solution and conjugated as described below. The resulting conjugate is purified by ultrafiltration before a final 0.2 micron filtration. Multiple process parameters such as pH, temperature, concentration and time are controlled in each step to produce a conjugate with desired properties.

[0809] Polysaccharide size reduction and oxidation

[0810] Purified pneumococcal capsule Ps powder was dissolved in water and filtered at 0.45 micron. The dissolved polysaccharide size was reduced by acid hydrolysis (by adding acetic acid to 200 mM, incubating at 90°C for 60 minutes, then neutralizing by adding cold potassium phosphate (pH 7) buffer to 400 mM).

[0811] The size-reduced polysaccharides were concentrated and diafiltered against water using a 5 kDa NMWCO tangential flow ultrafiltration membrane.

[0812] The polysaccharide solution was then adjusted to 22°C and pH 5 with sodium acetate buffer to minimize polysaccharide size reduction due to activation. 100 mM sodium metaperiodate solution was added to initiate polysaccharide activation. The oxidation reaction was carried out at 22°C for 2 hours.

[0813] The activated product was diafiltered against 10 mM potassium phosphate (pH 6.4) and then against water using a 5 kDa NMWCO tangential flow ultrafiltration membrane. The ultrafiltration was performed at 2-8°C.

[0814] Polysaccharide conjugation to CRM197

[0815] Purified CRM197 obtained by expression in Pseudomonas fluorescens as previously described (WO 2012 / 173876A1) was diafiltered using a 5 kDa NMWCO tangential flow ultrafiltration membrane with 2 mM phosphate (pH 7.2) buffer and filtered at 0.2 micron.

[0816] The activated polysaccharide was prepared at 6 mg Ps / mL and 5% w / v sucrose for lyophilization. CRM197 was prepared at 6 mg Pr / mL and 1% w / v sucrose for lyophilization.

[0817] The prepared Ps and CRM197 solutions were lyophilized separately. The lyophilized Ps and CRM197 materials were redissolved in an equal volume of DMSO. The polysaccharide was blended with the CRM197 solution to obtain a polysaccharide concentration of 3.0 g Ps / L and a polysaccharide to CRM197 mass ratio of 1.5. This mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate. Sodium cyanoborohydride (1 mol / mol polysaccharide repeating unit) was added and conjugation was performed at 22°C.

[0818] Reduction with sodium borohydride

[0819] Sodium borohydride (2 mol / mol polysaccharide repeating unit) was added after the conjugation reaction and incubated at 22°C for 1 hour. The batch was diluted to 150 mM sodium chloride and about 0.025% (w / v) polysorbate 20 at about 4°C. Potassium phosphate buffer was then added to neutralize the pH. The batch was concentrated and diafiltered using a 30 kDa NMWCO tangential flow ultrafiltration membrane using 150 mM sodium chloride, 25 mM potassium phosphate (pH 7) at about 4°C.

[0820] Final filtration and product storage

[0821] The batch was then concentrated and diafiltered at 4°C using a 300 kDa NMWCO tangential flow ultrafiltration membrane with 150 mM sodium chloride (pH 7.0) and 0.015% (w / v) polysorbate 20 containing 10 mM histidine.

[0822] The retentate batch was filtered at 0.2 micron (using a 0.5 micron prefilter) and then diluted with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0, and 0.015% (w / v) polysorbate 20, dispensed into aliquots and frozen at ≤ -60°C.

[0823] Embodiment 26

[0824] Preparation of serotype 14 conjugates for PCV23 (DMSO) multivalent studies using DMSO conjugation

[0825] The polysaccharide is dissolved, reduced in size to the target molecular weight, chemically activated and buffer exchanged by ultrafiltration. The activated polysaccharide and purified CRM197 are lyophilized and redissolved in DMSO separately. The redissolved polysaccharide is then combined with the CRM197 solution and conjugated as described below. The resulting conjugate is purified by diafiltration before final filtration at 0.2 microns. Multiple process parameters such as pH, temperature, concentration and time are controlled in each step to produce a conjugate with desired properties.

[0826] Polysaccharide size reduction and oxidation

[0827] The purified pneumococcal capsule Ps powder was dissolved in water and filtered at 0.45 micron. 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 to 200 bar / 6 times.

[0828] The size-reduced polysaccharides were concentrated and diafiltered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

[0829] The polysaccharide solution was then adjusted to 22°C and pH 5 with sodium acetate buffer to minimize polysaccharide size reduction due to activation. 100 mM sodium metaperiodate solution was added to initiate polysaccharide activation. The oxidation reaction was carried out at 22°C for 4 hours.

[0830] The activated product was diafiltered against 10 mM potassium phosphate (pH 6.4) and then against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane. The ultrafiltration was performed at 2-8°C.

[0831] Polysaccharide conjugation to CRM197

[0832] Purified CRM197 obtained by expression in Pseudomonas fluorescens as previously described (WO 2012 / 173876A1) was diafiltered using a 5 kDa NMWCO tangential flow ultrafiltration membrane with 2 mM phosphate (pH 7.2) buffer and filtered at 0.2 micron.

[0833] The activated polysaccharide was prepared at 6 mg Ps / mL and 5% w / v sucrose for lyophilization. CRM197 was prepared at 6 mg Pr / mL and 1% w / v sucrose for lyophilization.

[0834] The prepared Ps and CRM197 solutions were lyophilized separately. The lyophilized Ps and CRM197 materials were redissolved in an equal volume of DMSO. The polysaccharide was blended with the CRM197 solution to obtain a polysaccharide concentration of 1.8 g Ps / L and a polysaccharide to CRM197 mass ratio of 1.5. This mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate. Sodium cyanoborohydride (1 mol / mol polysaccharide repeating unit) was added and conjugation was performed at 22°C.

[0835] Reduction with sodium borohydride

[0836] Sodium borohydride (2 mol / mol polysaccharide repeating unit) was added after the conjugation reaction and incubated at 22°C for 1 hour. The batch was diluted to 150 mM sodium chloride and about 0.025% (w / v) polysorbate 20 at about 4°C. Potassium phosphate buffer was then added to neutralize the pH. The batch was dialyzed against 150 mM sodium chloride, 0.05% polysorbate 20 using a 300 kDa MWCO dialysis cassette at about 4°C for 22.5 hours.

[0837] Final filtration and product storage

[0838] The retentate batch was filtered at 0.2 micron (using a 0.5 micron pre-filter), dispensed into aliquots, and frozen at ≤ -60°C.

[0839] Embodiment 27

[0840] Preparation of serotype 14 conjugates for PCV22 and PCV23 (DMSO+Aq) multivalent studies using aqueous conjugation

[0841] The polysaccharide is dissolved, reduced in size, chemically activated and buffer exchanged by ultrafiltration. Purified CRM197 is then conjugated to the activated polysaccharide using nickel chloride in the reaction mixture, and the resulting conjugate is purified by ultrafiltration before a final 0.2 micron filtration. Multiple process parameters such as pH, temperature, concentration and time are controlled in each step to produce a conjugate with desired properties.

[0842] Polysaccharide size reduction and oxidation

[0843] Purified pneumococcal capsular polysaccharide powder was dissolved in water and filtered at 0.45 micron. The dissolved polysaccharide was homogenized to reduce the molecular weight. The homogenization pressure and the number of passes through the homogenizer were controlled to 200 bar / 6 times to reduce the size to the target molecular weight. The size-reduced polysaccharide was then concentrated and diafiltered with water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

[0844] The polysaccharide solution was then adjusted to 22°C and pH 5 with sodium acetate buffer to minimize polysaccharide size reduction due to activation. 100 mM sodium metaperiodate solution was added to initiate polysaccharide activation. The oxidation reaction was carried out at 22°C for 4 hours.

[0845] The activated product was diafiltered against 10 mM potassium phosphate (pH 6.4) using a 10 kDa NMWCO tangential flow ultrafiltration membrane. The ultrafiltration was performed at 2-8°C.

[0846] Polysaccharide conjugation to CRM197

[0847] The oxidized polysaccharide solution is mixed with water and 1.5M potassium phosphate (pH 7.0). The buffer pH is selected to improve the stability of the activated polysaccharide during the conjugation reaction. The purified CRM197 obtained by expression in Pseudomonas fluorescens as described above (WO 2012 / 173876A1) is filtered at 0.2 microns and combined with a buffered polysaccharide solution with a polysaccharide to CRM197 mass ratio of 1.0. This mass ratio is selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate. The concentrations of polysaccharide and phosphate are 3.8g / L and 100mM respectively. The polysaccharide concentration is selected to control the size of the resulting conjugate. The solution is then filtered at 0.2 microns. Nickel chloride is added to about 2mM using 100mM nickel chloride solution. Sodium cyanoborohydride (2 moles / mole polysaccharide repeating units) is added. Conjugation is carried out for 72 hours to maximize the consumption of polysaccharides and proteins.

[0848] Reduction with sodium borohydride

[0849] After the conjugation reaction, the batch was diluted to a polysaccharide concentration of about 3.5 g / L, cooled to 2-8° C., and filtered at 1.2 microns. The batch was diafiltered at 2-8° C. with 100 mM potassium phosphate (pH 7.0) using a 100 kDa NMWCO tangential flow ultrafiltration membrane. Subsequently, the batch recovered in the retentate was diluted to about 2.0 g polysaccharide / L and the pH was adjusted by adding 1.2 M sodium bicarbonate (pH 9.4). Sodium borohydride (1 mol / mol polysaccharide repeating unit) was added. Subsequently, 1.5 M potassium phosphate (pH 6.0) was added.

[0850] Final filtration and product storage

[0851] The batch was then concentrated and diafiltered using a 300 kDa NMWCO tangential flow ultrafiltration membrane with 150 mM sodium chloride, pH 7.0, containing 10 mM L-histidine at 4° C. The batch was then filtered at 0.2 microns.

[0852] The polysaccharide concentration of the batch was adjusted to 1.0 g / L with additional 150 mM sodium chloride (pH 7.0) buffer containing 10 mM L-histidine. The batch was divided into aliquots and frozen at ≤ -60°C.

[0853] Embodiment 28

[0854] Preparation of serotype 15A conjugates for PCV23 (DMSO) and PCV23 (DMSO + Aq) multivalent studies using DMSO conjugation

[0855] The polysaccharide is dissolved, reduced in size to the target molecular weight, chemically activated and buffer exchanged by ultrafiltration. The activated polysaccharide and purified CRM197 are lyophilized and redissolved in DMSO separately. The redissolved polysaccharide is then combined with the CRM197 solution and conjugated as described below. The resulting conjugate is purified by ultrafiltration before a final 0.2 micron filtration. Multiple process parameters such as pH, temperature, concentration and time are controlled in each step to produce a conjugate with desired properties.

[0856] Polysaccharide size reduction and oxidation

[0857] Purified pneumococcal capsule Ps powder was dissolved in water and filtered at 0.45 micron. 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 to 210 bar / 5 times.

[0858] The size-reduced polysaccharides were concentrated and diafiltered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

[0859] The polysaccharide solution was then adjusted to 22°C and pH 5 with sodium acetate buffer to minimize polysaccharide size reduction due to activation. 100 mM sodium metaperiodate solution was added to initiate polysaccharide activation. The oxidation reaction was carried out at 22°C for 20 hours.

[0860] The activated product was diafiltered against 10 mM potassium phosphate (pH 6.4) and then against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane. The ultrafiltration was performed at 2-8°C.

[0861] Polysaccharide conjugation to CRM197

[0862] Purified CRM197 obtained by expression in Pseudomonas fluorescens as previously described (WO 2012 / 173876A1) was diafiltered using a 5 kDa NMWCO tangential flow ultrafiltration membrane with 2 mM phosphate (pH 7.2) buffer and filtered at 0.2 micron.

[0863] The activated polysaccharide was prepared at 6 mg Ps / mL and 5% w / v sucrose for lyophilization. CRM197 was prepared at 6 mg Pr / mL and 1% w / v sucrose for lyophilization.

[0864] The prepared Ps and CRM197 solutions were lyophilized separately. The lyophilized Ps and CRM197 substances were redissolved in an equal volume of DMSO, which was preheated to 34°C. Sodium chloride was added to the polysaccharide solution to a concentration of 25mM. The polysaccharide was blended with the CRM197 solution to obtain a polysaccharide concentration of 5.0g Ps / L and a polysaccharide to CRM197 mass ratio of 2.0. This mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate. Sodium cyanoborohydride (1 mol / mol polysaccharide repeating unit) was added and conjugation was performed at 34°C.

[0865] Reduction with sodium borohydride

[0866] Sodium borohydride (2 mol / mol polysaccharide repeating unit) was added after the conjugation reaction and incubated for 1 hour at 34° C. The batch was diluted to 150 mM sodium chloride and approximately 0.025% (w / v) polysorbate 20 at approximately 4° C. Potassium phosphate buffer was then added to neutralize the pH.

[0867] Final filtration and product storage

[0868] The batch was then concentrated and diafiltered at 4°C using a 300 kDa NMWCO tangential flow ultrafiltration membrane with 150 mM sodium chloride (pH 7.0) and 0.015% (w / v) polysorbate 20 containing 10 mM histidine.

[0869] The retentate batch was filtered at 0.2 micron (using a 0.5 micron prefilter) and then diluted with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0, and 0.015% (w / v) polysorbate 20, dispensed into aliquots and frozen at ≤ -60°C.

[0870] Embodiment 29

[0871] Preparation of serotype 15A conjugates for PCV22 multivalent studies using DMSO conjugation

[0872] The polysaccharide is dissolved, reduced in size to the target molecular weight, chemically activated and buffer exchanged by ultrafiltration. The activated polysaccharide and purified CRM197 are lyophilized and redissolved in DMSO separately. The redissolved polysaccharide is then combined with the CRM197 solution and conjugated as described below. The resulting conjugate is purified by ultrafiltration before a final 0.2 micron filtration. Multiple process parameters such as pH, temperature, concentration and time are controlled in each step to produce a conjugate with desired properties.

[0873] Polysaccharide size reduction and oxidation

[0874] The purified pneumococcal capsule Ps powder was dissolved in water and filtered at 0.45 micron. 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 to 200 bar / 5 times.

[0875] The size-reduced polysaccharides were concentrated and diafiltered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

[0876] The polysaccharide solution was then adjusted to 22°C and pH 5 with sodium acetate buffer to minimize polysaccharide size reduction due to activation. 100 mM sodium metaperiodate solution was added to initiate polysaccharide activation. The oxidation reaction was carried out at 22°C for 20 hours.

[0877] The activated product was diafiltered against 10 mM potassium phosphate (pH 6.4) and then against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane. The ultrafiltration was performed at 2-8°C.

[0878] Polysaccharide conjugation to CRM197

[0879] Purified CRM197 obtained by expression in Pseudomonas fluorescens as previously described (WO 2012 / 173876A1) was diafiltered using a 5 kDa NMWCO tangential flow ultrafiltration membrane with 2 mM phosphate (pH 7.2) buffer and filtered at 0.2 micron.

[0880] The activated polysaccharide was prepared at 6 mg Pr / mL and 5% w / v sucrose for lyophilization. CRM197 was prepared at 6 mg Pr / mL and 1% w / v sucrose for lyophilization.

[0881] The prepared Ps and CRM197 solutions were lyophilized separately. The lyophilized Ps and CRM197 substances were redissolved in an equal volume of DMSO, which was preheated to 34°C. Sodium chloride was added to the polysaccharide solution to a concentration of 25mM. The polysaccharide was blended with the CRM197 solution to obtain a polysaccharide concentration of 5.0g Ps / L and a polysaccharide to CRM197 mass ratio of 2.0. This mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate. Sodium cyanoborohydride (1 mol / mol polysaccharide repeating unit) was added and conjugation was performed at 34°C.

[0882] Reduction with sodium borohydride

[0883] Sodium borohydride (2 mol / mol polysaccharide repeating unit) was added after the conjugation reaction and incubated at 22°C for 1 hour. The batch was diluted to 150 mM sodium chloride and about 0.025% (w / v) polysorbate 20 at about 4°C. Potassium phosphate buffer was then added to neutralize the pH. The batch was concentrated and diafiltered with 150 mM sodium chloride, 25 mM potassium phosphate (pH 7) using a 30 kDa NMWCO tangential flow ultrafiltration membrane at about 4°C.

[0884] Final filtration and product storage

[0885] The batch was then concentrated and diafiltered at 4°C using a 300 kDa NMWCO tangential flow ultrafiltration membrane with 150 mM sodium chloride (pH 7.0) and 0.015% (w / v) polysorbate 20 containing 10 mM histidine.

[0886] The retentate batch was filtered at 0.2 micron (using a 0.5 micron prefilter) and then diluted with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0, and 0.015% (w / v) polysorbate 20, dispensed into aliquots and frozen at ≤ -60°C.

[0887] Embodiment 30

[0888] Preparation of serotype 15C conjugates for PCV23 (DMSO) and PCV23 (DMSO + Aq) multivalent studies using DMSO conjugation

[0889] The polysaccharide derived from Streptococcus pneumoniae serotype 15B was dissolved, sized to the target molecular weight, hydrolyzed by mild base to release the O-acetyl groups, chemically activated and buffer exchanged by ultrafiltration. The activated polysaccharide and purified CRM197 were lyophilized and redissolved in DMSO separately. The redissolved polysaccharide was then combined with the CRM197 solution and conjugated as described below. The resulting conjugate was purified by ultrafiltration before a final 0.2 micron filtration. Multiple process parameters in each step, such as pH, temperature, concentration and time, were controlled to produce a conjugate with the desired properties.

[0890] Polysaccharide size reduction, alkaline hydrolysis and oxidation

[0891] Purified serotype 15B pneumococcal capsule Ps powder was dissolved in water and filtered at 0.45 micron. 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 to 300 bar / 5 times.

[0892] The size-reduced polysaccharides were concentrated and diafiltered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

[0893] The polysaccharide solution was heated to 60°C and sodium bicarbonate (pH 9) buffer was added to a final concentration of 50 mM. The batch was mixed and incubated at 60°C for 13 hours to release the O-acetyl groups. Potassium phosphate (pH 6) buffer 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 using a 10 kDa NMWCO tangential flow ultrafiltration membrane using water.

[0894] The polysaccharide solution was adjusted to 22°C and to pH 5 using sodium acetate buffer to minimize polysaccharide size reduction due to activation. 100 mM sodium metaperiodate solution was added to initiate polysaccharide activation. The oxidation reaction was carried out at 22°C for 2 hours.

[0895] The activated product was diafiltered against 10 mM potassium phosphate (pH 6.4) and then against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane. The ultrafiltration was performed at 2-8°C.

[0896] Polysaccharide conjugation to CRM197

[0897] Purified CRM197 obtained by expression in Pseudomonas fluorescens as previously described (WO 2012 / 173876A1) was diafiltered using a 5 kDa NMWCO tangential flow ultrafiltration membrane with 2 mM phosphate (pH 7.2) buffer and filtered at 0.2 micron.

[0898] The activated polysaccharide was prepared at 6 mg Ps / mL and 5% w / v sucrose for lyophilization. CRM197 was prepared at 6 mg Pr / mL and 1% w / v sucrose for lyophilization.

[0899] The prepared Ps and CRM197 solutions were lyophilized separately. The lyophilized Ps and CRM197 materials were redissolved in an equal volume of DMSO. The polysaccharide was blended with the CRM197 solution to obtain a polysaccharide concentration of 3.0 g Ps / L and a polysaccharide to CRM197 mass ratio of 1.75. This mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate. Sodium cyanoborohydride (1 mol / mol polysaccharide repeating unit) was added and conjugation was performed at 22°C.

[0900] Reduction with sodium borohydride

[0901] Sodium borohydride (2 mol / mol polysaccharide repeating unit) was added after the conjugation reaction and incubated at 22°C for 1 hour. The batch was diluted to 150 mM sodium chloride and about 0.025% (w / v) polysorbate 20 at about 4°C. Potassium phosphate buffer was then added to neutralize the pH. The batch was concentrated and diafiltered with 150 mM sodium chloride, 25 mM potassium phosphate (pH 7) using a 30 kDa NMWCO tangential flow ultrafiltration membrane at about 4°C.

[0902] Final filtration and product storage

[0903] The batch was then concentrated and diafiltered at 4°C using a 300 kDa NMWCO tangential flow ultrafiltration membrane with 150 mM sodium chloride (pH 7.0) and 0.015% (w / v) polysorbate 20 containing 10 mM histidine.

[0904] The retentate batch was filtered at 0.2 micron (using a 0.5 micron prefilter) and then diluted with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0, and 0.015% (w / v) polysorbate 20, dispensed into aliquots and frozen at ≤ -60°C.

[0905] Embodiment 31

[0906] Preparation of serotype 15C conjugates for PCV22 multivalent studies using DMSO conjugation

[0907] The polysaccharide derived from Streptococcus pneumoniae serotype 15B was dissolved, sized to the target molecular weight, hydrolyzed by mild base to release the O-acetyl groups, chemically activated and buffer exchanged by ultrafiltration. The activated polysaccharide and purified CRM197 were lyophilized and redissolved in DMSO separately. The redissolved polysaccharide was then combined with the CRM197 solution and conjugated as described below. The resulting conjugate was purified by ultrafiltration before a final 0.2 micron filtration. Multiple process parameters in each step, such as pH, temperature, concentration and time, were controlled to produce a conjugate with the desired properties.

[0908] Polysaccharide size reduction, alkaline hydrolysis and oxidation

[0909] Purified serotype 15B pneumococcal capsule Ps powder is dissolved in water and filtered at 0.45 micron. The dissolved polysaccharide is homogenized to reduce the molecular weight of Ps. The homogenization pressure and the number of times through the homogenizer are controlled to 300 bar / 5 times. The size-reduced polysaccharide solution is heated to 60°C, and sodium bicarbonate (pH 9.4) buffer is added to a final concentration of 50mM. The batch is mixed and incubated at 60°C for 12 hours to release the O-acetyl group. Potassium phosphate (pH 6) buffer is added to a final concentration of 150mM to neutralize the pH and cool the solution to ambient temperature. The solution is then concentrated and diafiltered using a 10kDa NMWCO tangential flow ultrafiltration membrane using water.

[0910] The polysaccharide solution was adjusted to 22°C and to pH 5 using sodium acetate buffer to minimize polysaccharide size reduction due to activation. 100 mM sodium metaperiodate solution was added to initiate polysaccharide activation. The oxidation reaction was carried out at 22°C for 2 hours.

[0911] The activated product was diafiltered against 10 mM potassium phosphate (pH 6.4) and then against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane. The ultrafiltration was performed at 2-8°C.

[0912] Polysaccharide conjugation to CRM197

[0913] Purified CRM197 obtained by expression in Pseudomonas fluorescens as previously described (WO 2012 / 173876A1) was diafiltered using a 5 kDa NMWCO tangential flow ultrafiltration membrane with 2 mM phosphate (pH 7.2) buffer and filtered at 0.2 micron.

[0914] The activated polysaccharide was prepared at 6 mg Ps / mL and 5% w / v sucrose for lyophilization. CRM197 was prepared at 6 mg Pr / mL and 1% w / v sucrose for lyophilization.

[0915] The prepared Ps and CRM197 solutions were lyophilized separately. The lyophilized Ps and CRM197 materials were redissolved in an equal volume of DMSO. The polysaccharide was blended with the CRM197 solution to obtain a polysaccharide concentration of 3.2 g Ps / L and a polysaccharide to CRM197 mass ratio of 1.75. This mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate. Sodium cyanoborohydride (1 mol / mol polysaccharide repeating unit) was added and conjugation was performed at 22°C.

[0916] Reduction with sodium borohydride

[0917] Sodium borohydride (2 mol / mol polysaccharide repeating unit) was added after the conjugation reaction and incubated at 22°C for 1 hour. The batch was diluted to 150 mM sodium chloride and about 0.025% (w / v) polysorbate 20 at about 4°C. Potassium phosphate buffer was then added to neutralize the pH. The batch was concentrated and diafiltered with 150 mM sodium chloride, 25 mM potassium phosphate (pH 7) using a 30 kDa NMWCO tangential flow ultrafiltration membrane at about 4°C.

[0918] Final filtration and product storage

[0919] The batch was then concentrated and diafiltered at 4°C using a 300 kDa NMWCO tangential flow ultrafiltration membrane with 150 mM sodium chloride (pH 7.0) and 0.015% (w / v) polysorbate 20 containing 10 mM histidine.

[0920] The retentate batch was filtered at 0.2 micron (using a 0.5 micron prefilter) and then diluted with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0, and 0.015% (w / v) polysorbate 20, dispensed into aliquots and frozen at ≤ -60°C.

[0921] Embodiment 32

[0922] Preparation of serotype 18C conjugates for PCV23 (DMSO) and PCV23 (DMSO + Aq) multivalent studies using DMSO conjugation

[0923] The polysaccharide is dissolved, reduced in size to the target molecular weight, chemically activated and buffer exchanged by ultrafiltration. The activated polysaccharide and purified CRM197 are lyophilized and redissolved in DMSO separately. The redissolved polysaccharide is then combined with the CRM197 solution and conjugated as described below. The resulting conjugate is purified by ultrafiltration before a final 0.2 micron filtration. Multiple process parameters such as pH, temperature, concentration and time are controlled in each step to produce a conjugate with desired properties.

[0924] Polysaccharide size reduction and oxidation

[0925] Purified pneumococcal capsule Ps powder was dissolved in water and filtered at 0.45 micron. The dissolved polysaccharide was reduced in size by acid hydrolysis (by adding acetic acid to 200 mM, incubating at 90°C for 160 minutes, then neutralizing by adding cold potassium phosphate (pH 7) buffer to 400 mM).

[0926] The size-reduced polysaccharides were concentrated and diafiltered against water using a 5 kDa NMWCO tangential flow ultrafiltration membrane.

[0927] The polysaccharide solution was then adjusted to 22°C and pH 5 with sodium acetate buffer to minimize polysaccharide size reduction due to activation. 100 mM sodium metaperiodate solution was added to initiate polysaccharide activation. The oxidation reaction was carried out at 22°C for 2 hours.

[0928] The activated product was diafiltered against 10 mM potassium phosphate (pH 6.4) and then against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane. The ultrafiltration was performed at 2-8°C.

[0929] Polysaccharide conjugation to CRM197

[0930] Purified CRM197 obtained by expression in Pseudomonas fluorescens as previously described (WO 2012 / 173876A1) was diafiltered using a 5 kDa NMWCO tangential flow ultrafiltration membrane with 2 mM phosphate (pH 7.2) buffer and filtered at 0.2 micron.

[0931] The activated polysaccharide was prepared at 6 mg Ps / mL and 5% w / v sucrose for lyophilization. CRM197 was prepared at 6 mg Pr / mL and 1% w / v sucrose for lyophilization.

[0932] The prepared Ps and CRM197 solutions were lyophilized separately. The lyophilized Ps and CRM197 materials were redissolved in an equal volume of DMSO. The polysaccharide was blended with the CRM197 solution to obtain a polysaccharide concentration of 3.49 g Ps / L and a polysaccharide to CRM197 mass ratio of 1.5. This mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate. Sodium cyanoborohydride (1 mol / mol polysaccharide repeating unit) was added and conjugation was performed at 22°C.

[0933] Reduction with sodium borohydride

[0934] Sodium borohydride (2 mol / mol polysaccharide repeating unit) was added after the conjugation reaction and incubated at 22°C for 3 hours. The batch was diluted to 150 mM sodium chloride and about 0.025% (w / v) polysorbate 20 at about 4°C. Potassium phosphate buffer was then added to neutralize the pH. The batch was concentrated and diafiltered with 150 mM sodium chloride, 25 mM potassium phosphate (pH 7) using a 30 kDa NMWCO tangential flow ultrafiltration membrane at about 4°C.

[0935] Final filtration and product storage

[0936] The batch was then concentrated and diafiltered at 4°C using a 300 kDa NMWCO tangential flow ultrafiltration membrane with 150 mM sodium chloride (pH 7.0) and 0.015% (w / v) polysorbate 20 containing 10 mM histidine.

[0937] The retentate batch was filtered at 0.2 micron (using a 0.5 micron prefilter) and then diluted with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0, and 0.015% (w / v) polysorbate 20, dispensed into aliquots and frozen at ≤ -60°C.

[0938] Embodiment 33

[0939] Preparation of serotype 18C conjugates for PCV22 multivalent studies using DMSO conjugation

[0940] The polysaccharide is dissolved, reduced in size to the target molecular weight, chemically activated and buffer exchanged by ultrafiltration. The activated polysaccharide and purified CRM197 are lyophilized and redissolved in DMSO separately. The redissolved polysaccharide is then combined with the CRM197 solution and conjugated as described below. The resulting conjugate is purified by ultrafiltration before a final 0.2 micron filtration. Multiple process parameters such as pH, temperature, concentration and time are controlled in each step to produce a conjugate with desired properties.

[0941] Polysaccharide size reduction and oxidation

[0942] Purified pneumococcal capsule Ps powder was dissolved in water and filtered at 0.45 micron. The dissolved polysaccharide was reduced in size by acid hydrolysis (by adding acetic acid to 200 mM, incubating at 90°C for 160 minutes, then neutralizing by adding cold potassium phosphate (pH 7) buffer to 400 mM).

[0943] The size-reduced polysaccharides were concentrated and diafiltered against water using a 5 kDa NMWCO tangential flow ultrafiltration membrane.

[0944] The polysaccharide solution was then adjusted to 22°C and pH 5 with sodium acetate buffer to minimize polysaccharide size reduction due to activation. 100 mM sodium metaperiodate solution was added to initiate polysaccharide activation. The oxidation reaction was carried out at 22°C for 2 hours.

[0945] The activated product was diafiltered against 10 mM potassium phosphate (pH 6.4) and then against water using a 5 kDa NMWCO tangential flow ultrafiltration membrane. The ultrafiltration was performed at 2-8°C.

[0946] Polysaccharide conjugation to CRM197

[0947] Purified CRM197 obtained by expression in Pseudomonas fluorescens as previously described (WO 2012 / 173876A1) was diafiltered using a 5 kDa NMWCO tangential flow ultrafiltration membrane with 2 mM phosphate (pH 7.2) buffer and filtered at 0.2 micron.

[0948] The activated polysaccharide was prepared at 6 mg Ps / mL and 5% w / v sucrose for lyophilization. CRM197 was prepared at 6 mg Pr / mL and 1% w / v sucrose for lyophilization.

[0949] The prepared Ps and CRM197 solutions were lyophilized separately. The lyophilized Ps and CRM197 materials were redissolved in an equal volume of DMSO. The polysaccharide was blended with the CRM197 solution to obtain a polysaccharide concentration of 2.49 g Ps / L and a polysaccharide to CRM197 mass ratio of 1.5. This mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate. Sodium cyanoborohydride (1 mol / mol polysaccharide repeating unit) was added and conjugation was performed at 22°C.

[0950] Reduction with sodium borohydride

[0951] Sodium borohydride (2 mol / mol polysaccharide repeating unit) was added after the conjugation reaction and incubated at 22°C for 3 hours. The batch was diluted to 150 mM sodium chloride and about 0.025% (w / v) polysorbate 20 at about 4°C. Potassium phosphate buffer was then added to neutralize the pH. The batch was concentrated and diafiltered with 150 mM sodium chloride, 25 mM potassium phosphate (pH 7) using a 30 kDa NMWCO tangential flow ultrafiltration membrane at about 4°C.

[0952] Final filtration and product storage

[0953] The batch was then concentrated and diafiltered at 4°C using a 300 kDa NMWCO tangential flow ultrafiltration membrane with 150 mM sodium chloride (pH 7.0) and 0.015% (w / v) polysorbate 20 containing 10 mM histidine.

[0954] The retentate batch was filtered at 0.2 micron (using a 0.5 micron prefilter) and then diluted with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0, and 0.015% (w / v) polysorbate 20, dispensed into aliquots and frozen at ≤ -60°C.

[0955] Embodiment 34

[0956] Preparation of serotype 19A conjugates for PCV23 (DMSO) and PCV23 (DMSO + Aq) multivalent studies using DMSO conjugation

[0957] The polysaccharide is dissolved, chemically activated and buffer exchanged by ultrafiltration. The activated polysaccharide and purified CRM197 are lyophilized and redissolved in DMSO separately. The redissolved polysaccharide is then combined with the CRM197 solution and conjugated as described below. The resulting conjugate is purified by ultrafiltration before a final 0.2 micron filtration. Multiple process parameters such as pH, temperature, concentration and time are controlled in each step to produce a conjugate with desired properties.

[0958] Polysaccharide oxidation

[0959] The purified pneumococcal capsule Ps powder was dissolved in water and filtered at 0.22 μm. The polysaccharide concentration was determined and diafiltered using a 10 kDa NMWCO tangential flow ultrafiltration membrane with water.

[0960] The polysaccharide solution was then adjusted to 22°C and pH 5 with sodium acetate buffer to minimize polysaccharide size reduction due to activation. 100 mM sodium metaperiodate solution was added to initiate polysaccharide activation. The oxidation reaction was carried out at 22°C for 20 hours.

[0961] The activated product was diafiltered against 10 mM potassium phosphate (pH 6.4) and then against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane. The ultrafiltration was performed at 2-8°C.

[0962] Polysaccharide conjugation to CRM197

[0963] Purified CRM197 obtained by expression in Pseudomonas fluorescens as previously described (WO 2012 / 173876A1) was diafiltered using a 5 kDa NMWCO tangential flow ultrafiltration membrane with 2 mM phosphate (pH 7.2) buffer and filtered at 0.2 micron.

[0964] The activated polysaccharide was prepared at 6 mg Ps / mL and 5% w / v sucrose for lyophilization. CRM197 was prepared at 6 mg Pr / mL and 1% w / v sucrose for lyophilization.

[0965] The prepared Ps and CRM197 solutions were lyophilized separately. The lyophilized Ps and CRM197 materials were redissolved in an equal volume of DMSO. The polysaccharide was blended with the CRM197 solution to obtain a polysaccharide concentration of 3.8 g Ps / L and a polysaccharide to CRM197 mass ratio of 1.33. This mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate. Sodium cyanoborohydride (1 mol / mol polysaccharide repeating unit) was added and conjugation was performed at 22°C.

[0966] Reduction with sodium borohydride

[0967] Sodium borohydride (2 mol / mol polysaccharide repeating unit) was added after the conjugation reaction and incubated at 22°C for 3 hours. The batch was diluted to 150 mM sodium chloride and about 0.025% (w / v) polysorbate 20 at about 4°C. Potassium phosphate buffer was then added to neutralize the pH. The batch was concentrated and diafiltered with 150 mM sodium chloride, 25 mM potassium phosphate (pH 7) using a 30 kDa NMWCO tangential flow ultrafiltration membrane at about 4°C.

[0968] Final filtration and product storage

[0969] The batch was then concentrated and diafiltered at 4°C using a 300 kDa NMWCO tangential flow ultrafiltration membrane with 150 mM sodium chloride (pH 7.0) and 0.015% (w / v) polysorbate 20 containing 10 mM histidine.

[0970] The retentate batch was filtered at 0.2 micron and then diluted with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0, and 0.015% (w / v) polysorbate 20, dispensed into aliquots, and frozen at ≤ -60°C.

[0971] Embodiment 35

[0972] Preparation of serotype 19A conjugates for PCV22 multivalent studies using DMSO conjugation

[0973] The polysaccharide is dissolved, chemically activated and buffer exchanged by ultrafiltration. The activated polysaccharide and purified CRM197 are lyophilized and redissolved in DMSO separately. The redissolved polysaccharide is then combined with the CRM197 solution and conjugated as described below. The resulting conjugate is purified by ultrafiltration before a final 0.2 micron filtration. Multiple process parameters such as pH, temperature, concentration and time are controlled in each step to produce a conjugate with desired properties.

[0974] Polysaccharide oxidation

[0975] The purified pneumococcal capsule Ps powder was dissolved in water and filtered at 0.22 μm. The polysaccharide concentration was determined and diafiltered using a 10 kDa NMWCO tangential flow ultrafiltration membrane with water.

[0976] The polysaccharide solution was then adjusted to 22°C and pH 5 with sodium acetate buffer to minimize polysaccharide size reduction due to activation. 100 mM sodium metaperiodate solution was added to initiate polysaccharide activation. The oxidation reaction was carried out at 22°C for 20 hours.

[0977] The activated product was diafiltered against 10 mM potassium phosphate (pH 6.4) and then against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane. The ultrafiltration was performed at 2-8°C.

[0978] Polysaccharide conjugation to CRM197

[0979] Purified CRM197 obtained by expression in Pseudomonas fluorescens as previously described (WO 2012 / 173876A1) was diafiltered using a 5 kDa NMWCO tangential flow ultrafiltration membrane with 2 mM phosphate (pH 7.2) buffer and filtered at 0.2 micron.

[0980] The activated polysaccharide was prepared at 6 mg Ps / mL and 5% w / v sucrose for lyophilization. CRM197 was prepared at 6 mg Pr / mL and 1% w / v sucrose for lyophilization.

[0981] The prepared Ps and CRM197 solutions were lyophilized separately. The lyophilized Ps and CRM197 materials were redissolved in an equal volume of DMSO. The polysaccharide was blended with the CRM197 solution to obtain a polysaccharide concentration of 3.8 g Ps / L and a polysaccharide to CRM197 mass ratio of 1.33. This mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate. Sodium cyanoborohydride (1 mol / mol polysaccharide repeating unit) was added and conjugation was performed at 22°C.

[0982] Reduction with sodium borohydride

[0983] Sodium borohydride (2 mol / mol polysaccharide repeating unit) was added after the conjugation reaction and incubated at 22°C for 3 hours. The batch was diluted to 150 mM sodium chloride and about 0.025% (w / v) polysorbate 20 at about 4°C. Potassium phosphate buffer was then added to neutralize the pH. The batch was concentrated and diafiltered with 150 mM sodium chloride, 25 mM potassium phosphate (pH 7) using a 30 kDa NMWCO tangential flow ultrafiltration membrane at about 4°C.

[0984] Final filtration and product storage

[0985] The batch was then concentrated and diafiltered at 4°C using a 300 kDa NMWCO tangential flow ultrafiltration membrane with 150 mM sodium chloride (pH 7.0) and 0.015% (w / v) polysorbate 20 containing 10 mM histidine.

[0986] The retentate batch was filtered at 0.2 micron and then diluted with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0, and 0.015% (w / v) polysorbate 20, dispensed into aliquots, and frozen at ≤ -60°C.

[0987] Embodiment 36

[0988] Preparation of serotype 19F conjugates for PCV23 (DMSO), PCV23 (DMSO + Aq), and PCV22 multivalent studies using DMSO conjugation

[0989] The polysaccharide is dissolved, reduced in size to the target molecular weight, chemically activated and buffer exchanged by ultrafiltration. The activated polysaccharide and purified CRM197 are lyophilized and redissolved in DMSO separately. The redissolved polysaccharide is then combined with the CRM197 solution and conjugated as described below. The resulting conjugate is purified by ultrafiltration before a final 0.2 micron filtration. Multiple process parameters such as pH, temperature, concentration and time are controlled in each step to produce a conjugate with desired properties.

[0990] Polysaccharide size reduction and oxidation

[0991] The purified pneumococcal capsule Ps powder was dissolved in water and filtered at 0.45 micron. 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 to 150 bar / 5 times.

[0992] The size-reduced polysaccharides were concentrated and diafiltered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

[0993] The polysaccharide was then adjusted to 4°C and to pH 5 with sodium acetate buffer to minimize polysaccharide size reduction due to activation. 100 mM sodium metaperiodate solution was added to initiate polysaccharide activation. The oxidation reaction was carried out at 4°C for 4 hours.

[0994] The activated product was diafiltered against 10 mM potassium phosphate (pH 6.4) and then against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane. The ultrafiltration was performed at 2-8°C.

[0995] Polysaccharide conjugation to CRM197

[0996] Purified CRM197 obtained by expression in Pseudomonas fluorescens as previously described (WO 2012 / 173876A1) was diafiltered using a 5 kDa NMWCO tangential flow ultrafiltration membrane with 2 mM phosphate (pH 7.2) buffer and filtered at 0.2 micron.

[0997] The activated polysaccharide was prepared at 6 mg Ps / mL and 5% w / v sucrose for lyophilization. CRM197 was prepared at 6 mg Pr / mL and 1% w / v sucrose for lyophilization.

[0998] The prepared Ps and CRM197 solutions were lyophilized separately. The lyophilized Ps and CRM197 materials were redissolved in an equal volume of DMSO. The polysaccharide was blended with the CRM197 solution to obtain a polysaccharide concentration of 2.0 g Ps / L and a polysaccharide to CRM197 mass ratio of 1.2. This mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate. Sodium cyanoborohydride (1 mol / mol polysaccharide repeating unit) was added and the conjugation was performed at 22°C.

[0999] Reduction with sodium borohydride

[1000] Sodium borohydride (2 mol / mol polysaccharide repeating unit) was added after the conjugation reaction and incubated at 22°C for 3 hours. The batch was diluted to 150 mM sodium chloride and about 0.025% (w / v) polysorbate 20 at about 4°C. Potassium phosphate buffer was then added to neutralize the pH. The batch was concentrated and diafiltered with 150 mM sodium chloride, 25 mM potassium phosphate (pH 7) using a 30 kDa NMWCO tangential flow ultrafiltration membrane at about 4°C. The retentate batch was filtered at 0.2 microns and then incubated at 22°C for 4.5 days.

[1001] Final filtration and product storage

[1002] The batch was then concentrated and diafiltered using a 300 kDa NMWCO tangential flow ultrafiltration membrane at 4°C using 150 mM sodium chloride, pH 7.0, containing 10 mM histidine.

[1003] The retentate batch was filtered at 0.2 micron and then diluted with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0, and 0.015% (w / v) polysorbate 20, dispensed into aliquots, and frozen at ≤ -60°C.

[1004] Embodiment 37

[1005] Preparation of serotype 22F conjugates for PCV23 (DMSO) multivalent studies using DMSO conjugation

[1006] The polysaccharide is dissolved, reduced in size to the target molecular weight, chemically activated and buffer exchanged by ultrafiltration. The activated polysaccharide and purified CRM197 are lyophilized and redissolved in DMSO separately. The redissolved polysaccharide is then combined with the CRM197 solution and conjugated as described below. The resulting conjugate is purified by ultrafiltration before a final 0.2 micron filtration. Multiple process parameters such as pH, temperature, concentration and time are controlled in each step to produce a conjugate with desired properties.

[1007] Polysaccharide size reduction and oxidation

[1008] The purified pneumococcal capsule Ps powder was dissolved in water and filtered at 0.45 micron. 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 to 810 bar / 5 times.

[1009] The size-reduced polysaccharides were concentrated and diafiltered against water using a 5 kDa NMWCO tangential flow ultrafiltration membrane.

[1010] The polysaccharide solution was then adjusted to 22°C and pH 5 with sodium acetate buffer to minimize polysaccharide size reduction due to activation. 100 mM sodium metaperiodate solution was added to initiate polysaccharide activation. The oxidation reaction was carried out at 22°C for 2 hours.

[1011] The activated product was diafiltered against 10 mM potassium phosphate (pH 6.4) and then against water using a 5 kDa NMWCO tangential flow ultrafiltration membrane. The ultrafiltration was performed at 2-8°C.

[1012] Polysaccharide conjugation to CRM197

[1013] Purified CRM197 obtained by expression in Pseudomonas fluorescens as previously described (WO 2012 / 173876A1) was diafiltered using a 5 kDa NMWCO tangential flow ultrafiltration membrane with 2 mM phosphate (pH 7.2) buffer and filtered at 0.2 micron.

[1014] The activated polysaccharide was prepared at 6 mg Ps / mL and 5% w / v sucrose for lyophilization. CRM197 was prepared at 6 mg Pr / mL and 1% w / v sucrose for lyophilization.

[1015] The prepared Ps and CRM197 solutions were lyophilized separately. The lyophilized Ps and CRM197 materials were redissolved in an equal volume of DMSO. The polysaccharide was blended with the CRM197 solution to obtain a polysaccharide concentration of 2.3 g Ps / L and a polysaccharide to CRM197 mass ratio of 1.5. This mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate. Sodium cyanoborohydride (1 mol / mol polysaccharide repeating unit) was added and conjugation was performed at 22°C.

[1016] Reduction with sodium borohydride

[1017] Sodium borohydride (2 mol / mol polysaccharide repeating unit) was added after the conjugation reaction and incubated for 1 hour at 22° C. The batch was diluted to 150 mM sodium chloride and approximately 0.025% (w / v) polysorbate 20 at approximately 4° C. Potassium phosphate buffer was then added to neutralize the pH.

[1018] Final filtration and product storage

[1019] The batch was then concentrated and diafiltered at 4°C using a 300 kDa NMWCO tangential flow ultrafiltration membrane with 150 mM sodium chloride (pH 7.0) and 0.015% (w / v) polysorbate 20 containing 10 mM histidine.

[1020] The retentate batch was filtered at 0.2 micron (using a 0.5 micron prefilter) and then diluted with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0, and 0.015% (w / v) polysorbate 20, dispensed into aliquots, and frozen at ≤ -60°C.

[1021] Embodiment 38

[1022] Preparation of serotype 22F conjugates for PCV22 and PCV23 (DMSO+Aq) multivalent studies using aqueous conjugation

[1023] The polysaccharide is dissolved, reduced in size, chemically activated and buffer exchanged by ultrafiltration. Purified CRM197 is then conjugated to the activated polysaccharide using nickel chloride in the reaction mixture, and the resulting conjugate is purified by ultrafiltration before a final 0.2 micron filtration. Multiple process parameters such as pH, temperature, concentration and time are controlled in each step to produce a conjugate with desired properties.

[1024] Polysaccharide size reduction and oxidation

[1025] Purified pneumococcal capsular polysaccharide powder was dissolved in water and filtered at 0.45 micron. The dissolved polysaccharide was homogenized to reduce the molecular weight. The homogenization pressure and the number of passes through the homogenizer were controlled to 350 bar / 5 times to reduce the size to the target molecular weight. The size-reduced polysaccharide was then concentrated and diafiltered with water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

[1026] The polysaccharide solution was then adjusted to 22°C and pH 5 with sodium acetate buffer to minimize polysaccharide size reduction due to activation. 100 mM sodium metaperiodate solution was added to initiate polysaccharide activation. The oxidation reaction was carried out at 22°C for 2 hours.

[1027] The activated product was diafiltered against 10 mM potassium phosphate (pH 6.4) using a 10 kDa NMWCO tangential flow ultrafiltration membrane. The ultrafiltration was performed at 2-8°C.

[1028] Polysaccharide conjugation to CRM197

[1029] The oxidized polysaccharide solution is mixed with water and 1.5M potassium phosphate (pH 7.0). The buffer pH is selected to improve the stability of the activated polysaccharide during the conjugation reaction. The purified CRM197 obtained by expression in Pseudomonas fluorescens as described above (WO 2012 / 173876A1) is filtered at 0.2 microns and combined with a buffered polysaccharide solution with a polysaccharide to CRM197 mass ratio of 0.6. This mass ratio is selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate. The concentrations of polysaccharide and phosphate are 7.5g / L and 100mM, respectively. The polysaccharide concentration is selected to control the size of the resulting conjugate. The solution is then filtered at 0.2 microns. Nickel chloride is added to about 2mM using a 100mM nickel chloride solution. Sodium cyanoborohydride (2 moles / mole polysaccharide repeating unit) is added. Conjugation is carried out for 120 hours to maximize the consumption of polysaccharides and proteins.

[1030] Reduction with sodium borohydride

[1031] After the conjugation reaction, the batch was diluted to a polysaccharide concentration of about 3.5 g / L, cooled to 2-8° C., and filtered at 1.2 microns. The batch was diafiltered at 2-8° C. with 100 mM potassium phosphate (pH 7.0) using a 100 kDa NMWCO tangential flow ultrafiltration membrane. Subsequently, the batch recovered in the retentate was diluted to about 2.0 g polysaccharide / L and the pH was adjusted by adding 1.2 M sodium bicarbonate (pH 9.4). Sodium borohydride (1 mol / mol polysaccharide repeating unit) was added. Subsequently, 1.5 M potassium phosphate (pH 6.0) was added.

[1032] Final filtration and product storage

[1033] The batch was then concentrated and diafiltered at 4°C using a 300 kDa NMWCO tangential flow ultrafiltration membrane against 150 mM sodium chloride, pH 7.0, containing 10 mM L-histidine.

[1034] The batch was filtered at 0.2 micron and adjusted to a polysaccharide concentration of 1.0 g / L with additional 150 mM sodium chloride (pH 7.0) buffer containing 10 mM L-histidine. The batch was divided into aliquots and frozen at ≤ -60°C.

[1035] ...

Claims

1. A multivalent immunogenic composition comprising a Streptococcus pneumoniae (S. pneumoniae) polysaccharide carrier protein conjugate, wherein each of the conjugates comprises a polysaccharide of a specific S. pneumoniae serotype conjugated to a carrier protein, and wherein the S. pneumoniae serotype is selected from the following: a) 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 12F, 14, 15A, DeOAc15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; b) 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; c) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, DeOAc15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; d) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; e) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; f) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, DeOAc15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; g) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; h) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; i) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; j) 1, 3, 4, 5, 6A, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; k) 1, 3, 4, 5, 6B, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; l) 1, 3, 4, 5, 6C, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; m) 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; n) 1, 3, 4, 5, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; o) 1, 3, 4, 5, 6C, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; p) 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; q)1, 3, 4, 5, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; r) 1, 3, 4, 5, 6C, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; s)1, 3, 4, 5, 6A, 7F, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; t) 1, 3, 4, 5, 6B, 7F, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; u) 1, 3, 4, 5, 6C, 7F, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; v) 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; w) 1, 3, 4, 5, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; x) 1, 3, 4, 5, 6C, 7F, 8, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; y) 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; z) 1, 3, 4, 5, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; aa) 1, 3, 4, 5, 6C, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; bb) 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15A, deO - 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; cc) 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; dd) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 12F, 14, 15A, deO - 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; ee) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; ff) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; gg) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 11A, 12F, 14, 15A, deO - 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; hh) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; ii) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; jj) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; kk) 1, 3, 4, 5, 6A, 7F, 9V, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; ll)1, 3, 4, 5, 6B, 7F, 9V, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; mm) 1, 3, 4, 5, 6C, 7F, 9V, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; nn) 1, 3, 4, 5, 6A, 7F, 8, 9V, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; oo)1, 3, 4, 5, 6B, 7F, 8, 9V, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; pp) 1, 3, 4, 5, 6C, 7F, 8, 9V, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; qq)1, 3, 4, 5, 6A, 7F, 8, 9V, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; rr)1, 3, 4, 5, 6B, 7F, 8, 9V, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; ss) 1, 3, 4, 5, 6C, 7F, 8, 9V, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; tt)1, 3, 4, 5, 6A, 7F, 9V, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; uu)1, 3, 4, 5, 6B, 7F, 9V, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; vv) 1, 3, 4, 5, 6C, 7F, 9V, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; ww)1, 3, 4, 5, 6A, 7F, 8, 9V, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; xx) 1, 3, 4, 5, 6B, 7F, 8, 9V, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; yy) 1, 3, 4, 5, 6C, 7F, 8, 9V, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; zz)1, 3, 4, 5, 6A, 7F, 8, 9V, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; aaa) 1, 3, 4, 5, 6B, 7F, 8, 9V, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B, and 39; and bbb)1, 3, 4, 5, 6C, 7F, 8, 9V, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39, wherein the composition does not comprise a polysaccharide carrier protein conjugate with a polysaccharide of any other S. pneumoniae serotype.

2. The multivalent immunogenic composition of claim 1, wherein at least one of the polysaccharide carrier protein conjugates is formed by a conjugation reaction comprising an aprotic solvent.

3. The multivalent immunogenic composition of claim 1, wherein each of the polysaccharide carrier protein conjugates is formed by a conjugation reaction comprising an aprotic solvent.

4. The multivalent immunogenic composition of claim 2 or claim 3, wherein the aprotic solvent is dimethyl sulfoxide (DMSO).

5. The multivalent immunogenic composition of any one of claims 1 to 4, wherein the carrier protein is selected from the group consisting of: outer membrane protein complex (OMPC), tetanus toxoid, diphtheria toxoid, protein D and CRM197.

6. The multivalent immunogenic composition of any one of claims 1 to 5, wherein the carrier protein is CRM197.

7. The multivalent immunogenic composition of any one of claims 1 to 6, wherein the composition further comprises an adjuvant.

8. The multivalent immunogenic composition of any one of claims 1 to 6, wherein the composition does not comprise an adjuvant.

9. Use of the multivalent immunogenic composition of any one of claims 1 to 8, 15, 18, 21, 30, 32, 34-35, 37-39, 41-43, 45-47 and 49 in the preparation of a medicament for inducing an immune response in a human patient.

10. Use of the multivalent immunogenic composition of any one of claims 1 to 8, 15, 18, 21, 30, 32, 34-35, 37-39, 41-43, 45-47 and 49 for the preparation of a medicament for inducing a protective immune response in a human patient.

11. Use of the multivalent immunogenic composition of any one of claims 1 to 8, 15, 18, 21, 30, 32, 34-35, 37-39, 41-43, 45-47 and 49 for the preparation of a medicament for inducing a protective immune response against Streptococcus pneumoniae in a human patient.

12. The use according to any one of claims 9 to 11, wherein the patient has previously been treated with a multivalent pneumococcal vaccine.

13. The use according to any one of claims 9 to 12, wherein the patient is between 6 weeks and 17 years of age.

14. Use of the multivalent immunogenic composition of any one of claims 1, 15, 18, 21, 30, 32, 34-35, 37-39, 41-43, 45-47 and 49 in the preparation of a medicament for preventing pneumococcal pneumonia and / or invasive pneumococcal disease in patients aged 6 weeks to 17 years.

15. A multivalent immunogenic composition comprising 23 different S. pneumoniae polysaccharide-carrier protein conjugates, wherein each of the conjugates comprises a capsular polysaccharide of a specific S. pneumoniae serotype conjugated to a carrier protein, wherein the S. pneumoniae serotypes are 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B and the carrier protein is CRM197, and wherein the composition does not comprise a polysaccharide-carrier protein conjugate with a polysaccharide of any other S. pneumoniae serotype.

16. The multivalent immunogenic composition of claim 15, wherein each of the polysaccharide carrier protein conjugates is formed by a conjugation reaction comprising an aprotic solvent, wherein the aprotic solvent is dimethyl sulfoxide (DMSO).

17. The multivalent immunogenic composition of claim 15, wherein the composition comprises an adjuvant.

18. A multivalent immunogenic composition comprising 24 different Streptococcus pneumoniae polysaccharide-carrier protein conjugates, wherein each of the conjugates comprises a polysaccharide of a specific Streptococcus pneumoniae serotype conjugated to a carrier protein, wherein the Streptococcus pneumoniae serotypes are 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B and the carrier protein is CRM197, and wherein the composition does not comprise a polysaccharide-carrier protein conjugate with a polysaccharide of any other Streptococcus pneumoniae serotype.

19. The multivalent immunogenic composition of claim 18, wherein each of the polysaccharide carrier protein conjugates is formed by a conjugation reaction comprising an aprotic solvent, wherein the aprotic solvent is dimethyl sulfoxide (DMSO).

20. The multivalent immunogenic composition of claim 18, wherein the composition comprises an adjuvant.

21. A multivalent immunogenic composition comprising 24 different Streptococcus pneumoniae polysaccharide-carrier protein conjugates, wherein each of the conjugates comprises a polysaccharide of a specific Streptococcus pneumoniae serotype conjugated to a carrier protein, wherein the Streptococcus pneumoniae serotypes are polysaccharides of 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B, wherein the carrier protein is CRM197, and wherein the composition does not comprise a polysaccharide-carrier protein conjugate with a polysaccharide of any other Streptococcus pneumoniae serotype.

22. The multivalent immunogenic composition of claim 21, wherein each of the polysaccharide carrier protein conjugates is formed by a conjugation reaction comprising an aprotic solvent, wherein the aprotic solvent is dimethyl sulfoxide (DMSO).

23. The multivalent immunogenic composition of claim 21, wherein the composition comprises an adjuvant.

24. The multivalent immunogenic composition of claim 1, wherein the S. pneumoniae serotype 15C polysaccharide is a de-O-acetylated S. pneumoniae serotype 15B polysaccharide.

25. The multivalent immunogenic composition of claim 15, wherein the S. pneumoniae serotype 15C polysaccharide is a de-O-acetylated S. pneumoniae serotype 15B polysaccharide.

26. The multivalent immunogenic composition of claim 18, wherein the S. pneumoniae serotype 15C polysaccharide is a de-O-acetylated S. pneumoniae serotype 15B polysaccharide.

27. The multivalent immunogenic composition of claim 24, wherein the de-O-acetylated 15B polysaccharide has an O-acetyl content of less than 5% per repeating unit.

28. The multivalent immunogenic composition of claim 25, wherein the de-O-acetylated 15B polysaccharide has an O-acetyl content of less than 5% per repeating unit.

29. The multivalent immunogenic composition of claim 26, wherein the de-O-acetylated 15B polysaccharide has an O-acetyl content of less than 5% per repeating unit.

30. A multivalent immunogenic composition comprising 23 different S. pneumoniae polysaccharide-carrier protein conjugates, wherein each of the conjugates comprises a polysaccharide of a specific S. pneumoniae serotype conjugated to a carrier protein, wherein the S. pneumoniae serotypes are 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, de-O-acetylated 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B and the carrier protein is CRM197, and wherein the composition does not comprise a polysaccharide-carrier protein conjugate with a polysaccharide of any other S. pneumoniae serotype.

31. The multivalent immunogenic composition of claim 30, wherein the de-O-acetylated 15B polysaccharide has an O-acetyl content of less than 5% per repeating unit.

32. A multivalent immunogenic composition comprising 24 different pneumococcal polysaccharide-carrier protein conjugates, wherein each of the conjugates comprises a polysaccharide of a specific pneumococcal serotype conjugated to a carrier protein, wherein the pneumococcal serotypes are 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, de-O-acetylated 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B, and the carrier protein is CRM197, and wherein the composition does not comprise a polysaccharide-carrier protein conjugate with a polysaccharide of any other pneumococcal serotype.

33. The multivalent immunogenic composition of claim 32, wherein the de-O-acetylated 15B polysaccharide has an O-acetyl content of less than 5% per repeating unit.

34. A multivalent immunogenic composition comprising a Streptococcus pneumoniae polysaccharide carrier protein conjugate, wherein each of the conjugates comprises a polysaccharide of a specific Streptococcus pneumoniae serotype conjugated to a carrier protein, wherein the Streptococcus pneumoniae serotypes consist essentially of 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B, and the carrier protein is CRM197.

35. A multivalent immunogenic composition comprising a pneumococcal polysaccharide carrier protein conjugate, wherein each of the conjugates comprises a polysaccharide of a specific pneumococcal serotype conjugated to a carrier protein, wherein the pneumococcal serotypes consist essentially of 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, de-O-acetylated 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B, and the carrier protein is CRM197.

36. The multivalent immunogenic composition of claim 35, wherein the de-O-acetylated 15B polysaccharide has an O-acetyl content of less than 5% per repeating unit.

37. A multivalent immunogenic composition comprising a Streptococcus pneumoniae polysaccharide carrier protein conjugate, wherein each of the conjugates comprises a polysaccharide of a specific Streptococcus pneumoniae serotype conjugated to a carrier protein, wherein the Streptococcus pneumoniae serotypes consist essentially of 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B, and the carrier protein is CRM197.

38. A multivalent immunogenic composition comprising a Streptococcus pneumoniae polysaccharide carrier protein conjugate, wherein each of the conjugates comprises a polysaccharide of a specific Streptococcus pneumoniae serotype conjugated to a carrier protein, wherein the Streptococcus pneumoniae serotypes consist essentially of 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B, and the carrier protein is CRM197.

39. A multivalent immunogenic composition comprising a pneumococcal polysaccharide carrier protein conjugate, wherein each of the conjugates comprises a polysaccharide of a specific pneumococcal serotype conjugated to a carrier protein, wherein the pneumococcal serotypes consist essentially of 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, de-O-acetylated 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B, and the carrier protein is CRM197.

40. The multivalent immunogenic composition of claim 39, wherein the de-O-acetylated 15B polysaccharide has an O-acetyl content of less than 5% per repeating unit.

41. A multivalent immunogenic composition comprising a Streptococcus pneumoniae polysaccharide carrier protein conjugate, wherein each of the conjugates comprises a polysaccharide of a specific Streptococcus pneumoniae serotype conjugated to a carrier protein, wherein the Streptococcus pneumoniae serotypes consist essentially of 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B, and the carrier protein is CRM197.

42. A multivalent immunogenic composition comprising a Streptococcus pneumoniae polysaccharide carrier protein conjugate, wherein each of the conjugates comprises a polysaccharide of a specific Streptococcus pneumoniae serotype conjugated to a carrier protein, wherein the Streptococcus pneumoniae serotypes consist of 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B, and the carrier protein is CRM197.

43. A multivalent immunogenic composition comprising a pneumococcal polysaccharide carrier protein conjugate, wherein each of the conjugates comprises a polysaccharide of a specific pneumococcal serotype conjugated to a carrier protein, wherein the pneumococcal serotypes consist of 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, de-O-acetylated 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B, and the carrier protein is CRM197.

44. The multivalent immunogenic composition of claim 43, wherein the de-O-acetylated 15B polysaccharide has an O-acetyl content of less than 5% per repeating unit.

45. A multivalent immunogenic composition comprising a Streptococcus pneumoniae polysaccharide carrier protein conjugate, wherein each of the conjugates comprises a polysaccharide of a specific Streptococcus pneumoniae serotype conjugated to a carrier protein, wherein the Streptococcus pneumoniae serotype consists of 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B, and the carrier protein is CRM197.

46. ​​A multivalent immunogenic composition comprising a pneumococcal polysaccharide carrier protein conjugate, wherein each of the conjugates comprises a polysaccharide of a specific pneumococcal serotype conjugated to a carrier protein, wherein the pneumococcal serotypes consist of 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B, and the carrier protein is CRM197.

47. A multivalent immunogenic composition comprising a pneumococcal polysaccharide carrier protein conjugate, wherein each of the conjugates comprises a polysaccharide of a specific pneumococcal serotype conjugated to a carrier protein, wherein the pneumococcal serotypes consist of 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, de-O-acetylated 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B, and the carrier protein is CRM197.

48. The multivalent immunogenic composition of claim 47, wherein the de-O-acetylated 15B polysaccharide has an O-acetyl content of less than 5% per repeating unit.

49. A multivalent immunogenic composition comprising a pneumococcal polysaccharide carrier protein conjugate, wherein each of the conjugates comprises a polysaccharide of a specific pneumococcal serotype conjugated to a carrier protein, wherein the pneumococcal serotypes consist of 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B, and the carrier protein is CRM197.

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