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

Abstract

To provide additional pneumococcal vaccine compositions which can provide protection against pneumococcal serotypes not present in currently available vaccines.SOLUTION: The invention is related to multivalent immunogenic compositions comprising more than one S. pneumoniae polysaccharide protein conjugates, where each of the conjugates comprises a polysaccharide from an S. pneumoniae serotype conjugated to a carrier protein. In some embodiments, at least one polysaccharide protein conjugate is formed by a conjugation reaction comprising an aprotic solvent. In other embodiment, each of the polysaccharide protein conjugates are formed by a conjugation reaction comprising an aprotic solvent. Also provided are methods for inducing a protective immune response in a human patient comprising administering the multivalent immunogenic compositions of the invention to the patient.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention provides a multivalent immunogenic composition containing different polysaccharide-protein conjugates. Each conjugate is conjugated to a carrier protein, preferably CRM197. Conjugated Streptococcus pneumoniae of different serotypes ( Capsules produced from Streptococcus pneumoniae The immunogenic composition comprises a polysaccharide that provides broad spectrum efficacy against pneumococcal disease.

[0002] This application is filed electronically in ASCII format with a Sequence Listing, which is hereby incorporated by reference in its entirety. The above ASCI dated December 3, 2019 includes the above. I copy is called 24683WOPCT-SEQTXT-03DEC2019, 6kg It has a size of bytes. [Background technology]

[0003] Streptococcus pneumoniae ae) is a gram-positive bacterium that is responsible for invasive bacterial diseases in infants and young children (e.g., pneumonia, bacteremia, Streptococcus pneumoniae is the most common cause of chronic respiratory syndrome (bronchitis, meningitis, and otitis media). The bacteria are encapsulated in a chemically bonded polysaccharide. There are over 90 known serotypes of Streptococcus pneumoniae. The capsule is the major virulence determinant of pneumococci because it protects the inner surface of the bacterium from complementation. Polysaccharides not only protect against T cell-dependent immune responses, but also have low immunogenicity by themselves. It is an antigen that, in most cases, is processed or bound to the MHC It is not presented on a molecular surface, but it does cross-link surface receptors on B cells. The immune system may be stimulated by alternative mechanisms, including

[0004] The multivalent pneumococcal polysaccharide vaccine, which has been licensed for many years, is used in adults, especially the elderly and high-risk individuals. However, it has been shown to be useful in preventing pneumococcal disease in infants and young children. Inadequate response to jugate pneumococcal polysaccharides. Invasive pneumococcal infection in infants and young children. The seven most frequently isolated serotypes causing disease at the time (4, 6B, 9V, 14 Pneumococcal conjugate vaccine Prevn containing 18C, 19F and 23F ar was first approved in the United States in February 2000. After widespread use of Prevnar in the United States, invasive pneumococcal disease in children was largely prevented by Prevnar. The incidence of serotypes present in the HIV-1 strain was significantly reduced by 100%. Disease Control and Prevention,MMWR Mor b Mortal Wkly Rep 2005,54(36):893-7 However, in some parts of the world, seroprevalence with Prevnar® is It has a limited spectrum and in the United States, some of the emerging serotypes (e.g., 19A) There is evidence that O'Brien et al., 2004, Am J Epidemiol 15 9:634-44;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-S1 Please see reference 89.

[0005] U.S. Patent Application Publication No. US2006 / 0228380 discloses serotypes 1, 3, 4, 5, and 6A 13-valent pneumococcal strain containing 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F It describes a polysaccharide-protein conjugate vaccine. China Patent Application Publication No. CN1 01590224A is for serotypes 1, 2, 4, 5, 6A, 6B, 7F, 9N, 9V, 14, 14-valent pneumococcal polysaccharide-protein conjugates containing 18C, 19A, 19F and 23F The article describes a vaccine that is effective against rabies.

[0006] Other PCVs cover serotypes 7, 10, 11 or 13 contained in PCV-15. (U.S. Patent Publication No. 2011 / 0195086), but immunity is not known for some serotypes. Interference has been observed (e.g., GSK's PCV-11 serotype 3). Pfizer's PCV-13 [PREVNAR (registered trademark)] )13], a lower response rate was observed against serotype 6B. ula et al., 2006, Lancet 367:740-48 and Kieninger et al. ,Safety and Immunologic Non-inferiority of 13-valent Pneumococcal Conjugate Vacc ine Compared to 7-valent Pneumococcal Co njugate Vaccine Given as a 4-Dose Series in Healthy Infants and Toddlers(48th An nual ICAAC / ISDA 46th Annual Meeting,Wash For more information, please see the article presented at the 2008 Symposium on Drugs and Drug Administration, Washington DC, October 25-28, 2008.

[0007] Current multivalent pneumococcal vaccines limit the risk of pneumococcal disease associated with the serotypes present in the vaccine. However, there are no currently available vaccines that could be effective in reducing the incidence of The prevalence of pneumococci expressing serotypes has been increasing. Additional pneumococcal vaccine combinations that may confer protection against pneumococcal serotypes not present in the current study The product is needed. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] U.S. Patent Application Publication No. US2006 / 0228380 [Patent Document 2] China Patent Application Publication No. CN101590224A [Patent Document 3] U.S. Patent Publication No. 2011 / 0195086 [Non-patent literature]

[0009] [Non-Patent Document 1] O'Brien et al., 2004, Am J Epidemiol 159:634-44; Whitney et al., 2003, N Engl J Med 348:1737-46 [Non-Patent Document 2] Kyaw et al., 2006, N Engl J Med 354:1455-63 [Non-Patent Document 3] ;Hicks et al., 2007, J Infect Dis 196:1346-54 [Non-Patent Document 4] Traore et al., 2009, Clin Infect Dis 48:S181-S189 [Non-Patent Document 5] Prymula et al., 2006, Lancet 367:740-48 [Non-Patent Document 6] Kieninger et al., Safety and Immunologic Non-inferiority of 13-valent Pneumococcal Conjugate Vaccine Compared to 7-valent Pneumococcal Conjugate Vaccine Given as a 4-Dose Series in Healthy Infants and Toddlers (Presented at the 48th Annual ICAAC / ISDA 46th Annual Meeting, Washington DC, October 25-28, 2008) Summary of the Invention

[0010] Overview of the Invention The present invention relates to a method for the treatment of Streptococcus pneumoniae (S. pneumoniae) The present invention provides a multivalent immunogenic composition comprising a conjugate polysaccharide protein conjugate, Each of the conjugates was conjugated to a carrier protein, Streptococcus cerevisiae. The polysaccharide-protein conjugates contain polysaccharides derived from S. pneumoniae serovars and are The polysaccharides of the Streptococcus pneumoniae serotype group are selected from the group consisting of: a)1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 12F, 14, 15A, D eOAc15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and and 35B; b)1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 12F, 14, 15A, 1 5C, 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, 33 F 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 3 5B; 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, 24 F, 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 3 5B; 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, 15 C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; n)1, 3, 4, 5, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15 C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; o)1, 3, 4, 5, 6C, 7F, 8, 9V, 10A, 12F, 14, 15A, 15 C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; p)1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 11A, 12F, 14, 15 A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; q)1, 3, 4, 5, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15 A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; r)1, 3, 4, 5, 6C, 7F, 8, 9V, 10A, 11A, 12F, 14, 15 A, 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, 15 B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; w)1, 3, 4, 5, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15 B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; x)1, 3, 4, 5, 6C, 7F, 8, 9V, 10A, 12F, 14, 15A, 15 B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; y)1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 11A, 12F, 14, 15 A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; z)1, 3, 4, 5, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15 A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; and aa)1, 3, 4, 5, 6C, 7F, 8, 9V, 10A, 11A, 12F, 14, 1 5A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and and 35B.

[0011] The present invention provides a multivalent immunogenic composition comprising 22 different polysaccharide-protein conjugates. wherein each of the conjugates is conjugated to a carrier protein. The polysaccharides are derived from the capsular polysaccharides of Streptococcus pneumoniae serovars Bacillus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 12F , 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, Made from 33F and 35B.

[0012] The present invention provides a multivalent immunogenic composition comprising 22 different polysaccharide-protein conjugates. wherein each of the conjugates is conjugated to a carrier protein. Contains polysaccharides derived from Streptococcus pneumoniae serovars and polysaccharide-protein conjugates Gates are 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35 The polysaccharides of the group of Streptococcus pneumoniae serotypes selected from the group consisting of B .

[0013] The present invention provides a multivalent immunogenic composition comprising 23 different polysaccharide-protein conjugates. wherein each of the conjugates is conjugated to a carrier protein. The polysaccharides are derived from the capsular polysaccharides of Streptococcus pneumoniae serovars Bacillus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 1 2F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24 It is made from 35F, 33F and 35B.

[0014] The present invention provides 23 different Streptococcus pneumoniae polysaccharide-protein conjugates. The present invention provides a multivalent immunogenic composition comprising a conjugate, wherein each of the conjugates is a carrier Protein-conjugated capsules from Streptococcus pneumoniae serovars Each different polysaccharide-protein conjugate contains a membrane polysaccharide and is a serovar that binds to the streptococcus of pneumoniae. Tococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A , 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, It contains polysaccharides derived from 24F, 33F and 35B, and the carrier protein is CRM197.

[0015] The present invention provides a multivalent immunogenic composition comprising 24 different polysaccharide-protein conjugates. wherein each of the conjugates is conjugated to a carrier protein. The polysaccharides are derived from the capsular polysaccharides of Streptococcus pneumoniae serovars Bacillus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 1 1A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23 It is made from F, 24F, 33F and 35B.

[0016] The present invention provides 24 different Streptococcus pneumoniae polysaccharide-protein conjugates. The present invention provides a multivalent immunogenic composition comprising a conjugate, wherein each of the conjugates is a carrier Protein-conjugated capsules from Streptococcus pneumoniae serovars Each of the different polysaccharide-protein conjugates contains a membrane polysaccharide. Bacillus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 1 1A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23 It contains polysaccharides derived from F, 24F, 33F and 35B, and the carrier protein is CRM197 It is.

[0017] The present invention provides a multivalent immunogenic composition comprising 24 different polysaccharide-protein conjugates. wherein each of the conjugates is conjugated to a carrier protein. The polysaccharides are derived from the capsular polysaccharides of Streptococcus pneumoniae serovars Bacillus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 1 1A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23 It is made from F, 24F, 33F and 35B.

[0018] The present invention provides 24 different Streptococcus pneumoniae polysaccharide-protein conjugates. The present invention provides a multivalent immunogenic composition comprising a conjugate, wherein each of the conjugates is a carrier Protein-conjugated capsules from Streptococcus pneumoniae serovars Each of the different polysaccharide-protein conjugates contains a membrane polysaccharide. Bacillus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 1 1A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23 It contains polysaccharides derived from F, 24F, 33F and 35B, and the carrier protein is CRM197. do.

[0019] The present invention provides a multivalent immunogenic composition comprising up to 33 different polysaccharide-protein conjugates. wherein each of the conjugates is conjugated to a carrier protein. Contains polysaccharides derived from selected Streptococcus pneumoniae serovars and a polysaccharide protein complex The jugates are 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F , 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, Streptococcus pneumoniae serovars selected from the group consisting of 33F and 35B , further selected from 7C, 9N, 16F, 21, 23A, 31, 34, 35F and 38 1, 2, 3, 4, 5, 6, 7, 8, or 9 additional Streptococcus pneumoniae It contains polysaccharides from the group including the N. nie serovar.

[0020] The present invention relates to a multivalent immunogenic composition comprising up to 30 different polysaccharide-protein conjugates. wherein each of the conjugates is conjugated to a carrier protein. Contains polysaccharides derived from selected Streptococcus pneumoniae serovars and a polysaccharide protein complex The jugates are 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F , 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, Streptococcus pneumoniae serovars selected from the group consisting of 33F and 35B , 1, 2, 3, 4 selected from 7C, 9N, 16F, 23A, 35F and 38; Contains polysaccharides from a group that includes five or six additional Streptococcus pneumoniae serotypes .

[0021] The present invention provides a multivalent immunogenic composition comprising up to 33 different polysaccharide-protein conjugates. wherein each of the conjugates is conjugated to a carrier protein. Contains polysaccharides derived from selected Streptococcus pneumoniae serovars and a polysaccharide protein complex The jugates are 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F , 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, Streptococcus pneumoniae serovars selected from the group consisting of 33F and 35B , further selected from 7C, 9N, 16F, 21, 23A, 31, 34, 35F and 38 1, 2, 3, 4, 5, 6, 7, 8, or 9 additional Streptococcus pneumoniae It contains polysaccharides of the S. nie serovar group.

[0022] The present invention relates to a multivalent immunogenic composition comprising up to 30 different polysaccharide-protein conjugates. wherein each of the conjugates is conjugated to a carrier protein. Contains polysaccharides derived from selected Streptococcus pneumoniae serovars and a polysaccharide protein complex The jugates are 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F , 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, Streptococcus pneumoniae serovars selected from the group consisting of 33F and 35B , 1, 2, 3, 4 selected from 7C, 9N, 16F, 23A, 35F and 38; Contains polysaccharides of five or six additional Streptococcus pneumoniae serotype groups.

[0023] In some embodiments, at least one of the polysaccharide protein conjugates is a non- Conjugation reactions involving protic solvents, such as dimethylsulfoxide (DMSO) In certain embodiments, each of the polysaccharide-protein conjugates is formed by It is formed by conjugation reactions involving aprotic solvents, such as DMSO. A method for preventing infection in a human patient comprising administering to the patient a multivalent immunogenic composition of the invention. Also provided are methods for inducing a regulatory immune response in a mammalian subject. The patient had been previously treated with multivalent pneumococcal vaccine.

[0024] The multivalent immunogenic compositions of the invention are useful in the treatment of multiple pneumococcal vaccine regimens. Thus, the present invention relates to a method for administering a multivalent immunogenic composition of the present invention to a patient. and further administering the multivalent pneumococcal vaccine to the patient, in any order. In certain embodiments, a method of inducing a protective immune response in a human patient is provided, the method comprising: In the case of pneumococcal polyvalent vaccines, multiple Streptococcus pneumoniae polysaccharide proteins are used. and a carrier protein conjugate comprising: The antibody comprises a polysaccharide derived from a Streptococcus pneumoniae serovar conjugated to In other embodiments, the multivalent pneumococcal vaccine comprises unconjugated capsular polysaccharides. The present invention also relates to a method for the preparation of a Streptococcus pneumoniae polysaccharide protein conjugate. The present invention provides a multivalent immunogenic composition comprising a conjugate, wherein each of the conjugates comprises a carrier. Conjugated to Streptococcus pneumoniae serovar Selected serotypes of Streptococcus pneumoniae contain polysaccharides and are highly conserved among other selected This results in cross-reactivity against serotypes. [Brief description of the drawings]

[0025] [Figure 1]Pre-immunization (Pre), post-dose 1 (PD1), 2 (PD2) and 3 (PD3) IgG antibody dilution titers measured by ECL for mice immunized with unadjuvanted PCV22 (PCV22 unadj) or PCV22 formulated with aluminum phosphate adjuvant (PCV22 / APA). From left to right: Pre PCV22 unadj, Pre PCV22 / APA, PD1 PCV22 unadj, PD1 PCV22 / APA, PD2 PCV22 unadj, PD2 PCV22 / APA, PD3 PCV22 unadj and PD3 PCV22 / APA. [Diagram 2] ECL dilution titer ratio of PCV22 / APA compared to non-adjuvanted PCV22 (PCV22 unadj) at PD3. [Diagram 3] Serotype-specific OPA dilution titers (pre-immune, PD1, PD2, PD3) for mice immunized with unadjuvanted PCV22 (PCV22 unadj) or PCV22 formulated with APA (PCV22 / APA). From left to right: Pre PCV22 unadj, Pre PCV22 / APA, PD1 PCV22 unadj, PD1 PCV22 / APA, PD2 PCV22 unadj, PD2 PCV22 / APA, PD3 PCV22 unadj and PD3 PCV22 / APA. [Figure 4] OPA dilution titer ratio of PCV22 / APA compared with non-adjuvanted PCV22 (PCV22 unadj) at PD3. [Diagram 5] PCV22-immunized mice are protected against intratracheal challenge with Streptococcus pneumoniae 24F. [Figure 6] Pre-immunization (Pre), PD1 and PD2 IgG antibody dilution titers measured by ECL for rabbits immunized with non-adjuvanted PCV22 or PCV22 / APA. Error bars represent 95% confidence intervals (CI) of the geometric mean titers (GMT). From left to right: Pre PCV22 unadj, Pre PCV22 / APA, PD1 PCV22 unadj, PD1 PCV22 / APA, PD2 PCV22 unadj and PD2 PCV22 / APA. [Figure 7] ECL GMT ratios of PCV22 / APA compared to non-adjuvanted PCV22 at PD2. Error bars represent 95% confidence intervals (CI). [Figure 8] Serotype-specific OPA dilution titers (pre-immunization "Pre" and PD2) for rabbits immunized with non-adjuvanted PCV22 or PCV22 / APA. Error bars represent the variation in functional antibody titers for five rabbits. [Figure 9A] Pre-immune (Pre), PD1, PD2 and PD3 IgG antibody dilution titers measured by ECL for IRM immunized with non-adjuvanted PCV23. Error bars represent 95% confidence intervals (CI) of the geometric mean titers (GMT). [Figure 9B] Pre-immunization (Pre), PD1, PD2 and PD3 IgG antibody dilution titers measured by ECL for IRM immunized with PCV23(DMSO) / APA. Error bars represent 95% confidence intervals (CI) of the geometric mean titers (GMT). [Figure 9C] Pre-immunization (Pre), PD1, PD2 and PD3 IgG antibody dilution titers measured by ECL for IRM immunized with PCV23(DMSO+Aq) / APA. Error bars represent 95% confidence intervals (CI) of the geometric mean titers (GMT). [Figure 9D] Pre-immunization (Pre), PD1, PD2 and PD3 IgG antibody dilution titers measured by ECL for IRM immunized with PCV15 / APA+PCV8 / APA. Error bars represent 95% confidence intervals (CI) of the geometric mean titers (GMT). [Figure 10A] Comparison of ECL antibody responses in IRM (8-9 per group) following vaccination with PCV23 in the presence or absence of APA. Symbols indicate proportions at PD1. GMT ratios with error bars representing 95% CI. [Figure 10B] Comparison of ECL antibody responses in IRM (8-9 per group) following vaccination with PCV23 in the presence or absence of APA. Symbols indicate ratios at PD2. GMT ratios with error bars representing 95% CI. [Figure 10C] Comparison of ECL antibody responses in IRM (8-9 per group) following vaccination with PCV23 in the presence or absence of APA. Symbols indicate proportions at PD3. GMT ratios with error bars representing 95% CI. [Figure 11A] Comparison of ECL antibody responses in IRM (9 per group) following vaccination with PCV23(DMSO+Aq) / APA or co-administered with PCV15 / APA+PCV8 / APA. Symbols indicate ratios at PD1. GMT ratios with error bars representing 95% CI. [Figure 11B] Comparison of ECL antibody responses in IRM (9 per group) following vaccination with PCV23(DMSO+Aq) / APA or co-administered with PCV15 / APA+PCV8 / APA. Symbols indicate ratios at PD2. GMT ratios with error bars representing 95% CI. [Figure 11C] Comparison of ECL antibody responses in IRM (9 per group) following vaccination with PCV23(DMSO+Aq) / APA or co-administered with PCV15 / APA+PCV8 / APA. Symbols indicate ratios at PD3. GMT ratios with error bars representing 95% CI. [Figure 12A] Comparison of enhanced ECL antibody responses in IRM (8-9 animals per group) after vaccination with non-adjuvanted PCV23, PCV23(DMSO) / APA, PCV23(DMSO+Aq) / APA or PCV15 / APA+PCV8 / APA. PD1 / Pre. Symbols are GMT ratios and error bars represent 95% CI. [Figure 12B] Comparison of enhanced ECL antibody responses in IRM (8-9 animals per group) after vaccination with non-adjuvanted PCV23, PCV23(DMSO) / APA, PCV23(DMSO+Aq) / APA or PCV15 / APA+PCV8 / APA. PD2 / Pre. Symbols are GMT ratios and error bars represent 95% CI. [Figure 12C]Comparison of enhanced ECL antibody responses in IRM (8-9 animals per group) after vaccination with non-adjuvanted PCV23, PCV23(DMSO) / APA, PCV23(DMSO+Aq) / APA or PCV15 / APA+PCV8 / APA. PD3 / Pre. Symbols are GMT ratios and error bars represent 95% CI. [Figure 12D] Comparison of enhanced ECL antibody responses in IRM (8–9 animals per group) after vaccination with non-adjuvanted PCV23, PCV23(DMSO) / APA, PCV23(DMSO+Aq) / APA or PCV15 / APA+PCV8 / APA. PD2 / PD1. Symbols are GMT ratios and error bars represent 95% CI. [Figure 12E] Comparison of enhanced ECL antibody responses in IRM (8–9 animals per group) after vaccination with non-adjuvanted PCV23, PCV23(DMSO) / APA, PCV23(DMSO+Aq) / APA or PCV15 / APA+PCV8 / APA. PD3 / PD2. Symbols are GMT ratios and error bars represent 95% CI. [Figure 13A] Pre-immunization (Pre), post-dose 1 (PD1) and 2 (PD2) IgG antibody dilution titers measured by ECL for New Zealand White rabbits immunized with PCV24 formulated with aluminum phosphate adjuvant (PCV24 / APA). Error bars represent 95% confidence intervals (CI) of the geometric mean titers (GMT). [Figure 13B] Serotype-specific OPA dilution titers (pre-immune and PD2) for NZWR immunized with PCV24 / APA. Error bars represent the variation in functional antibody titers for eight NZWR. [Figure 14A] Pre-immunization (Pre), post-dose 1 (PD1), 2 (PD2) and 3 (PD3) IgG antibody dilution titers measured by ECL in infant rhesus macaques (IRM) immunized with PCV24 formulated with aluminum phosphate adjuvant (PCV24 / APA). Error bars represent 95% confidence intervals (CI) of the geometric mean titers (GMT). [Figure 14B]Serotype-specific OPA dilution titers (pre-immune and PD3) for IRM immunized with PCV24 / APA. Error bars represent the variation in functional antibody titers among five IRM.

[0026] Detailed Description of the Invention The present invention provides a multivalent immunogenic composition comprising a pneumococcal polysaccharide-protein conjugate. wherein each of the conjugates is a sequence conjugated to a carrier protein. Contains polysaccharides derived from S. pneumoniae serovars , Streptococcus pneumoniae serotypes are as defined herein. do.

[0027] In some embodiments, the present invention provides a method for the preparation of a Streptococcus pneumoniae polysaccharide protein. The present invention provides a multivalent immunogenic composition comprising a protein conjugate, wherein the conjugate Each of these antibodies expresses Streptococcus pneumoniae conjugated to a carrier protein. The polysaccharide-protein conjugate comprises a polysaccharide derived from a serotype selected from the group consisting of: Polysaccharides of selected Streptococcus pneumoniae serotypes include: a)1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 12F, 14, 15A, D eOAc15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and and 35B; b)1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 12F, 14, 15A, 1 5C, 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, 33 F 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 3 5B; 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, 24 F, 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 3 5B; 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, 15 C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; n)1, 3, 4, 5, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15 C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; o)1, 3, 4, 5, 6C, 7F, 8, 9V, 10A, 12F, 14, 15A, 15 C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; p)1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 11A, 12F, 14, 15 A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; q)1, 3, 4, 5, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15 A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; r)1, 3, 4, 5, 6C, 7F, 8, 9V, 10A, 11A, 12F, 14, 15 A, 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, 15 B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; w)1, 3, 4, 5, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15 B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; x)1, 3, 4, 5, 6C, 7F, 8, 9V, 10A, 12F, 14, 15A, 15 B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; y)1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 11A, 12F, 14, 15 A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; z)1, 3, 4, 5, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15 A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; aa)1, 3, 4, 5, 6C, 7F, 8, 9V, 10A, 11A, 12F, 14, 1 5A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and and 35B; bb)1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15A, DeOA c15B, 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, De OAc15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 3 5B and 39; ee)1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 12F, 14, 15A, 15 C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; ff)1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 12F, 14, 15B, 18 C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; gg)1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 11A, 12F, 14, 15 A, DeOAc15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 3 3F, 35B and 39; hh)1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 11A, 12F, 14, 15 A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35 B and 39; ii)1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 11A, 12F, 14, 15 A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35 B and 39; jj)1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 11A, 12F, 14, 15 B, 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, 1 8C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; oo)1, 3, 4, 5, 6B, 7F, 8, 9V, 12F, 14, 15A, 15C, 1 8C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; pp)1, 3, 4, 5, 6C, 7F, 8, 9V, 12F, 14, 15A, 15C, 1 8C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; qq)1, 3, 4, 5, 6A, 7F, 8, 9V, 11A, 12F, 14, 15A, 1 5C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; rr)1, 3, 4, 5, 6B, 7F, 8, 9V, 11A, 12F, 14, 15A, 1 5C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; ss)1, 3, 4, 5, 6C, 7F, 8, 9V, 11A, 12F, 14, 15A, 1 5C, 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, 1 8C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; xx)1, 3, 4, 5, 6B, 7F, 8, 9V, 12F, 14, 15A, 15B, 1 8C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; yy)1, 3, 4, 5, 6C, 7F, 8, 9V, 12F, 14, 15A, 15B, 1 8C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; zz)1, 3, 4, 5, 6A, 7F, 8, 9V, 11A, 12F, 14, 15A, 1 5B, 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 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 and 39.

[0028] In some embodiments, the multivalent immunogenic composition comprises: i) 1, 3, 4, 5, 6A, 6 B, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 2 2F, 23B, 23F, 24F, 33F and 35B; or ii) 1, 3, 4, 5, 6 A, 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, 15 C, 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, 3 Streptococcus pneumoniae serotype selected from the group consisting of 3F and 35B In certain embodiments, the multivalent immunogenic compositions of the invention comprise multiple pneumococcal strains. 1. A method for producing a method for the production of a medicament for the preparation ... Each of the peptides expresses Streptococcus nucleus conjugated to a carrier protein. It contains polysaccharides derived from Streptococcus pneumoniae serotypes. :i)1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 12F, 14, 15A, 15 C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; or ii) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 1 5A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and and 35B; or iii) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11 A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F The composition comprises 24F, 33F and 35B. The composition is administered to mice, rabbits and / or monkeys. and killing vaccine-type bacterial strains at all doses tested. It was found that the antibody produced specific targets.

[0029] The multivalent immunogenic composition of the present invention is directed to vaccine-type Streptococcus pneumoniae serovar and / or to immunize patients against pneumococcal pneumoniae and / or to treat them with different complementary pneumococcal vaccines. Thus, the present invention provides a multivalent immunogenic composition of the present invention. administering to the patient a multivalent pneumococcal vaccine; The present invention provides a method for inducing a protective immune response in a human patient, comprising administering to a subject, in any order, In some embodiments, the multivalent immunogenic compositions of the invention may be used in combination with other multivalent pneumococcal vaccines. Administered to immunized patients.

[0030] In an embodiment of the invention, a conjugate derived from at least one pneumococcal serotype is The ester is prepared using reductive amination in an aprotic solvent such as DMSO. In one embodiment, the multivalent immunogenic composition is prepared by reductive amination in an aprotic solvent. The use of DMSO solvent includes pneumococcal conjugates, each of which is produced using Covalent attachment of polysaccharides to proteins by direct consumption of lysine residues on the surface of the carrier protein The covalent binding enhancement was achieved by using polysaccharide antigens conjugated in DMSO. The direct benefit of this approach is the increased stability of the polysaccharide-protein conjugates in the multivalent immunogenic composition. vinegar.

[0031] I. Definitions and Abbreviations The following abbreviations apply throughout the specification and appended claims:

[0032] APA Aluminum phosphate adjuvant APC antigen presenting cells CI Confidence Interval DMSO Dimethyl sulfoxide DS Polysaccharide-Protein Drug GMC geometric mean concentration GMT Geometric Mean Titer HPSEC High Performance Size Exclusion Chromatography IM Intramuscular or intramuscular IRM infant rhesus monkey LOS Lipooligosaccharide LPS lipopolysaccharide MALS multi-angle light scattering MBC Monovalent Bulk Conjugate Mn number average molecular weight MOPA multiplex opsonization assay MW molecular weight NMWCO Nominal Molecular Weight Cutoff NZWR New Zealand White Rabbit OPA opsonization assay PCV Pneumococcal Conjugate Vaccine PD1 administration 1 PD2 post-administration 2 PD3 post-injection 3 PnPs Pneumococcus polysaccharide Ps polysaccharide PS-20 Polysorbate-20 RI Refractive Index UV ultraviolet light w / v weight / volume So that the present invention may be more readily understood, certain scientific and technical terms are specifically defined below. All other terms used herein are intended to be construed as meaning the same unless specifically defined elsewhere in this specification. All technical and scientific terms have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs.

[0033] As used throughout this specification and the appended claims, the singular forms "a," "an," and "the" are used unless the context clearly dictates otherwise. "includes its plural objects, unless otherwise contradicted."

[0034] The word "or" is used unless the context clearly dictates one of the stated possibilities. In some cases, one or both possibilities may be indicated. "And / or" is used for emphasis.

[0035] "Aqueous solvent" when used in connection with conjugation such as reductive amination The term "aqueous conditions" refers to the use of water as the solvent for the conjugation reaction. The water may contain buffers and other components, provided that no organic solvents are present.

[0036] "Aprotic Solvent" when used in conjunction with conjugation such as reductive amination The terms "DMSO solvent" or "DMSO conditions" refer to a solvent for a conjugation reaction. aprotic solvent or solvents (or solvents, if applicable) as a solvent Aprotic solvents refer to the use of a combination of DMSO (water, ethanol, etc.). Aprotic solvents include some water, e.g., up to 1%, It may contain 2%, 5%, 10% or 20% water.

[0037] The term "comprises" when used in reference to the immunogenic compositions of the present invention includes adjuvants and and the inclusion of any other ingredients such as excipients, or one or more other ingredients not specifically listed. Addition of a polysaccharide-protein conjugate is meant. The term "consisting of" when used in reference to a mixture means that the mixture is made up of those particular streptococci. Other strains containing the Cas pneumoniae polysaccharide-protein conjugate and derived from different serotypes Mixtures not containing Streptococcus pneumoniae polysaccharide-protein conjugates "Consists essentially of" and its derivatives, such as "consists essentially of" or " "Consisting essentially of" includes the inclusion of any element or elements listed, and Other elements of a similar or different nature to the element may be included in the specified administration regimen, method or The optional inclusion of elements that do not materially change the basic or novel characteristics of the compound or composition (i.e. , which may or may not be included).

[0038] An "effective amount" of a composition of the invention is an amount that is effective to inhibit the proliferation of microorganisms, such as Streptococcus mutans, during a subsequent challenge. Induce antibodies that significantly reduce the infectivity or severity of Caspian virus infection means the dose required for

[0039] As used herein, the phrase "adapted for the prevention of pneumococcal disease" refers to pneumococcal disease in general. , Pneumococcal pneumonia, Pneumococcal meningitis, Pneumococcal bacteremia, Streptococcus pneumoniae Invasive disease caused by Escherichia coli and Streptococcus pneumoniae Otitis media caused by Streptococcus pyogenes, including but not limited to For the prevention of one or more diseases caused by any serotype of M. monier, a vaccine or or the immunogenic composition is approved by the US Food and Drug Administration This means that the product is approved by one or more regulatory agencies, such as the National Institute of Integrative Medicine (NII).

[0040] The "multivalent pneumococcal vaccine" is a vaccine that protects against multiple serotypes of Streptococcus pneumoniae. Multiple active substances that provide active immunity against a disease or condition caused by pneumococcal capsular polysaccharide or pneumococcal polysaccharide-protein conjugate) .

[0041] The term "polysaccharide" includes the terms "saccharide," "oligosaccharide," "polysaccharide," "liposaccharide," "lipooligosaccharide ( "Lipopolysaccharides (LPS)", "glycosylates", "conjugate carbohydrates", etc. Commonly used in immunology and bacterial vaccine technology, including but not limited to It is intended to include any antigenic glycoelement (or antigenic unit).

[0042] "Unadjuvanted" in the context of the vaccine or immunogenic composition of the present invention The term "juvanted" refers to PCV8, PCV15, PCV22, PCV23 and Pneumococcal polysaccharide compositions, including, but not limited to, PCV24, By adjuvant-free compositions is meant compositions that do not contain adjuvants.

[0043] "PCV8" is a variant of Streptococcus pneumoniae polysaccharide (PnP) serotype-8, -1 Immune system containing 0A, -12F, -15A, -15C, -23B, -24F and -35B means an immunogenic composition.

[0044] "PCV15" is a variant of Streptococcus pneumoniae polysaccharide (PnP) serotype-1, 3, -4, -5, -6A, -6B, -7F, -9V, -14, -18C, -19A, -1 By this is meant an immunogenic composition containing 9F, -22F, -23F and -33F.

[0045] "PCV22" is a Streptococcus pneumoniae polysaccharide (PnP) serotype-1, - 3, -4, -5, -6A, -6B, -7F, -9V, -10A, -12F, -14, -1 5A, -15C, -18C, -19A, -19F, -22F, -23B, -23F, -2 By this reference is meant an immunogenic composition comprising 4F, -33F and -35B.

[0046] "PCV23" is a Streptococcus pneumoniae polysaccharide (PnP) 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.

[0047] "PCV24" is a Streptococcus pneumoniae polysaccharide (PnP) serotype-1, 3, -4, -5, -6A, -6B, -7F, -8, -9V, -10A, -11A, -12 F, -14, -15A, -15C, -18C, -19A, -19F, -22F, -23B , -23F, -24F, -33F and -35B.

[0048] "CpG-containing nucleotide", "CpG-containing oligonucleotide", "CpG oligonucleotide" "Nucleotide" and similar terms refer to a 6-50 nucleotide sequence that contains an unmethylated CpG moiety. For example, Wang et al., 2003, Vaccine 21:4297. CpG-containing oligonucleotides include any synthetic nucleotide. These include modified oligonucleotides with interosidic linkages, modified bases and / or modified sugars. do.

[0049] As defined herein, an "adjuvant" is an agent that enhances the immunogenicity of the immunogenic composition of the present invention. Immunoadjuvants are substances that help to strengthen the immune system by inhibiting immune responses to antigens that are weakly immunogenic when administered alone. (e.g., induces no or only a weak antibody titer or cellular immune response) enhances an immune response to an antigen, increases antibody titers against an antigen, and / or enhances immunity in an individual Adjuvants can reduce the dose of antigen that is effective in achieving an immune response. They are often administered to enhance an immune response and are well known to those skilled in the art.

[0050] A "patient" (also referred to herein as a "subject") is a person who is In a preferred embodiment, the term refers to a mammal that can be infected with Cas pneumoniae. The subject is a human. The subject may be treated prophylactically or therapeutically. Prophylactic treatment includes treatment of pneumococcal disease. A vaccine that is effective in preventing or reducing the likelihood or severity of a disease or its effects, e.g., pneumococcal pneumonia. Immunity to Streptococcus pneumoniae infection or its severity of clinical effects Therapeutic treatments may be performed to reduce the incidence or prevent recurrence of the disease. This can be done using the multivalent immunogenic compositions of the invention as described herein. The disclosed compositions may be administered to the general population or to individuals at high risk of pneumococcal infection, such as the elderly or It can be administered to those who live with or care for elderly people.

[0051] Those who "require treatment" include those with previous exposure to or infection with Streptococcus pneumoniae those who have previously been vaccinated against Streptococcus pneumoniae, and those who are susceptible or anyone for whom it is desirable to reduce the possibility of infection, such as immunocompromised individuals, the elderly, children, Adult or healthy individuals are included.

[0052] A "stable" multivalent immunogenic composition is one that is at least as stable 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 A "stable" composition is one in which no significant change is observed over a period of months. and 37°C for 1 month, 3 months, 6 months, 12 months and / or 24 Typical tolerances for stability include those that exhibit the desired characteristics over a period of time including months. The criteria are: approximately 5%, approximately 10%, approximately 15% or more of the following: or less than about 20% variation in: (a) Streptococcus pneumoniae polysaccharide in composition; (b) the number average molecular weight (Mn) of the protein conjugate; (c) Weight average molecular weight (Mw) of the Bacillus pneumoniae polysaccharide-protein conjugate (d) the specific excitation wavelength, e.g., 280 nanometers; (e) the emission maximum of the composition as measured using protein fluorescence spectroscopy; and Fluorescence intensity in the composition measured using intrinsic protein fluorescence spectroscopy at an excitation wavelength. The term "stable" is also used in reference to individual pneumococcal conjugates within a multivalent immunogenic composition. In such use, the term refers to the formation of desired properties over time at a particular temperature. "refers to a conjugate that exhibits a specific property over a specified time period and at a specified time interval." fluctuates by no more than about 5%, about 10%, about 15%, or about 20% in the temperature range in which it is measured .

[0053] II. Multivalent immunogenic compositions The present invention provides a method for the preparation of a Streptococcus pneumoniae polysaccharide-protein conjugate. and a multivalent immunogenic composition comprising: Containing polysaccharides from Streptococcus pneumoniae serovars conjugated to a saccharide Various aspects and embodiments of the multivalent immunogenic compositions of the invention are described below.

[0054] In one embodiment (embodiment E1), the present invention provides a method for the preparation of a medicament conjugated to a carrier protein. Each contains capsular polysaccharide derived from a modified Streptococcus pneumoniae serovar. Polyvalent Immunoglobulin Containing Multiple Streptococcus pneumoniae Polysaccharide-Protein Conjugates The present invention provides an active ingredient composition, wherein the serotype of Streptococcus pneumoniae is 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, 15 C, 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, or alternatively, consisting essentially of them. In a subembodiment of embodiment E1, the immunogenic composition comprises other Streptococcus nucleus. -Monnier polysaccharide-protein conjugate-free.

[0055] As used herein, de-O-acetylated serotype 15B (DeOAc15B) pneumococcal polysaccharide is essentially identical to the serotype 15C pneumococcal polysaccharide and has a virtually identical NMR spectrum. (data not shown). saccharides and serotype 15C pneumococcal polysaccharides ranged from 0 to 5% or 0% per repeating unit, respectively. ~4% range or 0~3% range or 0~2% range or 0~1% range or 0 having an O-acetyl content in the range of ~0.5% or in the range of 0-0.1% or 0%. According to a report by Spencer BL et al., pneumococcal polysaccharide 15C is slightly O-acetylated. (Spencer, BL et al., Clin. Vac. Immunol. No. (2017) 24(8):1-13). Therefore, the multivalent immunogen In any of the embodiments of the composition, deacetylated serotype 15C is substituted for serotype 15C. 15B (DeOAc15B) can be used. The de-O-acetylation method is described, for example, in Raj et al. am et al., Clinical and Vaccine Immunology, 2007 , 14(9):1223-1227, and are known in the art.

[0056] Any of the multivalent immunogenic compositions of the invention, including embodiment E1 and any subembodiments thereof. In certain embodiments of any of the above, the composition further comprises a pharma- ceutically acceptable carrier.

[0057] Cross-reactivity In one embodiment, the present invention provides a method for the preparation of a Streptococcus pneumoniae polysaccharide protein. The present invention provides a multivalent immunogenic composition comprising a protein conjugate, wherein the conjugate These are Streptococcus serovar 6C-containing antibodies conjugated to a carrier protein. It contains polysaccharides derived from serovar S. pneumoniae and inhibits the blood Serotype 6C cross-reacts with Streptococcus pneumoniae serotypes 6A and 6B Brings sex.

[0058] In one embodiment, the present invention provides a method for the preparation of a Streptococcus pneumoniae polysaccharide protein. The present invention provides a multivalent immunogenic composition comprising a protein conjugate, wherein the conjugate These are Streptococcus serovar 6A-containing antibodies conjugated to a carrier protein. It contains polysaccharides derived from serovar S. pneumoniae and inhibits the blood Serotype 6A is specific for Streptococcus pneumoniae serotypes 6B and / or 6C. Provides cross-protection.

[0059] In one embodiment, the present invention provides a method for the preparation of a Streptococcus pneumoniae polysaccharide protein. The present invention provides a multivalent immunogenic composition comprising a protein conjugate, wherein the conjugate These are Streptococcus serovar 6B-containing antibodies conjugated to a carrier protein. It contains polysaccharides derived from serovar S. pneumoniae and inhibits the blood Serotype 6B is specific for serotypes 6A and / or 6C of Streptococcus pneumoniae. Provides cross-protection.

[0060] In one embodiment, the present invention provides a method for the preparation of a Streptococcus pneumoniae polysaccharide protein. The present invention provides a multivalent immunogenic composition comprising a protein conjugate, wherein the conjugate These are streptococcal antibodies containing serotype 15C conjugated to a carrier protein. Contains polysaccharides derived from Streptococcus pneumoniae serovars and is a Serotype 15C also provides cross-protection against serotype 15B of Streptococcus pneumoniae Drop.

[0061] In one embodiment, the present invention provides a method for the preparation of a Streptococcus pneumoniae polysaccharide protein. The present invention provides a multivalent immunogenic composition comprising a protein conjugate, wherein the conjugate These are streptococcal antibodies, including serotype 15B, conjugated to a carrier protein. Contains polysaccharides derived from Streptococcus pneumoniae serovars and is a Serotype 15B also provides cross-protection against serotype 15C of Streptococcus pneumoniae Drop.

[0062] Carrier Proteins In a particular embodiment of the invention, CRM197 is used as the carrier protein. CRM197 is a non-toxic mutant of diphtheria toxin having the following amino acid sequence: , toxoid): [ka]

[0063] In one embodiment, CRM197 is grown in a casamino acid and yeast extract based medium. Corynebacterium diphtheriae grown in In another embodiment, the present invention is isolated from a culture of C. phtheriae strain C7(β197). In this case, CRM197 was prepared according to the method described in U.S. Pat. No. 5,614,382. Typically, CRM197 is purified by ultrafiltration, ammonium sulfate precipitation, and In some embodiments, the product is purified by a combination of precipitation and ion exchange chromatography. In this regard, CRM197 is a Pfenex Expression Technology y (trademark) (Pfenex Inc., San Diego, CA) was used to It is produced in Domonas fluorescens (Pseudomonas fluorescens).

[0064] Other suitable carrier proteins include additional inactivated (killed) bacterial toxins, such as DT ( Diphtheria toxoid) or DT fragment B (DTFB), TT (tetanus toxoid) or or TT fragment C, pertussis toxoid, cholera toxoid (e.g., WO 2004 / 0 83251), E. coli LT, E. coli i) ST, and Pseudomonas aeruginosa inosa. Bacterial outer membrane proteins, e.g., outer membrane protein complexes. OMPC, porin, transferrin-binding protein, pneumococcal surface protein 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, pneumococcal pneumolysin (Kuo et al., 1995, Infect Immunol. un 63;2706-13), e.g., ply that has been detoxified in some way, e.g., dPL Y-GMBS (see WO 04 / 081515) or dPLY-formo formol, PhtX, e.g. PhtA, PhtB, PhtD, PhtE and and Pht protein fusions, such as PhtDE fusions, PhtBE fusions (WO 01 Other tanks may also be used. Proteins such as ovalbumin, keyhole limpet hemocyanin (KLH), bovine blood Serum albumin (BSA) or purified protein derivative of tuberculin (PPD), B (derived from N. meningitidis), PD (Haemophilus influenza enzae protein D; see, for example, EP 0 594 610 B), and have disclosed their immunologically functional equivalents, synthetic peptides (EP0378881 and EP042 7347), heat shock proteins (see WO 93 / 17712 and WO 94 / 03208), pertussis proteins (see WO 98 / 58668 and (see EP0471177), cytokines, lymphokines, growth factors or phosphatases. Lumon (see WO 91 / 01146), multiple antigens from various pathogens Artificial proteins containing human CD4+ T cell epitopes (Falugi et al., 2001, E ur J Immunol 31:3816-3824), e.g., N19 Protein (Baraldoi et al., 2004, Infect Immun 72:488 4-7), iron uptake proteins (see WO 01 / 72337 ), Clostridium difficile toxin A or B ( See WO 00 / 61761), and flagellin (Ben-Yedidia (see, for example, Wang et al., 1998, Immunol Lett 64:9) also uses carrier proteins It can be used as.

[0065] Other DT mutants, such as CRM176, CRM228, CRM45 (Uchida et al., 1973, J Biol Chem 218:3838-3844); CRM9, C RM45, CRM102, CRM103 and CRM107, and Nicholls and Youle, Genetically Engineered Toxins, F Others are described in "Maecel Dekker Inc., 1992" edited by Rankel. Those with mutations; deletion of Glu-148 or replacing it with Asp, Gln or Se r mutation and / or Ala 158 to Gly mutation and US4 ,709,017 or other mutations disclosed in US 4,950,740. Lys 516, Lys 526, Phe 530 and / or Lys 5 34 and a mutation in at least one of the residues of US 5,917,017 or US or those having other mutations disclosed in U.S. Patent No. 6,455,6735; Fragments disclosed in US Pat. No. 5,843,711 may also be used as carrier proteins. Such a DT mutant is a DTFB mutant containing a B fragment containing the epitope region. It can also be used to prepare

[0066] In certain embodiments, the carrier protein is an outer membrane protein complex (OMPC), Tetanus toxoid, diphtheria toxoid, protein D and CRM 197.

[0067] In some embodiments of the invention, the polysaccharide protein conjugates in the multivalent immunogenic composition A second carrier may be used in one or more of the conjugates. The second carrier protein is preferably an inorganic carrier. A protein that is non-toxic, non-reactive, and available in sufficient quantity and purity. The protein was also conjugated to high Streptococcus pneumoniae multimers to enhance the immunogenicity of the antigen. The carrier protein is conjugated or bound to the sugar. In one embodiment, the first carrier protein is conjugated to Each unconjugated capsular polysaccharide 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 first carrier protein is conjugated to the Capsular polysaccharides that are not conjugated to two or more carrier proteins (each capsular polysaccharide molecule are conjugated to a single carrier protein). In such an embodiment, For this reason, each capsular polysaccharide of the same serotype is typically conjugated to the same carrier protein. It has been done.

[0068] In embodiments of the invention, including embodiment E1 and any subembodiments thereof, a polysaccharide One or more of the serotypes (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, if applicable) 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 2 5, 26, 27, 28, 29, 30 or more) were conjugated to CRM197. In other embodiments of the invention, including embodiment E1 and any subembodiments thereof, In this case, each of the polysaccharide serotypes is conjugated to CRM197.

[0069] The formulation of the polysaccharide-protein conjugates of the present invention can be carried out using methods recognized in the art. For example, the individual pneumococcal conjugates can be prepared by the methods described in Such formulations can be prepared using physiologically acceptable vehicles. Examples of vehicles include water, buffered saline, polyols (e.g., glycerol, propylene glycol, glycol, liquid polyethylene glycol) and dextrose solutions. However, the present invention is not limited to these.

[0070] In a preferred embodiment, the vaccine composition comprises L-histidine containing sodium chloride. It is formulated in cytosine buffer.

[0071] In some embodiments of the invention, the multivalent immunogenic composition comprises a carrier protein and Adjuvant-conjugated Streptococcus pneumoniae serovar Multiple Streptococcus pneumoniae polysaccharide-protein conjugates containing capsular polysaccharide wherein the Streptococcus pneumoniae serovar is described herein. Suitable adjuvants for enhancing the effectiveness of the composition include the following: Includes but is not limited to: (1) Aluminum salts (alum), such as aluminum hydroxide and aluminum phosphate um, aluminum sulfate, etc.; (2) Oil-in-water emulsion formulations [other specific immunostimulants, e.g. muramyl peptides (as defined below) or bacterial cell wall components, e.g. (a) Model 110Y Microfluidizer (Microfluidics, New York, 1999) Formulated into submicron particles using a microfluidizer such as the Sigma-Aldrich Co., Ltd. 5% squalene, 0.5% Tween 80 and 0.5% S Pan 85 (optionally containing various amounts of MTP-PE) (b) submicron Microfluidized into a polysaccharide emulsion or a larger particle size emulsion 10% squalene, 0.4% Twe, vortexed to produce a en 80, containing 5% Pluronic blocked polymer L121 and thr-MDP (c) Ribi™ Adjuvant System (RAS) (Corixa, Ha Milton, MT) containing: 2% squalene, 0.2 % Tween 80, and one or more bacterial cell wall components selected from the group consisting of: 3-O-deacylated monophospholipid A (M PL™), trehalose dimycolate (TDM) and cell wall skeleton (CWS), Preferably, MPL+CWS (Detox™), and (d) Montanide (Montanide™). anide)ISA; (3) Saponin adjuvants, such as Quil A or STIMULON (trademark) QS-21 (Antigenics, Framingham, Mass.) (e.g., See U.S. Pat. No. 5,057,540), or particles produced therefrom, e.g. For example, ISCOMs (combinations of cholesterol, saponin, phospholipids and amphipathic proteins) (immunostimulating complex formed from the protein (which have substantially the same structure as ISCOMs except that they do not contain any proteins) may be used; (4) Bacterial lipopolysaccharides, synthetic lipid A analogues, e.g. aminoalkylglucosamine phosphatase Antioxidant compounds (AGPs) or their derivatives or analogues (which may be purchased from Corixa) and is described in U.S. Pat. No. 6,113,918; one such The AGP is 2-[(R)-3-tetradecanoyloxytetradecanoylamino]ethyl 2-Deoxy-4-O-phosphono-3-O-[(R)-3-tetradecanoyloxyte tetradecanoyl]-2-[(R)-3-tetradecanoyloxytetradecanoylamino ]-β-D-glucopyranoside [which was previously known as 529 (RC529)] Also known as acetaminophen (CA), and formulated in aqueous form or as a stable emulsion; (5) Synthetic polynucleotides, such as oligonucleotides containing CpG motifs. (U.S. Patent No. 6,207,646); (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., gamma interferon), granulocyte-macrophage colony stimulators Macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), tumor necrosis factor tumor necrosis factor (TNF), costimulatory molecules B7-1 and B7-2, and (7) Complement, for example, the trimer of complement component C3d.

[0072] In another embodiment, the adjuvant is a mixture of two, three or more of said adjuvants. or a mixture of more than 100% of the total lipids, e.g., SBAS2 (3-deacylated monophosphoryl lipid A and and QS21).

[0073] Muramyl peptides include N-acetyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanine-2-(1',2'diaminodiphenylphosphine) Palmitoyl-sn-glycero)-3-hydroxyphosphoryloxy)-ethylamine ( MTP-PE), but are not limited to these.

[0074] In certain embodiments, the adjuvant is an aluminum salt. The adjuvant may be an alum-precipitated or alum-adsorbed vaccine. Nitrate 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 Nick las, W. (1992;Aluminum salts.Research in I The aluminum salts are described in , hydrated alumina, alumina hydrate, alumina trihydrate (ATH), aluminum hydrate, Aluminum Trihydrate, Alhydrogel, Superphos perfos, Amphogel, Aluminum(III) hydroxide, Aluminum hydroxyphosphate sulfate, aluminum phosphate adjuvant (APA), amo These include rufous alumina, alumina trihydrate, or aluminum trihydroxide. However, the present invention is not limited to these.

[0075] APA is an aqueous suspension of aluminum hydroxyphosphate. APA is a mixture of aluminum chloride and Aluminum hydroxyphosphate was prepared by mixing aluminum and sodium phosphate in a 1:1 volume ratio. After the mixing process, the material is sized in a high shear mixer. The product is then dialyzed against saline to obtain a monodisperse particle size distribution. In one embodiment, the aluminum salt is used. Amounts are 10, 15, 20, 25, 30, 50, 70, 100, 125, 150, 200, 3 00, 500 or 700 μg, or 1, 1.2, 1.5, 2, 3, 5 mg or In yet another embodiment, the dose of alum salt is equal to or greater than 100 mg / kg. Per μg of protein.

[0076] In a particular embodiment, 50-200 mg of protein per mg of aluminum hydroxide. To adsorb proteins at a ratio of μg, commercially available Al(OH)3 [e.g. ,Denmark / Accurate Chemical and Scientific Alhydrogel, c Co., Westbury, NY; In another embodiment, a tannin is used. Protein adsorption depends on the pI (isoelectric pH) of the protein and the pH of the medium. Proteins with a low pI are more strongly positively charged than proteins with a higher pI. The aluminum salt is slowly released over a period of 2-3 weeks. They form a depot of antigens that are absorbed by the blood and are involved in non-specific activation of macrophages and complement activation. and / or stimulate innate immune mechanisms (possibly through stimulation by uric acid) For example, Lambrecht et al., 2009, Curr Opin Immunol. See nol 21:23.

[0077] Monovalent bulk aqueous conjugates are typically mixed together and diluted with 6B (which is the target concentration Dilute to a target concentration of 4 μg / mL for all serotypes except for serotype 1 (which can be diluted to 8 μg / mL). Once diluted, the batch is filter sterilized and an equal volume of aluminum phosphate adjuvant is added without Add aluminium adjuvant to target a final aluminium concentration of 250 μg / mL. The quantified and formulated batches are filled into single-use 0.5 mL / dose vials.

[0078] In certain embodiments, the adjuvant is a CpG-containing nucleotide sequence, e.g., Cp G-containing oligonucleotides, particularly CpG-containing oligodeoxynucleotides (CpG OD In another embodiment, the adjuvant is ODN 1826, It is available from Coley Pharmaceutical Company.

[0079] Methods for using CpG oligonucleotides are well known in the art, see, for example, Sur et al. 1999,J Immunol.162:6284-93;Verthelyi,200 6, Methods Mol Med. 127:139-58; and Yasuda et al. 2006,Crit Rev Ther Drug Carrier Syst.23: It is described in 89-110.

[0080] In an alternative embodiment, the immunogenic composition is as described herein, e.g., embodiment E1 or Any of the subembodiments thereof may be used to identify any of the Streptococcus pneumoniae strains. The present invention relates to a method for producing a medicament for the treatment of cancer, the method comprising:

[0081] formulation The multivalent immunogenic compositions of the invention may be packaged in single-dose vials, multi-dose vials or prefilled vials. It may be formulated as a filled glass or plastic syringe.

[0082] In another embodiment, the multivalent immunogenic composition of the invention is administered orally, but Therefore, the pharmaceutical composition is formulated in a form suitable for oral administration, i.e., as a solid or liquid formulation. Oral formulations include tablets, capsules, pills, granules, pellets, etc. Oral formulations include solutions, suspensions, dispersions, emulsions, oils, and the like.

[0083] Pharmaceutically acceptable carriers for liquid formulations may be aqueous or non-aqueous solutions, suspensions, emulsions or is an oil. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, etc. and injectable organic esters, such as ethyl oleate. Aqueous carriers include , water, alcoholic / aqueous solutions, emulsions or suspensions (including saline and buffered media) Examples of oils include those of animal, vegetable or synthetic origin, e.g. Peanut oil, soybean oil, olive oil, sunflower oil, fish liver oil, other fish oils, or milk. and lipids derived from eggs.

[0084] The multivalent immunogenic compositions of the present invention may be isotonic, hypotonic or hypertonic. Alternatively, it is often preferred that a composition for injection be substantially isotonic at the time of administration. Thus, for storage, the composition may preferably be isotonic or hypertonic. If it is hypertonic for storage, it can be diluted to an isotonic solution prior to administration.

[0085] The tonicity agent may be an ionic tonicity agent, such as a salt, or a non-ionic tonicity agent, such as a carbohydrate. Examples of ionic tonicity agents include NaCl, CaCl2, KCl, and MgCl 2. Examples of non-ionic tonicity agents include, but are not limited to, mannitol, ethyl cellulose, ethyl cellulose acetate, ethyl cellulose esters, ethyl cellulose esters, ethyl cellulose acetate ... These include, but are not limited to, ethanol, sorbitol and glycerol. do not have.

[0086] It is also preferred that at least one pharma- ceutically acceptable excipient is a buffer. For such purposes, for example, when the pharmaceutical composition is intended for infusion or injection, The composition is dissolved in a solution at a pH in the range of 4 to 10, for example at a pH of 5 to 9, for example at a pH of 6 to 8. It is often desirable to include a buffer capable of buffering the

[0087] Buffers include, for example, TRIS, acetate, glutamate, lactate, malate, etc. tartrate, phosphate, citrate, carbonate, glycinate, histidine selected from the group consisting of glycine, succinate and triethanolamine buffers It can be done.

[0088] The buffer should be a USP compatible buffer for parenteral use, especially if the pharmaceutical formulation is for parenteral use. For example, the buffer may be selected from monobasic acids, such as acetic acid, benzoic acid, glucose, dibasic acids, such as aconitic acid, adipic acid, ascorbic acid, acetic acid, carbonic acid, glutamic acid, malic acid, succinic acid and tartaric acid; polybasic acids, such as kueh and bases, such as ammonia, diethanolamine, glycine, It may be selected from the group consisting of triethanolamine and TRIS.

[0089] Parenteral vehicles (for subcutaneous, intravenous, intraarterial, or intramuscular injection) include sodium chloride solution. solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution and fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, Examples include those based on Ringer's dextrose. With or without the addition of an active agent and other pharma- ceutically acceptable adjuvants, Examples include sterile liquids such as water and oils. Generally, water, saline, aqueous dextrose, etc. sugars and related sugar solutions, glycols, e.g. propylene glycol or polyethylene glycol Cole, Polysorbate 80 (PS-80), Polysorbate 20 (PS-20) and Poloxamer 188 (P188) is a preferred liquid carrier, especially for injectable solutions. Examples of oils include those of animal, vegetable or synthetic origin, e.g., peanut oil, soybean oil. These include olive oil, sunflower oil, fish liver oil, other fish oils, or lipids derived from milk or eggs. Can be obtained.

[0090] The formulations of the invention may include a surfactant. Preferred surfactants include but not limited to: poloxamer-188 (P188; Pluronic ;F68 NF), polyoxyethylene sorbitan ester surfactants (generally Twee n (Tween)), especially PS-20 and PS-80; DOWFAX (trademark Ethylene oxide (EO), propylene oxide (PO) and and / or copolymers of butylene oxide (BO), e.g. linear EO / PO blocks Copolymer; Octoxynol [which is a repeating ethoxy(oxy-1,2-ethanediyl ) groups, but are generally or t-octylphenoxypolyethoxyethanol) are of particular interest; Ctylphenoxy)polyethoxyethanol (IGEPAL CA-630 / NP-40 ); phospholipids, such as phosphatidylcholine (lecithin); nonylphenol ethoxylate glyceryl, lauryl, cetyl, stearyl, etc. Polyoxyethylene fatty ethers derived from oleyl alcohol (Brij interface) activators), such as triethylene glycol monolauryl ether (Br ij 30); and sorbitan esters (commonly known as SPAN), e.g. For example, sorbitan trioleate (Span 85) and sorbitan monolaurate. A preferred surfactant for incorporation into the formulation is PS-80.

[0091] Mixtures of surfactants may be used, for example PS-80 / Span 85 mixtures. Diethylene sorbitan esters, such as polyoxyethylene sorbitan monooleate ( PS-80) and octoxynol, e.g. t-octylphenoxypolyethoxyethano A combination with Triton X-100 is also suitable. In addition to RES 9, polyoxyethylene sorbitan ester and / or octoxynol Includes

[0092] The preferred amount (wt %) of the surfactant is as follows: 0.01 to 1%, particularly about 0. 0.001 to 1% polyoxyethylene sorbitan ester (e.g., PS-80); 0.1%, especially 0.005-0.02%, of octyl or nonylphenoxy polyoxyethylene Triton X-100 or other compounds in the Triton series Surfactant); 0.1 to 20%, preferably 0.1 to 10%, particularly 0.1 to 1% or about 0.5% polyoxyethylene ether (e.g., Laureth 9).

[0093] In certain embodiments, the composition contains 250 μg / mL APA (aluminum phosphate). adjuvant), histidine (20 mM), saline (150 mM) and It essentially consists of 0.2% PS-20 (pH 5.8). PS-20 is 0.005% to 0 0.3% (w / v). In another embodiment, PS-20 can range from 0. In another embodiment, the PS -20 may range from 0.05% to 0.8% (w / v). For example, PS-20 may range from 0.05% to 0.2% (w / v). A mixture (blend) of up to 24 serotypes is administered in 100 mL of ... The mixture is then mixed with APA and ethanol in the presence or absence of an antimicrobial preservative. It consists of combining with saline.

[0094] In certain embodiments, the multivalent immunogenic composition comprises a Streptococcus pneumoniae Polysaccharide-protein conjugate, and further, 20-80 mM histidine (pH 5.8 ) and 150 mM NaCl, where each of the conjugates is a carrier tank. Polysaccharides derived from Streptococcus pneumoniae serovars conjugated to proteins Contains polysaccharide-protein conjugates of Streptococcus pneumoniae in serum The types include any of the serotype combinations described herein. In some embodiments, In this case, the multivalent immunogenic composition further contains 0.2% to 0.8% w / v polysorbate 20. Included.

[0095] The polyvalent immunogenic composition PCV24 is formulated in an aprotic solvent (also known as DMSO chemistry). Streptococcus pneumoniae polysaccharide (PnP) serotype-I was isolated using reductive amination with 1, -3, -4, -5, -6A, -6B, -7F, -8, -9V, -10A, -11A, -12F, -14, -15A, -15C, -18C, -19A, -19F, -22F, - CRM197 protein was individually added to -23B, -23F, -24F, -33F and -35B. and 20 mM L-histidine (pH 5.8) , 150 mM NaCl and 0.1% w / v polysorbate-20 (PS-20) In each case, 4 μg / mL or 8 μg / mL of each polysaccharide serotype (total polysaccharide concentration was 96 μg / m It is formulated at 100 μg / mL or 192 μg / mL and is called "PCV24 unadjuvanted." In another specific embodiment, the multivalent immunogenic composition PCV24 comprises 20 mM L- Histidine (pH 5.8), 150 mM NaCl and 0.2% w / v polysorbate In PS-20, further aluminum phosphate adjuvant [Al]2 50 μg / mL, and 4 μg / mL of each polysaccharide serotype (total polysaccharide concentration is 96 μg / mL This is called "PCV24 / APA".

[0096] The choice of surfactant may need to be optimized for different pharmaceuticals and drugs. For multivalent vaccines containing 15 or more serotypes, PS-20 and P188 are preferred. The choice of chemical method used to prepare the conjugate also plays a role in the stabilization of the formulation. In particular, the various polysaccharide-protein conjugates in a multivalent composition can be The conjugation reaction used to prepare the compound includes both an aqueous solvent and a DMSO solvent. When used in combination, individual surfactant systems result in significant differences in stability. The improvement in stability of mercaptoglyceride was observed when polysorbate 20 alone or poloxamer 188 and polysorbate 20 were used together. It was recognized in combination with an oar.

[0097] The exact mechanism by which a particular surfactant protects a biotherapeutic is unknown. Possible stabilization mechanisms are not fully understood and cannot be predicted in advance. are due to preferential hydration, preferential exclusion, air / liquid interfacial competition between the biotherapeutic material and the surface, surface tension, and / or boundaries to mask hydrophobic patches that act as seeds for aggregation. Direct conjugation of the surfactant with the biotherapeutic agent is included.

[0098] Poloxamers may also be used in the compositions of the present invention. Poloxamers are polyoxyethylene Polyoxypropylene flanked by two hydrophilic chains of ethylene (poly(ethylene oxide)) A non-ionic triblock copolymer consisting of a central hydrophobic chain of poly(propylene oxide) Poloxamer is also known under the trade name Pluronic®. The length of the polymer block can be customized to achieve slightly different properties. There are many different poloxamers with different molecular weights. These copolymers are generally named after the letter "P" (for poloxamer) followed by three digits. where the first two digits x 100 is the polyoxypropylene core. The last digit x 10 indicates the percentage of polyoxyethylene content. (For example, P407 is a polyoxypropylene with a molecular weight of 4,000 g / mol and It is a poloxamer with 70% polyoxyethylene content. In trade names, the symbols for these copolymers are the letters ( L = liquid, P = paste, F = flake (solid)) followed by two or three digits. The first digit in the numerical display (two digits in the case of three-digit numbers) x 300 is the number of the hydrophobic substance. indicates the approximate molecular weight of the polymer, and the last digit x 10 indicates the percentage of polyoxyethylene content. (For example, L61 is a polyoxypropylene with a molecular weight of 1,800 g / mol and 10% (Pluronic® having a polyoxyethylene content of 1.0 to 1.0 g / g). See US Pat. No. 3,740,421.

[0099] Specific examples of poloxamers include those with the general formula: HO(CHO) a (C3H6O) b (C2H4O ) a H, where the a and b blocks have the following values: [Table 1]

[0100] Preferably, the poloxamer is generally between 1,100 and 17,400 Da, between 7,500 and It has a molecular weight of 15,000 Da or in the range of 7,500 to 10,000 Da. The poloxamer may be selected from poloxamer 188 or poloxamer 407. The final concentration of xamers ranged from 0.001% to 5% (weight / volume) or 0.025% to 1% (weight / volume). In a particular embodiment, the polyol is propylene glycol; The final concentration is 1% to 20% (weight / volume). The primary concentration is triethylene glycol 400, with a final concentration of 1%-20% (weight / volume).

[0101] Suitable polyols for the formulations of the present invention are polymeric polyols, especially polyether diols. propylene glycol and polyethylene glycol, polyethylene glycol Examples include, but are not limited to, propylene glycol methyl ether. Polyethylene glycol is available in a range of monomer molecular weights from about 425 to about 2,700. Polyethylene glycol and polyethylene glycol monomethyl ether are from about 200 to about 35,0 00 molecular weight range, such as PEG200, PEG300, PEG400, EG1000, PEG MME 550, PEG MME 600, PEG MME 2 000, PEG MME 3350 and PEG MME4000 (including but not limited to The preferred polyethylene glycol is polyethylene glycol glycerol. The final concentration of polyol in the formulation of the present invention is 1% to 20% (by weight). / volume) or 6% to 20% (weight / volume).

[0102] The formulation also contains a pH buffered saline solution. The buffer may be, for example, TRIS, acetate, Glutamate, lactate, malate, tartrate, phosphate, citrate, potassium carbonate, glycinate, histidine, glycine, succinate, HEPES(4-( 2-hydroxyethyl)-1-piperazineethanesulfonic acid), MOPS (3-(N-Mo 2-(N-morpholino)propanesulfonic acid), MES (2-(N-morpholino)ethanesulfonic acid) and triethanolamine buffer. The buffer may be selected from the group consisting of 4 to 100% ethanolamine buffer. The solution may be buffered to a pH in the range of 10, 5.2 to 7.5, or 5.8 to 7.0. In a particular embodiment, the buffer is selected from the group consisting of phosphate, succinate, histidine, M ES, MOPS, HEPES, acetate or citrate. The buffer may further comprise, for example, a buffer solution according to USP for parenteral use, particularly if the pharmaceutical formulation is for parenteral use. In one embodiment, the concentration of the buffer is 1 In another embodiment, the concentration of the buffer ranges from 1 mM to 100 mM. In another embodiment, the concentration of the buffer ranges from 10 mM to 80 mM. In a particular embodiment, the buffer is in the range of 5 mM to 50 mM or 5 mM to 50 mM. - histidine at a final concentration of 5mM to 50mM or sucrose at a final concentration of 1mM to 10mM In a particular embodiment, the histidine buffer is at a maximum concentration of 20 mM ± 2 mM. This is the final concentration.

[0103] Although saline solutions (e.g., solutions containing NaCl) are preferred, other salts suitable for formulation include , CaCl2, KCl, and MgCl2 and combinations thereof, including but not limited to Instead of salt, it contains sucrose, trehalose, mannitol, and soybean oil. Non-ionic, including but not limited to, sorbitol and glycerol A tonicity agent may be used. Suitable salt concentration ranges include 20 mM to 500 mM or 40 mM to In one embodiment, the dietary fiber concentration includes, but is not limited to, 170 mM. The saline is NaCl, which may be present at a concentration of 25 mM to 170 mM, as desired.

[0104] In a preferred embodiment, the formulation is an L-histidine buffer containing sodium chloride. Includes fur.

[0105] In another embodiment, the pharmaceutical composition is administered in a controlled release system. For example, the agent may be delivered by intravenous infusion, a transdermal patch, liposomes, or other administration systems. In another embodiment, the compound can be administered using any suitable form of administration, such as, for example, microspheres or In the implant or implant, polymeric materials are used.

[0106] The amount of conjugate in each dose of the composition may be adjusted to provide immunoprotection without significant adverse effects. The amount of the antibody that induces a mitogenic response is selected as the amount required to induce a mitogenic response. Such an amount may vary depending on the pneumococcal serotype. Generally, for polysaccharide-based conjugates, each dose is 0.08-100 μg of each polysaccharide. In some embodiments of the invention, the dosage of each polysaccharide conjugate is 0.0 In other embodiments, the dose of each conjugate is 1-5 μg, 0.4-4μg, 0.4-3μg, 0.4-2μg or 0.4-1μg. In this embodiment, the dose of one or more polysaccharide conjugates is 100, 150, 200, 250, 300, 400, 500 or 750ng, or 0.4, 0.5, 0.6, 0.7, 0.75, 0.8, 0.9, 1, 1.5, 2, 3, 4, 5, 6, 7, 7.5, 8 , 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 22, 25, 30, 4 0, 50, 60, 70, 80, 90 or 100 μg.

[0107] In some embodiments of the compositions of the present invention, all of the polysaccharide conjugates are present in the same amount. In other embodiments, the polysaccharide conjugates are present in the composition in different amounts. (i.e., at least one polysaccharide conjugate is present in the composition) (wherein one or more of the polysaccharide conjugates are present in an amount different from that of the other polysaccharide conjugates).

[0108] The optimal amounts of the individual immunogenic composition components are determined based on a set of criteria, including the observation of an appropriate immune response in a subject. For example, in another embodiment, the human vaccine The dosage for inoculation is determined by extrapolation from animal studies to human data. In this case, the dosage is empirically determined.

[0109] The compositions of the invention include one or more proteins derived from Streptococcus pneumoniae. Examples of Streptococcus pneumoniae proteins suitable for incorporation include those disclosed in International Patent Publication No. The compounds identified in application publication numbers WO 02 / 083855 and WO 02 / 053761 are This includes those that are

[0110] In certain embodiments, the compositions of the present invention can be prepared by one or more methods known to those of skill in the art. For example, parenterally, transmucosally, transdermally, intramuscularly, intravenously, intradermally, intranasally, subcutaneously, intraperitoneally, In one embodiment, the composition of the present invention is administered to a subject and formulated accordingly. The liquid preparation is injected superficially, intramuscularly, intravenously, intraarterially, subcutaneously, or into the respiratory mucosa. The liquid preparation for injection includes solutions and the like.

[0111] III. Manufacturing method Capsular polysaccharides from Streptococcus pneumoniae can be isolated using standard techniques known to those of skill in the art. For example, polysaccharides can be isolated from bacteria and prepared by known methods (e.g., see European Patent Nos. EP497524 and EP497525), preferably By microfluidization achieved using a homogenizer or by chemical In one embodiment, each strain may be sized by selective hydrolysis. Putococcus pneumoniae polysaccharide serovars are grown in soy-based media. The individual polysaccharides are purified by standard procedures including centrifugation, precipitation and ultrafiltration. For example, No. 2008 / 0286838 and U.S. Pat. No. 5,847,112 To reduce the viscosity of the polysaccharide sample, and / or to conjugate Using techniques such as mechanical or chemical sizing to improve the filterability of the product It is possible to size the polysaccharides by chemical hydrolysis using acetic acid. Mechanical sizing can be performed using a high pressure homogenizing shear.

[0112] The purified polysaccharide can be chemically activated to obtain a polysaccharide capable of reacting with a carrier protein. It is possible. The purified polysaccharides can be linked to a linker. Each capsular polysaccharide can be activated or Once attached to the linker, they are individually conjugated to proteins to form glycoconjugates. Polysaccharide conjugates are produced by known coupling techniques. It is possible.

[0113] The polysaccharide is coupled to a linker, the free end of which is an ester group. Thus, the linker can form a polysaccharide-linker intermediate having at least one terminal The other end is the linker that reacts with the polysaccharide to form the polysaccharide-linker. - intermediates are selected so as to be capable of forming

[0114] The polysaccharide can be coupled to the linker using primary amine groups on the polysaccharide. In the case of , the linker typically has ester groups at both ends. The coupling was achieved by reacting one of the groups with the primary amine groups of the polysaccharide by nucleophilic acyl substitution. The reaction allows the polysaccharide to be coupled to the linker via an amide bond. Thus, the linker is a polysaccharide first linker. A first ester group for reacting with a primary amine group of a carrier molecule A typical linker is an adipic acid linker that provides a second ester group for the The compound is carboxylic acid N-hydroxysuccinimide diester (SIDEA).

[0115] Coupling can also be done indirectly, i.e., coupling to a linker. This can be accomplished by using an additional linker that is used to derivatize the polysaccharide prior to linking.

[0116] The polysaccharide can be coupled to an additional linker using the carbonyl group at the reducing end of the polysaccharide. This coupling is carried out in two steps: (a1) the cleavage of a carbonyl group; with an additional linker, and (a2) reacting the free end of the additional linker with the linker. In these embodiments, the additional linker is typically The saccharide has primary amine groups at both ends, allowing one of the primary amine groups to be attached to the carboxyl group of the polysaccharide. It is possible to carry out step (a1) by reacting with a carboxyl group by reductive amination. The primary amine groups are used because they are reactive with the carbonyl groups of the polysaccharide. The same primary amine group is typically also present as an additional linker. The reaction is carried out by attaching the polysaccharide to additional linkers via C-N bonds. A coupled polysaccharide-additional linker intermediate is produced.

[0117] The polysaccharide can be linked to additional linkers using different groups on the polysaccharide, particularly the carboxyl groups. This coupling can be carried out in two steps: (a1) coupling the group with an additional linker, and (a2) reacting the free end of the additional linker with the linker. In this case, the additional linker typically has a second linker at both ends. It has a primary amine group, which allows one of the primary amine groups to couple with the carboxyl group of the polysaccharide. It is possible to carry out step (a1) by reacting with AC activation. A primary amine group is used that is reactive with the DAC-activated carboxyl group. The same primary amine group is typically present at both ends of the additional linker. The reaction produces polysaccharide- An additional linker intermediate is generated.

[0118] In one embodiment, chemical activation of the polysaccharide followed by reductive amination Conjugation to a carrier protein by the method described in U.S. Pat. No. 4,365,170, US Pat. ,673,574 and 4,902,506, U.S. Patent Application Publication Nos. 2006 / 0 228380, 2007 / 184072, 2007 / 0231340 and 2007 / 0 184071, as well as WO2006 / 110381, WO2008 / 079653 and and WO2008 / 143709. The chemical method comprises: Any oxidizing agent that oxidizes terminal hydroxyl groups to aldehydes, such as periodate Iodates) (including sodium periodate, potassium periodate or periodic acid) and The reaction may include activation of pneumococcal polysaccharides by reaction with Together they result in random oxidative cleavage of vicinal hydroxyl groups of the carbohydrate.

[0119] Coupling to carrier proteins is by direct amination of the lysyl groups of the protein. For example, conjugation can be achieved by the reaction of an activated polysaccharide with a carrier protein. By reacting the mixture with a reducing agent such as sodium cyanoborohydride, The conjugation reaction can take place in aqueous solution or in the presence of DMSO. For example, US2015 / 0231270, US2011 / 0195086 and EP 04 See 71 177 B1. The unreacted aldehyde is then quenched with sodium borohydride. The compound is capped by the addition of a strong reducing agent such as

[0120] Reductive amination involves two steps: (1) oxidation of polysaccharides to reactive aldehydes; (2) forming an imine ( The method includes reducing the Schiff base to form a stable amine conjugate bond. Prior to this, the polysaccharide may, if desired, be size-reduced by mechanical methods (e.g., homogenization) or Chemical hydrolysis can be used. Chemical hydrolysis can be carried out using acetic acid. The process may involve reaction with a periodate. For purposes of this invention, "periodate" refers to The term includes both periodate and periodic acid. Metaperiodite (IO4 - ) and orthoperiodite (IO6 -) and various salts of periodate (e.g., sodium periodate and potassium periodate). In one embodiment, the capsular polysaccharide comprises Preferably, the oxidation is carried out in the presence of sodium periodate (NaIO4). In an embodiment, the capsular polysaccharide is oxidized in the presence of orthoperiodate, preferably periodate. It is oxidized in the presence of acid.

[0121] In one embodiment, the oxidizing agent is an oxidizing agent that selectively oxidizes primary hydroxyls. In the presence of a stable nitroxyl or nitroxide radical compound, such as piperidinium pyrrolidine-N-oxy or pyrrolidine-N-oxy compounds (see WO 2014 / 097099) In the reaction, the actual oxidant is the N In one embodiment, the stable nitroxyl or The nitroxide radical compound is piperidine-N-oxy or pyrrolidine-N-oxy. In one embodiment, the stable nitroxy or nitroxide compound is The dicarboxylic acid compound is TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxy). or the PROXYL (2,2,5,5-tetramethyl-1-pyrrolidinyloxy) moiety In one embodiment, the stable nitroxyl radical compound is TEMP O or a derivative thereof. In one embodiment, the oxidizing agent contains an N-halo moiety. In one embodiment, the oxidizing agent is N-chlorosuccinimide, N -Bromosuccinimide, N-iodosuccinimide, dichloroisocyanuric acid, 1,3 ,5-Trichloro-1,3,5-triazinane-2,4,6-trione, Dibromoisocyanate Anuric 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 oxidizing agent is selected from the group consisting of N-chloro It is a succinimide.

[0122] In certain embodiments, the oxidizing agent is 2,2,6,6-tetramethyl-1-piperidinyl oxidizing agent. TEMPO free radical and N-chlorosuccinimide as a co-oxidant NCS) (described in WO 2014 / 097099). In one embodiment, the glycoconjugates derived from Streptococcus pneumoniae are capable of: a) converting sugars into aqueous solutions; 2,2,6,6-Tetramethyl-1-piperidinyloxy (TEMPO) and N-chlorosuccinimide (NCS) to obtain an activated sugar, and b) reacting the activated sugar with one or more The method includes reacting the amine group-containing carrier protein with the amine group-containing carrier protein (hereinafter, the method is referred to as "TE These are available via MPO / NCS-reductive amination.

[0123] Optionally, the oxidation reaction is quenched by the addition of a quenching agent. Vicinal diols, 1,2-amino alcohols, amino acids, glutathione, sulfites, bis Sulfate, dithionite, metabisulfite, thiosulfate, phosphite, hypophos Phyto- or phosphorous acid (e.g., glycerol, ethylene glycol, propane-1,2 -diol, butane-1,2-diol or butane-2,3-diol, ascorbic acid The acid may be selected from the group consisting of aryl, ... and aryl acids.

[0124] In certain embodiments, the present invention relates to conjugation reactions in aprotic solvents. Utilizing serotype 8 Streptococcus pneumoniae polysaccharide-protein conjugates The present invention provides a method for the preparation of a compound, wherein the conjugation reaction does not involve the use of cyanoborohydrides. In other embodiments, the conjugation reaction involves Schiff base reduction or reductive amine reduction. In other embodiments, the protein is a tetanus toxoid, a diphtheria toxoid, or a combination thereof. In yet another embodiment, the protein is CRM1. 97. In other embodiments, the conjugation reaction is a reductive amination. In other embodiments, the reductive amination is carried out in dimethylsulfoxide (DMSO). can be.

[0125] In some embodiments, the oxidized polysaccharide prior to conjugation is between 30 kDa and 1,000 kDa. The molecular weight was determined by multi-angle light scattering (MALS) and refractive index detection. It can be calculated by size exclusion chromatography (SEC) combined with a radioisotope detector (RI). In some embodiments, the polysaccharide has a molecular weight of 50 kDa to 300 kDa. In some embodiments, the polysaccharide has a molecular weight of 50 kDa to 1,000 kDa. In additional embodiments, the polysaccharide has a molecular weight of 70 kDa to 900 kDa. In an embodiment, the polysaccharide has a molecular weight of between 100 kDa and 800 kDa. In one embodiment, the polysaccharide has a molecular weight of 200 kDa to 600 kDa. For example, polysaccharides are 100kDa to 1,000kDa, 100kDa to 900kDa, and 100kDa to 1,000kDa. Da~800kDa, 100kDa~700kDa, 100kDa~600kDa, 10 0kDa~500kDa, 100kDa~400kDa, 100kDa~300kDa, 150kDa~1,000kDa, 150kDa~900kDa, 150kDa~800 kDa, 150kDa~700kDa, 150kDa~600kDa, 150kDa~5 00kDa, 150kDa~400kDa, 150kDa~300kDa, 200kDa ~1,000kDa, 200kDa~900kDa, 200kDa~800kDa, 20 0kDa~700kDa, 200kDa~600kDa, 200kDa~500kDa, 200kDa~400kDa, 200kDa~300, 250kDa~1,000kDa , 250kDa~900kDa, 250kDa~800kDa, 250kDa~700k Da, 250kDa~600kDa, 250kDa~500kDa, 250kDa~40 0kDa, 250kDa~350kDa, 300kDa~1,000kDa, 300kDa a~900kDa, 300kDa~800kDa, 300kDa~700kDa, 300 kDa~600kDa, 300kDa~500kDa, 300kDa~400kDa, 4 00kDa~1,000kDa, 400kDa~900kDa, 400kDa~800k Da, 400kDa-700kDa, 400kDa-600kDa, or 500kDa It has a molecular weight of ~600 kDa.

[0126] The second step of the conjugation process involves the use of a reducing agent to react the activated polysaccharide with the carrier protein. to form a stable conjugate bond by reducing the imine (Schiff base) bond between (so-called reductive amination). Suitable reducing agents include cyanoborohydrides (e.g. For example, sodium cyanoborohydride or sodium borohydride. 1 In one embodiment, the reducing agent is sodium cyanoborohydride.

[0127] In certain embodiments, the reductive amination reaction is carried out in an aprotic solvent (or In one embodiment, the reduction reaction is carried out in DMSO or The procedure is carried out in DMF (dimethylformamide) solvent. Activated polysaccharides and carrier proteins If the products have been lyophilized, DMSO or DMF solvent is required to reconstitute them. In one embodiment, the aprotic solvent is DMSO.

[0128] At the end of the reduction reaction, there may be unreacted aldehyde groups remaining in the conjugate. which can be capped using a suitable capping agent. In this case, the capping agent is sodium borohydride (NaBH4). The compounds include sodium or zinc borohydride in the presence of Bronsted or Lewis acids. or sodium triacetoxyborohydride, amine boranes, such as pyridine borane, 2-Picoline borane, 2,6-diborane-methanol, dimethylamine-borane, tB uMe'PrN-BH3, benzylamine-BH3 or 5-ethyl-2-methylpyridin Conjugation (reduction) After reaction, and optional capping, the glycoconjugates can be synthesized using a variety of methods known to those of skill in the art. The polysaccharide-protein conjugate can be purified (enriched with respect to the amount of the polysaccharide-protein conjugate) by the techniques These techniques include dialysis, concentration / diafiltration, tangential flow filtration, and precipitation. / Elution, column chromatography (ion exchange chromatography, multimodal ion exchange chromatography, DEAE or hydrophobic interaction chromatography) and In one embodiment, the glycoconjugates are subjected to diafiltration and depth filtration. Purification by ion exchange or size exclusion chromatography It is manufactured.

[0129] Glycoconjugates prepared using reductive amination in aprotic solvents are polyvalent pneumococcal conjugates. It is commonly used in adjuvant vaccines. Therefore, all serotypes are aprotic. In certain embodiments of multivalent compositions that are not prepared in an aqueous solvent, The reduction reaction for the serotypes of 2-(N-morpholino)ethanesulfonic acid), HEPES(4-(2-hydroxyethyl )-1-piperazineethanesulfonic acid), Bis-Tris, ADA (N-(2-acetamido do)iminodiacetic acid), PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid )), MOPSO (3-morpholino-2-hydroxypropanesulfonic acid), BES (N ,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid), MOPS(3- (N-morpholino)propanesulfonic acid), DIPSO(3-bis(2-hydroxyethyl) (N-morpholino-2-hydroxypropane-1-sulfonic acid), MOBS (4-(N-morpholino-2-hydroxypropane-1-sulfonic acid), N-(2-hydroxyethyl)piperazine- N'-(2-hydroxypropanesulfonic acid), POPSO (piperazine-1,4-bis(2-hydroxypropanesulfonic acid)), (2-hydroxy-3-propanesulfonic acid), TEA (triethanolamine), EPPS (4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid) or Bicine (pH 6.0-8.5, 7.0-8.0 or 7.0-7.5) ] is carried out.

[0130] Streptococcus nucleoids can be prepared using reductive amination in aprotic solvents. Streptococcus pneumoniae capsular polysaccharide-protein conjugates are Serotypes 1, 3, 4, 5, 6A, 6B, 6C, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 2 Polysaccharides include, but are not limited to, 4F, 33F and 35B. These may be obtained by fragmentation (e.g., by hydrolysis) of purified polysaccharides. This is conveniently done by purification of fragments of the desired size, usually followed by purification.

[0131] In certain embodiments, Streptococcus pneumoniae 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 3 One or more of the pneumococcal polysaccharide-protein conjugates of 5B may be reduced in an aprotic solvent. In certain embodiments, the multivalent immunogenic composition is prepared using reactive amination. Each of the conjugates in the study was prepared using reductive amination in an aprotic solvent. In certain embodiments, polysaccharides of one or more serotypes in the multivalent compositions of the invention is conjugated to a carrier protein using reductive amination in aprotic solvents and one or more serotype polysaccharides are conjugated using reductive amination in an aqueous medium. In certain embodiments, the multivalent compositions of the invention comprise a multivalent antibody of two or more serotypes. Sugars are conjugated to carrier proteins using reductive amination in aprotic solvents In other embodiments, the three or more, four or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 1 9 or more, 20 or more, 21 or more, 22 or more, 23 or more, or 24 or more serotypes is conjugated to a carrier protein using reductive amination in aprotic solvents In certain embodiments, the multivalent compositions of the invention include antibodies from one or more serotypes. Polysaccharides can be used to bind carrier proteins using other chemical properties that may be present in aprotic or aqueous solvents. It is conjugated to protein.

[0132] Thus, the present invention provides a streptococcus conjugated to a carrier protein. Several Streptococcus pneumoniae strains, each containing capsular polysaccharides from S. pneumoniae serovars The present invention relates to a multivalent immunogenic composition comprising a monier polysaccharide protein conjugate, Here, the serotypes of Streptococcus pneumoniae are as described herein (i.e., in Section II). 10, No. 6, pp. 111-115, 2002, entitled "Multivalent Immunogenic Compositions," in which one or more polysaccharides Protein Conjugation of Streptococcus pneumoniae Polysaccharide to Carrier Proteins The conjugation reaction is carried out in an aprotic solvent. In an embodiment, at least 30%, 30%, or 30% of the pneumococcal serotypes in the multivalent immunogenic composition are 5%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 8 5%, 90%, 95% or 100% is conjugated in an aprotic solvent. The remaining serotypes are conjugated using alternative chemistries and / or in aqueous media. will be done.

[0133] The use of DMSO as a solvent during the reductive amination of polysaccharide-protein conjugates , with respect to their serotype, as expected compared to the same conjugates produced under aqueous conditions In addition, it has been found to provide excellent stability and enhanced immunogenicity (U.S. Pat. App. No. 6,333,633). (See 2 / 463,216 and 62 / 555,444).

[0134] In certain embodiments of the invention, the total polysaccharide concentration in the composition is from about 0.02 to about 0. In certain embodiments of the present invention, the total polysaccharide concentration in the composition is 288 mg / mL. In a specific embodiment of the present invention, The total polysaccharide concentration in the composition is about 0.04 to about 0.192 mg / mL. In the present invention, the total polysaccharide concentration in the composition is about 0.065 to about 0.096 mg / mL, about 0 .070~approx. 0.080mg / mL, approx. 0.065~approx. 0.080mg / mL, approx. 0.0 70~about 0.085mg / mL, about 0.110~about 0.128mg / mL, about 0.110 ~about 0.175mg / mL, about 0.10 to about 0.175mg / mL, about 0.110 to about 0 .170mg / mL, about 0.115 to about 0.15mg / mL, 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 0.165mg / mL, about 0.140 to about 0.170mg / m L, 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 is about 0.065 to about 0.180 mg / mL.

[0135] One or more or all of the polysaccharide-protein conjugates in the multivalent immunogenic composition are non- In embodiments of the invention that are prepared in a protic solvent, the total polysaccharide concentration in the composition is stable for at least 4 weeks at 37°C, at least 4 weeks at 25°C, or at least 12 weeks at 4°C.

[0136] One or more or all of the polysaccharide-protein conjugates in the multivalent immunogenic composition are non- In certain embodiments of the present invention that are prepared in a protic solvent, the composition The average molecular weight (Mw ) (average of all conjugates in the composition) is about 2,000 to about 6,500 kDa, about 2,500~6,000kDa, 3,000~5,500kDa, 3,500~ Approximately 5,000 kDa, approximately 3,500 to approximately 4,500 kDa, approximately 3,500 to approximately 4,700 kDa, about 3,500 to about 4,600kDa, about 3,500 to about 4,500kDa, about 3 ,500~about 4,400 kDa, about 3,500~about 4,300 kDa, about 3,500~about 4,200kDa, approximately 3,600~4,700kDa, approximately 3,600~4,600k Da, about 3,600 to about 4,500 kDa, about 3,600 to about 4,400 kDa, about 3, 600 to approximately 4,300kDa, approximately 3,600 to approximately 4,200kDa, approximately 3,700 to approximately 4 ,700kDa, about 3,700 to about 4,600kDa, about 3,700 to about 4,500kD a, about 3,700 to about 4,400kDa, about 3,700 to about 4,300kDa, about 3,7 00~about 4,200kDa, about 3,800~about 4,700kDa, about 3,800~about 4, 600kDa, approx. 3,800~approx. 4,500kDa, approx. 3,800~approx. 4,400kDa , about 3,800 to about 4,300kDa, about 3,800 to about 4,200kDa, about 3,90 0 to about 4,700 kDa, about 3,900 to about 4,600 kDa, about 3,900 to about 4,5 00kDa, about 3,900 to about 4,400kDa, about 3,900 to about 4,300kDa, Or about 3,900 to about 4,200 kDa.

[0137] The polysaccharide-protein conjugate in the multivalent immunogenic composition is dissolved in an aprotic solvent. In a particular embodiment of the invention, the composition (for a single serotype) The Mw of each Streptococcus pneumoniae polysaccharide-protein conjugate is approximately 1,000 to approx. 10,000 kDa, approx. 1500 to approx. 5,500 kDa, approx. 1500 to approx. 5,600kDa, approx. 1500~approx. 5,700kDa, approx. 1500~approx. 5,800kDa , about 1500 to about 5,900 kDa, about 1500 to about 6000 kDa, about 1,000 to about 5,500kDa, approx. 1,000 to approx. 5,000kDa, approx. 1,000 to approx. 4,000k Da, about 1,000 to about 4,500 kDa, about 1,000 to about 4,000 kDa, or In another embodiment, the molecular weight of the single molecule in the composition is about 1,000 to about 3,500 kDa. The Mw of the conjugates from one serotype is approximately 1,000 kDa, approximately 1,100 kDa, and approximately 1 200kDa, approx. 1,300kDa, approx. 1,400kDa, approx. 1,500kDa, approx. 600kDa, approx. 1,700kDa, 1,800kDa, approx. 1,900kDa, approx. 2,0 00kDa, approx. 2,100kDa, approx. 2,200kDa, approx. 2,300kDa, approx. 2,4 2,500kDa, approx. 2,600kDa, approx. 2,700kDa, approx. 2,800kDa 00kDa, approx. 2,900kDa, approx. 3,000kDa, approx. 3,100kDa, approx. 3,2 00kDa, approx. 3,300kDa, approx. 3,400kDa, approx. 3,500kDa, approx. 3,6 00kDa, approx. 3,700kDa, approx. 3,800kDa, approx. 3,900kDa, approx. 4,0 00kDa, approx. 4,100kDa, approx. 4,200kDa, approx. 4,300kDa, approx. 4,4 00kDa, approx. 4,500kDa, approx. 4,600kDa, approx. 4,700kDa, approx. 4,8 00kDa, approx. 4,900kDa, approx. 5,000kDa, approx. 5,100kDa, approx. 5,2 00 kDa, about 5,300 kDa, about 5,400 kDa or about 5,500 kDa .

[0138] In certain embodiments of the invention, the polysaccharide-protein complex in the multivalent immunogenic composition The conjugate is prepared in an aprotic solvent. Streptococcus pneumoniae serotype-specific conjugates prepared in aqueous media (the number of polysaccharide serotypes produced in aprotic solvent divided by the total number of polysaccharide serotypes) where the total is calculated by adding the (including those manufactured in Japan) is more than 50% (i.e., more than 50%) or more than 60% or greater than 70%, or greater than 80%, or greater than 90%, or 100%.

[0139] In a particular embodiment of the invention, the serotype 3 polysaccharide-protein conjugate in the composition The conjugate is prepared in an aprotic solvent, and the Mw of the conjugate is about 1,000 to about 5,000 kDa, or about 1,000 to about 4,000 kDa, or 1,000 to about 3, 000 kDa, or about 1,000 to about 2,500 kDa, or about 1,000 to about 2, 000kDa.

[0140] One or more or all of the polysaccharide-protein conjugates in the multivalent immunogenic composition are non- In certain embodiments of the present invention that are prepared in a protic solvent, the composition Number average molecular weight (Mn) of Leptococcus pneumoniae polysaccharide-protein conjugate ( The average of all conjugates in the composition is about 900 to about 3,000 kDa, about 1,000 ~3,000kDa, ~1,000~2,500kDa, ~1,500~2,50 0kDa, about 1,800 to about 2,500kDa, about 1,900 to about 2,500kDa, or about 2,000 to about 2,500 kDa.

[0141] One or more or all of the polysaccharide-protein conjugates in the multivalent immunogenic composition are non- In certain embodiments of the present invention that are prepared in a protic solvent, the composition The Mn of each Leptococcus pneumoniae polysaccharide-protein conjugate (single blood Regarding the pure type, about 700 to about 7,000 kDa, about 1,000 to about 6,000 kDa a, about 1,000 to about 5,000kDa, about 1,000 to about 4,000kDa, about 1,0 00 to about 3,000 kDa, about 900 to about 5,500 kDa, about 900 to about 5,000 kDa Da, about 900 to about 4,500 kDa, about 900 to about 4,000 kDa, about 900 to about 3 ,500 kDa, or about 900 to about 3,000 kDa.

[0142] In an embodiment of the present invention, the composition comprises Streptococcus pneumoniae polysaccharides. The Mw and / or Mn of the protein conjugates can be maintained for more than 4 weeks at 37°C and for 4 weeks at 25°C. stable for more than 12 weeks at 4°C.

[0143] In an embodiment of the invention, the polysaccharide concentration, Mw and / or Mn are determined by HPSEC U Determined using V / MALS / RI.

[0144] One or more or all of the polysaccharide-protein conjugates in the multivalent immunogenic composition are non- In some embodiments of the invention prepared in a protic solvent, Emission maximum of the composition measured using intrinsic protein fluorescence spectroscopy at excitation wavelength in nm In some embodiments, the maximum emission is about 335 nm to about 342 nm. The fluorescence intensity remains stable at 37 °C for at least one week. In some embodiments, the emission maximum remains at about 335 nm to about 342 nm, and the fluorescence Light intensity is stable for one week at 37°C.

[0145] In some embodiments, all of the pneumococcal polysaccharide conjugates in the multivalent composition is prepared using reductive amination in DMSO. Multivalent compositions containing polysaccharide conjugates, all prepared using DMSO, are used as adjuvants. Does not include

[0146] Without wishing to be bound by any particular theory, glycoconjugates prepared in DMSO One possible mechanism for the enhanced immunogenicity observed in is the association of carbohydrates (capsular polysaccharides) with This involves increasing the number of bonds between lysine residues on the surface of the carrier protein, which Provides additional attachment points between the protein and the polysaccharide, imparting stability and enhancing peptide-carbohydrate bonds. This would counteract the chemical depolymerization or decomposition of the copolymer. erization of Saccharide-CRM197 Conjugate Vaccines in Brown F,Corbel M,Griffiths E (ed.): Physico-Chemical Procedures for the Characterization of Vaccines.Dev.Biol.B See Asel, Karger, 2000, vol 103, pp. 93-104. Following the increase in polysaccharide-protein bonds generated during conjugation in DMSO solvent, The additional benefit would be an additional opportunity for successful presentation of peptide-carbohydrate to T cells. Possible mechanisms for the enhanced immunogenicity observed upon conjugation in DMSO solvent This may be due to the denaturation of CRM197 in organic solvents. Exposing selective lysines for T cell-dependent responses to various peptide epitopes Increases the likelihood of glycopeptide presentation on the APC surface. Avci et al., 2011, Na See True Medicine 17:1602-1610.

[0147] Conjugation in organic solvents to generate modified CRM197 in the conjugate Another advantage of this approach is the reduced immunological interference of antibodies against the native CRM197 epitope. The number of polysaccharide-protein bonds formed during conjugation in DMSO solvent is likely to be reduced. Another advantage of the increase is the larger size of the glycoprotein, which leads to enhanced immunogenicity. The compositions of the present invention are important in inducing responses in humans. is believed to provide important advantages.

[0148] In certain embodiments, the conjugation reaction is carried out by reductive amination, For greater conjugation efficiency and to facilitate the removal of free cyanide, Nickel is used to promote the oxidation of the transition metal. and the cleavage of protein amino and formaldehyde groups by sodium cyanoborohydride. It is known to improve the reductive methylation of aldehydes (S Gidley et al., Biol. chem J.1982,203:331-334;Jentoft et al. Anal Bi Ochem. 1980, 106: 186-190). The addition of nickel inhibits residual shear. Complexing the diamine with the amine increases the consumption of protein during conjugation, resulting in a larger This results in the formation of larger, potentially more immunogenic conjugates.

[0149] Differences in initial cyanide levels in sodium cyanoborohydride reagent lots were also observed. Conjugate size and conjugate P The addition of nickel results in variations in product properties such as the ratio of cyanide to CRM197. Complexation to eliminate lot-to-lot variations in sodium cyanoborohydride Reduced conjugation inconsistencies.

[0150] A suitable alternative chemical method is 1-cyano-4-dimethylaminopyridinium tetrafluoroborate. This involves the activation of sugars with cyanate esters (CDAP) to form cyanate esters. The activated sugar can then be attached to a carrier protein, either directly or via a spacer (linker) group. For example, the spacer can be coupled to a maleimide-activated support. body proteins (e.g., using GMBS) or haloacetylated carrier proteins (e.g. , iodoacetimide [e.g., ethyl iodoacetimide HCl] or N-succinimide with imidyl bromoacetate, SIAB, SIA, or SBAP) Thiolated polysaccharides that can be coupled to supports via the thioether bonds obtained after the reaction Preferably, the cyanate ester may be cystamine or cysteamine to obtain The hexanediamine or adipic acid (which may be prepared by CDAP chemistry, if desired) is Amino-derivatized sugars are coupled to carboxylic acid dihydrazides (ADHs) via carbodiimides. (e.g., EDAC or EDC) chemical methods to convert carboxyl groups on the protein carrier Such conjugates are conjugated to carrier proteins via an ELISA kit. Patent application publication numbers WO 93 / 15760, WO 95 / 08348 and WO 96 / 2 9094, and Chu et al., 1983, Infect. Immunity 40:24 It is described in 5-256.

[0151] Other suitable conjugation techniques include carbodiimide, hydrazide, active ester, norbornyl ester, Bornane, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, Many of these are described in International Patent Application Publication No. WO 98 / 42721. The conjugates are formed by reaction of the free hydroxyl groups of the sugars with CDI. Carbonyl linker (Bethell et al., 1979, J. Biol. Chem. 25 4:2572-4; Hearn et al., 1981, J. Chromatogr. 218:50 9-18), which then reacts with the protein to form A carbamate bond is formed by reduction of the anomeric terminus to a primary hydroxyl group, Optional protection / deprotection of primary hydroxyl groups, primary hydroxyl groups and Reaction with CDI to form a CDI carbamate intermediate and cleavage of amino groups on proteins This may involve coupling of a CDI carbamate intermediate to

[0152] After conjugation of the capsular polysaccharide to a carrier protein, the polysaccharide-protein conjugate is The conjugate is purified (with respect to the amount of 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 trays. Includes purification, ultrafiltration, precipitation / elution, column chromatography and depth filtration. See, for example, U.S. Patent No. 6,146,902.

[0153] After the individual glycoconjugates are purified, they are combined to formulate the immunogenic compositions of the invention. These pneumococcal conjugates can be manufactured by separate methods and packaged in a single dosage form. It can be formulated into a bulk formulation.

[0154] An alternative method for characterizing the glycoconjugates of the present invention is to characterize the carrier conjugated to the sugar. This is due to the number of lysine residues in the protein (e.g., CRM197), The extent of conjugated lysine (degree of conjugation) can be characterized as the extent of conjugation of the lysine. Evidence for lysine modification of a carrier protein by covalent attachment to a sugar can be obtained by routine methods known to those of skill in the art. Conjugation can be performed by the bioanalysis of the conjugate material. A reduction in the number of lysine residues recovered compared to the carrier protein starting material used in synthesis. In a preferred embodiment, the glycoconjugates of the present invention are measured by lysine consumption. The degree of conjugation was 2–15, 2–13, 2–10, 2–8, 2–6, 2–5, 2~4, 3~15, 3~13, 3~10, 3~8, 3~6, ​​3~5, 3~4, 5~15, 5 to 10, 8 to 15, 8 to 12, 10 to 15, or 10 to 12. In the present invention, the degree of conjugation of the glycoconjugates 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 another embodiment, the degree of conjugation of the glycoconjugate of the present invention is 4 to 7. In some such embodiments, the carrier protein is CRM197.

[0155] The glycoconjugates of the compositions of the invention have a polysaccharide to carrier protein (Ps:Pr) ratio (w / w) In some embodiments, the glycoconjugates in the composition may also be characterized by The ratio of polysaccharide to carrier protein (w / w) is 0.5 to 3.0 (e.g., about 0.5, about 0.6, Approximately 0.7, approximately 0.8, approximately 0.9, approximately 1.0, approximately 1.1, approximately 1.2, approximately 1.3, approximately 1.4, Approximately 1.5, approximately 1.6, approximately 1.7, approximately 1.8, approximately 1.9, approximately 2.0, approximately 2.1, approximately 2.2, About 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9 or about 3. In another embodiment, the polysaccharide to carrier protein ratio (w / w) is 0.5 to 2.0. .5, 0.5~1.5, 0.8~2.5, 0.5~1.0, 1.0~1.5, 1.0~2 .0, 0.8~2.4, 0.8~2.3, 0.8~2.2, 0.8~2.1, 0.8~2 .0, 0.8~1.9, 0.8~1.8, 0.8~1.7, 0.8~1.6, 0.8~1 .5, 0.8~1.4, 0.8~1.3, 0.9~2.4, 0.9~2.3, 0.9~2 .2, 0.9~2.1, 0.9~2.0, 0.9~1.9, 0.9~1.8, 0.9~1 .7, 0.9~1.6, 0.9~1.5, 0.9~1.4, 0.9~1.3, 0.9~1 .2, 1.0~2.4, 1.0~2.3, 1.0~2.2, 1.0~2.1, 1.0~2 .0, 1.0~1.9, 1.0~1.8, 1.0~1.7, 1.0~1.6, 1.0~1 In other embodiments, the ratio is 0.5, 1.0 to 1.4, 1.0 to 1.3, or 1.0 to 1.2. In most cases, the sugar to carrier protein ratio (w / w) is 0.8-1.2. In an embodiment, the carrier protein is CRM197. Covalently bound to a carrier protein Free saccharides that are not (covalently conjugated) but are still present in the glycoconjugate composition The glycoconjugates and immunogenic compositions of the present invention may contain the free saccharide non-covalently bound to the glycoconjugate. They may be covalently associated (e.g., noncovalently bound to glycoconjugates) or adsorbed. (It may be attached to, or incorporated within or by, the

[0156] In certain embodiments, the sugar to carrier protein ratio (w / w) is about 1.0 to about 2.0, about 1.25 to about 1.75, or about 1.3 to about 1.7 In another embodiment, the saccharide to carrier protein ratio (w / w) of serotype 15A is about 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7 or 1.8.

[0157] In certain embodiments, the sugar to carrier protein ratio (w / w) is about 1.0 to about 2.0, about 1.25 to about 1.75, or about 1.3 to about 1.7 In another embodiment, the saccharide to carrier protein ratio (w / w) of serotype 15C is about 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7 or 1.8.

[0158] In certain embodiments, the sugar to carrier protein ratio (w / w) is about 1.0 to about 2.0, about 1.25 to about 1.75, or about 1.3 to about 1.7 In another embodiment, the saccharide to carrier protein ratio (w / w) of serotype 33F is about 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7 or 1.8.

[0159] In certain embodiments, the sugar to carrier protein ratio (w / w) is about 1.25 to about 2.25, about 1.25 to about 2.0, or about 1.3 to about 1.8. In another embodiment, the saccharide to carrier protein ratio (w / w) of serotype 35B is about 1. 2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2.0.

[0160] In certain embodiments, the sugar to carrier protein ratio (w / w) is about 0.5 to about 1.5, about 0.75 to about 1.25, or about 0.8 to about 1.0 In another embodiment, the saccharide to carrier protein ratio (w / w) of serotype 24F is about 0.5. , 0.6, 0.7, 0.8, 0.9 or 1.0.

[0161] In a preferred embodiment, the glycoconjugate composition comprises about 50% by weight of polysaccharides, Contains less than 45%, 40%, 35%, 30%, 25%, 20% or 15% free polysaccharides. In a preferred embodiment, the glycoconjugate composition comprises less than about 25% of the total amount of polysaccharides. In a preferred embodiment, the glycoconjugate composition comprises free polysaccharides in an amount comparable to the total amount of polysaccharides. In a preferred embodiment, the glycoconjugate composition contains less than about 20% free polysaccharides. , containing less than about 15% free polysaccharides as compared to the total amount of polysaccharides.

[0162] IV.How to use Embodiments of the invention also include (i) (a) therapeutic (e.g., human treatment); (b) pharmaceutical; c) suppression of infection with Streptococcus pneumoniae; (d) suppression of infection with Streptococcus (e) induction of an immune response or a protective immune response against Streptococcus pneumoniae; (f) prevention of recurrent Streptococcus pneumoniae infections; (g) prevention of streptococcus, including prevention of related complications including brain damage, hearing loss, and seizures. (h) reducing the progression, onset or severity of pathological symptoms associated with S. pneumoniae infection; (i) reducing the likelihood of infection with Leptococcus pneumoniae, or Treatment and prevention of pneumococcal diseases, including pneumococcal bacteremia, pneumococcal meningitis, otitis media and sinusitis or for use in delaying the onset, severity or progression of (iii) for use as a medicament or composition, or in the manufacture of a medicament therefor; The present invention includes one or more of the multivalent immunogenic compositions described herein for use in In these uses, the polyvalent pneumococcal polysaccharide-conjugate compositions of the invention may optionally be and may be used in combination with one or more adjuvants or without adjuvant.

[0163] Thus, the present invention provides a method for administering to a patient in need of treatment a multivalent immunogenic pneumococcal polysaccharide-protein of the invention. The present invention relates to a method for treating Streptococcus nucleus infection, comprising administering one or more of the following compositions to a patient: The present invention provides a method for the prophylactic treatment of (i.e., protection against) pneumococcal infection or pneumococcal disease. Provide.

[0164] The compositions and formulations of the present invention are intended to be administered by systemic or mucosal routes. and administering the same to a human subject susceptible to infection, such as pneumococcal infection, to prevent or treat the subject. It can be used to

[0165] In one embodiment, the present invention provides an immunologically effective amount of a multivalent immunogenic composition of the present invention. The present invention relates to a method for inducing an immune response against Streptococcus pneumoniae, comprising administering to a patient In another embodiment, the present invention provides a method for administering a multivalent immunogen of the present invention. The present invention relates to a method for treating a human against pneumococcal infection, comprising administering to the human an immunologically effective amount of a composition comprising the steps of: A method for inoculating the plant is provided.

[0166] Thus, in one aspect, the present invention provides a multivalent immunogenic composition of the invention (i.e. That is, any of the multivalent immunogenic compositions described herein, e.g., the "multivalent immunogenic compositions" described above. and administering to the patient a multivalent immunogenic composition (such as a multivalent immunogenic composition described in Section II entitled "Multivalent Immunogenic Compositions"). (1) inducing an immune response in a human patient; and (2) generating protective immunity in a human patient. (3) induce an immune response in human patients against infection with Streptococcus pneumoniae or (4) vaccinating a human patient against Streptococcus pneumoniae. The present invention provides a method for reducing the likelihood of infection.

[0167] In one embodiment, the present invention is directed to infants (under 1 year old), toddlers (about 12-24 months of age), and Prevention of pneumococcal pneumonia and / or invasive pneumococcal disease in children (approximately 2 to 5 years of age) A method is provided.

[0168] In another embodiment, the present invention relates to a method for treating pneumococcal pneumonia in patients between 6 weeks and 17 years of age. and / or methods for preventing invasive pneumococcal disease.

[0169] In another embodiment, the present invention relates to a method for treating pneumococcal pneumonia in patients between 6 months and 17 years of age. and / or methods for preventing invasive pneumococcal disease.

[0170] In another embodiment, the present invention provides a method for treating pneumococcal pneumonia and Streptococcus pneumoniae in adults aged 18 years or older. and / or methods for preventing invasive pneumococcal disease.

[0171] In another embodiment, the present invention relates to a method for treating pneumococcal pneumonia and Streptococcus pneumoniae in adults 50 years of age or older. and / or methods for preventing invasive pneumococcal disease.

[0172] In another embodiment, the present invention provides a method for treating pneumococcal pneumonia and Streptococcus pneumoniae in adults 65 years of age and older. and / or methods for preventing invasive pneumococcal disease.

[0173] In another embodiment, the present invention provides a method for the preparation of a Streptococcus pneumoniae strain comprising the steps of: Stocks, namely, 1, 3, 4, 5, 6A, 6B, 6C, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 18C, 19A, 19F, 22F, 23B, 2 Pneumococcal pneumonia caused by one or more of 3F, 24F, 33F and 35B and / or Or a method for preventing invasive pneumococcal disease is provided.

[0174] In one embodiment of the method, the composition comprises a plurality of Streptococcus pneumoniae strains. and a polysaccharide-protein conjugate, each of which is a carrier. Protein-conjugated Streptococcus pneumoniae serovar-derived polysaccharides The serotypes of Streptococcus pneumoniae include serotypes 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 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, In another embodiment of the method, the composition comprises a plurality of Streptococcus pneumoniae polysaccharide protein conjugate, Each of the conjugates contains streptococcus conjugated to a carrier protein. It contains polysaccharides derived from Streptococcus pneumoniae serotypes. Serotypes 1, 3, 4, 5, 6A, 6B, 6C, 7F, 8, 9V, 10A, 12F, 14, 1 5A, 15B, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 3 3F and 35B or serotypes 1, 3, 4, 5, 6A, 6B, 6C, 7F, 8, 9V, 1 0A, 11A, 12F, 14, 15A, 15B, 15C, 18C, 19A, 19F, 22 F, 23B, 23F, 24F, 33F and 35B. The composition comprises a plurality of Streptococcus pneumoniae polysaccharide-protein conjugates. a gate, wherein each of the conjugates is conjugated to a carrier protein. Contains polysaccharides derived from serovar Streptococcus pneumoniae, S. pneumoniae serotypes are serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, and 10 A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F , 24F, 33F and 35B or serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9 V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F , 23B, 23F, 24F, 33F and 35B. The composition comprises a plurality of Streptococcus pneumoniae polysaccharide-protein conjugates. a gate, wherein each of the conjugates is conjugated to a carrier protein. Contains polysaccharides derived from serovar Streptococcus pneumoniae, S. pneumoniae serotypes are serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, and 10 A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F , 24F, 33F and 35B or serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9 V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F , 23B, 23F, 24F, 33F and 35B. The composition comprises a plurality of Streptococcus pneumoniae polysaccharide-protein conjugates. wherein each of the conjugates is conjugated to a carrier protein. Contains polysaccharides derived from Streptococcus pneumoniae serovars and is S. pneumoniae serotypes are serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 1 2F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24 In another embodiment of the method, the composition comprises a plurality of 4. The method of claim 1, further comprising administering to said patient a number of Streptococcus pneumoniae polysaccharide-protein conjugates comprising the steps of: Each of the conjugates comprises a streptococcal antibody conjugated to a carrier protein. Contains polysaccharides derived from Streptococcus pneumoniae serovars and inhibits the activity of Streptococcus pneumoniae serum. The types are serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35 B or serovars.

[0175] Pneumococcal conjugate vaccines containing serotype 6A polysaccharide have some protection against serotype 6C. It has been shown that cross-protection can be achieved (Cooper et al., Vaccine 29( 2011) 7207-7211). Thus, in some embodiments of the method The present invention also does not include serotype 6C polysaccharide conjugates, but instead serotype 6A polysaccharide conjugates. Conjugates or multivalent immunogenic compositions comprising serotype 6A and 6B polysaccharide conjugates In another embodiment, the immunogenic composition is a combination of serotypes 6A, 6B and and 6C pneumococcal polysaccharide conjugates.

[0176] In certain embodiments of the method, the multivalent immunogenic composition is selected from the group consisting of: A pneumococcal conjugate comprising a polysaccharide of a group of Streptococcus pneumoniae serotypes selected from the group Contains Jugate: a)1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 12F, 14, 15A, D eOAc15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and and 35B; b)1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 12F, 14, 15A, 1 5C, 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, 33 F 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 3 5B; 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, 24 F, 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 3 5B; 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, 15 C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; n)1, 3, 4, 5, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15 C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; o)1, 3, 4, 5, 6C, 7F, 8, 9V, 10A, 12F, 14, 15A, 15 C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; p)1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 11A, 12F, 14, 15 A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; q)1, 3, 4, 5, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15 A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; r)1, 3, 4, 5, 6C, 7F, 8, 9V, 10A, 11A, 12F, 14, 15 A, 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, 15 B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; w)1, 3, 4, 5, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15 B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; x)1, 3, 4, 5, 6C, 7F, 8, 9V, 10A, 12F, 14, 15A, 15 B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; y)1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 11A, 12F, 14, 15 A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; z)1, 3, 4, 5, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15 A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; and aa)1, 3, 4, 5, 6C, 7F, 8, 9V, 10A, 11A, 12F, 14, 1 5A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and and 35B.

[0177] In another embodiment of the method, the composition comprises a plurality of Streptococcus pneumoniae. and polysaccharide-protein conjugates, each of which is a carrier. Polysaccharides from Streptococcus pneumoniae serovars conjugated to protein The serotypes of Streptococcus pneumoniae include serotypes: i) 1, 3, 4, 5, 6 A, 6B, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19 F, 22F, 23B, 23F, 24F, 33F and 35B; or ii) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 1 9A, 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 3 5B; or iv) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 1 2F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24 Includes F, 33F and 35B.

[0178] Pneumococcal conjugate vaccines containing serotype 10A polysaccharide have some protection against serotype 39. It has also been shown that cross-protection against Thus, in some embodiments of the method, the present invention also provides a method for detecting serotype Does not contain the 10A polysaccharide conjugate, but instead contains the serotype 39 polysaccharide conjugate In another embodiment, the immunogenic composition is a serum In certain embodiments of the method, the method comprises the step of: In the present invention, the serotype of Streptococcus pneumoniae is selected from the group consisting of: The following serotype groups may be selected: bb)1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15A, DeOA c15B, 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, De OAc15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 3 5B and 39; ee)1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 12F, 14, 15A, 15 C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; ff)1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 12F, 14, 15B, 18 C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; gg)1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 11A, 12F, 14, 15 A, DeOAc15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 3 3F, 35B and 39; hh)1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 11A, 12F, 14, 15 A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35 B and 39; ii)1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 11A, 12F, 14, 15 A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35 B and 39; jj)1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 11A, 12F, 14, 15 B, 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, 1 8C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; oo)1, 3, 4, 5, 6B, 7F, 8, 9V, 12F, 14, 15A, 15C, 1 8C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; pp)1, 3, 4, 5, 6C, 7F, 8, 9V, 12F, 14, 15A, 15C, 1 8C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; qq)1, 3, 4, 5, 6A, 7F, 8, 9V, 11A, 12F, 14, 15A, 1 5C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; rr)1, 3, 4, 5, 6B, 7F, 8, 9V, 11A, 12F, 14, 15A, 1 5C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; ss)1, 3, 4, 5, 6C, 7F, 8, 9V, 11A, 12F, 14, 15A, 1 5C, 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, 1 8C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; xx)1, 3, 4, 5, 6B, 7F, 8, 9V, 12F, 14, 15A, 15B, 1 8C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; yy)1, 3, 4, 5, 6C, 7F, 8, 9V, 12F, 14, 15A, 15B, 1 8C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; zz)1, 3, 4, 5, 6A, 7F, 8, 9V, 11A, 12F, 14, 15A, 1 5B, 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 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 and 39.

[0179] Streptococcus pneumoniae serotype 15B capsular polyclonal antibodies covalently linked to carrier proteins Immunogenic conjugates containing carbohydrates have been shown to be somewhat effective against serotype 15C and / or serotype 15A. It has also been shown that it may confer some cross-protection (see WO 2015 / 110942). Thus, in some embodiments of the method, the present invention also provides a method for the detection of blood does not contain the 15C (or de-O-acetylated 15B) polysaccharide conjugates, but instead contains a serotype 15B polysaccharide conjugate (i.e., the serotype 15B polysaccharide is substantially deoxyribonucleic acid -non-acetylated) multivalent immunogenic composition. For example, the immunogenic composition may be composed of serotypes 15B and 15C (or de-O-acetylated 15B). Includes pneumococcal polysaccharide conjugates.

[0180] The compositions of the invention are useful in preventing streptococcal infection in patients previously vaccinated with a multivalent pneumococcal vaccine. are useful in methods for conferring complementary protection against Coccus pneumoniae. In this use, the compositions of the invention are administered to a patient with a particular vaccine against which the patient has not previously been vaccinated. It is possible to confer protection against Streptococcus pneumoniae serotypes and to prevent disease Additional vaccination against Streptococcus pneumoniae serotypes against which the individual was previously vaccinated It is possible that the vaccine may provide substantial protection or that the patient has not been previously vaccinated. Streptococcus pneumoniae serotype and the streptococci against which the patient had previously been vaccinated It is possible to confer protection against both Tococcus pneumoniae serotypes.

[0181] Thus, the present invention provides a method for the treatment of a patient comprising administering to the patient a multivalent immunogenic composition. Inducing an immune response against Streptococcus pneumoniae by vaccination The present invention provides a method for inducing a protective immune response against a plurality of Streptococcus and a S. pneumoniae polysaccharide-protein conjugate, Conjugate is a Streptococcus pneumoniae conjugated to a carrier protein. The patient had previously been infected with Streptococcus pneumoniae. In an embodiment of this aspect of the invention, the multivalent immunogenic composition The product can be any multivalent immunogenic composition described herein. In certain embodiments, the multivalent immunogenic composition comprises a subject previously treated with a multivalent pneumococcal vaccine. The multivalent immunogenic vaccine is administered to patients who have been treated with Streptococcus pneumoniae. Any vaccine indicated for the prevention of pneumococcal disease caused by multiple serotypes of It's possible.

[0182] In certain embodiments of the method, the patient is i.4, 6B, 9V, 14, 18C, 19F and 23F; ii.4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 6A, 7F and and 19A; iii.1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F; iv.4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 6A, 7F, 19A, 22F and 33F; 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 vi.4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 6A, 7F, 19A, 22F, 33F, 8, 10A, 11A, 12F and 15B one or more serotypes of Streptococcus pneumoniae in the group selected from the group consisting of A multivalent pneumococcal vaccine indicated for the prevention of pneumococcal disease caused by the serotype He was being treated by.

[0183] In certain embodiments of the method, the multivalent pneumococcal vaccine comprises multiple polysaccharide proteins. Conjugates, wherein the polysaccharide-protein conjugate is conjugated to a carrier protein. It contains polysaccharides derived from conjugated Streptococcus pneumoniae serovars. In an embodiment, the multivalent pneumococcal vaccine is conjugated to a carrier protein. It contains multiple pneumococcal capsular polysaccharides that are not

[0184] In an additional embodiment of the method, the patient is receiving PREVNAR® 13 [pulmonary Streptococcus pneumoniae 13-valent conjugate vaccine (diphtheria CRM197 protein), Pfiz The patient had been previously treated at ER, Inc., Philadelphia, PA, USA.

[0185] In another embodiment of the method, the patient is administered PNEUMOVAX® 23 [ Pneumococcal Vaccine Polyvalent nt), Merck & Co., Inc., Kenilworth, NJ He was being treated.

[0186] In yet another embodiment of the method, the patient is receiving SYNFLORIX® Pneumococcal polysaccharide conjugate vaccine (adsorbed), GlaxoSmithKline Bio Previously treated with rheumatoid arthritis medicine, Rixensart, Belgium It was being done.

[0187] In an embodiment of the method, the multivalent immunogenic composition of the invention is administered to a medical professional, e.g. Patients received multivalent pneumococcal vaccine according to a treatment regimen provided by their physician In certain embodiments, the multivalent immunogenic antibody of the present invention is administered to the patient at any time thereafter. The composition is administered to the patient about one month to about five years after the patient receives the multivalent pneumococcal vaccine. 1 month to 1 year after the patient received the multivalent pneumococcal vaccine, or months to 2 years, 1 month to 3 years, 1 month to 4 years, 1 month to 6 months, 2 months to 6 months, 2 months to 1 year, 1 year to 5 years, 6 months to 5 years, 6 months to 4 years, 6 months to 3 years, 6 months to 2 years, 6 months The treatment is administered to the patient after 1-1 years, 1-4 years, 1-3 years, or 1-2 years. In some embodiments, the multivalent immunogenic composition is administered to a patient after the patient has received a multivalent pneumococcal vaccine. About 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, 9 months, 10 months, 11 months, 1 year, 1.25 years, 1.5 years, 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 1 year, about 3.75 years, about 4 years, about 4.25 years, about 4.5 years, about 4.75 years or about 5 years later It is administered to the patient.

[0188] In another embodiment, the present invention provides a method for the treatment of a cancer by administering to a patient a multivalent immunogenic composition of the present invention. and administering to the patient a multivalent pneumococcal vaccine, in any order. (2) induce a protective immune response in a human patient; 3) Vaccinate human patients against infection with Streptococcus pneumoniae or (4) reducing the likelihood of Streptococcus pneumoniae infection in a human patient. For example, a method for administering a multivalent pneumococcal vaccine to a patient is provided. Alternatively, the multivalent immunogenic composition of the present invention may be administered to the patient first. The composition is administered to a patient, and then a multivalent pneumococcal vaccine is administered to the patient. The vaccine is intended to treat pulmonary infections caused by multiple serotypes of Streptococcus pneumoniae. It may be any vaccine indicated for the prevention of pneumococcal disease.

[0189] In certain embodiments of the method, the patient is administered a multivalent immunogenic composition of the invention and i.4, 6B, 9V, 14, 18C, 19F and 23F; ii.4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 6A, 7F and and 19A; iii.1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F; iv.4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 6A, 7F, 19A, 22F and 33F; 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 vi.4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 6A, 7F, 19A, 22F, 33F, 8, 10A, 11A, 12F and 15B one or more Streptococcus pneumoniae serotypes selected from the group consisting of Multivalent pneumococcal vaccines are indicated for the prevention of pneumococcal disease caused by Streptococcus pneumoniae type 1 and for treatment. will be done.

[0190] In certain embodiments of the method, the multivalent pneumococcal vaccine comprises Streptococcus pneumoniae. pneumoniae serotypes 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 7 F, 19A, 22F, 33F, 2, 8, 9N, 10A, 11A, 12F, 15B, 17F and 20A capsular polysaccharide.

[0191] In certain embodiments of the method, the multivalent pneumococcal vaccine comprises multiple polysaccharide proteins. Conjugates, wherein the polysaccharide-protein conjugate is conjugated to a carrier protein. It contains polysaccharides derived from conjugated Streptococcus pneumoniae serovars. In an embodiment, the multivalent pneumococcal vaccine is conjugated to a carrier protein. not contain multiple Streptococcus pneumoniae capsular polysaccharides.

[0192] In an additional embodiment of the method, the patient is treated with a multivalent immunogenic composition of the invention. Treatment, and PREVNAR® 13 [Pneumococcal 13-valent Conjugate Vaccine ( Diphtheria CRM197 protein), Pfizer, Inc., Philadelphia In one embodiment, the patients receive treatments from the following countries: First, the patient is administered PREVNAR® 13, followed by administration of the multivalent immunogenic composition of the present invention. In an alternative embodiment, the multivalent immunogenic composition of the present invention is first administered to the patient. The patient is then administered PREVNAR® 13.

[0193] In another embodiment of the method, the patient is treated with a multivalent immunogenic composition of the invention; and PNEUMOVAX® 23 [Pneumococcal Vaccine Multivalent (Pneumococcal ccol Vaccine Polyvalent), Merck & Co., Inc. ., Kenilworth, NJ] in any order. In this method, PNEUMOVAX® 23 is first administered to the patient, and then the In an alternative embodiment, the multivalent immunogenic composition of the present invention is first administered to the patient. The immunogenic composition is administered to the patient, and then PNEUMOVAX® 23 is administered to the patient. Give.

[0194] In yet another embodiment of the method, the patient is administered a multivalent immunogenic composition of the invention. and SYNFLORIX (trademark) [pneumococcal polysaccharide conjugate vaccine ( adsorption), GlaxoSmithKline Biologicals sa, Rix In one embodiment, the patient is treated with 100 mg of ensart, 100 mg of serotonin ... In this case, SYNFLORIX™ is administered to the patient first, followed by administration of the multivalent immunogen of the present invention. In an alternative embodiment, the multivalent immunogenic composition of the present invention is first administered to the patient. The composition is administered to the patient and then SYNFLORIX™ is administered to the patient.

[0195] In some embodiments of the method, the multivalent immunogenic composition and the multivalent pneumococcal vaccine are As used herein, "co-administration" is not limited to simultaneous administration of two compositions. In some embodiments, the multivalent immunization is administered in any order, including one after the other. The primary composition and the multivalent pneumococcal vaccine are administered intramuscularly or subcutaneously into separate anatomical sites. The vaccine is administered at two different sites, for example, on two different arms.

[0196] In some embodiments of the method, the administration of the multivalent immunogenic composition of the invention and the multivalent pulmonary The length of time between administrations of the pneumococcal vaccine is from about 4 weeks to about 1 year. In this case, the period ranges from about one month to about five years.

[0197] In one embodiment, a patient is first administered a multivalent pneumococcal vaccine, followed by administration of the present invention. In an alternative embodiment, the patient is first administered a multivalent immunogenic composition of the present invention. The multivalent immunogenic composition is administered to the patient, and then the multivalent pneumococcal vaccine is administered to the patient.

[0198] Also, (1) administering to a patient a multivalent immunogenic composition of the invention; (2) waiting for a predetermined time to elapse; and (3) administering a multivalent pneumococcal vaccine to patients; Inducing an immune response against Streptococcus pneumoniae in a patient, Also provided are methods of vaccinating or inducing a protective immune response. In the present invention, the multivalent immunogenic composition comprises a Streptococcus neutrophil antibody as described herein. and any combination of Monnier polysaccharide protein conjugates, The coccal vaccine is a vaccine against Streptococcus pneumoniae caused by multiple serotypes. The vaccine may be any vaccine indicated for the prevention of a disease.

[0199] The present invention also provides a method for producing a semiconductor device comprising the steps of: (1) administering a multivalent pneumococcal vaccine to a patient; (2) waiting for a predetermined time to elapse; and (3) administering to a patient a multivalent immunogenic composition of the invention. Inducing an immune response against Streptococcus pneumoniae in a patient, Alternatively, methods of vaccinating or inducing a protective immune response are provided.

[0200] In this method, the multivalent immunogenic composition comprises a streptococcus erythropoietin antibody as described herein. The present invention may include any combination of Bacillus pneumoniae polysaccharide-protein conjugates. Multivalent pneumococcal vaccines protect against multiple serotypes of Streptococcus pneumoniae. The vaccine may be any vaccine indicated for the prevention of a disease caused by a virus.

[0201] In some embodiments of the method, the multivalent pneumococcal vaccine comprises a plurality of streptococci. and a Bacillus pneumoniae polysaccharide-protein conjugate, wherein the polysaccharide-protein conjugate The conjugate is a Streptococcus neutrophil conjugated to a carrier protein. In an alternative embodiment, the multivalent pneumococcal vaccine comprises a capsular polysaccharide derived from the Monnier serotype. Streptococcus pneumoniae not conjugated to a carrier protein Contains capsular polysaccharides.

[0202] Any embodiment of the method of the present invention (i.e., any of the methods described herein) In the method, one or more additional doses of a multivalent immunogenic composition of the invention are administered to the patient. In such a method, the patient may further comprise administering to the patient a multivalent immunization therapy according to the invention as described above. Those who have already received a multivalent pneumococcal vaccine before receiving the first dose of the immunogenic composition Alternatively, the subject may be administered a multivalent immunogenic composition of the present invention prior to stimulating the immune system. It is possible that you have not been vaccinated against Leptococcus pneumoniae. Thus, in one embodiment, the present invention relates to a method for treating Streptococcus pneumoniae-induced This vaccine is administered to patients who have received a multivalent pneumococcal vaccine that is intended to prevent pneumococcal disease. Two or more doses of the multivalent immunogenic composition of the invention are administered. In patients who have not been previously treated with any vaccine indicated for the prevention of coccal disease Two or more doses of the multivalent immunogenic composition of the invention are administered.

[0203] In an embodiment of the method, the two or more doses are of 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. It belongs to something.

[0204] In certain embodiments of any of these methods, the multivalent immunogenic composition of the invention Two, three or four doses are administered to the patient. In certain embodiments, the patient is Being immunocompromised (due to the immunosuppressive regimen following cell transplantation).

[0205] In some embodiments, the time between administration of each dose of the multivalent immunogenic composition of the invention The length of the immunogenic composition of the present invention is about 4 weeks to about 1 year. The length of time between administration of each dose of the compound is from about 1 month to about 5 years.

[0206] In any embodiment of the method of the present invention, the patient treated with the composition of the present invention is In certain embodiments, the human patient is an infant (about 6 weeks to 12 months of age). In certain embodiments, a human patient is an infant (about 12-24 months) or a child (about 2-5 years). The compositions of the present invention are suitable for use in older children, adolescents and adults (e.g., 18-45 years of age). , 18-50, 18-55, 18-60 or 18-65 years old) In other embodiments of any of the methods of the invention, the patient is between about 2 years old and about 18 years old. In other embodiments of any of the methods of the invention, the patient is 18 years of age or older.

[0207] In another embodiment of the method of the invention, the patient is an infant and is administered the multivalent immunogenic composition of the invention. Administer 1, 2 or 3 doses of the composition to the infant. The length of time between administration of each dose can be varied. However, an example dosing schedule would be a dose administered at 2 months of age, followed by another dose at 4 months of age. The dosing schedule for infants includes administering a final dose at 6 months of age. Another example would be administering a dose at 2 months of age, followed by a further dose at 3 months of age. Another example of a dosing schedule for this would be to give a dose at 2 months of age, followed by a dose at 3 months of age. and finally a final dose at 6 months of age. may be administered additional "booster" doses of the multivalent immunogenic composition of the invention when the infant becomes a toddler. For example, an infant may receive a dose at 2 months of age, followed by a further dose at 4 months of age, Finally, at 6 months of age, a final dose is administered, and then, when the infant reaches toddler age, multiple doses of the present invention are administered. An additional "booster" dose of the monovalent immunogenic composition is administered at 11-15 months of age.

[0208] In one embodiment, two doses of a multivalent immunogenic composition of the invention are administered to an infant.

[0209] In one embodiment, three doses of a multivalent immunogenic composition of the invention are administered to an infant.

[0210] In one embodiment, three doses of the multivalent immunogenic composition of the invention are administered to a patient, The first and second doses were administered at 2 to 10 months of age, and the third dose was administered at 11 to 15 months of age. Give.

[0211] In one embodiment, four doses of the multivalent immunogenic composition of the invention are administered to a patient, The first dose was administered at 2 months of age, the second dose at 4 months of age, and the third dose at 6 months of age. and the fourth dose is administered at 11 to 15 months of age.

[0212] In another embodiment of the method of the invention, the human patient is elderly. In some embodiments of any of the methods of the invention, the patient is 50 years of age or older. In some embodiments, the patient is 55 years of age or older. In some embodiments, the patient is 60 years of age or older. In other embodiments, the patient is 65 years of age or older. In an embodiment, the patient is 70 years of age or older.

[0213] In some embodiments of any of the methods of the invention, the subject is treated with an immunogenic composition of the invention. The patient being treated is immunocompromised.

[0214] In some embodiments of any of the methods of the invention, the multivalent immunogenic composition of the invention In certain embodiments, the vaccine is administered in combination with a vaccine against influenza. The influenza vaccine is a "senior influenza vaccine," i.e. for older people, e.g. This is a high-dose influenza vaccine suitable for people aged 5 years and older.

[0215] The present invention relates to the multivalent immunogenic pneumococcal polysaccharide-protein conjugates described herein. A method for treating pneumococcal infection comprising administering to a patient an immunologically effective amount of any of the GATE compositions. The present invention provides a method for inducing a protective immune response in a patient against a particular vaccine (e.g. For example, optimal amounts of components for a multivalent immunogenic composition of the invention may be determined by determining whether or not adequate immunological responses are achieved in a subject. This can be confirmed by standard studies involving observation of responses. For example, in another embodiment, For vaccines, human vaccination doses are determined by extrapolation from animal studies to human data. In another embodiment, dosages are empirically determined.

[0216] The methods of the invention are applicable to the treatment of invasive infections (meningitis, pneumonia and bacteremia) and non-invasive infections. (acute otitis media and sinusitis) It is used to prevent and / or alleviate the major clinical symptoms caused by monier. It is possible.

[0217] Administration of the compositions of the present invention may be by injection via intramuscular, intraperitoneal, intradermal or subcutaneous routes; or by oral route. Administration may include one or more of oral / alimentary tract, respiratory or urogenital mucosal administration. In one embodiment, intranasal administration is used to treat pneumonia or otitis media (because Therefore, nasopharyngeal carriage of pneumococcus can be prevented more effectively and therefore the infection can be prevented at an early stage. In certain embodiments, the composition of the present invention is administered to the patient intramuscularly or subcutaneously.

[0218] All publications cited herein are incorporated by reference in their entirety for purposes of illustration only and are not intended to be limiting unless otherwise specified. The materials are incorporated herein by reference for purposes of description and disclosure.

[0219] Various embodiments of the present invention are described herein with reference to the accompanying drawings, in which: The present invention is not limited to those precise embodiments, but is intended to be illustrative and not restrictive of the invention as defined in the appended claims. Various changes and modifications may be made thereto without departing from the scope or spirit of the disclosure. It should be understood that the above may be performed by

[0220] The following examples are illustrative of the present invention and are not intended to be limiting thereof.

[0221] Example 1 Production of Streptococcus pneumoniae capsular polysaccharide Methods for culturing pneumococci are well known in the art. See, e.g., Chase, 1967, Meth. thods of Immunology and Immunochemistry 1:52. Methods for producing pneumococcal capsular polysaccharides are also well known in the art. See, for example, European Patent No. EP 0 497 524 B1. The process described below generally According to the method described in European Patent No. EP 0 497 524 B1, all Generally applicable to pneumococcal serotypes.

[0222] Serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14 , 15A, 15B, 18C, 19A, 19F, 22F, 23F, 33F and 35B lungs Isolates of Streptococcus pneumoniae strains were obtained from the Merck Culture Collection (Merck Culture Collection). The serotype 23B strain was obtained from the US Centers for Disease Control and Prevention ( Department of Disease Control and Prevention and the University of Alabama University of Alabama Birmingham Serotype 24F strains were obtained from the Merck Culture Collection (Merck CultureCollection and the University of Alabama at Birmingham (University of Alabama at Birmingham) The data was obtained from the City of Alabama Birmingham. To distinguish between subtypes, the Quellung reaction was performed using specific antisera. See, e.g., U.S. Patent No. 5,847,112. The resulting isolates were Contains soy peptone, yeast extract and glucose (no hemin) Further cloning was performed by successive plating on agar plates consisting of a nutrient-rich medium in two steps. In the case of serotype 7F, the agar plates used also contained hemin. Clonal isolates of each serotype were cultured in soy peptone, yeast extract, HEPES, and sodium chloride. Contains thorium, sodium bicarbonate, potassium phosphate, glucose and glycerol. Further expansion in liquid culture using animal component-free medium to create a pre-master cell bank was prepared.

[0223] The production of each serotype of pneumococcal polysaccharide involves cell growth and batch production fermentation followed by The primary purification step consisted of chemical inactivation prior to downstream purification. thawed cell bank vials of soy peptone or soy peptone ultrafiltrate, Yeast extract or yeast extract ultrafiltrate, HEPES, sodium chloride, sodium bicarbonate A pre-sterilized, animal component-free growth medium containing cellulose, potassium phosphate and glucose. The growth was carried out using shake flasks or culture bottles containing The mixture was incubated in a sealed shake flask or bottle with minimal gas exchange while controlling the temperature. The cell growth cultures were subjected to growth. During cell growth of these serotypes, temperature, pH, pressure and The mixing and agitation were controlled. Sparging was not used, so there was no airflow over the The overlay was also controlled. After a suitable confluency was obtained, a portion of the cell growth culture was cultured in soybean peptone or soybean peptone. Ammonium sulfate ultrafiltrate, yeast extract or yeast extract ultrafiltrate, sodium chloride, calcium phosphate A pre-sterilized, animal component-free growth medium containing sodium and glucose The temperature, pH, pressure and agitation were controlled. No sparging was used. This allows for controlled airflow overlay.

[0224] Once glucose is nearly consumed, the batch is deactivated by the addition of phenol, a chemical deactivator. The fermentation was terminated. Pure phenol was added to a final concentration of 0.8–1.2% to dissolve the cells in The cells were inactivated and the capsular polysaccharides were released from the cell walls. A primary inactivation is carried out, in which case the temperature and agitation are kept under control. After the primary inactivation, The mixture was transferred to another vessel and incubated at controlled temperature and agitation with additional identified This was achieved by maintaining the bacteria for a period of time, which resulted in complete inactivation. or by verification of phenol concentration and time specified. The inactivated broth was clarified.

[0225] Example 2 Purification of pneumococcal polysaccharides The purification process of pneumococcal polysaccharides involves several steps: centrifugation, depth filtration, concentration / diafiltration. The procedure consisted of a polymerization procedure and a precipitation step. The procedure was carried out at room temperature.

[0226] Inactivation broth from fermenter cultures of Streptococcus pneumoniae was treated with cationic Polymers [e.g., BPA-1000, TRETOLITE® (Baker Hughes Inc., Houston, TX), Spectrum 8160, Poly (ethyleneimine) and Millipore pDADMAC. The soluble polymer bound impurities, proteins, nucleic acids and cell debris. After this, the flocculated solids were removed by centrifugation and multiple depth filtration steps. Concentrate and use 100kDa~500kDa MWCO (molecular weight cut-off) filters The diafiltration was performed using Tris, MgCl Diafiltration was performed using 2 buffer and sodium phosphate buffer. This step removed residual nucleic acids and proteins.

[0227] Further removal of impurities was achieved by denaturing the polysaccharide in sodium acetate and phenol with alcohol and This was achieved by reprecipitation with phenol and / or isopropanol. During the process, sodium acetate and phenol (liquid) in sodium phosphate buffer were added. phenol (dichlorophenol or solid phenol) is added to the diafiltered retentate. The polysaccharides were then fractionated in two steps. In the first step, a low proportion of Alcohol is added to the preparation to precipitate cell debris and other unwanted impurities while preserving the crude The purified polysaccharide was allowed to remain in solution. Impurities were removed by centrifugation and a subsequent depth filtration step. Additional isopropanol or denatured alcohol was then added to the batch. The polysaccharide was recovered from the solution by centrifugation. The precipitated polysaccharide pellet was recovered and powdered. It was triturated, dried to a powder and stored frozen at -70°C.

[0228] Example 3 Serotype 1 Conjugates for PCV23(DMSO) Multivalent Studies Using DMSO Conjugation Production of jugate The polysaccharides are dissolved, sized to a target molecular weight, chemically activated, and purified by ultrafiltration through a buffer. The activated polysaccharide and purified CRM197 were separately lyophilized and washed with dimethyl sulfoxide. The polysaccharide solution was then redissolved in DMSO. The redissolved polysaccharide solution and CRM197 solution were then mixed together. The resulting conjugate was dialyzed and then conjugated as described below. After further purification, the product was subjected to a final 0.2 micron filtration. In order to obtain the desired results, several process parameters within each step, e.g. pH, temperature, concentration, and time was controlled.

[0229] Size reduction and oxidation of polysaccharides The purified pneumococcal Ps capsule powder was dissolved in water and filtered at 0.45 microns. The polysaccharide was homogenized to reduce the molecular weight of Ps. The number of passes through the homogenizer was controlled at 250 bar / 5 passes.

[0230] Size-reduced polysaccharides were concentrated and filtered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane. and diafiltered.

[0231] The polysaccharide solution was then adjusted to 22°C and pH 5 with sodium acetate buffer to activate The size reduction of polysaccharides due to the activation was minimized. The reaction was initiated by the addition of the thorium solution and allowed to proceed for 15 hours at 22°C.

[0232] The activated product was filtered using a 10 kDa NMWCO tangential flow ultrafiltration membrane with 10 mM phosphate buffer. The mixture was diafiltered against potassium chloride (pH 6.4) and then diafiltered with 5 kDa N The solution was diafiltered against water using a MWCO tangential flow ultrafiltration membrane. Filtration was carried out at 2 to 8°C.

[0233] Polysaccharide conjugation to CRM197 As previously described (WO 2012 / 173876 A1), Pseudomonas Expression in Pseudomonas fluorescens The purified CRM197 obtained by the above procedure was filtered using a 5 kDa NMWCO tangential flow ultrafiltration membrane. Diafilter against 2 mM phosphate (pH 7.2) buffer, Filtered at 0.2 micron.

[0234] Activated polysaccharide at 10% w / v sucrose concentration at 2.5 mg Ps / mL for lyophilization CRM197 was frozen at 6 mg Pr / mL in a 1% w / v sucrose concentration. It was formulated for freeze-drying.

[0235] The formulated Ps and CRM197 solutions were lyophilized separately. The CRM197 and CRM201 substances were separately redissolved in an equal volume of DMSO. The polysaccharide solution and CRM197 were mixed to obtain a polysaccharide concentration and a polysaccharide to CRM197 mass ratio of 1.5. The polysaccharide to CRM197 ratio in the resulting conjugate was controlled. The mass ratio was selected so that sodium cyanoborohydride (per mole of polysaccharide repeating unit) (1 mol per 1000 mg) was added and conjugation was allowed to proceed at 22°C.

[0236] Reduction with sodium borohydride After the conjugation reaction, sodium borohydride (2 moles per mole of polysaccharide repeating unit) was added. ) was added and incubated at 22°C for 1 hour. The batch was diluted in 150 mM sodium chloride containing 20 at about 4°C. The pH was neutralized by adding potassium phosphate buffer. The solution was prepared using 150 mM sodium chloride, 0.05% (w / v) polysorbate 20 The batch was dialyzed against 100% ethanol at about 4° C. for 3 days.

[0237] Final Filtration and Product Storage The batch was filtered to 0.2 microns (with a 0.5 micron prefilter) and divided into aliquots. The mixture was then frozen at -60°C or below.

[0238] Example 4 Serotype 1 Conjugation for PCV23 (DMSO+Aq) Multivalent Studies Using Aqueous Conjugation Manufacturing of conjugate The polysaccharides are dissolved, sized, chemically activated, and buffer exchanged by ultrafiltration. The purified CRM197 was then coupled to an activated polysaccharide in a reaction mixture using nickel chloride. The resulting conjugate was purified by ultrafiltration and then added to the final 0. Each step was repeated to obtain a conjugate with the desired properties. Several process parameters were controlled, such as pH, temperature, concentration and time.

[0239] Size reduction and oxidation of polysaccharides Purified pneumococcal capsular polysaccharide powder was dissolved in water and filtered at 0.45 microns. The polysaccharides were homogenized to reduce their molecular weight. The homogenization pressure was adjusted to reduce the size to the target molecular weight. The pressure and number of passes through the homogenizer were controlled at 250 bar / 5 passes. Small polysaccharides were concentrated and diafiltered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane. It was filtered.

[0240] The polysaccharide solution was then adjusted to 22°C and pH 5 with sodium acetate buffer to activate The size reduction of polysaccharides due to the activation was minimized. The reaction was initiated by the addition of the thorium solution and allowed to proceed for 15 hours at 22°C.

[0241] The activated product was filtered using a 10 kDa NMWCO tangential flow ultrafiltration membrane with 10 mM phosphate buffer. The ultrafiltration was performed at 2 to 8°C. So I went.

[0242] Polysaccharide conjugation to CRM197 The oxidized polysaccharide solution was mixed with water and 1.5 M potassium phosphate (pH 7.0). The selected buffer pH improves the stability of the activated polysaccharide during the conjugation reaction. As previously described (WO 2012 / 173876 A 1) Purified CRM197 obtained by expression in Pseudomonas fluorescens The resulting conjugate was combined with the buffered polysaccharide solution at a polysaccharide to CRM197 weight ratio of 0.5. The mass ratio was chosen so that the ratio of polysaccharide to CRM197 in the gel was controlled. The concentrations of phosphate were 6.9 g / L and 100 mM, respectively. The polysaccharide concentration was chosen so that the size of the conjugates could be controlled. The solution was used to add nickel chloride to approximately 2 mM. (2 moles per mole of polysaccharide repeating unit) was added. This maximizes the consumption of polysaccharide and protein. Conjugation was allowed to proceed for 120 hours as described above.

[0243] Reduction with sodium borohydride After the conjugation reaction, the batch was diluted to a polysaccharide concentration of approximately 3.5 g / L and cooled to 2-8°C. The solution was cooled and filtered at 1.2 microns using a 100 kDa NMWCO tangential flow ultrafiltration membrane. The batch was then diaphragmed against 100 mM potassium phosphate (pH 7.0) at 2-8°C. The batch recovered in the retentate was then diluted to approximately 2.0 g polysaccharide / L. The pH was adjusted by adding 1.2 M sodium bicarbonate (pH 9.4). Sodium urate (1 mole per mole of polysaccharide repeating unit) was added. Then 1.5M phosphate Potassium was added (pH 6.0).

[0244] Final Filtration and Product Storage The batch was then concentrated and filtered using a 300 kDa NMWCO tangential flow ultrafiltration membrane for 15 min. Permeability to 10 mM L-histidine in 0 mM sodium chloride (pH 7.0) at 4°C Polysorbate 20 was added to the retentate batch to a concentration of 0.05% (w / v) and the The filter was 0.2 micron filtered.

[0245] 150 mM sodium chloride ( The batch was adjusted to a polysaccharide concentration of 1.0 g / L with additional 10 mM L-histidine in 100 mL of 1000 mM NaCl (pH 7.0). The batch was divided into aliquots and frozen at -60°C or below.

[0246] Example 5 Preparation of serotype 1 conjugates for PCV22 multivalent studies using aqueous conjugation manufacturing The polysaccharides are dissolved, sized, chemically activated, and buffer exchanged by ultrafiltration. The purified CRM197 was then coupled to an activated polysaccharide in a reaction mixture using nickel chloride. The resulting conjugate was purified by ultrafiltration and then added to the final 0. Each step was repeated to obtain a conjugate with the desired properties. Several process parameters were controlled, such as pH, temperature, concentration and time.

[0247] Size reduction and oxidation of polysaccharides Purified pneumococcal capsular polysaccharide powder was dissolved in water and filtered at 0.45 microns. The polysaccharides were homogenized to reduce their molecular weight. The homogenization pressure was adjusted to reduce the size to the target molecular weight. The pressure and number of passes through the homogenizer were controlled at 250 bar / 5 passes. Small polysaccharides were concentrated and diafiltered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane. It was filtered.

[0248] The polysaccharide solution was then adjusted to 22°C and pH 5 with sodium acetate buffer to activate The size reduction of polysaccharides due to the activation was minimized. The reaction was initiated by the addition of the thorium solution and allowed to proceed for 15 hours at 22°C.

[0249] The activated product was filtered using a 10 kDa NMWCO tangential flow ultrafiltration membrane with 10 mM phosphate buffer. The ultrafiltration was performed at 2 to 8°C. So I went.

[0250] Polysaccharide conjugation to CRM197 The oxidized polysaccharide solution was mixed with water and 1.5 M potassium phosphate (pH 7.0). The selected buffer pH improves the stability of the activated polysaccharide during the conjugation reaction. As previously described (WO 2012 / 173876 A 1) Purified CRM197 obtained by expression in Pseudomonas fluorescens The resulting conjugate was combined with the buffered polysaccharide solution at a polysaccharide to CRM197 weight ratio of 0.5. The mass ratio was chosen so that the ratio of polysaccharide to CRM197 in the gel was controlled. The concentrations of phosphate were 6.9 g / L and 100 mM, respectively. The polysaccharide concentration was selected so that the size of the conjugates was controlled. The nickel chloride was diluted to approximately 2 mM using a 100 mM nickel chloride solution. Sodium cyanoborohydride (2 moles per mole of polysaccharide repeat unit) was added. Conjugation was allowed to proceed for 120 hours to maximize polysaccharide and protein consumption. I did.

[0251] Reduction with sodium borohydride After the conjugation reaction, the batch was diluted to a polysaccharide concentration of approximately 3.5 g / L and cooled to 2-8°C. The solution was cooled and filtered at 1.2 microns using a 100 kDa NMWCO tangential flow ultrafiltration membrane. The batch was then diaphragmed against 100 mM potassium phosphate (pH 7.0) at 2-8°C. The batch recovered in the retentate was then diluted to approximately 2.0 g polysaccharide / L. The pH was adjusted by adding 1.2 M sodium bicarbonate (pH 9.4). Sodium urate (1 mole per mole of polysaccharide repeating unit) was added. Then 1.5M phosphate Potassium was added (pH 6.0).

[0252] Final Filtration and Product Storage The batch was then concentrated and filtered using a 300 kDa NMWCO tangential flow ultrafiltration membrane for 15 min. Permeability to 10 mM L-histidine in 0 mM sodium chloride (pH 7.0) at 4°C Polysorbate 20 was added to the retentate batch to a concentration of 0.05% (w / v) and the The filter was 0.2 micron filtered.

[0253] 150 mM sodium chloride ( The batch was adjusted to a polysaccharide concentration of 1.0 g / L with additional 10 mM L-histidine in 100 mL of 1000 mM NaCl (pH 7.0). The batch was divided into aliquots and frozen at -60°C or below.

[0254] Example 6 Serotype 3 Conjugates for PCV23(DMSO) Multivalent Studies Using DMSO Conjugation Production of jugate The polysaccharides are dissolved, sized to the target molecular weight, chemically activated, and purified by ultrafiltration through a buffer. The activated polysaccharide and purified CRM197 were separately lyophilized and washed with dimethyl sulfoxide. The polysaccharide solution was then redissolved in DMSO. The redissolved polysaccharide solution and CRM197 solution were then mixed together. were combined and conjugated as described below. The resulting conjugate was purified by ultrafiltration. The conjugates were purified by filtration and then subjected to a final 0.2 micron filtration. To obtain the gate, several process parameters within each step, such as pH, temperature, Concentration and time were controlled.

[0255] Size reduction and oxidation of polysaccharides The purified pneumococcal Ps capsule powder was dissolved in water and filtered at 0.45 microns. The polysaccharide was homogenized to reduce the molecular weight of Ps. The number of passes was controlled to 810 bar / 6 passes, then 900 bar / 3 passes.

[0256] Size-reduced polysaccharides were concentrated and filtered against water using a 5 kDa NMWCO tangential flow ultrafiltration membrane. The mixture was diafiltered.

[0257] The polysaccharide solution was then adjusted to 22°C and pH 5 with sodium acetate buffer to activate The size reduction of polysaccharides due to the activation was minimized. The reaction was initiated by the addition of the thorium solution and allowed to proceed for 12 hours at 22°C.

[0258] The activated product was diafiltered against 10 mM potassium phosphate (pH 6.4). The mixture was then diaphragmed against water using a 5 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was carried out at 2-8°C.

[0259] Polysaccharide conjugation to CRM197 As previously described (WO 2012 / 173876 A1), Pseudomonas Purified CRM197 expressed in C. fluorescens was analyzed using a 5 kDa NMW CO tangential flow ultrafiltration was used to filter 2 mM phosphate (pH 7.2) buffer. The solution was diafiltered through 0.2 microns.

[0260] Activated polysaccharide at 10% w / v sucrose concentration at 2.5 mg Ps / mL for lyophilization CRM197 was frozen at 6 mg Pr / mL in a 1% w / v sucrose concentration. It was formulated for freeze-drying.

[0261] The formulated Ps and CRM197 solutions were lyophilized separately. and CRM197 material were separately redissolved in an equal volume of DMSO. The polysaccharide solution and CRM197 were mixed to obtain a polysaccharide concentration and a polysaccharide to CRM197 mass ratio of 1.35. The polysaccharide to CRM197 ratio in the resulting conjugate was controlled. The mass ratio was selected so that the saccharide repeat unit was controlled. (1 mole per unit) was added and conjugation was allowed to proceed at 22°C.

[0262] Reduction with sodium borohydride After the conjugation reaction, sodium borohydride (2 moles per mole of polysaccharide repeating unit) was added. ) was added and incubated at 22°C for 1 hour. The batch was diluted in 150 mM sodium chloride containing 20 at about 4°C. Potassium phosphate buffer was added to neutralize the pH.

[0263] Final Filtration and Product Storage The batch was then concentrated and filtered using a 300 kDa NMWCO tangential flow ultrafiltration membrane to a concentration of 0. 150 mM sodium chloride (pH 7) containing 15% (w / v) polysorbate 20 The solution was diafiltered against 10 mM histidine in 0.5% NaCl (pH 7.0) at 4°C.

[0264] The retentate batch was filtered to 0.2 microns (with a 0.5 micron prefilter) and then 150 mM sodium chloride (pCl) containing 0.015% (w / v) polysorbate 20 The solution was diluted with additional 10 mM histidine in 100 mM NaCl (H7.0), dispensed into aliquots and stored at -60°C. Frozen below.

[0265] Example 7 Polyvalent Synthesis of PCV22 and PCV23 (DMSO+Aq) Using Aqueous Conjugation Production of serotype 3 conjugates for research The polysaccharides are dissolved, sized, chemically activated, and buffer exchanged by ultrafiltration. The purified CRM197 was then coupled to an activated polysaccharide in a reaction mixture using nickel chloride. The resulting conjugate was purified by ultrafiltration and then added to the final 0. Each step was repeated to obtain a conjugate with the desired properties. Several process parameters were controlled, such as pH, temperature, concentration and time.

[0266] Size reduction and oxidation of polysaccharides Purified pneumococcal capsular polysaccharide powder was dissolved in water and filtered at 0.45 microns. The polysaccharides were homogenized to reduce their molecular weight. The homogenization pressure was adjusted to reduce the size to the target molecular weight. The pressure and number of passes through the homogenizer were controlled at 380 bar / 5 passes. Small polysaccharides were concentrated and diafiltered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane. It was filtered.

[0267] The polysaccharide solution was then adjusted to 22°C and pH 5 with sodium acetate buffer to activate The size reduction of polysaccharides due to the activation was minimized. The reaction was initiated by the addition of the thorium solution and allowed to proceed for 12 hours at 22°C.

[0268] The activated product was filtered using a 10 kDa NMWCO tangential flow ultrafiltration membrane with 10 mM phosphate buffer. The ultrafiltration was performed at 2 to 8°C. So I went.

[0269] Polysaccharide conjugation to CRM197 The oxidized polysaccharide solution was mixed with water and 1.5 M potassium phosphate (pH 6.0). The selected buffer pH improves the stability of the activated polysaccharide during the conjugation reaction. As previously described (WO 2012 / 173876 A 1) Purified CRM197 obtained by expression in Pseudomonas fluorescens 0.2 micron filtered and combined with the buffered polysaccharide solution at a polysaccharide to CRM197 weight ratio of 0.6. The mass ratio was adjusted so that the polysaccharide to CRM197 ratio in the resulting conjugate was controlled. The concentrations of polysaccharide and phosphate were 4.1 g / L and 150 mM, respectively. The polysaccharide concentration was selected so as to control the size of the resulting conjugate. The solution was then filtered through a 0.2 micron filter. Nickel was added to approximately 2 mM. Sodium cyanoborohydride (per mole of polysaccharide repeating unit) The conjugates were added at 0.25 g / ml for maximum consumption of polysaccharide and protein. The polymerization was allowed to proceed for 120 hours at 10°C.

[0270] Reduction with sodium borohydride After the conjugation reaction, the batch was diluted to a polysaccharide concentration of approximately 3.5 g / L and cooled to 2-8°C. The solution was cooled and filtered at 1.2 microns using a 100 kDa NMWCO tangential flow ultrafiltration membrane. The batch was then diaphragmed against 100 mM potassium phosphate (pH 7.0) at 2-8°C. The batch recovered in the retentate was then diluted to approximately 2.0 g polysaccharide / L. The pH was adjusted by adding 1.2 M sodium bicarbonate (pH 9.4). Sodium urate (1 mole per mole of polysaccharide repeating unit) was added. Then 1.5M phosphate Potassium was added (pH 6.0).

[0271] Final Filtration and Product Storage The batch was then concentrated and filtered using a 300 kDa NMWCO tangential flow ultrafiltration membrane for 15 min. Permeability to 10 mM L-histidine in 0 mM sodium chloride (pH 7.0) at 4°C The batch was filtered at 0.2 micron.

[0272] Additional 10 mM L-hyaluronan in 150 mM sodium chloride (pH 7.0) buffer. The batch was adjusted with stigmine to a polysaccharide concentration of 1.0 g / L. The batch was divided into aliquots and Frozen at -60°C or below.

[0273] Example 8 Serotype 4 Conjugates for PCV23(DMSO) Multivalent Studies Using DMSO Conjugation Production of jugate The polysaccharides are dissolved, sized to the target molecular weight, chemically activated, and purified by ultrafiltration through a buffer. The activated polysaccharide and purified CRM197 were separately lyophilized and redissolved in DMSO. The reconstituted polysaccharide solution was then combined with the CRM197 solution and mixed as described below. The resulting conjugate was purified by dialysis and then added to the final 0. Each step was repeated to obtain a conjugate with the desired properties. Several process parameters were controlled, such as pH, temperature, concentration and time.

[0274] Size reduction and oxidation of polysaccharides The purified pneumococcal Ps capsule powder was dissolved in water and filtered at 0.45 microns. The polysaccharide was homogenized to reduce the molecular weight of Ps. The number of passes was controlled at 300 bar / 5 passes.

[0275] Size-reduced polysaccharides were concentrated and filtered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane. and diafiltered.

[0276] The polysaccharide solution was then adjusted to 50°C and pH 4.1 with sodium acetate buffer to obtain polysaccharides. The sugars were partially deketalized. The polysaccharide solution was then cooled to 22°C before activation. Activation was initiated by the addition of 100 mM sodium metaperiodate solution. was allowed to proceed for 4 hours at 22°C.

[0277] The activated product was filtered using a 10 kDa NMWCO tangential flow ultrafiltration membrane with 10 mM phosphate buffer. The mixture was diafiltered against potassium chloride (pH 6.4) and then diafiltered with 5 kDa N The solution was diafiltered against water using a MWCO tangential flow ultrafiltration membrane. Filtration was carried out at 2 to 8°C.

[0278] Polysaccharide conjugation to CRM197 As previously described (WO 2012 / 173876 A1), Pseudomonas Purified CRM197 expressed in C. fluorescens was analyzed using a 5 kDa NMW CO tangential flow ultrafiltration was used to filter 2 mM phosphate (pH 7.2) buffer. The solution was diafiltered through 0.2 microns.

[0279] Activated polysaccharides are formulated for lyophilization at 6 mg Ps / mL in a 5% w / v sucrose concentration CRM197 was lyophilized at 6 mg Pr / mL with a 1% w / v sucrose concentration. It was formulated for use.

[0280] The formulated Ps and CRM197 solutions were lyophilized separately. The CRM197 and CRM201 substances were separately redissolved in an equal volume of DMSO. The polysaccharide solution and CRM197 were mixed to obtain a polysaccharide concentration and a polysaccharide to CRM197 mass ratio of 2.0. The polysaccharide to CRM197 ratio in the resulting conjugate was controlled. The mass ratio was selected so that sodium cyanoborohydride (per mole of polysaccharide repeating unit) (1 mol per 1000 mg) was added and conjugation was allowed to proceed at 22°C.

[0281] Reduction with sodium borohydride After the conjugation reaction, sodium borohydride (2 moles per mole of polysaccharide repeating unit) was added. ) was added and incubated at 22°C for 1 hour. The batch was diluted in 150 mM sodium chloride containing 20 at about 4°C. The pH was neutralized by adding potassium phosphate buffer. The solution was prepared using 150 mM sodium chloride, 0.05% (w / v) polysorbate 20 The batch was dialyzed against 100% ethanol at about 4° C. for 3 days.

[0282] Final Filtration and Product Storage The batch was filtered to 0.2 microns (with a 0.5 micron prefilter) and divided into aliquots. The mixture was then frozen at -60°C or below.

[0283] Example 9 PCV23 (DMSO+Aq) and PCV22 polyvalent lab using aqueous conjugation Production of serotype 4 conjugates for research The polysaccharides are dissolved, sized, chemically activated, and buffer exchanged by ultrafiltration. The purified CRM197 was then coupled to an activated polysaccharide in a reaction mixture using nickel chloride. The resulting conjugate was purified by ultrafiltration and then added to the final 0. Each step was repeated to obtain a conjugate with the desired properties. Several process parameters were controlled, such as pH, temperature, concentration and time.

[0284] Size reduction and oxidation of polysaccharides Purified pneumococcal capsular polysaccharide powder was dissolved in water and filtered at 0.45 microns. The polysaccharides were homogenized to reduce their molecular weight. The homogenization pressure was adjusted to reduce the size to the target molecular weight. The pressure and number of passes through the homogenizer were controlled at 300 bar / 5 passes. Small polysaccharides were concentrated and diafiltered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane. It was filtered.

[0285] The polysaccharide solution was then adjusted to 50°C and pH 4.1 with sodium acetate buffer to obtain polysaccharides. The sugars were partially deketalized. The polysaccharide solution was then cooled to 22°C before activation. Activation was initiated by the addition of 100 mM sodium metaperiodate solution. was allowed to proceed for 4 hours at 22°C.

[0286] The activated product was filtered using a 10 kDa NMWCO tangential flow ultrafiltration membrane with 10 mM phosphate buffer. The ultrafiltration was performed at 2 to 8°C. So I went.

[0287] Polysaccharide conjugation to CRM197 The oxidized polysaccharide solution was mixed with water and 1.5 M potassium phosphate (pH 7.0). The selected buffer pH improves the stability of the activated polysaccharide during the conjugation reaction. As previously described (WO 2012 / 173876 A 1) Purified CRM197 obtained by expression in Pseudomonas fluorescens 0.2 micron filtered and combined with the buffered polysaccharide solution at a polysaccharide to CRM197 weight ratio of 0.5 The mass ratio was adjusted so that the polysaccharide to CRM197 ratio in the resulting conjugate was controlled. The concentrations of polysaccharide and phosphate were 8.3 g / L and 100 mM, respectively. The polysaccharide concentration was selected so as to control the size of the resulting conjugate. The solution was filtered at 0.2 microns using 100 mM nickel chloride solution. Nickel was added to approximately 2 mM. Sodium cyanoborohydride (per mole of polysaccharide repeating unit) was added. The conjugate was added at 200 mL / min to maximize the consumption of polysaccharide and protein. The polymerization was allowed to proceed for 120 hours.

[0288] Reduction with sodium borohydride After the conjugation reaction, the batch was diluted to a polysaccharide concentration of approximately 3.5 g / L and cooled to 2-8°C. The solution was cooled and filtered at 1.2 microns using a 100 kDa NMWCO tangential flow ultrafiltration membrane. The batch was then diaphragmed against 100 mM potassium phosphate (pH 7.0) at 2-8°C. The batch recovered in the retentate was then diluted to approximately 2.0 g polysaccharide / L. The pH was adjusted by adding 1.2 M sodium bicarbonate (pH 9.4). Sodium urate (1 mole per mole of polysaccharide repeating unit) was added. Then 1.5M phosphate Potassium was added (pH 6.0).

[0289] Final Filtration and Product Storage The batch was then concentrated and filtered using a 300 kDa NMWCO tangential flow ultrafiltration membrane for 15 min. Permeability to 10 mM L-histidine in 0 mM sodium chloride (pH 7.0) at 4°C The batch was then 0.2 micron filtered.

[0290] Additional 10 mM L-hyaluronan in 150 mM sodium chloride (pH 7.0) buffer. The batch was adjusted with stigmine to a polysaccharide concentration of 1.0 g / L. The batch was divided into aliquots and Frozen at -60°C or below.

[0291] Example 10 Serotype 5 Conjugates for PCV23(DMSO) Multivalent Studies Using DMSO Conjugation Production of jugate The polysaccharides are dissolved, sized to the target molecular weight, chemically activated, and purified by ultrafiltration through a buffer. The activated polysaccharide and purified CRM197 were separately lyophilized and redissolved in DMSO. The reconstituted polysaccharide solution was then combined with the CRM197 solution and mixed as described below. The resulting conjugate was purified by dialysis and then added to the final 0. Each step was repeated to obtain a conjugate with the desired properties. Several process parameters were controlled, such as pH, temperature, concentration and time.

[0292] Size reduction and oxidation of polysaccharides The purified pneumococcal Ps capsule powder was dissolved in water and filtered at 0.45 microns. The polysaccharide was homogenized to reduce the molecular weight of Ps. The number of passes was controlled to 600 bar / 5 passes.

[0293] Size-reduced polysaccharides were concentrated and filtered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane. and diafiltered.

[0294] The polysaccharide solution was then adjusted to 4°C and pH 4.1 with sodium acetate buffer to determine the activity. The size reduction of polysaccharides due to activation was minimized. The reaction was initiated by the addition of sodium solution and allowed to proceed for 4 hours at 4°C.

[0295] The activated product was filtered using a 10 kDa NMWCO tangential flow ultrafiltration membrane in 10 mM acetic acid Diafiltered against sodium (pH 4.1) and then 5 kDa N The solution was diafiltered against water using a MWCO tangential flow ultrafiltration membrane. Filtration was carried out at 2 to 8°C.

[0296] Polysaccharide conjugation to CRM197 As previously described (WO 2012 / 173876 A1), Pseudomonas Purified CRM197 expressed in C. fluorescens was analyzed using a 5 kDa NMW CO tangential flow ultrafiltration was used to filter 2 mM phosphate (pH 7.2) buffer. The solution was diafiltered through 0.2 microns.

[0297] Activated polysaccharides are formulated for lyophilization at 6 mg Ps / mL in a 5% w / v sucrose concentration CRM197 was lyophilized at 6 mg Pr / mL with a 1% w / v sucrose concentration. It was formulated for use.

[0298] The formulated Ps and CRM197 solutions were lyophilized separately. The polysaccharide solution was dissolved in an equal volume of DMSO. Polysaccharide concentration was 2.0 g Ps / L and 1.0 g Ps / L. The polysaccharide solution was mixed with the CRM197 solution to obtain a polysaccharide to CRM197 mass ratio of 0.5. The quality control was carried out so that the polysaccharide to CRM197 ratio in the resulting conjugate was controlled. The ratio of sodium cyanoborohydride (1 mole per mole of polysaccharide repeating unit) was chosen. and conjugation was allowed to proceed at 22°C.

[0299] Dilution and Neutralization 150 mM sodium chloride containing approximately 0.025% (w / v) polysorbate 20 The batch was diluted at about 4°C during the dilution. Potassium phosphate buffer was then added to neutralize the pH. A 300 kDa NMWCO dialysis cassette was used to dialyze 150 mM sodium chloride, The batch was dialysed against 0.05% (w / v) polysorbate 20 at approximately 4° C. for 3 days.

[0300] Final Filtration and Product Storage The retentate batch was filtered to 0.2 microns (through a 0.5 micron prefilter) and aliquoted. The mixture was dispensed into aliquots and frozen at -60°C or below.

[0301] Example 11 Polyvalent Synthesis of PCV22 and PCV23 (DMSO+Aq) Using Aqueous Conjugation Production of serotype 5 conjugates for research The polysaccharides are dissolved, sized, chemically activated, and buffer exchanged by ultrafiltration. The purified CRM197 was then coupled to an activated polysaccharide in a reaction mixture using nickel chloride. The resulting conjugate was purified by ultrafiltration and then added to the final 0. Each step was repeated to obtain a conjugate with the desired properties. Several process parameters were controlled, such as pH, temperature, concentration and time.

[0302] Size reduction and oxidation of polysaccharides Purified pneumococcal capsular polysaccharide powder was dissolved in water and filtered at 0.45 microns. The polysaccharides were homogenized to reduce their molecular weight. The homogenization pressure was adjusted to reduce the size to the target molecular weight. The pressure and number of passes through the homogenizer were controlled at 600 bar / 5 passes. Small polysaccharides were concentrated and diafiltered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane. It was filtered.

[0303] The polysaccharide solution was then adjusted to 4°C and pH 4.1 with sodium acetate buffer to determine the activity. The size reduction of polysaccharides due to activation was minimized. The reaction was initiated by the addition of sodium solution and allowed to proceed for 4 hours at 4°C.

[0304] The activated product was filtered using a 10 kDa NMWCO tangential flow ultrafiltration membrane in 10 mM acetic acid. The diafiltration was performed against sodium (pH 4.1). Ultrafiltration was performed at 2-8°C. So I went.

[0305] Polysaccharide conjugation to CRM197 The oxidized polysaccharide solution was mixed with water and 1.5 M potassium phosphate (pH 6.0). The selected buffer pH improves the stability of the activated polysaccharide during the conjugation reaction. As previously described (WO 2012 / 173876 A 1) Purified CRM197 obtained by expression in Pseudomonas fluorescens The resulting conjugate was combined with the buffered polysaccharide solution at a polysaccharide to CRM197 weight ratio of 0.4. The mass ratio was chosen so that the ratio of polysaccharide to CRM197 in the gel was controlled. The concentrations of phosphate were 3.8 g / L and 150 mM, respectively. The polysaccharide concentration was selected so that the size of the conjugates was controlled. The nickel chloride was diluted to approximately 2 mM using a 100 mM nickel chloride solution. Sodium cyanoborohydride (2 moles per mole of polysaccharide repeat unit) was added. Conjugation was allowed to proceed for 96 hours to maximize polysaccharide and protein consumption. Ta.

[0306] Purification and Neutralization After the conjugation reaction, the batch was diluted to a polysaccharide concentration of approximately 3.5 g / L and cooled to 2-8°C. The solution was cooled and filtered at 1.2 microns using a 100 kDa NMWCO tangential flow ultrafiltration membrane. The batch was then diaphragmed against 300 mM sodium bicarbonate (pH 9.3) at 2-8°C. The batch recovered in the retentate was then filtered with 1.5M potassium phosphate. The solution was neutralized with 100% alcohol (pH 6.0).

[0307] Final Filtration and Product Storage The batch was then concentrated and filtered using a 300 kDa NMWCO tangential flow ultrafiltration membrane for 15 min. Permeability to 10 mM L-histidine in 0 mM sodium chloride (pH 7.0) at 4°C Polysorbate 20 was added to the retentate batch to a concentration of 0.05% (w / v) and the The filter was 0.2 micron filtered.

[0308] 150 mM sodium chloride ( The batch was adjusted to a polysaccharide concentration of 1.0 g / L with an additional 10 mM L-histidine in 100 mL of 1000 mM NaCl (pH 7.0). The batch was divided into aliquots and frozen at -60°C or below.

[0309] Example 12 PCV23(DMSO) and PCV23(DMS) using DMSO conjugation O+Aq) and production of serotype 6A conjugates for PCV22 multivalent studies The polysaccharides were dissolved, chemically activated, and buffer exchanged by ultrafiltration. and purified CRM197 were separately lyophilized and redissolved in DMSO. The polysaccharide solution and CRM197 solution were combined and conjugated as described below. The resulting conjugate was purified by ultrafiltration and then subjected to a final 0.2 micron filtration. Several process steps within each step are optimized to obtain conjugates with desired properties. Parameters such as pH, temperature, concentration and time were controlled.

[0310] Size reduction and oxidation of polysaccharides The purified pneumococcal Ps capsule powder was dissolved in water and filtered at 0.45 microns. The polysaccharide was homogenized to reduce the molecular weight of Ps. The number of passes was controlled at 200 bar / 5 passes. The size-reduced polysaccharide was concentrated and reduced to 10 kDa NM. The solution was diafiltered against water using a WCO tangential flow ultrafiltration membrane.

[0311] The polysaccharide solution was then adjusted to 22°C and pH 5 with sodium acetate buffer to activate The size reduction of polysaccharides due to the activation was minimized. The reaction was initiated by the addition of the thorium solution and allowed to proceed for 2 hours at 22°C.

[0312] The activated product was diafiltered against 10 mM potassium phosphate (pH 6.4). The mixture was then diaphragmed against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was carried out at 2-8°C.

[0313] Polysaccharide conjugation to CRM197 As previously described (WO 2012 / 173876 A1), Pseudomonas Purified CRM197 expressed in C. fluorescens was analyzed using a 5 kDa NMW CO tangential flow ultrafiltration was used to filter 2 mM phosphate (pH 7.2) buffer. The mixture was diafiltered.

[0314] Activated polysaccharides are formulated for lyophilization at 6 mg Ps / mL in a 5% w / v sucrose concentration CRM197 was lyophilized at 6 mg Pr / mL with a 1% w / v sucrose concentration. It was formulated for use.

[0315] The formulated Ps and CRM197 solutions were lyophilized separately. The CRM197 and CRM201 substances were separately redissolved in an equal volume of DMSO. The polysaccharide solution and CRM197 were mixed to obtain a polysaccharide concentration and a polysaccharide to CRM197 mass ratio of 1.4. The polysaccharide to CRM197 ratio in the resulting conjugate was controlled. The mass ratio was selected so that sodium cyanoborohydride (per mole of polysaccharide repeating unit) (1 mol per 1000 mg) was added and conjugation was allowed to proceed at 22°C.

[0316] Reduction with sodium borohydride After the conjugation reaction, sodium borohydride (2 moles per mole of polysaccharide repeating unit) was added. ) was added and incubated at 22°C for 3 hours. The batch was then diluted with 150 mM sodium chloride containing phosphate 20 at approximately 4°C. The pH was neutralized by adding potassium triphosphate buffer. The batch was concentrated and 30 kDa NMWC Using a tangential flow ultrafiltration membrane, 150 mM sodium chloride, 25 mM potassium phosphate The solution was diafiltered against 100% ethanol (pH 7) at approximately 4°C.

[0317] Final Filtration and Product Storage The batch was then concentrated and filtered using a 300 kDa NMWCO tangential flow ultrafiltration membrane to a concentration of 0. 150 mM sodium chloride (pH 7) containing 15% (w / v) polysorbate 20 The solution was diafiltered against 10 mM histidine in 0.5% NaCl (pH 7.0) at 4°C.

[0318] The retentate batch was filtered at 0.2 microns and then diluted with 0.015% (w / v) Polysorbate. An additional 10 mM in 150 mM sodium chloride (pH 7.0) containing phosphate 20 The mixture was diluted with histidine, dispensed into aliquots, and frozen at -60°C or below.

[0319] Example 13 PCV23(DMSO) and PCV23(DMS) using DMSO conjugation Preparation of serotype 6B conjugates for (O / Aq) multivalent studies The polysaccharides are dissolved, sized to the target molecular weight, chemically activated, and purified by ultrafiltration through a buffer. The activated polysaccharide and purified CRM197 were separately lyophilized and redissolved in DMSO. The reconstituted polysaccharide solution was then combined with the CRM197 solution and mixed as described below. The resulting conjugate was purified by ultrafiltration and then Each was filtered through a 0.2 micron filter to obtain a conjugate with the desired properties. Several process parameters were controlled within the process, such as pH, temperature, concentration and time.

[0320] Size reduction and oxidation of polysaccharides The purified pneumococcal Ps capsule powder was dissolved in water and filtered at 0.45 microns. The polysaccharide was homogenized to reduce the molecular weight of Ps. The number of passes was controlled at 200 bar / 5 passes.

[0321] Size-reduced polysaccharides were concentrated and filtered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane. and diafiltered.

[0322] The polysaccharide solution was then adjusted to 22°C and pH 5 with sodium acetate buffer to activate The size reduction of polysaccharides due to the activation was minimized. The reaction was initiated by the addition of the thorium solution and allowed to proceed for 2 hours at 22°C.

[0323] The activated product was diafiltered against 10 mM potassium phosphate (pH 6.4). The mixture was then diaphragmed against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was carried out at 2-8°C.

[0324] Polysaccharide conjugation to CRM197 As previously described (WO 2012 / 173876 A1), Pseudomonas Purified CRM197 expressed in C. fluorescens was analyzed using a 5 kDa NMW CO tangential flow ultrafiltration was used to filter 2 mM phosphate (pH 7.2) buffer. The solution was diafiltered through 0.2 microns.

[0325] Activated polysaccharides are formulated for lyophilization at 6 mg Ps / mL in a 5% w / v sucrose concentration CRM197 was lyophilized at 6 mg Pr / mL with a 1% w / v sucrose concentration. It was formulated for use.

[0326] The formulated Ps and CRM197 solutions were lyophilized separately. and CRM197 material were separately redissolved in an equal volume of DMSO. The polysaccharide solution and CRM197 were mixed to obtain a polysaccharide concentration and a polysaccharide to CRM197 mass ratio of 1.35. The polysaccharide to CRM197 ratio in the resulting conjugate was controlled. The mass ratio was selected so that the saccharide repeat unit was controlled. (1 mole per unit) was added and conjugation was allowed to proceed at 22°C.

[0327] Reduction with sodium borohydride After the conjugation reaction, sodium borohydride (2 moles per mole of polysaccharide repeating unit) was added. ) was added and incubated at 22°C for 3 hours. The batch was diluted in 150 mM sodium chloride containing 20 at about 4°C. The pH was neutralized by adding potassium phosphate buffer. The batch was concentrated to a concentration of 30 kDa NMW. CO tangential flow ultrafiltration was performed using a 150 mM sodium chloride, 25 mM potassium phosphate The solution was diafiltered against 100 ml of ethanol (pH 7) at approximately 4°C.

[0328] Final Filtration and Product Storage The batch was then concentrated and filtered using a 300 kDa NMWCO tangential flow ultrafiltration membrane to a concentration of 0. 150 mM sodium chloride (pH 7) containing 15% (w / v) polysorbate 20 The solution was diafiltered against 10 mM histidine in 0.5% NaCl (pH 7.0) at 4°C.

[0329] The retentate batch was filtered at 0.2 microns and then diluted with 0.015% (w / v) Polysorbate. An additional 10 mM in 150 mM sodium chloride (pH 7.0) containing phosphate 20 The mixture was diluted with histidine, dispensed into aliquots, and frozen at -60°C or below.

[0330] Example 14 Serotype 6B conjugates for PCV22 multivalent studies using DMSO conjugation Manufacturing of The polysaccharides are dissolved, sized to the target molecular weight, chemically activated, and purified by ultrafiltration through a buffer. The activated polysaccharide and purified CRM197 were separately lyophilized and redissolved in DMSO. The reconstituted polysaccharide solution was then combined with the CRM197 solution and mixed as described below. The resulting conjugate was purified by ultrafiltration and then Each was filtered through a 0.2 micron filter to obtain a conjugate with the desired properties. Several process parameters were controlled within the process, such as pH, temperature, concentration and time.

[0331] Size reduction and oxidation of polysaccharides The purified pneumococcal Ps capsule powder was dissolved in water and filtered at 0.45 microns. The polysaccharide was homogenized to reduce the molecular weight of Ps. The number of passes was controlled at 200 bar / 5 passes. The size-reduced polysaccharide was concentrated and reduced to 10 kDa NM. The solution was diafiltered against water using a WCO tangential flow ultrafiltration membrane.

[0332] The polysaccharide solution was then adjusted to 22°C and pH 5 with sodium acetate buffer to activate The size reduction of polysaccharides due to the activation was minimized. The reaction was initiated by the addition of the thorium solution and allowed to proceed for 2 hours at 22°C.

[0333] The activated product was diafiltered against 10 mM potassium phosphate (pH 6.4). The mixture was then diaphragmed against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was carried out at 2-8°C.

[0334] Polysaccharide conjugation to CRM197 As previously described (WO 2012 / 173876 A1), Pseudomonas Purified CRM197 expressed in C. fluorescens was analyzed using a 5 kDa NMW CO tangential flow ultrafiltration was used to filter 2 mM phosphate (pH 7.2) buffer. The solution was diafiltered through 0.2 microns.

[0335] Activated polysaccharides are formulated for lyophilization at 6 mg Ps / mL in a 5% w / v sucrose concentration CRM197 was lyophilized at 6 mg Pr / mL with a 1% w / v sucrose concentration. It was formulated for use.

[0336] The formulated Ps and CRM197 solutions were lyophilized separately. and CRM197 substances were separately redissolved in an equal volume of DMSO. The polysaccharide solution and CRM197 were mixed to obtain a polysaccharide concentration and a polysaccharide to CRM197 mass ratio of 1.35. The polysaccharide to CRM197 ratio in the resulting conjugate was controlled. The mass ratio was selected so that the saccharide repeat unit was controlled. (1 mole per unit) was added and conjugation was allowed to proceed at 22°C.

[0337] Reduction with sodium borohydride After the conjugation reaction, sodium borohydride (2 moles per mole of polysaccharide repeating unit) was added. ) was added and incubated at 22°C for 3 hours. The batch was diluted in 150 mM sodium chloride containing 20 at about 4°C. The pH was neutralized by adding potassium phosphate buffer. The batch was concentrated to a concentration of 30 kDa NMW. CO tangential flow ultrafiltration was performed using a 150 mM sodium chloride, 25 mM potassium phosphate The solution was diafiltered against 100 ml of ethanol (pH 7) at approximately 4°C.

[0338] Final Filtration and Product Storage The batch was then concentrated and filtered using a 300 kDa NMWCO tangential flow ultrafiltration membrane to a concentration of 0. 150 mM sodium chloride (pH 7) containing 15% (w / v) polysorbate 20 The solution was diafiltered against 10 mM histidine in 0.5% NaCl (pH 7.0) at 4°C.

[0339] The retentate batch was filtered at 0.2 microns and then diluted with 0.015% (w / v) Polysorbate. An additional 10 mM in 150 mM sodium chloride (pH 7.0) containing phosphate 20 The mixture was diluted with histidine, dispensed into aliquots, and frozen at -60°C or below.

[0340] Example 15 PCV23(DMSO) and PCV23(DMS) using DMSO conjugation Preparation of serotype 7F conjugates for (O+Aq) multivalent studies The polysaccharides are dissolved, sized to the target molecular weight, chemically activated, and purified by ultrafiltration through a buffer. The activated polysaccharide and purified CRM197 were separately lyophilized and redissolved in DMSO. The reconstituted polysaccharide solution was then combined with the CRM197 solution and mixed as described below. The resulting conjugate was purified by ultrafiltration and then Each was filtered through a 0.2 micron filter to obtain a conjugate with the desired properties. Several process parameters were controlled within the process, such as pH, temperature, concentration and time.

[0341] Size reduction and oxidation of polysaccharides The purified pneumococcal Ps capsule powder was dissolved in water and filtered at 0.45 microns. The polysaccharide was homogenized to reduce the molecular weight of Ps. The number of passes was controlled at 150 bar / 7 passes. The size-reduced polysaccharide was concentrated and reduced to 10 kDa NM. The solution was diafiltered against water using a WCO tangential flow ultrafiltration membrane.

[0342] The polysaccharide solution was then adjusted to 4°C and pH 5 with sodium acetate buffer for activation. The polysaccharides were activated with 100 mM sodium metaperiodate. The oxidation reaction was initiated by the addition of the sodium solution and allowed to proceed for 4 h at 4 °C.

[0343] The activated product was diafiltered against 10 mM potassium phosphate (pH 6.4). The mixture was then diaphragmed against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was carried out at 2-8°C.

[0344] Polysaccharide conjugation to CRM197 As previously described (WO 2012 / 173876 A1), Pseudomonas Purified CRM197 expressed in C. fluorescens was analyzed using a 5 kDa NMW CO tangential flow ultrafiltration was used to filter 2 mM phosphate (pH 7.2) buffer. The solution was diafiltered through 0.2 microns.

[0345] Activated polysaccharides are formulated for lyophilization at 6 mg Ps / mL in a 5% w / v sucrose concentration CRM197 was lyophilized at 6 mg Pr / mL with a 1% w / v sucrose concentration. It was formulated for use.

[0346] The formulated Ps and CRM197 solutions were lyophilized separately. The CRM197 and CRM201 substances were separately redissolved in an equal volume of DMSO. The polysaccharide solution and CRM197 were mixed to obtain a polysaccharide concentration and a polysaccharide to CRM197 mass ratio of 1.5. The polysaccharide to CRM197 ratio in the resulting conjugate was controlled. The mass ratio was selected so that sodium cyanoborohydride (per mole of polysaccharide repeating unit) (1 mol per 1000 mg) was added and conjugation was allowed to proceed at 22°C.

[0347] Reduction with sodium borohydride After the conjugation reaction, sodium borohydride (2 moles per mole of polysaccharide repeating unit) was added. ) was added and incubated at 22°C for 3 hours. The batch was diluted in 150 mM sodium chloride containing 20 at about 4°C. The pH was neutralized by adding potassium phosphate buffer. The batch was concentrated to a concentration of 30 kDa NMW. CO tangential flow ultrafiltration was performed using a 150 mM sodium chloride, 25 mM potassium phosphate The solution was diafiltered against 100 ml of ethanol (pH 7) at approximately 4°C.

[0348] Final Filtration and Product Storage The batch was then concentrated and filtered using a 300 kDa NMWCO tangential flow ultrafiltration membrane to a concentration of 0. 150 mM sodium chloride (pH 7) containing 15% (w / v) polysorbate 20 The solution was diafiltered against 10 mM histidine in 0.5% NaCl (pH 7.0) at 4°C.

[0349] The retentate batch was filtered at 0.2 microns and then diluted with 0.015% (w / v) Polysorbate. An additional 10 mM in 150 mM sodium chloride (pH 7.0) containing phosphate 20 The mixture was diluted with histidine, dispensed into aliquots, and frozen at -60°C or below.

[0350] Example 16 Serotype 7F conjugates for PCV22 multivalent studies using DMSO conjugation Manufacturing of The polysaccharides are dissolved, sized to the target molecular weight, chemically activated, and purified by ultrafiltration through a buffer. The activated polysaccharide and purified CRM197 were separately lyophilized and redissolved in DMSO. The reconstituted polysaccharide solution was then combined with the CRM197 solution and mixed as described below. The resulting conjugate was purified by ultrafiltration and then Each was filtered through a 0.2 micron filter to obtain a conjugate with the desired properties. Several process parameters were controlled within the process, such as pH, temperature, concentration and time.

[0351] Size reduction and oxidation of polysaccharides The purified pneumococcal Ps capsule powder was dissolved in water and filtered at 0.45 microns. The polysaccharide was homogenized to reduce the molecular weight of Ps. The number of passes was controlled at 150 bar / 7 passes. The size-reduced polysaccharide was concentrated and reduced to 10 kDa NM. The solution was diafiltered against water using a WCO tangential flow ultrafiltration membrane.

[0352] The polysaccharide solution was then adjusted to 4°C and pH 5 with sodium acetate buffer for activation. The polysaccharides were activated with 100 mM sodium metaperiodate. The oxidation reaction was initiated by the addition of the sodium solution and allowed to proceed for 4 h at 4 °C.

[0353] The activated product was diafiltered against 10 mM potassium phosphate (pH 6.4). The mixture was then diaphragmed against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was carried out at 2-8°C.

[0354] Polysaccharide conjugation to CRM197 As previously described (WO 2012 / 173876 A1), Pseudomonas Purified CRM197 expressed in C. fluorescens was analyzed using a 5 kDa NMW CO tangential flow ultrafiltration was used to filter 2 mM phosphate (pH 7.2) buffer. The solution was diafiltered through 0.2 microns.

[0355] Activated polysaccharides are formulated for lyophilization at 6 mg Ps / mL in a 5% w / v sucrose concentration CRM197 was lyophilized at 6 mg Pr / mL with a 1% w / v sucrose concentration. It was formulated for use.

[0356] The formulated Ps and CRM197 solutions were lyophilized separately. and CRM197 material were separately redissolved in an equal volume of DMSO. The polysaccharide solution and CRM197 were mixed to obtain a polysaccharide concentration and a polysaccharide to CRM197 mass ratio of 1.5. The polysaccharide to CRM197 ratio in the resulting conjugate was controlled. The mass ratio was chosen so that sodium cyanoborohydride (1 mole of polysaccharide repeating unit) (1 mole per unit time) was added and conjugation was allowed to proceed at 22°C.

[0357] Reduction with sodium borohydride After the conjugation reaction, sodium borohydride (2 moles per mole of polysaccharide repeating unit) was added. ) was added and incubated at 22°C for 3 hours. The batch was diluted in 150 mM sodium chloride containing 20 at about 4°C. The pH was neutralized by adding potassium phosphate buffer. The batch was concentrated to a concentration of 30 kDa NMW. CO tangential flow ultrafiltration was performed using a 150 mM sodium chloride, 25 mM potassium phosphate The solution was diafiltered against 100 ml of ethanol (pH 7) at approximately 4°C.

[0358] Final Filtration and Product Storage The batch was then concentrated and filtered using a 300 kDa NMWCO tangential flow ultrafiltration membrane to a concentration of 0. 150 mM sodium chloride (pH 7) containing 15% (w / v) polysorbate 20 The solution was diafiltered against 10 mM histidine in 0.5% NaCl (pH 7.0) at 4°C.

[0359] The retentate batch was filtered at 0.2 microns and then diluted with 0.015% (w / v) Polysorbate. An additional 10 mM in 150 mM sodium chloride (pH 7.0) containing phosphate 20 The mixture was diluted with histidine, dispensed into aliquots, and frozen at -60°C or below.

[0360] Example 17 PCV23(DMSO) and PCV23(DMS) using DMSO conjugation Preparation of serotype 8 conjugates for (O+Aq) multivalent studies The polysaccharides are dissolved, sized to the target molecular weight, chemically activated, and purified by ultrafiltration through a buffer. The activated polysaccharide and purified CRM197 were separately lyophilized and redissolved in DMSO. The reconstituted polysaccharide solution was then combined with the CRM197 solution and mixed as described below. The resulting conjugate was purified by ultrafiltration and then Each was filtered through a 0.2 micron filter to obtain a conjugate with the desired properties. Several process parameters were controlled within the process, such as pH, temperature, concentration and time.

[0361] Size reduction and oxidation of polysaccharides The purified pneumococcal Ps capsule powder was dissolved in water and filtered at 0.45 microns. The polysaccharide was homogenized to reduce the molecular weight of Ps. The number of passes was controlled at 600 bar / 6 passes. The size-reduced polysaccharide was concentrated and had a 5 kDa NMW CO was diafiltered against water using a tangential flow ultrafiltration membrane.

[0362] The polysaccharide solution was then adjusted to 22°C and pH 5 with sodium acetate buffer to activate The size reduction of polysaccharides due to the activation was minimized. The reaction was initiated by the addition of the thorium solution and allowed to proceed for 4 hours at 22°C.

[0363] The activated product was diafiltered against 10 mM potassium phosphate (pH 6.4). The mixture was then diaphragmed against water using a 5 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was carried out at 2-8°C.

[0364] Polysaccharide conjugation to CRM197 As previously described (WO 2012 / 173876 A1), Pseudomonas Purified CRM197 expressed in C. fluorescens was analyzed using a 5 kDa NMW CO tangential flow ultrafiltration was used to filter 2 mM phosphate (pH 7.2) buffer. The solution was diafiltered through 0.2 microns.

[0365] Activated polysaccharides are formulated for lyophilization at 6 mg Ps / mL in a 5% w / v sucrose concentration CRM197 was lyophilized at 6 mg Pr / mL with a 1% w / v sucrose concentration. It was formulated for use.

[0366] The formulated Ps and CRM197 solutions were lyophilized separately. The CRM197 and CRM201 substances were separately redissolved in an equal volume of DMSO. The polysaccharide solution and CRM197 were mixed to obtain a polysaccharide concentration and a polysaccharide to CRM197 mass ratio of 1.5. The polysaccharide to CRM197 ratio in the resulting conjugate was controlled. After mixing, the conjugation reaction was allowed to proceed at 22°C.

[0367] Reduction with sodium borohydride After the conjugation reaction, sodium borohydride (2 moles per mole of polysaccharide repeating unit) was added. ) was added and incubated at 22°C for 1 hour. The batch was diluted in 150 mM sodium chloride containing 20 at about 4°C. Potassium phosphate buffer was added to neutralize the pH.

[0368] Final Filtration and Product Storage The batch was then concentrated and filtered using a 300 kDa NMWCO tangential flow ultrafiltration membrane to a concentration of 0. 150 mM sodium chloride (pH 7) containing 15% (w / v) polysorbate 20 The solution was diafiltered against 10 mM histidine in 0.5% NaCl (pH 7.0) at 4°C.

[0369] The retentate batch was filtered to 0.2 microns (with a 0.5 micron prefilter) and then 150 mM sodium chloride (pCl) containing 0.015% (w / v) polysorbate 20 The solution was diluted with additional 10 mM histidine in 100 mM NaCl (H7.0), dispensed into aliquots and stored at -60°C. Frozen below.

[0370] Example 18 Serotype 9V Conjugate for PCV23(DMSO) Multivalent Studies Using DMSO Conjugation Manufacturing of conjugate The polysaccharides are dissolved, sized to the target molecular weight, chemically activated, and purified by ultrafiltration through a buffer. The activated polysaccharide and purified CRM197 were separately lyophilized and redissolved in DMSO. The reconstituted polysaccharide solution was then combined with the CRM197 solution and mixed as described below. The resulting conjugate was purified by ultrafiltration and then Each was filtered through a 0.2 micron filter to obtain a conjugate with the desired properties. Several process parameters were controlled within the process, such as pH, temperature, concentration and time.

[0371] Size reduction and oxidation of polysaccharides The purified pneumococcal Ps capsule powder was dissolved in water and filtered at 0.45 microns. The polysaccharide was homogenized to reduce the molecular weight of Ps. The number of passes was controlled at 230 bar / 5.5 passes. The size-reduced polysaccharide was concentrated and reduced to 10 kDa. The solution was diafiltered against water using a NMWCO tangential flow ultrafiltration membrane.

[0372] The polysaccharide solution was then adjusted to 22°C and pH 5 with sodium acetate buffer to activate The size reduction of polysaccharides due to the activation was minimized. The reaction was initiated by the addition of the thorium solution and allowed to proceed for 6 hours at 22°C.

[0373] The activated product was diafiltered against 10 mM potassium phosphate (pH 6.4). The mixture was then diaphragmed against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was carried out at 2-8°C.

[0374] Polysaccharide conjugation to CRM197 As previously described (WO 2012 / 173876 A1), Pseudomonas Purified CRM197 expressed in C. fluorescens was analyzed using a 5 kDa NMW CO tangential flow ultrafiltration was used to filter 2 mM phosphate (pH 7.2) buffer. The solution was diafiltered through 0.2 microns.

[0375] Activated polysaccharides are formulated for lyophilization at 6 mg Ps / mL in a 5% w / v sucrose concentration CRM197 was lyophilized at 6 mg Pr / mL with a 1% w / v sucrose concentration. It was formulated for use.

[0376] The formulated Ps and CRM197 solutions were lyophilized separately. The CRM197 and CRM201 substances were separately redissolved in an equal volume of DMSO. The polysaccharide solution and CRM197 were mixed to obtain a polysaccharide concentration and a polysaccharide to CRM197 mass ratio of 1.3. The polysaccharide to CRM197 ratio in the resulting conjugate was controlled. The mass ratio was selected so that sodium cyanoborohydride (per mole of polysaccharide repeating unit) (1 mol per 1000 mg) was added and conjugation was allowed to proceed at 22°C.

[0377] Reduction with sodium borohydride After the conjugation reaction, sodium borohydride (2 moles per mole of polysaccharide repeating unit) was added. ) was added and incubated at 22°C for 3 hours. The batch was diluted in 150 mM sodium chloride containing 20 at about 4°C. The pH was neutralized by adding potassium phosphate buffer. The batch was concentrated to a concentration of 30 kDa NMW. CO tangential flow ultrafiltration was performed using a 150 mM sodium chloride, 25 mM potassium phosphate The solution was diafiltered against 100 ml of ethanol (pH 7) at approximately 4°C.

[0378] Final Filtration and Product Storage The batch was then concentrated and filtered using a 300 kDa NMWCO tangential flow ultrafiltration membrane to a concentration of 0. 150 mM sodium chloride (pH 7) containing 15% (w / v) polysorbate 20 The solution was diafiltered against 10 mM histidine in 0.5% NaCl (pH 7.0) at 4°C.

[0379] The retentate batch was filtered to 0.2 microns (with a 0.5 micron prefilter) and diluted to 0.01 150 mM sodium chloride (pH 7.0) containing 5% (w / v) polysorbate 20 ) and dispense into aliquots and freeze at 60°C or lower. did.

[0380] Example 19 Preparation of serotype 9V conjugates for PCV22 multivalent studies using aqueous conjugation Construction The polysaccharides are dissolved, sized, chemically activated, and buffer exchanged by ultrafiltration. The purified CRM197 was then coupled to an activated polysaccharide in a reaction mixture using nickel chloride. The resulting conjugate was purified by ultrafiltration and then added to the final 0. Each step was repeated to obtain a conjugate with the desired properties. Several process parameters were controlled, such as pH, temperature, concentration and time.

[0381] Size reduction and oxidation of polysaccharides Purified pneumococcal capsular polysaccharide powder was dissolved in water and filtered at 0.45 microns. The polysaccharides were homogenized to reduce their molecular weight. The homogenization pressure was adjusted to reduce the size to the target molecular weight. The pressure and number of passes through the homogenizer were controlled at 100 bar / 5 passes. Small polysaccharides were concentrated and diafiltered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane. It was filtered.

[0382] The polysaccharide solution was then adjusted to 22°C and pH 5 with sodium acetate buffer to activate The size reduction of polysaccharides due to the activation was minimized. The reaction was initiated by the addition of the thorium solution and allowed to proceed for 6 hours at 22°C.

[0383] The activated product was filtered using a 10 kDa NMWCO tangential flow ultrafiltration membrane with 10 mM phosphate buffer. The ultrafiltration was performed at 2 to 8°C. So I went.

[0384] Polysaccharide conjugation to CRM197 The oxidized polysaccharide solution was mixed with water and 1.5 M potassium phosphate (pH 7.0). The selected buffer pH improves the stability of the activated polysaccharide during the conjugation reaction. As previously described (WO 2012 / 173876 A 1) Purified CRM197 obtained by expression in Pseudomonas fluorescens 0.2 micron filtered and combined with the buffered polysaccharide solution at a polysaccharide to CRM197 weight ratio of 0.7 The mass ratio was adjusted so that the polysaccharide to CRM197 ratio in the resulting conjugate was controlled. The concentrations of polysaccharide and phosphate were 10.0 g / L and 100 mM, respectively. The polysaccharide concentration was chosen to control the size of the resulting conjugates. The solution was then 0.2 micron filtered. The salt was removed using 100 mM nickel chloride solution. Nickel chloride was added to approximately 2 mM. Sodium cyanoborohydride (1 mole of polysaccharide repeating unit) was added to approximately 2 mM. The conjugates were added at 200 ng / ml for maximum consumption of polysaccharide and protein. The polymerization was allowed to proceed for 120 hours.

[0385] Reduction with sodium borohydride After the conjugation reaction, the batch was diluted to a polysaccharide concentration of approximately 3.5 g / L and cooled to 2-8°C. The solution was cooled and filtered at 1.2 microns using a 100 kDa NMWCO tangential flow ultrafiltration membrane. The batch was then diaphragmed against 100 mM potassium phosphate (pH 7.0) at 2-8°C. The batch recovered in the retentate was then diluted to approximately 2.0 g polysaccharide / L. The pH was adjusted by adding 1.2 M sodium bicarbonate (pH 9.4). Sodium urate (1 mole per mole of polysaccharide repeating unit) was added. Then 1.5M phosphate Potassium was added (pH 6.0).

[0386] Final Filtration and Product Storage The batch was then concentrated and filtered using a 300 kDa NMWCO tangential flow ultrafiltration membrane for 15 min. Permeability to 10 mM L-histidine in 0 mM sodium chloride (pH 7.0) at 4°C Polysorbate 20 was added to the retentate batch to a concentration of 0.05% (w / v) and the The filter was 0.2 micron filtered.

[0387] 150 mM sodium chloride ( The batch was adjusted to a polysaccharide concentration of 1.0 g / L with an additional 10 mM L-histidine in 100 mL of 1000 mM NaCl (pH 7.0). The batch was divided into aliquots and frozen at -60°C or below.

[0388] Example 20 Serotype 9V for PCV23(DMSO+Aq) multivalent studies using aqueous conjugation Conjugate Production The polysaccharides are dissolved, sized, chemically activated, and buffer exchanged by ultrafiltration. The purified CRM197 was then coupled to an activated polysaccharide in a reaction mixture using nickel chloride. The resulting conjugate was purified by ultrafiltration and then added to the final 0. Each step was repeated to obtain a conjugate with the desired properties. Several process parameters were controlled, such as pH, temperature, concentration and time.

[0389] Size reduction and oxidation of polysaccharides Purified pneumococcal capsular polysaccharide powder was dissolved in water and filtered at 0.45 microns. The polysaccharides were homogenized to reduce their molecular weight. The homogenization pressure was adjusted to reduce the size to the target molecular weight. The pressure and number of passes through the homogenizer were controlled at 100 bar / 5 passes. Small polysaccharides were concentrated and diafiltered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane. It was filtered.

[0390] The polysaccharide solution was then adjusted to 22°C and pH 5 with sodium acetate buffer to activate The size reduction of polysaccharides due to the activation was minimized. The reaction was initiated by the addition of the thorium solution and allowed to proceed for 6 hours at 22°C.

[0391] The activated product was filtered using a 10 kDa NMWCO tangential flow ultrafiltration membrane with 10 mM phosphate buffer. The ultrafiltration was performed at 2 to 8°C. So I went.

[0392] Polysaccharide conjugation to CRM197 The oxidized polysaccharide solution was mixed with water and 1.5 M potassium phosphate (pH 7.0). The selected buffer pH improves the stability of the activated polysaccharide during the conjugation reaction. As previously described (WO 2012 / 173876 A 1) Purified CRM197 obtained by expression in Pseudomonas fluorescens 0.2 micron filtered and combined with the buffered polysaccharide solution at a polysaccharide to CRM197 weight ratio of 0.7 The mass ratio was adjusted so that the polysaccharide to CRM197 ratio in the resulting conjugate was controlled. The concentrations of polysaccharide and phosphate were 10.0 g / L and 100 mM, respectively. The polysaccharide concentration was chosen to control the size of the resulting conjugates. The solution was then 0.2 micron filtered. The salt was removed using 100 mM nickel chloride solution. Nickel chloride was added to approximately 2 mM. Sodium cyanoborohydride (1 mole of polysaccharide repeating unit) was added to approximately 2 mM. The conjugates were added at 200 ng / ml for maximum consumption of polysaccharide and protein. The polymerization was allowed to proceed for 120 hours.

[0393] Reduction with sodium borohydride After the conjugation reaction, the batch was diluted to a polysaccharide concentration of approximately 3.5 g / L and cooled to 2-8°C. The solution was cooled and filtered at 1.2 microns using a 100 kDa NMWCO tangential flow ultrafiltration membrane. The batch was then diaphragmed against 100 mM potassium phosphate (pH 7.0) at 2-8°C. The batch recovered in the retentate was then diluted to approximately 2.0 g polysaccharide / L. The pH was adjusted by adding 1.2 M sodium bicarbonate (pH 9.4). Sodium urate (1 mole per mole of polysaccharide repeating unit) was added. Then 1.5M phosphate Potassium was added (pH 6.0).

[0394] Final Filtration and Product Storage The batch was then concentrated and filtered using a 300 kDa NMWCO tangential flow ultrafiltration membrane for 15 min. Permeability to 10 mM L-histidine in 0 mM sodium chloride (pH 7.0) at 4°C Polysorbate 20 was added to the retentate batch to a concentration of 0.05% (w / v) and the The filter was 0.2 micron filtered.

[0395] 150 mM sodium chloride ( The batch was adjusted to a polysaccharide concentration of 1.0 g / L with an additional 10 mM L-histidine in 100 mL of 1000 mM NaCl (pH 7.0). The batch was divided into aliquots and frozen at -60°C or below.

[0396] Example 21 Serotype 10A conjugates for PCV23 multivalent studies using DMSO conjugation Production of The polysaccharides are dissolved, sized to the target molecular weight, chemically activated, and purified by ultrafiltration through a buffer. The activated polysaccharide and purified CRM197 were separately lyophilized and redissolved in DMSO. The reconstituted polysaccharide solution was then combined with the CRM197 solution and mixed as described below. The resulting conjugate was purified by ultrafiltration and then Each was filtered through a 0.2 micron filter to obtain a conjugate with the desired properties. Several process parameters were controlled within the process, such as pH, temperature, concentration and time.

[0397] Size reduction and oxidation of polysaccharides The purified pneumococcal Ps capsule powder was dissolved in water and filtered at 0.45 microns. The polysaccharide was homogenized to reduce the molecular weight of Ps. The number of passes was controlled at 615 bar / 5 passes.

[0398] Size-reduced polysaccharides were concentrated and filtered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane. and diafiltered.

[0399] The polysaccharide solution was then adjusted to 22°C and pH 5 with sodium acetate buffer to activate The size reduction of polysaccharides due to the activation was minimized. The reaction was initiated by the addition of the thorium solution and allowed to proceed for 2 hours at 22°C.

[0400] The activated product was diafiltered against 10 mM potassium phosphate (pH 6.4). The mixture was then diaphragmed against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was carried out at 2-8°C.

[0401] Polysaccharide conjugation to CRM197 As previously described (WO 2012 / 173876 A1), Pseudomonas Purified CRM197 expressed in C. fluorescens was analyzed using a 5 kDa NMW CO tangential flow ultrafiltration was used to filter 2 mM phosphate (pH 7.2) buffer. The solution was diafiltered through 0.2 microns.

[0402] Activated polysaccharides are formulated for lyophilization at 6 mg Ps / mL in a 5% w / v sucrose concentration CRM197 was lyophilized at 6 mg Pr / mL with a 1% w / v sucrose concentration. It was formulated for use.

[0403] The formulated Ps and CRM197 solutions were lyophilized separately. The CRM197 and CRM201 substances were separately redissolved in an equal volume of DMSO. The polysaccharide solution and CRM197 were mixed to obtain a polysaccharide concentration and a polysaccharide to CRM197 mass ratio of 1.6. The polysaccharide to CRM197 ratio in the resulting conjugate was controlled. The mass ratio was selected so that sodium cyanoborohydride (per mole of polysaccharide repeating unit) (1 mol per 1000 mg) was added and conjugation was allowed to proceed at 22°C.

[0404] Reduction with sodium borohydride After the conjugation reaction, sodium borohydride (2 moles per mole of polysaccharide repeating unit) was added. ) was added and incubated at 22°C for 1 hour. The batch was diluted in 150 mM sodium chloride containing 20 at about 4°C. Potassium phosphate buffer was added to neutralize the pH.

[0405] Final Filtration and Product Storage Concentrate the batch to 0.015 µL using a 300 kDa NMWCO tangential flow ultrafiltration membrane. 150 mM sodium chloride (pH 7.0) containing % (w / v) polysorbate 20 The solution was diafiltered against 10 mM histidine in 0.5 mL of 0.1% NaCl at 4°C.

[0406] The retentate batch was filtered to 0.2 microns (with a 0.5 micron prefilter) and then 150 mM sodium chloride (pCl) containing 0.015% (w / v) polysorbate 20 The solution was diluted with additional 10 mM histidine in 100 mM NaCl (H7.0), dispensed into aliquots and stored at -60°C. Frozen below.

[0407] Example 22 Serotypes for PCV23 (DMSO+Aq) multivalent studies using DMSO conjugation Preparation of 10A conjugate The polysaccharides are dissolved, sized to the target molecular weight, chemically activated, and purified by ultrafiltration through a buffer. The activated polysaccharide and purified CRM197 were separately lyophilized and redissolved in DMSO. The reconstituted polysaccharide solution was then combined with the CRM197 solution and mixed as described below. The resulting conjugate was purified by ultrafiltration and then Each was filtered through a 0.2 micron filter to obtain a conjugate with the desired properties. Several process parameters were controlled within the process, such as pH, temperature, concentration and time.

[0408] Size reduction and oxidation of polysaccharides The purified pneumococcal Ps capsule powder was dissolved in water and filtered at 0.45 microns. The polysaccharide was homogenized to reduce the molecular weight of Ps. The number of passes was controlled at 600 bar / 5 passes. The size-reduced polysaccharide was concentrated and reduced to 10 kDa NM. The solution was diafiltered against water using a WCO tangential flow ultrafiltration membrane.

[0409] The polysaccharide solution was then adjusted to 22°C and pH 5 with sodium acetate buffer to activate The size reduction of polysaccharides due to the activation was minimized. The reaction was initiated by the addition of the thorium solution and allowed to proceed for 2 hours at 22°C.

[0410] The activated product was diafiltered against 10 mM potassium phosphate (pH 6.4). The mixture was then diaphragmed against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was carried out at 2-8°C.

[0411] Polysaccharide conjugation to CRM197 As previously described (WO 2012 / 173876 A1), Pseudomonas Purified CRM197 expressed in C. fluorescens was analyzed using a 5 kDa NMW CO tangential flow ultrafiltration was used to filter 2 mM phosphate (pH 7.2) buffer. The solution was diafiltered through 0.2 microns.

[0412] Activated polysaccharides are formulated for lyophilization at 6 mg Ps / mL in a 5% w / v sucrose concentration CRM197 was lyophilized at 6 mg Pr / mL with a 1% w / v sucrose concentration. It was formulated for use.

[0413] The formulated Ps and CRM197 solutions were lyophilized separately. The CRM197 and CRM201 substances were separately redissolved in an equal volume of DMSO. The polysaccharide solution and CRM197 were mixed to obtain a polysaccharide concentration and a polysaccharide to CRM197 mass ratio of 1.6. The polysaccharide to CRM197 ratio in the resulting conjugate was controlled. The mass ratio was selected so that sodium cyanoborohydride (per mole of polysaccharide repeating unit) (1 mol per 1000 mg) was added and conjugation was allowed to proceed at 22°C.

[0414] Reduction with sodium borohydride After the conjugation reaction, sodium borohydride (2 moles per mole of polysaccharide repeating unit) was added. ) was added and incubated at 22°C for 1 hour. The batch was diluted in 150 mM sodium chloride containing 20 at about 4°C. Potassium phosphate buffer was added to neutralize the pH.

[0415] Final Filtration and Product Storage The batch was then concentrated and diluted with 150 mM sodium chloride, 25 mM potassium phosphate (p H7) followed by diafiltration against 300 kDa NMWCO tangential flow. Using an ultrafiltration membrane, 150 ml of 0.015% (w / v) polysorbate 20 was filtered. Dialysis was performed at 4°C against 10 mM histidine in 10 mM sodium chloride (pH 7.0). Refurgitated.

[0416] The retentate batch was filtered to 0.2 microns (with a 0.5 micron prefilter) and then 150 mM sodium chloride (pCl) containing 0.015% (w / v) polysorbate 20 The solution was diluted with additional 10 mM histidine in 100 mM NaCl (H7.0), dispensed into aliquots and stored at -60°C. Frozen below.

[0417] Example 23 Serotype 10A conjugates for PCV22 multivalent studies using DMSO conjugation Production of The polysaccharides are dissolved, sized to the target molecular weight, chemically activated, and purified by ultrafiltration through a buffer. The activated polysaccharide and purified CRM197 were separately lyophilized and redissolved in DMSO. The reconstituted polysaccharide solution was then combined with the CRM197 solution and mixed as described below. The resulting conjugate was purified by ultrafiltration and then Each was filtered through a 0.2 micron filter to obtain a conjugate with the desired properties. Several process parameters were controlled within the process, such as pH, temperature, concentration and time.

[0418] Size reduction and oxidation of polysaccharides The purified pneumococcal Ps capsule powder was dissolved in water and filtered at 0.45 microns. The polysaccharide was homogenized to reduce the molecular weight of Ps. The number of passes was controlled at 600 bar / 5 passes. The size-reduced polysaccharide was concentrated and reduced to 10 kDa NM. The solution was diafiltered against water using a WCO tangential flow ultrafiltration membrane.

[0419] The polysaccharide solution was then adjusted to 22°C and pH 5 with sodium acetate buffer to activate The size reduction of polysaccharides due to the activation was minimized. The reaction was initiated by the addition of the thorium solution and allowed to proceed for 2 hours at 22°C.

[0420] The activated product was diafiltered against 10 mM potassium phosphate (pH 6.4). The mixture was then diaphragmed against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was carried out at 2-8°C.

[0421] Polysaccharide conjugation to CRM197 As previously described (WO 2012 / 173876 A1), Pseudomonas Purified CRM197 expressed in C. fluorescens was analyzed using a 5 kDa NMW CO tangential flow ultrafiltration was used to filter 2 mM phosphate (pH 7.2) buffer. The solution was diafiltered through 0.2 microns.

[0422] Activated polysaccharides are formulated for lyophilization at 6 mg Ps / mL in a 5% w / v sucrose concentration CRM197 was lyophilized at 6 mg Pr / mL with a 1% w / v sucrose concentration. It was formulated for use.

[0423] The formulated Ps and CRM197 solutions were lyophilized separately. The CRM197 and CRM201 substances were separately redissolved in an equal volume of DMSO. The polysaccharide solution and CRM197 were mixed to obtain a polysaccharide concentration and a polysaccharide to CRM197 mass ratio of 1.75. The polysaccharide to CRM197 ratio in the resulting conjugate was controlled. The mass ratio was chosen so that sodium cyanoborohydride (1 mole of polysaccharide repeating unit) (1 mole per unit time) was added and conjugation was allowed to proceed at 22°C.

[0424] Reduction with sodium borohydride After the conjugation reaction, sodium borohydride (2 moles per mole of polysaccharide repeating unit) was added. ) was added and incubated at 22°C for 1 hour. The batch was diluted in 150 mM sodium chloride containing 20 at about 4°C. The pH was neutralized by adding potassium phosphate buffer. The batch was concentrated to a concentration of 30 kDa NMW. CO tangential flow ultrafiltration was performed using a 150 mM sodium chloride, 25 mM potassium phosphate The solution was diafiltered against 100 ml of ethanol (pH 7) at approximately 4°C.

[0425] Final Filtration and Product Storage The batch was then concentrated and filtered using a 300 kDa NMWCO tangential flow ultrafiltration membrane to a concentration of 0. 150 mM sodium chloride (pH 7) containing 15% (w / v) polysorbate 20 The solution was diafiltered against 10 mM histidine in 0.5% NaCl (pH 7.0) at 4°C.

[0426] The retentate batch was filtered to 0.2 microns (with a 0.5 micron prefilter) and then 150 mM sodium chloride (pCl) containing 0.015% (w / v) polysorbate 20 The solution was diluted with additional 10 mM histidine in 100 mM NaCl (H7.0), dispensed into aliquots and stored at -60°C. Frozen below.

[0427] Example X Serotype 11A conjugates for PCV24 multivalent studies using DMSO conjugation Production of The polysaccharides are dissolved, sized to the target molecular weight, chemically activated, and purified by ultrafiltration through a buffer. The activated polysaccharide and purified CRM197 were separately lyophilized and redissolved in DMSO. The reconstituted polysaccharide solution was then combined with the CRM197 solution and mixed as described below. The resulting conjugate was purified by ultrafiltration and then Each was filtered through a 0.2 micron filter to obtain a conjugate with the desired properties. Several process parameters were controlled within the process, such as pH, temperature, concentration and time.

[0428] Size reduction and oxidation of polysaccharides The purified pneumococcal Ps capsule powder was dissolved in water and filtered at 0.45 microns. The polysaccharide was homogenized to reduce the molecular weight of Ps. The number of passes was controlled at 800 bar / 8 passes. The size-reduced polysaccharide was concentrated and had a 5 kDa NMW CO was diafiltered against water using a tangential flow ultrafiltration membrane.

[0429] The polysaccharide solution was then adjusted to 22°C and pH 5 with sodium acetate buffer to activate The size reduction of polysaccharides due to the activation was minimized. The reaction was initiated by the addition of the thorium solution and allowed to proceed for 2 hours at 22°C.

[0430] The activated product was diafiltered against 10 mM potassium phosphate (pH 6.4). The mixture was then diaphragmed against water using a 5 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was carried out at 2-8°C.

[0431] Polysaccharide conjugation to CRM197 Pseudomonas as previously described (WO 2012 / 173876 A1). Purified CRM197 expressed in C. fluorescens was analyzed using a 5 kDa NMW CO tangential flow ultrafiltration was used to filter 2 mM phosphate (pH 7.2) buffer. The solution was diafiltered through 0.2 microns.

[0432] Activated polysaccharides are formulated for lyophilization at 6 mg Ps / mL in a 5% w / v sucrose concentration CRM197 was lyophilized at 6 mg Pr / mL with a 1% w / v sucrose concentration. It was formulated for use.

[0433] The formulated Ps and CRM197 solutions were lyophilized separately. The CRM197 and CRM201 substances were separately redissolved in an equal volume of DMSO. The polysaccharide solution and CRM197 were mixed to obtain a polysaccharide concentration and a polysaccharide to CRM197 mass ratio of 1.5. The polysaccharide to CRM197 ratio in the resulting conjugate was controlled. The mass ratio was selected so that sodium cyanoborohydride (per mole of polysaccharide repeating unit) (1 mol per 1000 mg) was added and conjugation was allowed to proceed at 22°C.

[0434] Reduction with sodium borohydride After the conjugation reaction, sodium borohydride (2 moles per mole of polysaccharide repeating unit) was added. ) was added and incubated at 22°C for 1 hour. The batch was then diluted with 150 mM sodium chloride containing phosphate 20 at approximately 4°C. The pH was neutralized by adding potassium acid buffer.

[0435] Final Filtration and Product Storage The batch was then concentrated and filtered using a 300 kDa NMWCO tangential flow ultrafiltration membrane to a concentration of 0. 150 mM sodium chloride (pH 7) containing 15% (w / v) polysorbate 20 The solution was diafiltered against 10 mM histidine in 0.5% NaCl (pH 7.0) at 4°C.

[0436] The retentate batch was filtered to 0.2 microns (through a 0.5 micron prefilter) and then filtered through a 150 ml Addition in M ​​sodium chloride (pH 7.0, 0.015% (w / v) polysorbate 20) The mixture was diluted with additional 10 mM histidine, dispensed into aliquots, and frozen at -60°C or colder.

[0437] Example 24 PCV23(DMSO) and PCV23(DMS) using DMSO conjugation Preparation of serotype 12 F conjugates for (O+Aq) multivalent studies The polysaccharides are dissolved, sized to the target molecular weight, chemically activated, and purified by ultrafiltration through a buffer. The activated polysaccharide and purified CRM197 were separately lyophilized and redissolved in DMSO. The reconstituted polysaccharide solution was then combined with the CRM197 solution and mixed as described below. The resulting conjugate was purified by ultrafiltration and then Each was filtered through a 0.2 micron filter to obtain a conjugate with the desired properties. Several process parameters were controlled within the process, such as pH, temperature, concentration and time.

[0438] Size reduction and oxidation of polysaccharides The purified pneumococcal Ps capsule powder was dissolved in water and filtered at 0.45 microns. The resulting polysaccharides were size-reduced by acid hydrolysis. The acid hydrolysis was carried out by adding acetic acid to 200 mM. , and incubated at 80°C for 155 minutes, followed by addition of cold potassium phosphate (pH 7) buffer. This was neutralized by adding

[0439] Size-reduced polysaccharides were concentrated and filtered against water using a 5 kDa NMWCO tangential flow ultrafiltration membrane. The mixture was diafiltered.

[0440] The polysaccharide solution was then adjusted to 22°C and pH 5 with sodium acetate buffer to activate The size reduction of polysaccharides due to the activation was minimized. The reaction was initiated by the addition of the thorium solution and allowed to proceed for 2 hours at 22°C.

[0441] The activated product was diafiltered against 10 mM potassium phosphate (pH 6.4). The mixture was then diaphragmed against water using a 5 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was carried out at 2-8°C.

[0442] Polysaccharide conjugation to CRM197 As previously described (WO 2012 / 173876 A1), Pseudomonas Purified CRM197 expressed in C. fluorescens was analyzed using a 5 kDa NMW CO tangential flow ultrafiltration was used to filter 2 mM phosphate (pH 7.2) buffer. The solution was diafiltered through 0.2 microns.

[0443] Activated polysaccharides are formulated for lyophilization at 6 mg Ps / mL in a 5% w / v sucrose concentration CRM197 was lyophilized at 6 mg Pr / mL with a 1% w / v sucrose concentration. It was formulated for use.

[0444] The formulated Ps and CRM197 solutions were lyophilized separately. The CRM197 and CRM201 substances were separately redissolved in an equal volume of DMSO. The polysaccharide solution and CRM197 were mixed to obtain a polysaccharide concentration and a polysaccharide to CRM197 mass ratio of 1.8. The polysaccharide to CRM197 ratio in the resulting conjugate was controlled. The mass ratio was selected so that sodium cyanoborohydride (per mole of polysaccharide repeating unit) (1 mol per 1000 mg) was added and conjugation was allowed to proceed at 22°C.

[0445] Reduction with sodium borohydride After the conjugation reaction, sodium borohydride (2 moles per mole of polysaccharide repeating unit) was added. ) was added and incubated at 22°C for 1 hour. The batch was diluted in 150 mM sodium chloride containing 20 at about 4°C. Potassium phosphate buffer was added to neutralize the pH.

[0446] Final Filtration and Product Storage The batch was then concentrated and filtered using a 300 kDa NMWCO tangential flow ultrafiltration membrane to a concentration of 0. 150 mM sodium chloride (pH 7) containing 15% (w / v) polysorbate 20 The solution was diafiltered against 10 mM histidine in 0.5% NaCl (pH 7.0) at 4°C.

[0447] The retentate batch was filtered to 0.2 microns (with a 0.5 micron prefilter) and then 150 mM sodium chloride (pCl) containing 0.015% (w / v) polysorbate 20 The solution was diluted with additional 10 mM histidine in 100 mM NaCl (H7.0), dispensed into aliquots and stored at -60°C. Frozen below.

[0448] Example 25 PCV23(DMSO) and PCV23(DMS) using DMSO conjugation Preparation of serotype 12 F conjugates for (O+Aq) multivalent studies The polysaccharides are dissolved, sized to the target molecular weight, chemically activated, and purified by ultrafiltration through a buffer. The activated polysaccharide and purified CRM197 were separately lyophilized and redissolved in DMSO. The reconstituted polysaccharide solution was then combined with the CRM197 solution and mixed as described below. The resulting conjugate was purified by ultrafiltration and then Each was filtered through a 0.2 micron filter to obtain a conjugate with the desired properties. Several process parameters were controlled within the process, such as pH, temperature, concentration and time.

[0449] Size reduction and oxidation of polysaccharides The purified pneumococcal Ps capsule powder was dissolved in water and filtered at 0.45 microns. The resulting polysaccharides were size-reduced by acid hydrolysis. The acid hydrolysis was carried out by adding acetic acid to 200 mM. The mixture was incubated at 90°C for 60 minutes, and then cooled potassium phosphate buffer (pH 7) was added. This was done by adding 5-aminoethyl 3-phenylindole to 400 mM to neutralize.

[0450] Size-reduced polysaccharides were concentrated and filtered against water using a 5 kDa NMWCO tangential flow ultrafiltration membrane. The mixture was diafiltered.

[0451] The polysaccharide solution was then adjusted to 22°C and pH 5 with sodium acetate buffer to activate The size reduction of polysaccharides due to the activation was minimized. The reaction was initiated by the addition of the thorium solution and allowed to proceed for 2 hours at 22°C.

[0452] The activated product was diafiltered against 10 mM potassium phosphate (pH 6.4). The mixture was then diaphragmed against water using a 5 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was carried out at 2-8°C.

[0453] Polysaccharide conjugation to CRM197 As previously described (WO 2012 / 173876 A1), Pseudomonas Purified CRM197 expressed in C. fluorescens was analyzed using a 5 kDa NMW CO tangential flow ultrafiltration was used to filter 2 mM phosphate (pH 7.2) buffer. The solution was diafiltered through 0.2 microns.

[0454] Activated polysaccharides are formulated for lyophilization at 6 mg Ps / mL in a 5% w / v sucrose concentration CRM197 was lyophilized at 6 mg Pr / mL with a 1% w / v sucrose concentration. It was formulated for use.

[0455] The formulated Ps and CRM197 solutions were lyophilized separately. The CRM197 and CRM201 substances were separately redissolved in an equal volume of DMSO. The polysaccharide solution and CRM197 were mixed to obtain a polysaccharide concentration and a polysaccharide to CRM197 mass ratio of 1.5. The polysaccharide to CRM197 ratio in the resulting conjugate was controlled. The mass ratio was selected so that sodium cyanoborohydride (per mole of polysaccharide repeating unit) (1 mol per 1000 mg) was added and conjugation was allowed to proceed at 22°C.

[0456] Reduction with sodium borohydride After the conjugation reaction, sodium borohydride (2 moles per mole of polysaccharide repeating unit) was added. ) was added and incubated at 22°C for 1 hour. The batch was diluted in 150 mM sodium chloride containing 20 at about 4°C. The pH was neutralized by adding potassium phosphate buffer. The batch was concentrated to a concentration of 30 kDa NMW. CO tangential flow ultrafiltration was performed using a 150 mM sodium chloride, 25 mM potassium phosphate The solution was diafiltered against 100 ml of ethanol (pH 7.0) at approximately 4°C.

[0457] Final Filtration and Product Storage The batch was then concentrated and filtered using a 300 kDa NMWCO tangential flow ultrafiltration membrane to a concentration of 0. 150 mM sodium chloride (pH 7) containing 15% (w / v) polysorbate 20 The solution was diafiltered against 10 mM histidine in 0.5% NaCl (pH 7.0) at 4°C.

[0458] The retentate batch was filtered to 0.2 microns (with a 0.5 micron prefilter) and then 150 mM sodium chloride (pCl) containing 0.015% (w / v) polysorbate 20 The solution was diluted with additional 10 mM histidine in 100 mM NaCl (H7.0), dispensed into aliquots and stored at -60°C. Frozen below.

[0459] Example 26 Serotype 14 Conjugates for PCV23(DMSO) Multivalent Studies Using DMSO Conjugation Manufacturing of conjugate The polysaccharides are dissolved, sized to the target molecular weight, chemically activated, and purified by ultrafiltration through a buffer. The activated polysaccharide and purified CRM197 were separately lyophilized and redissolved in DMSO. The reconstituted polysaccharide solution was then combined with the CRM197 solution and mixed as described below. The resulting conjugate was purified by dialysis and then added to the final 0. Each step was repeated to obtain a conjugate with the desired properties. Several process parameters were controlled, such as pH, temperature, concentration and time.

[0460] Size reduction and oxidation of polysaccharides The purified pneumococcal Ps capsule powder was dissolved in water and filtered at 0.45 microns. The polysaccharide was homogenized to reduce the molecular weight of Ps. The number of passes was controlled at 200 bar / 6 passes.

[0461] Size-reduced polysaccharides were concentrated and filtered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane. and diafiltered.

[0462] The polysaccharide solution was then adjusted to 22°C and pH 5 with sodium acetate buffer to activate The size reduction of polysaccharides due to the activation was minimized. The reaction was initiated by the addition of the thorium solution and allowed to proceed for 4 hours at 22°C.

[0463] The activated product was diafiltered against 10 mM potassium phosphate (pH 6.4). The mixture was then diaphragmed against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was carried out at 2-8°C.

[0464] Polysaccharide conjugation to CRM197 As previously described (WO 2012 / 173876 A1), Pseudomonas Purified CRM197 expressed in C. fluorescens was analyzed using a 5 kDa NMW CO tangential flow ultrafiltration was used to filter 2 mM phosphate (pH 7.2) buffer. The solution was diafiltered through 0.2 microns.

[0465] Activated polysaccharides are formulated for lyophilization at 6 mg Ps / mL in a 5% w / v sucrose concentration CRM197 was lyophilized at 6 mg Pr / mL with a 1% w / v sucrose concentration. It was formulated for use.

[0466] The formulated Ps and CRM197 solutions were lyophilized separately. The CRM197 and CRM201 substances were separately redissolved in an equal volume of DMSO. The polysaccharide solution and CRM197 were mixed to obtain a polysaccharide concentration and a polysaccharide to CRM197 mass ratio of 1.5. The polysaccharide to CRM197 ratio in the resulting conjugate was controlled. The mass ratio was selected so that sodium cyanoborohydride (per mole of polysaccharide repeating unit) (1 mol per 1000 mg) was added and conjugation was allowed to proceed at 22°C.

[0467] Reduction with sodium borohydride After the conjugation reaction, sodium borohydride (2 moles per mole of polysaccharide repeating unit) was added. ) was added and incubated at 22°C for 1 hour. The batch was diluted in 150 mM sodium chloride containing 20 at about 4°C. The pH was neutralized by adding potassium phosphate buffer. 300 kDa MWCO dialysis cassette for 150 mM sodium chloride, 0.05% polysorbate 20, The batch was dialysed at approximately 4° C. for 22.5 hours.

[0468] Final Filtration and Product Storage The retentate batch was filtered to 0.2 microns (through a 0.5 micron prefilter) and aliquoted. The mixture was dispensed into aliquots and frozen at -60°C or below.

[0469] Example 27 Polyvalent Synthesis of PCV22 and PCV23 (DMSO+Aq) Using Aqueous Conjugation Production of serotype 14 conjugates for research The polysaccharides are dissolved, sized, chemically activated, and buffer exchanged by ultrafiltration. The purified CRM197 was then coupled to an activated polysaccharide in a reaction mixture using nickel chloride. The resulting conjugate was purified by ultrafiltration and then added to the final 0. Each step was repeated to obtain a conjugate with the desired properties. Several process parameters were controlled, such as pH, temperature, concentration and time.

[0470] Size reduction and oxidation of polysaccharides Purified pneumococcal capsular polysaccharide powder was dissolved in water and filtered at 0.45 microns. The polysaccharides were homogenized to reduce their molecular weight. The homogenization pressure was adjusted to reduce the size to the target molecular weight. The pressure and number of passes through the homogenizer were controlled at 200 bar / 6 passes. Small polysaccharides were concentrated and diafiltered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane. It was filtered.

[0471] The polysaccharide solution was then adjusted to 22°C and pH 5 with sodium acetate buffer to activate The size reduction of polysaccharides due to the activation was minimized. The reaction was initiated by the addition of the thorium solution and allowed to proceed for 4 hours at 22°C.

[0472] The activated product was filtered using a 10 kDa NMWCO tangential flow ultrafiltration membrane with 10 mM phosphate buffer. The ultrafiltration was performed at 2 to 8°C. So I went.

[0473] Polysaccharide conjugation to CRM197 The oxidized polysaccharide solution was mixed with water and 1.5 M potassium phosphate (pH 7.0). The selected buffer pH improves the stability of the activated polysaccharide during the conjugation reaction. As previously described (WO 2012 / 173876 A 1) Purified CRM197 obtained by expression in Pseudomonas fluorescens 0.2 micron filtered and combined with the buffered polysaccharide solution at a polysaccharide to CRM197 weight ratio of 1.0. The mass ratio was adjusted so that the polysaccharide to CRM197 ratio in the resulting conjugate was controlled. The concentrations of polysaccharide and phosphate were 3.8 g / L and 100 mM, respectively. The polysaccharide concentration was selected so as to control the size of the resulting conjugate. The solution was then filtered through a 0.2 micron filter. Nickel was added to approximately 2 mM. Sodium cyanoborohydride (per mole of polysaccharide repeating unit) The conjugates were added at 0.25 g / ml for maximum consumption of polysaccharide and protein. Incubation was allowed to proceed for 72 hours.

[0474] Reduction with sodium borohydride After the conjugation reaction, the batch was diluted to a polysaccharide concentration of approximately 3.5 g / L and cooled to 2-8°C. The solution was cooled and filtered at 1.2 microns using a 100 kDa NMWCO tangential flow ultrafiltration membrane. The batch was then diaphragmed against 100 mM potassium phosphate (pH 7.0) at 2-8°C. The batch recovered in the retentate was then diluted to approximately 2.0 g polysaccharide / L. The pH was adjusted by adding 1.2 M sodium bicarbonate (pH 9.4). Sodium urate (1 mole per mole of polysaccharide repeating unit) was added. Then 1.5M phosphate Potassium was added (pH 6.0).

[0475] Final Filtration and Product Storage The batch was then concentrated and filtered using a 300 kDa NMWCO tangential flow ultrafiltration membrane for 15 min. Permeability to 10 mM L-histidine in 0 mM sodium chloride (pH 7.0) at 4°C The batch was then 0.2 micron filtered.

[0476] Additional 10 mM L-hyaluronan in 150 mM sodium chloride (pH 7.0) buffer. The batch was adjusted with stigmine to a polysaccharide concentration of 1.0 g / L. The batch was divided into aliquots and Frozen at -60°C or below.

[0477] Example 28 PCV23(DMSO) and PCV23(DMS) using DMSO conjugation Preparation of serotype 15A conjugates for (O+Aq) multivalent studies The polysaccharides are dissolved, sized to the target molecular weight, chemically activated, and purified by ultrafiltration through a buffer. The activated polysaccharide and purified CRM197 were separately lyophilized and redissolved in DMSO. The reconstituted polysaccharide solution was then combined with the CRM197 solution and mixed as described below. The resulting conjugate was purified by ultrafiltration and then Each was filtered through a 0.2 micron filter to obtain a conjugate with the desired properties. Several process parameters were controlled within the process, such as pH, temperature, concentration and time.

[0478] Size reduction and oxidation of polysaccharides The purified pneumococcal Ps capsule powder was dissolved in water and filtered at 0.45 microns. The polysaccharide was homogenized to reduce the molecular weight of Ps. The number of passes was controlled at 210 bar / 5 passes.

[0479] Size-reduced polysaccharides were concentrated and filtered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane. and diafiltered.

[0480] The polysaccharide solution was then adjusted to 22°C and pH 5 with sodium acetate buffer to activate The size reduction of polysaccharides due to the activation was minimized. The reaction was initiated by the addition of the thorium solution and allowed to proceed for 20 hours at 22°C.

[0481] The activated product was diafiltered against 10 mM potassium phosphate (pH 6.4). The mixture was then diaphragmed against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was carried out at 2-8°C.

[0482] Polysaccharide conjugation to CRM197 As previously described (WO 2012 / 173876 A1), Pseudomonas Purified CRM197 expressed in C. fluorescens was analyzed using a 5 kDa NMW CO tangential flow ultrafiltration was used to filter 2 mM phosphate (pH 7.2) buffer. The solution was diafiltered through 0.2 microns.

[0483] Activated polysaccharides are formulated for lyophilization at 6 mg Ps / mL in a 5% w / v sucrose concentration CRM197 was lyophilized at 6 mg Pr / mL with a 1% w / v sucrose concentration. It was formulated for use.

[0484] The formulated Ps and CRM197 solutions were lyophilized separately. The CRM197 and CRM201 substances were individually redissolved in an equal volume of DMSO preheated to 34°C. Sodium chloride was added to the sugar solution to a concentration of 25 mM. Polysaccharide concentration of 5.0 g Ps / L. The polysaccharide solution and CRM197 solution were mixed together to obtain a polysaccharide to CRM197 mass ratio of 2.0 and 2.0. The polysaccharide to CRM197 ratio in the resulting conjugate was controlled. The mass ratios were chosen so that sodium cyanoborohydride (1 mole per mole of polysaccharide repeating unit) mol) was added and conjugation was allowed to proceed at 34°C.

[0485] Reduction with sodium borohydride After the conjugation reaction, sodium borohydride (2 moles per mole of polysaccharide repeating unit) was added. ) was added and incubated at 34°C for 1 hour. The batch was diluted in 150 mM sodium chloride containing 20 at about 4°C. Potassium phosphate buffer was added to neutralize the pH.

[0486] Final Filtration and Product Storage The batch was then concentrated and filtered using a 300 kDa NMWCO tangential flow ultrafiltration membrane to a concentration of 0. 150 mM sodium chloride (pH 7) containing 15% (w / v) polysorbate 20 The solution was diafiltered against 10 mM histidine in 0.5% NaCl (pH 7.0) at 4°C.

[0487] The retentate batch was filtered to 0.2 microns (with a 0.5 micron prefilter) and then 150 mM sodium chloride (pCl) containing 0.015% (w / v) polysorbate 20 The solution was diluted with additional 10 mM histidine in 100 mM NaCl (H7.0), dispensed into aliquots and stored at -60°C. Frozen below.

[0488] Example 29 Serotype 15A conjugates for PCV22 multivalent studies using DMSO conjugation Production of The polysaccharides are dissolved, sized to the target molecular weight, chemically activated, and purified by ultrafiltration through a buffer. The activated polysaccharide and purified CRM197 were separately lyophilized and redissolved in DMSO. The reconstituted polysaccharide solution was then combined with the CRM197 solution and mixed as described below. The resulting conjugate was purified by ultrafiltration and then Each was filtered through a 0.2 micron filter to obtain a conjugate with the desired properties. Several process parameters were controlled within the process, such as pH, temperature, concentration and time.

[0489] Size reduction and oxidation of polysaccharides The purified pneumococcal Ps capsule powder was dissolved in water and filtered at 0.45 microns. The polysaccharide was homogenized to reduce the molecular weight of Ps. The number of passes was controlled at 200 bar / 5 passes.

[0490] Size-reduced polysaccharides were concentrated and filtered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane. and diafiltered.

[0491] The polysaccharide solution was then adjusted to 22°C and pH 5 with sodium acetate buffer to activate The size reduction of polysaccharides due to the activation was minimized. The reaction was initiated by the addition of the thorium solution and allowed to proceed for 20 hours at 22°C.

[0492] The activated product was diafiltered against 10 mM potassium phosphate (pH 6.4). The mixture was then diaphragmed against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was carried out at 2-8°C.

[0493] Polysaccharide conjugation to CRM197 As previously described (WO 2012 / 173876 A1), Pseudomonas Purified CRM197 expressed in C. fluorescens was analyzed using a 5 kDa NMW CO tangential flow ultrafiltration was used to filter 2 mM phosphate (pH 7.2) buffer. The solution was diafiltered through 0.2 microns.

[0494] Activated polysaccharides are formulated for lyophilization at 6 mg Ps / mL in a 5% w / v sucrose concentration CRM197 was lyophilized at 6 mg Pr / mL with a 1% w / v sucrose concentration. It was formulated for use.

[0495] The formulated Ps and CRM197 solutions were lyophilized separately. The CRM197 and CRM201 substances were individually redissolved in an equal volume of DMSO preheated to 34°C. Sodium chloride was added to the sugar solution to a concentration of 25 mM. Polysaccharide concentration of 5.0 g Ps / L. The polysaccharide solution and CRM197 solution were mixed together to obtain a polysaccharide to CRM197 mass ratio of 2.0 and 2.0. The polysaccharide to CRM197 ratio in the resulting conjugate was controlled. The mass ratios were chosen so that sodium cyanoborohydride (1 mole per mole of polysaccharide repeating unit) mol) was added and conjugation was allowed to proceed at 34°C.

[0496] Reduction with sodium borohydride After the conjugation reaction, sodium borohydride (2 moles per mole of polysaccharide repeating unit) was added. ) was added and incubated at 22°C for 1 hour. The batch was diluted in 150 mM sodium chloride containing 20 at about 4°C. The pH was neutralized by adding potassium phosphate buffer. The batch was concentrated to a concentration of 30 kDa NMW. CO tangential flow ultrafiltration was performed using a 150 mM sodium chloride, 25 mM potassium phosphate The solution was diafiltered against 100 ml of ethanol (pH 7) at approximately 4°C.

[0497] Final Filtration and Product Storage The batch was then concentrated and filtered using a 300 kDa NMWCO tangential flow ultrafiltration membrane to a concentration of 0. 150 mM sodium chloride (pH 7) containing 15% (w / v) polysorbate 20 The solution was diafiltered against 10 mM histidine in 0.5% NaCl (pH 7.0) at 4°C.

[0498] The retentate batch was filtered to 0.2 microns (with a 0.5 micron prefilter) and then 150 mM sodium chloride (pCl) containing 0.015% (w / v) polysorbate 20 The solution was diluted with additional 10 mM histidine in 100 mM NaCl (H7.0), dispensed into aliquots and stored at -60°C. Frozen below.

[0499] Example 30 PCV23(DMSO) and PCV23(DMS) using DMSO conjugation Preparation of serotype 15C conjugates for (O+Aq) multivalent studies Streptococcus pneumoniae ae) Polysaccharide derived from serotype 15B is dissolved, sized to a target molecular weight, and O-acetylated. It is subjected to mild base hydrolysis to liberate the aryl groups, chemically activated, and purified by ultrafiltration. The activated polysaccharide and purified CRM197 were lyophilized separately and resuspended in DMSO. The redissolved polysaccharide solution was then mixed with the CRM197 solution and the mixture was stirred for 1 hour at 37°C. The resulting conjugate was purified by ultrafiltration and then A final 0.2 micron filtration was performed to obtain a conjugate with the desired properties. In addition, several process parameters within each step, such as pH, temperature, concentration and time, can be controlled. did.

[0500] Size reduction, base hydrolysis and oxidation of polysaccharides Purified serotype 15B pneumococcal capsular Ps powder was dissolved in water and filtered at 0.45 microns. The dissolved polysaccharide was homogenized to reduce the molecular weight of Ps. The number of passes through the sieve was controlled at 300 bar / 5 passes.

[0501] Size-reduced polysaccharides were concentrated and filtered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane. and diafiltered.

[0502] 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 incubated at 60° C. for 13 hours with mixing to obtain O The acetyl group was released. Potassium phosphate (pH 6) buffer was added to a final concentration of 136 mM. The pH was neutralized by adding 100 to 100 mM NaOH and the solution was cooled to ambient temperature. The solution was then concentrated to 10 kDa. The solution was diafiltered against water using a NMWCO tangential flow ultrafiltration membrane.

[0503] The polysaccharide solution was adjusted to 22°C and pH 5 with sodium acetate buffer to determine the activation Polysaccharide size reduction was minimized. Polysaccharide activation was performed with 100 mM sodium metaperiodate. The oxidation reaction was initiated by the addition of the ethanol solution and allowed to proceed for 2 h at 22 °C.

[0504] The activated product was diafiltered against 10 mM potassium phosphate (pH 6.4). The mixture was then diaphragmed against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was carried out at 2-8°C.

[0505] Polysaccharide conjugation to CRM197 As previously described (WO 2012 / 173876 A1), Pseudomonas Purified CRM197 expressed in C. fluorescens was analyzed using a 5 kDa NMW CO tangential flow ultrafiltration was used to filter 2 mM phosphate (pH 7.2) buffer. The solution was diafiltered through 0.2 microns.

[0506] Activated polysaccharides are formulated for lyophilization at 6 mg Ps / mL in a 5% w / v sucrose concentration CRM197 was lyophilized at 6 mg Pr / mL with a 1% w / v sucrose concentration. It was formulated for use.

[0507] The formulated Ps and CRM197 solutions were lyophilized separately. The CRM197 and CRM201 substances were separately redissolved in an equal volume of DMSO. The polysaccharide solution and CRM197 were mixed to obtain a polysaccharide concentration and a polysaccharide to CRM197 mass ratio of 1.75. The polysaccharide to CRM197 ratio in the resulting conjugate was controlled. The mass ratio was chosen so that sodium cyanoborohydride (1 mole of polysaccharide repeating unit) (1 mole per unit time) was added and conjugation was allowed to proceed at 22°C.

[0508] Reduction with sodium borohydride After the conjugation reaction, sodium borohydride (2 moles per mole of polysaccharide repeating unit) was added. ) was added and incubated at 22°C for 1 hour. The batch was diluted in 150 mM sodium chloride containing 20 at about 4°C. The pH was neutralized by adding potassium phosphate buffer. The batch was concentrated to a concentration of 30 kDa NMW. CO tangential flow ultrafiltration was performed using a 150 mM sodium chloride, 25 mM potassium phosphate The solution was diafiltered against 100 ml of ethanol (pH 7) at approximately 4°C.

[0509] Final Filtration and Product Storage The batch was then concentrated and filtered using a 300 kDa NMWCO tangential flow ultrafiltration membrane to a concentration of 0. 150 mM sodium chloride (pH 7) containing 15% (w / v) polysorbate 20 The solution was diafiltered against 10 mM histidine in 0.5% NaCl (pH 7.0) at 4°C.

[0510] The retentate batch was filtered to 0.2 microns (with a 0.5 micron prefilter) and then 150 mM sodium chloride (pCl) containing 0.015% (w / v) polysorbate 20 The solution was diluted with additional 10 mM histidine in 100 mM NaCl (H7.0), dispensed into aliquots and stored at -60°C. Frozen below.

[0511] Example 31 Serotype 15C conjugates for PCV22 multivalent studies using DMSO conjugation Production of Streptococcus pneumoniae ae) Polysaccharide derived from serotype 15B is dissolved, sized to a target molecular weight, and O-acetylated. It is subjected to mild base hydrolysis to liberate the aryl groups, chemically activated, and purified by ultrafiltration. The activated polysaccharide and purified CRM197 were lyophilized separately and resuspended in DMSO. The redissolved polysaccharide solution was then mixed with the CRM197 solution and the mixture was stirred for 1 hour at 37°C. The resulting conjugate was purified by ultrafiltration and then A final 0.2 micron filtration was performed to obtain a conjugate with the desired properties. In addition, several process parameters within each step, such as pH, temperature, concentration and time, can be controlled. did.

[0512] Size reduction, base hydrolysis and oxidation of polysaccharides Purified serotype 15B pneumococcal capsular Ps powder was dissolved in water and filtered at 0.45 microns. The dissolved polysaccharide was homogenized to reduce the molecular weight of Ps. The number of passes through the sizer was controlled at 300 bar / 5 passes, and the size-reduced polysaccharide solution was heated to 60°C. The mixture was heated and sodium bicarbonate (pH 9.4) buffer was added to a final concentration of 50 mM. The mixture was incubated at 60°C for 12 hours with mixing to release the O-acetyl groups. Potassium phosphate (pH 6) buffer was added to a final concentration of 150 mM to moderate the pH. The solution was then concentrated and diluted with 10 kDa NMWCO tangential flow chromatograph. It was diafiltered against water using an ultrafiltration membrane.

[0513] The polysaccharide solution was adjusted to 22°C and pH 5 with sodium acetate buffer to determine the activation Polysaccharide size reduction was minimized. Polysaccharide activation was performed with 100 mM sodium metaperiodate. The oxidation reaction was initiated by the addition of the ethanol solution and allowed to proceed for 2 h at 22 °C.

[0514] The activated product was diafiltered against 10 mM potassium phosphate (pH 6.4). The mixture was then diaphragmed against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was carried out at 2-8°C.

[0515] Polysaccharide conjugation to CRM197 As previously described (WO 2012 / 173876 A1), Pseudomonas Purified CRM197 expressed in C. fluorescens was analyzed using a 5 kDa NMW CO tangential flow ultrafiltration was used to filter 2 mM phosphate (pH 7.2) buffer. The solution was diafiltered through 0.2 microns.

[0516] Activated polysaccharides are formulated for lyophilization at 6 mg Ps / mL in a 5% w / v sucrose concentration CRM197 was lyophilized at 6 mg Pr / mL with a 1% w / v sucrose concentration. It was formulated for use.

[0517] The formulated Ps and CRM197 solutions were lyophilized separately. The CRM197 and CRM201 substances were separately redissolved in an equal volume of DMSO. The polysaccharide solution and CRM197 were mixed to obtain a polysaccharide concentration and a polysaccharide to CRM197 mass ratio of 1.75. The polysaccharide to CRM197 ratio in the resulting conjugate was controlled. The mass ratio was chosen so that sodium cyanoborohydride (1 mole of polysaccharide repeating unit) (1 mole per unit time) was added and conjugation was allowed to proceed at 22°C.

[0518] Reduction with sodium borohydride After the conjugation reaction, sodium borohydride (2 moles per mole of polysaccharide repeating unit) was added. ) was added and incubated at 22°C for 1 hour. The batch was diluted in 150 mM sodium chloride containing 20 at about 4°C. The pH was neutralized by adding potassium phosphate buffer. The batch was concentrated to a concentration of 30 kDa NMW. CO tangential flow ultrafiltration was performed using a 150 mM sodium chloride, 25 mM potassium phosphate The solution was diafiltered against 100 ml of ethanol (pH 7) at approximately 4°C.

[0519] Final Filtration and Product Storage The batch was then concentrated and filtered using a 300 kDa NMWCO tangential flow ultrafiltration membrane to a concentration of 0. 150 mM sodium chloride (pH 7) containing 15% (w / v) polysorbate 20 The solution was diafiltered against 10 mM histidine in 0.5% NaCl (pH 7.0) at 4°C.

[0520] The retentate batch was filtered to 0.2 microns (with a 0.5 micron prefilter) and then 150 mM sodium chloride (pCl) containing 0.015% (w / v) polysorbate 20 The solution was diluted with additional 10 mM histidine in 100 mM NaCl (H7.0), dispensed into aliquots and stored at -60°C. Frozen below.

[0521] Example 32 PCV23(DMSO) and PCV23(DMS) using DMSO conjugation Preparation of serotype 18C conjugates for (O+Aq) multivalent studies The polysaccharides are dissolved, sized to the target molecular weight, chemically activated, and purified by ultrafiltration through a buffer. The activated polysaccharide and purified CRM197 were separately lyophilized and redissolved in DMSO. The reconstituted polysaccharide solution was then combined with the CRM197 solution and mixed as described below. The resulting conjugate was purified by ultrafiltration and then Each was filtered through a 0.2 micron filter to obtain a conjugate with the desired properties. Several process parameters were controlled within the process, such as pH, temperature, concentration and time.

[0522] Size reduction and oxidation of polysaccharides The purified pneumococcal Ps capsule powder was dissolved in water and filtered at 0.45 microns. The resulting polysaccharides were size-reduced by acid hydrolysis. The acid hydrolysis was carried out by adding acetic acid to 200 mM. , and incubated at 90°C for 160 minutes, then cooled potassium phosphate (pH 7) buffer This was neutralized by adding

[0523] Size-reduced polysaccharides were concentrated and filtered against water using a 5 kDa NMWCO tangential flow ultrafiltration membrane. The mixture was diafiltered.

[0524] The polysaccharide solution was then adjusted to 22°C and pH 5 with sodium acetate buffer to activate The size reduction of polysaccharides due to the activation was minimized. The reaction was initiated by the addition of the thorium solution and allowed to proceed for 2 hours at 22°C.

[0525] The activated product was diafiltered against 10 mM potassium phosphate (pH 6.4). The mixture was then diaphragmed against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was carried out at 2-8°C.

[0526] Polysaccharide conjugation to CRM197 As previously described (WO 2012 / 173876 A1), Pseudomonas Purified CRM197 expressed in C. fluorescens was analyzed using a 5 kDa NMW CO tangential flow ultrafiltration was used to filter 2 mM phosphate (pH 7.2) buffer. The solution was diafiltered through 0.2 microns.

[0527] Activated polysaccharides are formulated for lyophilization at 6 mg Ps / mL in a 5% w / v sucrose concentration CRM197 was lyophilized at 6 mg Pr / mL with a 1% w / v sucrose concentration. It was formulated for use.

[0528] The formulated Ps and CRM197 solutions were lyophilized separately. and CRM197 material were separately redissolved in an equal volume of DMSO. The polysaccharide solution and CRM197 were mixed to obtain a polysaccharide concentration and a polysaccharide to CRM197 mass ratio of 1.5. The polysaccharide to CRM197 ratio in the resulting conjugate was controlled. The mass ratio was chosen so that sodium cyanoborohydride (1 mole of polysaccharide repeating unit) (1 mole per unit time) was added and conjugation was allowed to proceed at 22°C.

[0529] Reduction with sodium borohydride After the conjugation reaction, sodium borohydride (2 moles per mole of polysaccharide repeating unit) was added. ) was added and incubated at 22°C for 3 hours. The batch was diluted in 150 mM sodium chloride containing 20 at about 4°C. The pH was neutralized by adding potassium phosphate buffer. The batch was concentrated to a concentration of 30 kDa NMW. CO tangential flow ultrafiltration was performed using a 150 mM sodium chloride, 25 mM potassium phosphate The solution was diafiltered against 100 ml of ethanol (pH 7) at approximately 4°C.

[0530] Final Filtration and Product Storage The batch was then concentrated and filtered using a 300 kDa NMWCO tangential flow ultrafiltration membrane to a concentration of 0. 150 mM sodium chloride (pH 7) containing 15% (w / v) polysorbate 20 The solution was diafiltered against 10 mM histidine in 0.5% NaCl (pH 7.0) at 4°C.

[0531] The retentate batch was filtered to 0.2 microns (with a 0.5 micron prefilter) and then 150 mM sodium chloride (pCl) containing 0.015% (w / v) polysorbate 20 The solution was diluted with additional 10 mM histidine in 100 mM NaCl (H7.0), dispensed into aliquots and stored at -60°C. Frozen below.

[0532] Example 33 Serotype 18C conjugates for PCV22 multivalent studies using DMSO conjugation Production of The polysaccharides are dissolved, sized to the target molecular weight, chemically activated, and purified by ultrafiltration through a buffer. The activated polysaccharide and purified CRM197 were separately lyophilized and redissolved in DMSO. The reconstituted polysaccharide solution was then combined with the CRM197 solution and mixed as described below. The resulting conjugate was purified by ultrafiltration and then Each was filtered through a 0.2 micron filter to obtain a conjugate with the desired properties. Several process parameters were controlled within the process, such as pH, temperature, concentration and time.

[0533] Size reduction and oxidation of polysaccharides The purified pneumococcal Ps capsule powder was dissolved in water and filtered at 0.45 microns. The resulting polysaccharides were size-reduced by acid hydrolysis. The acid hydrolysis was carried out by adding acetic acid to 200 mM. , and incubated at 90°C for 160 minutes, then cooled potassium phosphate (pH 7) buffer This was neutralized by adding

[0534] Size-reduced polysaccharides were concentrated and filtered against water using a 5 kDa NMWCO tangential flow ultrafiltration membrane. The mixture was diafilte...

Claims

1. Streptococcus pneumoniae (S. pneumoniae) polysaccharide protein conjugate A multivalent immunogenic composition comprising a conjugate, each of which is coupled to a carrier tag. Polysaccharides from Streptococcus pneumoniae serovars conjugated to protein - Patents.com wherein the polysaccharide-protein conjugate comprises a) 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 12F, 14, 15A, D eOAc15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and and 35B; b) 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 12F, 14, 15A, 1 5C, 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, 33 F 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 3 5B; 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, 24 F, 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 3 5B; 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, 15 C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; n) 1, 3, 4, 5, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15 C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; o) 1, 3, 4, 5, 6C, 7F, 8, 9V, 10A, 12F, 14, 15A, 15 C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; p) 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 11A, 12F, 14, 15 A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; q) 1, 3, 4, 5, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15 A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; r) 1, 3, 4, 5, 6C, 7F, 8, 9V, 10A, 11A, 12F, 14, 15 A, 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, 15 B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; w) 1, 3, 4, 5, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15 B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; x) 1, 3, 4, 5, 6C, 7F, 8, 9V, 10A, 12F, 14, 15A, 15 B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; y) 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 11A, 12F, 14, 15 A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; z) 1, 3, 4, 5, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15 A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; aa) 1, 3, 4, 5, 6C, 7F, 8, 9V, 10A, 11A, 12F, 14, 1 5A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 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 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, de O-15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35 B and 39; ee) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 12F, 14, 15A, 15 C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and ３９； ff) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 12F, 14, 15B, 18 C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; gg) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 11A, 12F, 14, 15 A, deO-15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33 F, 35B and 39; hh) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 11A, 12F, 14, 15 A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35 B and 39; ii) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 11A, 12F, 14, 15 A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35 B and 39; jj) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 11A, 12F, 14, 15 B, 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, 1 8C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; oo) 1, 3, 4, 5, 6B, 7F, 8, 9V, 12F, 14, 15A, 15C, 1 8C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; pp) 1, 3, 4, 5, 6C, 7F, 8, 9V, 12F, 14, 15A, 15C, 1 8C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; qq) 1, 3, 4, 5, 6A, 7F, 8, 9V, 11A, 12F, 14, 15A, 1 5C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; rr) 1, 3, 4, 5, 6B, 7F, 8, 9V, 11A, 12F, 14, 15A, 1 5C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; ss) 1, 3, 4, 5, 6C, 7F, 8, 9V, 11A, 12F, 14, 15A, 1 5C, 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, 1 8C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; xx) 1, 3, 4, 5, 6B, 7F, 8, 9V, 12F, 14, 15A, 15B, 1 8C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; yy) 1, 3, 4, 5, 6C, 7F, 8, 9V, 12F, 14, 15A, 15B, 1 8C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; zz) 1, 3, 4, 5, 6A, 7F, 8, 9V, 11A, 12F, 14, 15A, 1 5B, 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 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 Call 39 The polysaccharides of the Streptococcus pneumoniae serotype group are selected from the group consisting of Multivalent immunogenic compositions.

2. The immunogenic composition is not limited to any other Streptococcus pneumoniae (S. pneum oniae serotype polysaccharide-protein conjugates. The multivalent immunogenic composition of claim 1.

3. At least one of the polysaccharide-protein conjugates is a conjugate that includes an aprotic solvent. The multivalent immunogenic composition of claim 1 or 2, formed by a gating reaction.

4. Each of the polysaccharide-protein conjugates comprises an aprotic solvent. The multivalent immunogenic composition of claim 1 or 2, which is formed by a polymerization reaction.

5. 5. The method according to claim 3, wherein the aprotic solvent is dimethylsulfoxide (DMSO). A multivalent immunogenic composition of claim 1.

6. Carrier proteins include outer membrane protein complex (OMPC), tetanus toxoid, diphtheria 1 to 5, which are selected from the group consisting of atoxoid, protein D and CRM197.

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

7. The multivalent immunogen according to any one of claims 1 to 6, wherein the carrier protein is CRM197. sexual composition.

8. The multivalent immunogenic composition according to any one of claims 1 to 7, wherein the composition further comprises an adjuvant. Composition.

9. The multivalent immunogenic composition according to any one of claims 1 to 7, wherein the composition does not contain an adjuvant. Composition.

10. 10. A method for treating a patient comprising administering to said patient a multivalent immunogenic composition according to any one of claims 1 to 9. A method for inducing an immune response in a human patient.

11. 10. A method for treating a patient comprising administering to said patient a multivalent immunogenic composition according to any one of claims 1 to 9. A method for inducing a protective immune response in a human patient.

12. 10. A method for treating a patient comprising administering to said patient a multivalent immunogenic composition according to any one of claims 1 to 9. In human patients, against Streptococcus pneumoniae (S. pneumoniae) A method for inducing a protective immune response against a virulent pathogen.

13. 13. The method according to claim 10, wherein the patient has been previously treated with a multivalent pneumococcal vaccine. The method described in paragraph .

14. The method of any one of claims 10 to 13, wherein the patient is between 6 weeks and 17 years old.

15. A method for the prevention and treatment of a disease comprising administering the multivalent immunogenic composition of claim 1 to a patient aged between 6 weeks and 17 years. Methods for preventing pneumococcal pneumonia and / or invasive pneumococcal disease in patients aged between 12 weeks and 17 years 。

16. 23 different S. pneumoniae polysaccharides A multivalent immunogenic composition comprising a protein conjugate, Each of them is a Streptococcus pneumoniae antibody conjugated to a carrier protein. Each of the different polysaccharide-protein conjugates contains a capsular polysaccharide derived from the avian type. Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23 B, 23F, 24F, 33F and 35B, and the carrier protein is CRM1 97. A multivalent immunogenic composition.

17. The immunogenic composition is not limited to any other Streptococcus pneumoniae (S. pneum oniae serotype polysaccharide-protein conjugates. The multivalent immunogenic composition of claim 16.

18. Each of the polysaccharide-protein conjugates comprises an aprotic solvent. and the aprotic solvent is dimethylsulfoxide (DMSO). The multivalent immunogenic composition of claim 16.

19. The multivalent immunogenic composition of claim 16 , wherein the composition comprises an adjuvant.

20. 24 different S. pneumoniae polysaccharides A multivalent immunogenic composition comprising a protein conjugate, Each of them is a Streptococcus pneumoniae antibody conjugated to a carrier protein. Each of the different polysaccharide-protein conjugates contains a capsular polysaccharide derived from the avian type. Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22 F, 23B, 23F, 24F, 33F and 35B polysaccharides, A multivalent immunogenic composition, which is CRM197.

21. The immunogenic composition is not limited to any other Streptococcus pneumoniae (S. pneum oniae serotype polysaccharide-protein conjugates. The multivalent immunogenic composition of claim 20.

22. Each of the polysaccharide-protein conjugates comprises an aprotic solvent. and the aprotic solvent is dimethylsulfoxide (DMSO). The multivalent immunogenic composition of claim 20.

23. 21. The multivalent immunogenic composition of claim 20, wherein the composition comprises an adjuvant.

24. 24 different S. pneumoniae polysaccharides A multivalent immunogenic composition comprising a protein conjugate, Each of them is a Streptococcus pneumoniae antibody conjugated to a carrier protein. Each of the different polysaccharide-protein conjugates contains a capsular polysaccharide derived from the avian type. Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22 F, 23B, 23F, 24F, 33F and 35B polysaccharides, A multivalent immunogenic composition, which is CRM197.

25. The immunogenic composition is not limited to any other Streptococcus pneumoniae (S. pneum oniae serotype polysaccharide-protein conjugates.

25. The multivalent immunogenic composition of claim 24.

26. Each of the polysaccharide-protein conjugates comprises an aprotic solvent. and the aprotic solvent is dimethylsulfoxide (DMSO).

25. The multivalent immunogenic composition of claim 24.

27. 25. The multivalent immunogenic composition of claim 24, wherein the composition comprises an adjuvant.

28. Up to 30 different S. pneumoniae strains A multivalent immunogenic composition comprising a polysaccharide-protein conjugate, Each of them is a Streptococcus pneumoniae conjugated to a carrier protein. and up to 30 polysaccharide-protein conjugates comprising the polysaccharide from serotype E, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 1 5C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B The polysaccharides of the Streptococcus pneumoniae serotype group are selected from the group consisting of Multivalent immunogenic compositions.

29. Up to 30 polysaccharide-protein conjugates are available, including 7C, 9N, 16F, 23A, 35F and 1, 2, 3, 4, 5 or 6 additional Streptococcus selected from 38. The multivalent vaccine of claim 28, further comprising a S. pneumoniae serotype. Immunogenic composition.

30. Up to 30 different S. pneumoniae strains A multivalent immunogenic composition comprising a polysaccharide-protein conjugate, Each of them is a Streptococcus pneumoniae conjugated to a carrier protein. and up to 30 polysaccharide-protein conjugates comprising the polysaccharide from serotype E, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 1 5B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B The polysaccharides of the Streptococcus pneumoniae serotype group are selected from the group consisting of Multivalent immunogenic compositions.

31. Up to 30 polysaccharide-protein conjugates are available, including 7C, 9N, 16F, 23A, 35F and 1, 2, 3, 4, 5 or 6 additional Streptococcus selected from 38. - The multivalent vaccine of claim 30, further comprising a S. pneumoniae serotype. Immunogenic composition.

32. The multivalent immunoglobulin according to any one of claims 28 to 31, wherein the carrier protein is CRM197. Epidemiogenic composition.

33. Each of the polysaccharide-protein conjugates comprises an aprotic solvent. and the aprotic solvent is dimethylsulfoxide (DMSO). The multivalent immunogenic composition according to any one of claims 28 to 32.

34. The multivalent immunogenic composition according to any one of claims 28 to 33, wherein the composition comprises an adjuvant. Composition.

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