Compositions Comprising Streptococcus pneumoniae Polysaccharide-Protein Conjugates and Methods of Use 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 THE INVENTION The present invention provides multivalent immunogenic compositions 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 (pneumococcus) The immunogenic composition is composed of polysaccharides and provides broad spectrum efficacy against pneumococcal disease.

[0002] This application relates to the Sequence Listing submitted electronically in ASCII format, which is hereby incorporated by reference in its entirety. The above ASCI dated December 3, 2019 I copy is entitled 24683WOPCT-SEQTXT-03DEC2019, 6 kg 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 serotype specificity. It is encapsulated by chemically bonded polysaccharides. There are more than 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 complement Polysaccharides not only protect against T cell-dependent immune responses but also have low immunogenicity. antigens, most often processed and bound to 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 those at high risk. However, it has been shown to be useful in preventing pneumococcal disease in infants and young children. Inadequate response to conjugated pneumococcal polysaccharides. Invasive pneumococcus 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. Prevnar® After widespread use of Prevnar in the United States, invasive pneumococcal disease in children was The incidence of serotypes present in the HIV-1 strain is significantly reduced. Disease Control and Prevention,MMWR Mor b Mortal Wkly Rep 2005,54(36):893-7 However, in some parts of the world, the prevalence of serotypes with Prevnar® is It has 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 See 89.

[0005] U.S. Patent Application Publication No. US2006 / 0228380 identifies serotypes 1, 3, 4, 5, and 6A 13-valent pneumococcal strains containing 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F describes a polysaccharide-protein conjugate vaccine. Chinese Patent Application Publication No. CN1 01590224A is serotype 1, 2, 4, 5, 6A, 6B, 7F, 9N, 9V, 14, 14-valent pneumococcal polysaccharide-protein conjugate containing 18C, 19A, 19F, and 23F The following vaccines are listed:

[0006] Other PCVs cover 7, 10, 11 or 13 serotypes contained in PCV-15. (U.S. Patent Publication No. 2011 / 0195086), but immunity is limited to some serotypes. Interference has been observed (e.g., with serotype 3 in GSK's PCV-11). Pfizer's PCV-13 [PREVNAR® )13], a lower response rate to serotype 6B has been observed. 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 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. effective in reducing incidence. However, there are serologic factors not present in currently available vaccines. The prevalence of pneumococci expressing Streptococcus pneumoniae is increasing. Additional pneumococcal vaccine combinations that may provide 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] Chinese 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 treating Streptococcus pneumoniae (S. pneumoniae) The present invention provides a multivalent immunogenic composition comprising a conjugate (bacterial) polysaccharide protein conjugate, wherein the conjugate Each of the conjugates was conjugated to a carrier protein containing Streptococcus erythrocytosis. The polysaccharide-protein conjugate contains a polysaccharide derived from a S. pneumoniae serovar, and is selected from the group consisting of: The polysaccharides of the Streptococcus pneumoniae serovar 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 Yobi 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 contain capsular polysaccharides derived from Streptococcus pneumoniae serovars. Bacillus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, and 12F , 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, Manufactured 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 containing polysaccharides of the group of Streptococcus pneumoniae serovars 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 contain capsular polysaccharides derived from 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 F, 33F and 35B.

[0014] The present invention provides 23 different Streptococcus pneumoniae polysaccharide-protein conjugates. and a multivalent immunogenic composition comprising a conjugate, wherein each of the conjugates is a carrier. Protein-conjugated pods from Streptococcus pneumoniae serovars Each different polysaccharide-protein conjugate contains a membrane polysaccharide, which is Tococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, and 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 contain capsular polysaccharides derived from 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 Manufactured from F, 24F, 33F and 35B.

[0016] The present invention provides 24 different Streptococcus pneumoniae polysaccharide-protein conjugates. and a multivalent immunogenic composition comprising a conjugate, wherein each of the conjugates is a carrier. Protein-conjugated pods from Streptococcus pneumoniae serovars Each different polysaccharide-protein conjugate 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 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 contain capsular polysaccharides derived from 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 Manufactured from F, 24F, 33F and 35B.

[0018] The present invention provides 24 different Streptococcus pneumoniae polysaccharide-protein conjugates. and a multivalent immunogenic composition comprising a conjugate, wherein each of the conjugates is a carrier. Protein-conjugated pods from Streptococcus pneumoniae serovars Each different polysaccharide-protein conjugate 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 serotype 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 strains It contains polysaccharides from a group including the serovar N. ie.

[0020] The present invention provides 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 serotype selected from the group consisting of 33F and 35B , further 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 serotype 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 strains Contains polysaccharides of the S. nie serovar group.

[0022] The present invention provides 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 serotype selected from the group consisting of 33F and 35B , further 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 dimethyl sulfoxide (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 present invention. Also provided are methods for inducing a regulatory immune response. The patient had previously been treated with multivalent pneumococcal vaccine.

[0024] The multivalent immunogenic compositions of the invention are useful for the treatment of pneumococcal pneumoniae in combination with different complementary pneumococcal vaccine regimens. Thus, the present invention provides a method for administering a multivalent immunogenic composition of the present invention to a patient. and further administering a multivalent pneumococcal vaccine to the patient, in any order. In certain embodiments, a method for inducing a protective immune response in a human patient is provided. In this regard, polyvalent pneumococcal vaccines contain multiple Streptococcus pneumoniae polysaccharide proteins. and a carrier protein conjugate, each of which is The antibody comprises a polysaccharide derived from Streptococcus pneumoniae serovar conjugated to In other embodiments, the multivalent pneumococcal vaccine comprises unconjugated capsular polysaccharides. The present invention also provides a method for the preparation of Streptococcus pneumoniae polysaccharide protein conjugates. and a multivalent immunogenic composition comprising a conjugate, wherein each of the conjugates comprises a carrier. Streptococcus pneumoniae serovar conjugated to body proteins Selected serotypes of Streptococcus pneumoniae contain polysaccharides and are highly susceptible to other selected This results in cross-reactivity against serotypes. [Brief explanation 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. [Figure 2] ECL dilution titer ratio of PCV22 / APA compared with non-adjuvanted PCV22 (PCV22 unadj) at PD3. [Figure 3] Serotype-specific OPA dilution titers (pre-immunization, 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. [Figure 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 unadjuvanted 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 ratio of PCV22 / APA compared with 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-immunization (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 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 dogs per group) after vaccination with PCV23 in the presence or absence of APA. Symbols indicate ratios at PD1. GMT ratios with error bars representing 95% CI. [Figure 10B] Comparison of ECL antibody responses in IRM (8-9 dogs per group) after 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 dogs per group) after vaccination with PCV23 in the presence or absence of APA. Symbols indicate ratios at PD3. GMT ratios with error bars representing 95% CI. [Figure 11A] Comparison of ECL antibody responses in IRM (9 per group) after 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) after 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) after 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 post-dose 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 for 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 multivalent immunogenic compositions comprising pneumococcal polysaccharide-protein conjugates. wherein each of the conjugates is a sequence conjugated to a carrier protein. Contains polysaccharides derived from serovars of Streptococcus pneumoniae (S. pneumoniae) , Streptococcus pneumoniae serotypes are as defined herein do.

[0027] In some embodiments, the present invention provides a method for producing a Streptococcus pneumoniae polysaccharide protein. The present invention provides a multivalent immunogenic composition comprising a protein conjugate, wherein the conjugate Each of these antibodies is a Streptococcus pneumoniae antibody conjugated to a carrier protein. and 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 Yobi 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 Call 39.

[0028] In some embodiments, the multivalent immunogenic composition comprises: i) 1, 3, 4, 5, 6A, 6B, 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. a Enterococcus pneumoniae polysaccharide-protein conjugate, Each of the peptides was conjugated to a carrier protein containing Streptococcus nucleus. It contains polysaccharides derived from Streptococcus pneumoniae serotypes, and the serotypes of Streptococcus pneumoniae are 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 contains 24F, 33F and 35B. The composition is administered to mice, rabbits and / or monkeys. immunogenic in humans and capable of killing vaccine-type bacterial strains at all doses tested It was found that the antibody produced

[0029] The multivalent immunogenic compositions of the present invention are directed to vaccine-type Streptococcus pneumoniae serovars to immunize patients against pneumococcal pneumonia 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 compound, and further 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 a therapeutically effective amount of a compound selected from the group consisting of 2-amino-3-methyl-2-propanol, ... In some embodiments, the multivalent immunogenic compositions of the present invention may be used in combination with other multivalent pneumococcal vaccines previously described. It is administered to immunized patients.

[0030] In an embodiment of the invention, a conjugate derived from at least one pneumococcal serotype is The esters are prepared using reductive amination in aprotic solvents 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 prepared using Covalent attachment of polysaccharides to proteins by direct consumption of lysine residues on the surface of the carrier protein Enhanced covalent binding was observed in polysaccharides containing conjugated polysaccharide antigens in DMSO. The direct benefit of this approach is the enhanced stability of polysaccharide-protein conjugates in multivalent immunogenic compositions. vinegar.

[0031] I. Definitions and Abbreviations The following abbreviations apply throughout this specification and the 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 monkeys 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-administration 3 PnPs Pneumococcus polysaccharide Ps polysaccharide PS-20 Polysorbate-20 RI refractive index UV ultraviolet light w / v weight / volume In order that the present invention may be more readily understood, certain scientific and technical terms are specifically defined below. Unless specifically defined elsewhere in this specification, all other terms used herein are 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 in the appended claims, the singular forms "a," "the," and "the" are used unless the context clearly dictates otherwise. "a" 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 are indicated. "And / or" is used for emphasis.

[0035] "Aqueous solvent" or "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 connection with conjugation reactions such as reductive amination The terms "solvent," "DMSO solvent," or "DMSO conditions" refer to a solvent for a conjugation reaction. aprotic solvent or solvents (or solvents, if applicable) as solvents The aprotic solvent refers to the use of a combination of water (DMSO) and ethanol. 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. It refers to the addition of a sugar-protein conjugate. Multivalent polysaccharide-protein conjugate 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 Caspian virus polysaccharide-protein conjugate and derived from different serotypes Mixtures not containing Streptococcus pneumoniae polysaccharide-protein conjugates "consisting essentially of" and its derivatives, such as "consisting essentially of" or " "Consisting essentially of" includes the inclusion of any element or group of elements listed, and Other elements of a similar or different nature to the element may be used in combination with 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 present invention is an amount that can be used to treat a microorganism, such as a streptococcus, 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 "indicated 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 nucleus, including but not limited to For the prevention of one or more diseases caused by any serotype of S. monnier, 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 authorities, such as the National Institute of Infectious Diseases (NIID).

[0040] "Multivalent pneumococcal vaccine" is a vaccine that protects against multiple serotypes of Streptococcus pneumoniae. Multiple active substances (e.g., pneumococcal capsular polysaccharide or pneumococcal polysaccharide-protein conjugate) .

[0041] The term "polysaccharide" includes "sugar," "oligosaccharide," "polysaccharide," "liposaccharide," "lipooligosaccharide ( LOS), lipopolysaccharides (LPS), glycosylates, complex 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 "immunized" refers to PCV8, PCV15, PCV22, PCV23, and Pneumococcal polysaccharide compositions, including, but not limited to, PCV24, It refers to a composition that does not contain an adjuvant.

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

[0044] "PCV15" is a Streptococcus pneumoniae polysaccharide (PnP) serotype-1, 3, -4, -5, -6A, -6B, -7F, -9V, -14, -18C, -19A, -1 By "immunogenic compositions" is meant immunogenic compositions 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 "immunogenic composition" is meant an immunogenic composition containing -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 nucleotides," "CpG-containing oligonucleotides," "CpG oligonucleotides" "CpG nucleotide" and similar terms refer to a nucleotide sequence of 6 to 50 nucleotides containing an unmethylated CpG moiety. For example, Wang et al., 2003, Vaccine 21:4297. CpG-containing oligonucleotides include any synthetic nucleotide. Modified oligonucleotides include those 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. Immune adjuvants are substances that help to enhance the immune system's immunity when administered alone. (e.g., induces no or only a weak antibody or cellular immune response) enhances the immune response to an antigen, increases antibody titers against the antigen, and / or enhances the immune response in an individual Adjuvants can reduce the dose of antigen that is effective in achieving an immune response. They are often administered to enhance immune responses 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 Caspian virus. The subject is a human. The subject may be treated prophylactically or therapeutically. Prophylactic treatment is the treatment of pneumococcal disease. sufficient protection to reduce 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 treatment may be performed to reduce the incidence or prevent recurrence. 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 people 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 infected persons, or anyone for whom it is desirable to reduce the possibility of infection, such as immunocompromised persons, the elderly, children, Adult or healthy individuals are included.

[0052] A "stable" multivalent immunogenic composition is one that remains stable at refrigerated temperatures (e.g., 2-8°C or 4°C) for at least 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 for a period of 2 months. and 37°C for 1 month, 3 months, 6 months, 12 months and / or 24 hours. 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 about 20% or less variation in: (a) Streptococcus pneumoniae polysaccharide in composition; (b) the number average molecular weight (Mn) of the protein conjugate; (b) the number average molecular weight (Mn) of the streptococcus in the composition; (c) Weight-average molecular weight (Mw) of Bacillus pneumoniae polysaccharide-protein conjugate (d) the specific excitation wavelength, e.g., 280 nanometers, at which the specific time is detected; (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 a desired characteristic over time at a particular temperature. " refers to a conjugate that exhibits a specific property, such property lasting for a specified time and duration. fluctuates by no more than about 5%, about 10%, about 15%, or about 20% at the temperature .

[0053] II. Multivalent immunogenic compositions The present invention provides a plurality of Streptococcus pneumoniae polysaccharide-protein conjugates. and a multivalent immunogenic composition comprising: Containing a polysaccharide derived from Streptococcus pneumoniae serovar conjugated to a protein 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 conjugating a medicament 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 antimicrobial 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 consisting essentially of them. In a subembodiment of embodiment E1, the immunogenic composition is an immunogenic composition that ... -Does not contain Monnier polysaccharide protein conjugates.

[0055] As used herein, de-O-acetylated serotype 15B (DeOAc15B) pneumococcal polysaccharide is substantially identical to the serotype 15C pneumococcal polysaccharide and has a virtually identical NMR spectrum. (data not shown). The saccharide and serotype 15C pneumococcal polysaccharides ranged from 0 to 5% per repeating unit or 0 ~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 may be substituted for serotype 15C. 15B (DeOAc15B) can be used. De-O-acetylation methods are described, for example, in Raj et al. am et al., Clinical and Vaccine Immunology, 2007 ,14(9):1223-1227, and are well known in the art.

[0056] Any of the multivalent immunogenic compositions of the invention, including embodiment E1 and any subembodiments thereof. In any particular embodiment, the composition further comprises a pharmaceutically acceptable carrier.

[0057] Cross-reactivity In one embodiment, the present invention provides a method for producing 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 Streptococcus pneumoniae serotypes and inhibits the blood Serotype 6C cross-reacts with Streptococcus pneumoniae serotypes 6A and 6B brings about sex.

[0058] In one embodiment, the present invention provides a method for producing 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 Streptococcus pneumoniae serotypes and inhibits the blood Serotype 6A is against Streptococcus pneumoniae serotypes 6B and / or 6C Provides cross-protection.

[0059] In one embodiment, the present invention provides a method for producing 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 Streptococcus pneumoniae serotypes and inhibits the blood Serotype 6B is against Streptococcus pneumoniae serotypes 6A and / or 6C Provides cross-protection.

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

[0061] In one embodiment, the present invention provides a method for producing 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, Serotype 15B also provides cross-protection against Streptococcus pneumoniae serotype 15C. 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 (i.e. , 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 compound is isolated from a culture of the C. phtheriae strain C7(β197). In this case, CRM197 was prepared according to the method described in U.S. Pat. No. 5,614,382. CRM197 is typically 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 Pfenex Inc., San Diego, CA It is produced in Pseudomonas fluorescens (Pseudomonas fluorescens).

[0064] Other suitable carrier proteins include additional inactivated 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 (E. coli) LT, E. coli (E. i) ST, and Pseudomonas aeruginosa inosa. Bacterial outer membrane proteins, e.g., outer membrane protein complexes. Coalescence membrane protein (OMPC), porin, transferrin-binding protein, pneumococcal surface protein Pseudomonas pneumoniae adhesin A (PspA; see WO 02 / 091998), pneumococcal adhesin PsaA, a 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 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 (see WO 03 / 54007) may also be used. Proteins such as ovalbumin, keyhole limpet hemocyanin (KLH), bovine blood serum albumin (BSA) or purified protein derivative of tuberculin (PPD), Por B (derived from N. meningitidis), PD (Haemophilus influenza enzae protein D; see, for example, EP 0 594 610 B), and have been developed into 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 hormonal factors 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 ( WO 00 / 61761), and flagellin (Ben-Yedidia (see, e.g., Wang et al., 1998, Immunol Lett 64:9) also acts as a carrier protein 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 Other publications in the journal "Maecel Dekker Inc., 1992" 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 mutation of at least one residue of U.S. Pat. No. 5,917,017 or U.S. Pat. or those with 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 present invention, the polysaccharide protein conjugates in the multivalent immunogenic composition A second carrier may be used for 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 purified using high Streptococcus pneumoniae multimers to enhance the immunogenicity of the antigen. The carrier protein is conjugated or attached to a 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 No capsular polysaccharide is conjugated to two or more carrier proteins (each capsular polysaccharide molecule are conjugated to a single carrier protein). For example, 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) are 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 art-recognized methods. For example, individual pneumococcal conjugates can be prepared by the methods described in the present application. Such a vehicle can be formulated using a physiologically acceptable vehicle to produce a Examples of vehicles include water, buffered saline, polyols (e.g., glycerol, propylene glycol, glycol, liquid polyethylene glycol) and dextrose solution, 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 thiazolidine 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 polysaccharides wherein the Streptococcus pneumoniae serovar is described herein. Suitable adjuvants to enhance the effectiveness of the composition include: These include, but are 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, Newton) Formulated into submicron particles using a microfluidizer such as 5% squalene, 0.5% Tween 80 and 0.5% S Pan 85 (which may optionally contain various amounts of MTP-PE) (b) submicron-sized particles containing MF59 (International Patent Application Publication No. WO 90 / 14837); Microfluidized into a granular 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 from the group consisting of: U.S. 3-O-deacylated monophospholipid A (M) described in Patent No. 4,912,0945 PL™), trehalose dimycolate (TDM) and cell wall skeleton (CWS), preferably Preferably, MPL+CWS (Detox™), and (d) Montanide (Montanide anide)ISA; (3) Saponin adjuvants, such as Quil A or STIMULON (trademark) QS-21 (Antigenics, Framingham, MA) (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) and Iscomatrix® (protein (which have substantially the same structure as ISCOMs except that they do not contain proteins) can be used; (4) Bacterial lipopolysaccharides, synthetic lipid A analogues, e.g., aminoalkylglucosamine Antioxidant phosphate (AGP) or its derivatives or analogues (which may be obtained 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 benzodiazepines, which are formulated in aqueous form or as stable emulsions; (5) Synthetic polynucleotides, such as oligonucleotides containing CpG motifs Do (U.S. Patent No. 6,207,646); (6) Cytokines, such as interleukins (e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, 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, e.g., the trimer of complement component C3d.

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

[0073] Muramyl peptides include N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanine-2-(1',2'diaminodiphenylmethane) 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 can be an alum-precipitated vaccine or an alum-adsorbed vaccine. Sodium 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 Immunology 143:489-493. Aluminum salts include , 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), These include rufous alumina, alumina trihydrate, or aluminum trihydroxy. However, the present invention is not limited to these.

[0075] APA is an aqueous suspension of aluminum hydroxyphosphate. APA is a compound containing aluminum chloride. 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: 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 of recombinant alum salt. Per μg of protein.

[0076] In certain embodiments, 50 to 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 point 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 salts are slowly released over a period of 2-3 weeks. They form a depot of antigens that are absorbed by the cells and are involved in the nonspecific activation of macrophages and complement activation. and / or stimulate innate immune mechanisms (possibly due to stimulation by uric acid) (See, e.g., Lambrecht et al., 2009, Curr Opin Immunol. See Nol 21:23.

[0077] Monovalent bulk aqueous conjugates are typically mixed together and 6B (which is the target concentration Diluted to a target concentration of 4 μg / mL for all serotypes except for 100 (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. Add adjuvant to target a final aluminum concentration of 250 μg / mL. The purified 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, especially CpG-containing oligodeoxynucleotides (CpG ODs) 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: 89-110.

[0080] In an alternative embodiment, the immunogenic composition is an immunogenic composition as described herein, e.g., embodiment E1 or any of the subembodiments thereof. It contains a polysaccharide-protein conjugate and does not contain an adjuvant.

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

[0082] In another embodiment, the multivalent immunogenic composition of the present invention is administered orally, but Therefore, it 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 and polyethylene glycol. and injectable organic esters such as ethyl oleate. , 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 compositions 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 before 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, methyl ... Examples of suitable glycerols include, but are not limited to, ethanol, sorbitol, and glycerol. do not have.

[0086] It is also preferred that at least one pharmaceutically acceptable excipient is a buffer. For example, when the pharmaceutical composition is intended for infusion or injection, The composition may be dissolved 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, and maltodextrin. tartrate, phosphate, citrate, carbonate, glycinate, histidine selected from the group consisting of amine, glycine, succinate and triethanolamine buffers It can be done.

[0088] The buffer should be a parenteral USP compatible buffer, especially if the pharmaceutical formulation is intended 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 surfactants, such as those based on Ringer's dextrose. with or without the addition of an active agent and other pharmaceutically acceptable adjuvants. Examples include sterile liquids such as water and oil. Generally, water, saline, aqueous dextrose, sugars and related sugar solutions, glycols such as 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 fats derived from milk or eggs. It can be obtained.

[0090] The formulations of the present invention may include a surfactant. Preferred surfactants include: Examples include, but are 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 may vary in the number of groups. or t-octylphenoxypolyethoxyethanol) are of particular interest; Ctylphenoxy)polyethoxyethanol (IGEPAL CA-630 / NP-40 ); phospholipids, such as phosphatidylcholine (lecithin); nonylphenol ethoxylate acrylates, such as the Tergitol (trademark) NP series; lauryl, cetyl, stearyl and Polyoxyethylene fatty ethers derived from olefins and 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 Les 9, polyoxyethylene sorbitan ester and / or octoxynol Includes

[0092] The preferred amount (wt%) of surfactant is as follows: 0.01 to 1%, particularly about 0 0.1% polyoxyethylene sorbitan ester (e.g., PS-80); 0.001 to 0.1%, especially 0.005-0.02%, of octyl or nonylphenoxy polyoxyethylene ethanol (e.g., 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 of 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 In another embodiment, the PS-20 may range from 0.3% (w / v). In another embodiment, the PS -20 may range from 0.05% to 0.8% (w / v). For example, PS-20 may be in the range of 0.05% to 0.2% (w / v). A mixture (blend) of up to 24 serotypes is prepared in 100% ethanol, saline, and PS-20. The mixture is then mixed with APA and PEG in the presence or absence of an antimicrobial preservative. It consists of combining it with saline.

[0094] In certain embodiments, the multivalent immunogenic composition is an antibody against 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 this case, the multivalent immunogenic composition further contains 0.2% to 0.8% w / v polysorbate 20. Included.

[0095] The multivalent immunogenic composition PCV24 was prepared in an aprotic solvent (also known as DMSO chemistry). Streptococcus pneumoniae polysaccharide (PnP) serotype-I using reductive amination 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 / mL, respectively) It is formulated at 192 μg / mL (1 μg / mL or 192 μg / mL) and is called "PCV24 unadjuvant." In another specific embodiment, the multivalent immunogenic composition PCV24 contains 20 mM L- Histidine (pH 5.8), 150 mM NaCl and 0.2% w / v polysorbate in Pretz-20 (PS-20), and also in the form of aluminum phosphate adjuvant [Al]2 50 μg / mL of each polysaccharide serotype at 4 μg / mL (total polysaccharide concentration 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 the multivalent composition can be The conjugation reaction used to prepare the compound contains 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 dextrin was observed when polysorbate 20 was used alone or in combination with poloxamer 188. It was recognized in combination with an oar.

[0097] The exact mechanism by which certain surfactants protect biotherapeutics is unclear. Possible stabilization mechanisms are not fully understood and cannot be predicted in advance. are due to factors such as 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 aggregation seeds. Direct binding of surfactants to biotherapeutics 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 nonionic 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 the same properties. These copolymers are generally designated by the letter "P" (for poloxamer) followed by three digits. where the first two digits multiplied by 100 is the polyoxypropylene core. The last digit x 10 indicates the percentage of polyoxyethylene content. (e.g., P407 is a polyoxypropylene with a molecular weight of 4,000 g / mol and It is a poloxamer with a polyoxyethylene content of 70%. In trade names, the symbols for these copolymers are letters ( L = liquid, P = paste, F = flake (solid)) followed by a two or three digit number 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 1000 ppm or less). See US Pat. No. 3,740,421.

[0099] Specific examples of poloxamers include those with the general formula: HO(C2H4O) 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 the oxamer was 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 solution is polyethylene 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. glycols, such as propylene glycol and polyethylene glycol, polyethylene glycol Examples include, but are not limited to, propylene glycol monomethyl ether. Polyethylene 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 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, maleate, tartrate, phosphate, citrate, carbonate, glycinate, histidine, glycine, succinate, HEPES(4-( 2-hydroxyethyl)-1-piperazineethanesulfonic acid), MOPS (3-(N-molybdenum-2-hydroxyethyl)-1-piperazineethanesulfonic acid), MES (2-(N-morpholino)ethanesulfonic acid) and triethanolamine buffer. The buffer may be selected from the group consisting of 4 to 1000 mg of 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 certain embodiments, the buffer contains phosphate, succinate, histidine, M ES, MOPS, HEPES, acetate or citrate. The buffer may further comprise, for example, a buffer containing a soluble phosphate buffer according to USP 1000 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 certain embodiments, 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. 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 sodium Non-ionics, including but not limited to sorbitol and glycerol Tonicity agents may be used. Suitable salt concentration ranges include 20 mM to 500 mM or 40 mM to In one embodiment, the dietary fiber content includes, but is not limited to, 170 mM. The brine is NaCl, which may optionally be present at a concentration of 25 mM to 170 mM.

[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 may be administered using any suitable form of administration, such as a microsphere or Polymeric materials are used in implants.

[0106] The amount of conjugate in each dose of the composition may be adjusted to provide immunoprotection without significant adverse effects. The amount is selected to induce a protective 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, 0.4-4 μg, 0.4-3 μg, 0.4-2 μg, or 0.4-1 μg. In some embodiments, the dose of one or more polysaccharide conjugates is 100, 150, 200, 250, 300, 400, 500 or 750 ng, or 0.4, 0.5, 0.6, 0.7, 0.75, 0.8, 0.9, 1, 1.5, 2, 3, 4, 5, 6, 7, 7.5, 8 , 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 22, 25, 30, 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 equal amounts. 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 different amounts).

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

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

[0110] In certain embodiments, the compositions of the present invention can be prepared by one or more methods known to those skilled in the art. For example, parenterally, transmucosally, transdermally, intramuscularly, intravenously, intradermally, intranasally, subcutaneously, intraperitoneally, In one embodiment, the compositions of the present invention are administered to a subject and formulated accordingly. The product is injected into the skin, muscles, veins, arteries, subcutaneously, or into the respiratory mucosa as a liquid preparation. Liquid preparations for injection include solutions.

[0111] III. Manufacturing method Capsular polysaccharides from Streptococcus pneumoniae can be isolated using standard techniques known to those skilled 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 Putococcus pneumoniae polysaccharide serovars are grown in soy-based media. The individual polysaccharides are purified by standard procedures including centrifugation, precipitation and ultrafiltration. U.S. Patent Application Publication No. 2008 / 0286838 and U.S. Patent No. 5,847,112 To reduce the viscosity of the polysaccharide sample, and / or conjugates Use techniques such as mechanical or chemical sizing to improve the filterability of the product. Chemical hydrolysis can be carried out 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 that can react with a carrier protein. 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, and the free end of the linker is an ester group. Thus, the linker may have at least one terminal The other end is a linker that reacts with the polysaccharide to form a polysaccharide-linker. - intermediates are formed.

[0114] The polysaccharide can be coupled to the linker using primary amine groups on the polysaccharide. In this case, the linker typically has an ester group at both ends. The cuprate was prepared 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 the first linker of the polysaccharide. A first ester group to react with primary amine groups on the carrier molecule A typical linker is a bifunctional 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 done 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 carbonyl group with an additional linker, and (a2) connecting the free end of the additional linker to the linker. In these embodiments, the additional linker is typically has primary amine groups at both ends, thereby attaching one of the primary amine groups to the cap of the polysaccharide. Step (a1) can be carried out by reacting the carboxyl group with the carboxyl group by reductive amination. The primary amine groups are used, which are reactive with the carbonyl groups of the polysaccharide. The same primary amine group is typically a hydroxylamino group. The reaction occurs at both ends of the polysaccharide to form additional linkers via C-N bonds. A coupled polysaccharide-additional linker intermediate is produced.

[0117] The polysaccharide may 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) connecting the free end of the additional linker to the linker. In this case, the additional linker typically has a first linker at both ends. It has a primary amine group, which allows one of the primary amine groups to interface with the carboxyl group of the polysaccharide. The reaction by AC activation makes it possible to carry out step (a1). 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 results in a polysaccharide- An additional linker intermediate is generated.

[0118] In one embodiment, chemical activation of the polysaccharide followed by reductive amination Conjugation to carrier proteins by the method described in U.S. Pat. No. 4,365,170, 4 ,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. 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 involve activation of pneumococcal polysaccharides by reaction with Together, they result in random oxidative cleavage of adjacent 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 occur 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 capping is carried out 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 be optionally 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" The term includes both periodate and periodic acid. Metaperiodite (IO4 - ) and orthoperiodate (IO6 -) and various salts of periodate (e.g., sodium periodate and potassium periodate). In one embodiment, the capsular polysaccharide comprises: Preferably, it is oxidized in the presence of sodium periodate (NaIO4). In an embodiment, the capsular polysaccharide is lyophilized 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 piperidin pyrrolidine-N-oxy or pyrrolidine-N-oxy compounds (see WO 2014 / 097099) In the reaction, the actual oxidant is N in the catalytic cycle. In one embodiment, the stable nitroxy or The nitroxide radical compound is piperidine-N-oxy or pyrrolidine-N-oxy. In one embodiment, the stable nitroxyl or nitroxide compound The dicarboxylic acid compound is TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxy) or 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 Annuric acid, 1,3,5-tribromo-1,3,5-triazinane-2,4,6-trione , diiodoisocyanuric acid and 1,3,5-triiodo-1,3,5-triazinane- Preferably, the oxidizing agent is selected from the group consisting of N-chloro-2,4,6-trione. It is succinimide.

[0122] In certain embodiments, the oxidizing agent is 2,2,6,6-tetramethyl-1-piperidinyl oxidizing agent. TEMPO free radical and N-chlorosuccinimide ( NCS) (described in WO 2014 / 097099). In one embodiment, the glycoconjugates derived from Streptococcus pneumoniae are prepared by: 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 a carrier protein containing an amine group (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 acid).

[0124] In certain embodiments, the present invention provides a method for carrying out a conjugation reaction in an aprotic solvent. Utilizing serotype 8 Streptococcus pneumoniae polysaccharide-protein conjugates The present invention provides a method for preparing 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 amino acid reduction. In other embodiments, the protein is tetanus toxoid, diphtheria toxoid, or the like. 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 dimethyl sulfoxide (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. 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 some embodiments, the polysaccharide has a molecular weight of between 100 kDa and 800 kDa. In another embodiment, the polysaccharide has a molecular weight of 200 kDa to 600 kDa. For example, polysaccharides range from 100kDa to 1,000kDa, 100kDa to 900kDa, and 100kDa. 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 to 700kDa, 400kDa to 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 combine 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., Examples include sodium cyanoborohydride or sodium borohydride. 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 a mixture of carboxylic acid solvents. The procedure is carried out in DMF (dimethylformamide) solvent. Activated polysaccharides and carrier proteins If the products have been lyophilized, they may require DMSO or DMF solution to be reconstituted. In one embodiment, the aprotic solvent is DMSO.

[0128] At the end of the reduction reaction, unreacted aldehyde groups may remain in the conjugate. which can be capped using a suitable capping agent. In 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 prepared by various methods known to those skilled in the art. The polysaccharide-protein conjugate can be purified (enriched with respect to the amount of 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 complex carbohydrates are subjected to diafiltration and depth filtration. Purification by ion exchange chromatography 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 involving multivalent compositions that are not prepared in an organic 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 nucleus can be prepared using reductive amination in aprotic solvents. Streptococcus pneumoniae capsular polysaccharide-protein conjugates 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., hydrolysis) of purified polysaccharides. The fragments are conveniently formed by cleavage, usually followed by purification to obtain fragments of the desired size.

[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 this 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 conjugated to a carrier protein using reductive amination in aprotic solvents Polysaccharides of one or more serotypes are conjugated using reductive amination in aqueous media. In certain embodiments, the multivalent compositions of the present invention comprise 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 conjugated to a carrier protein using reductive amination in aprotic solvents In certain embodiments, the multivalent compositions of the present invention comprise antibodies from more than one serotype. Polysaccharides can be used to form carrier proteins using other chemical properties that may be present in aprotic or aqueous solvents. It is conjugated to proteins.

[0132] Thus, the present invention provides a streptococcus conjugated to a carrier protein. Multiple Streptococcus pneumoniae strains, each containing capsular polysaccharides from a S. pneumoniae serovar and a multivalent immunogenic composition comprising a monier polysaccharide-protein conjugate, Here, the serotypes of Streptococcus pneumoniae are those described herein (i.e., in Section II). and the like), wherein 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%, ... 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 serotypes, as expected compared to the same conjugates produced under aqueous conditions It has also been found to provide excellent stability and enhanced immunogenicity (U.S. Patent Application No. 6 See 2 / 463,216 and 62 / 555,444).

[0134] In certain embodiments of the present 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 certain embodiments of the present invention, the concentration is about 0.03 to about 0.192 mg / mL. 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 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 prepared in a protic solvent, the storage The average molecular weight (Mw) of all Leptococcus pneumoniae polysaccharide-protein conjugates ) (average of all conjugates in the composition) is about 2,000 to about 6,500 kDa, about 2,500 to approximately 6,000 kDa, approximately 3,000 to approximately 5,500 kDa, approximately 3,500 to 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 to approximately 4,400 kDa, approximately 3,500 to approximately 4,300 kDa, approximately 3,500 to approximately 4,200kDa, approximately 3,600~4,700kDa, approximately 3,600~4,600k Da, approximately 3,600 to approximately 4,500 kDa, approximately 3,600 to approximately 4,400 kDa, approximately 3, 600 to about 4,300kDa, about 3,600 to about 4,200kDa, about 3,700 to about 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, approximately 3,800 to approximately 4,500kDa, approximately 3,800 to approximately 4,400kDa , about 3,800 to about 4,300kDa, about 3,800 to about 4,200kDa, about 3,90 0 to approximately 4,700 kDa, approximately 3,900 to approximately 4,600 kDa, approximately 3,900 to approximately 4,5 00kDa, approximately 3,900 to approximately 4,400kDa, approximately 3,900 to approximately 4,300kDa, Or about 3,900 to about 4,200 kDa.

[0137] The polysaccharide-protein conjugate in the multivalent immunogenic composition is 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 approximately 10,000 kDa, approximately 1,500 to approximately 5,500 kDa, approximately 1,500 to approximately 5,600kDa, approximately 1500 to approximately 5,700kDa, approximately 1500 to approximately 5,800kDa , about 1500 to about 5,900 kDa, about 1500 to about 6,000 kDa, about 1,000 to about 5,500kDa, approx. 1,000 to approx. 5,000kDa, approx. 1,000 to approx. 4,000kDa 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, approximately 1,300kDa, approximately 1,400kDa, approximately 1,500kDa, approximately 1, 600kDa, approximately 1,700kDa, 1,800kDa, approximately 1,900kDa, approximately 2,0 00kDa, approximately 2,100kDa, approximately 2,200kDa, approximately 2,300kDa, approximately 2,4 00kDa, approximately 2,500kDa, approximately 2,600kDa, approximately 2,700kDa, approximately 2,8 00kDa, approximately 2,900kDa, approximately 3,000kDa, approximately 3,100kDa, approximately 3,2 00kDa, approximately 3,300kDa, approximately 3,400kDa, approximately 3,500kDa, approximately 3,6 00kDa, approximately 3,700kDa, approximately 3,800kDa, approximately 3,900kDa, approximately 4,0 00kDa, approximately 4,100kDa, approximately 4,200kDa, approximately 4,300kDa, approximately 4,4 00kDa, approximately 4,500kDa, approximately 4,600kDa, approximately 4,700kDa, approximately 4,8 00kDa, approximately 4,900kDa, approximately 5,000kDa, approximately 5,100kDa, approximately 5,2 00kDa, about 5,300kDa, about 5,400kDa or about 5,500kDa .

[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 solvents The percentage of polysaccharide serotypes produced in aprotic solvents divided by the total number of polysaccharide serotypes. where the total is calculated by adding the (including those manufactured in 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, 000kDa, or about 1,000 to about 2,500kDa, 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 prepared in a protic solvent, the storage Number average molecular weight (Mn) of Leptococcus pneumoniae polysaccharide-protein conjugate ( The average of all conjugates in the composition is approximately 900 to 3,000 kDa, and approximately 1,000 ~approximately 3,000kDa, approximately 1,000~approximately 2,500kDa, approximately 1,500~approximately 2,50 0kDa, approximately 1,800 to approximately 2,500kDa, approximately 1,900 to approximately 2,500kDa, or 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 prepared in a protic solvent, the storage Mn of each Leptococcus pneumoniae polysaccharide-protein conjugate (single blood) (relating to 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 approximately 3,000 kDa, approximately 900 to approximately 5,500 kDa, approximately 900 to approximately 5,000 kDa Da, approximately 900 to approximately 4,500 kDa, approximately 900 to approximately 4,000 kDa, approximately 900 to approximately 3 ,500 kDa, or about 900 to about 3,000 kDa.

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

[0143] In an embodiment of the present 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 present 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 from 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 fluorescent Light intensity is stable for 1 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 adjuvants. does not include

[0146] Without being bound by any particular theory, glycoconjugates prepared in DMSO One possible mechanism for the enhanced immunogenicity observed in This involves increasing the number of bonds between lysine residues on the surface of the carrier protein, which Provides additional points of attachment between the protein and the polysaccharide, imparting stability and enhancing the peptide-carbohydrate bond. This will resist chemical depolymerization or degradation of the polymer. 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 formed during conjugation in DMSO solvent The additional benefit would be an additional opportunity for successful peptide-carbohydrate presentation 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, which may be an additional factor for polysaccharide binding. Exposing specific 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 Nature Medicine 17:1602-1610.

[0147] Conjugation in organic solvents to generate modified CRM197 in the conjugate Another advantage of CRM197 is the reduced immunological interference of antibodies against the native CRM197 epitope. The polysaccharide-protein bond formed during conjugation in DMSO solvent 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 bring significant benefits.

[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 transition metal to form a stable complex with cyanide. and the removal of protein amino groups and formaldehyde 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 amide 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 among 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 eliminates lot variations in sodium cyanoborohydride Reduced inconsistencies in conjugation.

[0150] A suitable alternative chemical method is 1-cyano-4-dimethylaminopyridinium tetrafluoroborate. This involves the activation of sugars with cyanate (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 an amino group on a maleimide-activated substrate. body proteins (e.g., using GMBS) or haloacetylated carrier proteins (e.g., , iodoacetimide [e.g., ethyl iodoacetimide HCl] or N-succinic acid Imidyl bromoacetate or SIAB, or 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 amine (which may be prepared by CDAP chemistry if desired) is hexanediamine or adipic acid. The amino-derivatized sugar is coupled to carboxylic acid dihydrazide (ADH) and then to a carbodiimide. (e.g., EDAC or EDC) chemical methods to deprotect carboxyl groups on protein carriers. Such conjugates are designated International Patent Office. Patent application publication numbers WO 93 / 15760, WO 95 / 08348 and WO 96 / 2 9094, and Chu et al., 1983, Infect. Immunity 40:24 5-256.

[0151] Other suitable conjugation techniques include carbodiimides, hydrazides, active esters, norbornanes, and the like. Bornane, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, Many of these are described in International Patent Application Publication No. WO 98 / 42721. The conjugate is formed by the reaction of the free hydroxyl group of the sugar with CDI. a 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 Carbamate bond formation occurs by reduction of the anomeric terminus to a primary hydroxyl group, Optionally, protection / deprotection of primary hydroxyl groups, primary hydroxyl groups and Reaction with CDI to form a CDI carbamate intermediate and 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 filtration, 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 present invention. These pneumococcal conjugates can be manufactured by different methods and combined into 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), which The extent of conjugated lysine can be characterized as the extent of conjugation (degree of conjugation). Evidence for lysine modification of carrier proteins by covalent attachment to sugars can be obtained by routine methods known to those skilled in the art. Conjugation can be performed by analyzing the amino acids of the conjugates. 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 used to measure lysine consumption. The conjugation degrees were 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 above, the degree of conjugation of the glycoconjugates of the present invention is about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14 or about 15. In another embodiment, the degree of conjugation of the glycoconjugates 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 present invention have a polysaccharide to carrier protein (Ps:Pr) ratio (wt / wt) of 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, Approximately 2.3, approximately 2.4, approximately 2.5, approximately 2.6, approximately 2.7, approximately 2.8, approximately 2.9 or approximately 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 some cases, the sugar to carrier protein ratio (w / w) is 0.8 to 1.2. In an embodiment, the carrier protein is CRM197. Covalently bound to a carrier protein Free sugars that are not (covalently conjugated) but are still present in the glycoconjugate composition The glycoconjugates and immunogenic compositions of the present invention may contain free sugars non-covalently bound to the glycoconjugates. They may be covalently associated (e.g., non-covalently 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 other embodiments, the saccharide to carrier protein ratio (w / w) for 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% of the total amount of polysaccharides. Contains less than 45%, 40%, 35%, 30%, 25%, 20% or 15% free polysaccharides. In a preferred embodiment, the glycoconjugate composition contains less than about 25% of the total amount of polysaccharides. In a preferred embodiment, the glycoconjugate composition contains free polysaccharides in a proportion of 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 compared to the total amount of polysaccharides.

[0162] IV.How to use Embodiments of the present invention also include (i) (a) therapeutic (e.g., human treatment), (b) pharmaceutical, (c) c) suppression of infection by Streptococcus pneumoniae, (d) 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 streptococcal disease, including prevention of brain damage, hearing loss, and related complications, including 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 (ii) pneumococcal pneumonia, pneumonia 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 (iv) in the manufacture of a medicament therefor; The multivalent immunogenic compositions may comprise 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 and may be used in combination with one or more adjuvants or without adjuvants.

[0163] Thus, the present invention provides a method for administering to a patient in need thereof a multivalent immunogenic pneumococcal polysaccharide-protein of the present invention. and administering one or more of the following protein conjugate compositions to treat Streptococcus nucleus. and providing a method for the prophylactic treatment of (i.e., protection against) pneumococcal infection or pneumococcal disease. To provide.

[0164] The compositions and formulations of the present invention are intended to be used to administer such compositions or formulations via 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 the multivalent immunogenic composition of the present invention. to induce an immune response against Streptococcus pneumoniae, comprising administering to the patient In another embodiment, the present invention provides a method for producing a multivalent immunogen of the present invention. A method for treating a human against pneumococcal infection, comprising administering to the human an immunologically effective amount of a composition comprising the compound of formula (I) and (II). A method for inoculating cutin is provided.

[0166] Thus, in one aspect, the present invention provides a multivalent immunogenic composition of the present invention (i.e. That is, any of the multivalent immunogenic compositions described herein, e.g., the "multivalent immunogen" described above. 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; (2) producing 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 old), or Prevention of pneumococcal pneumonia and / or invasive pneumococcal disease in children (approximately 2-5 years of age) A method is provided.

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

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

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

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

[0172] In another embodiment, the present invention provides a method for the treatment of pneumococcal pneumonia and pneumonia 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: Strains, 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 Also provided is a method for preventing invasive pneumococcal disease.

[0174] In one embodiment of the method, the composition comprises a plurality of Streptococcus pneumoniae strains. and a polysaccharide-protein conjugate, wherein each of the conjugates is a carrier. Protein-conjugated Streptococcus pneumoniae serovar-derived polyclonal antibodies Contains sugars, and the serotypes of Streptococcus pneumoniae are 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 a 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. In one embodiment, the composition comprises a plurality of Streptococcus pneumoniae polysaccharide-protein conjugates. gates, 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. In one embodiment, the composition comprises a plurality of Streptococcus pneumoniae polysaccharide-protein conjugates. gates, 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 multiple Streptococcus pneumoniae polysaccharide-protein conjugates. wherein each of the conjugates is conjugated to a carrier protein. Contains polysaccharides derived from Streptococcus pneumoniae serovars, and Serotypes of S. pneumoniae 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 a number of Streptococcus pneumoniae polysaccharide-protein conjugates, wherein Each of the conjugates is a streptococcal antibody conjugated to a carrier protein. Contains polysaccharides derived from Streptococcus pneumoniae serovars and is a serovar of Streptococcus pneumoniae 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 efficacy 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 - Patent Application 20070122999 In another embodiment, the immunogenic composition is a composition comprising serotypes 6A, 6B and 6C. and 6C pneumococcal polysaccharide conjugates.

[0176] In certain embodiments of the method, the multivalent immunogenic composition is selected from the group consisting of: Pneumococcal conjugates containing polysaccharides of the Streptococcus pneumoniae serotype group selected from Contains: a)1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 12F, 14, 15A, D eOAc15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and Yobi 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 strains. and polysaccharide-protein conjugates, wherein each of the conjugates is a carrier protein. Protein-conjugated polysaccharides from Streptococcus pneumoniae serovars 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 vaccine containing serotype 10A polysaccharide has some efficacy 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 serotypes Does not contain 10A polysaccharide conjugates, but instead contains serotype 39 polysaccharide conjugates In another embodiment, the immunogenic composition is a serum In certain embodiments of the method, the method comprises the use of pneumococcal polysaccharide conjugates of types 10A and 39. In the present invention, the serotype of Streptococcus pneumoniae is selected from the group consisting of: Selected serotype groups include: 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 Call 39.

[0179] Streptococcus pneumoniae serotype 15B capsular polynucleotides 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). Therefore, in some embodiments of the method, the present invention also provides a method for treating blood It does not contain purified 15C (or de-O-acetylated 15B) polysaccharide conjugates, but instead containing a serotype 15B polysaccharide conjugate (i.e., the serotype 15B polysaccharide is substantially deoxyribonucleotide-containing). In another embodiment, the use of a multivalent immunogenic composition is provided. For example, the immunogenic composition may comprise serotypes 15B and 15C (or de-O-acetylated 15B). Includes pneumococcal polysaccharide conjugates.

[0180] The compositions of the present invention are effective in preventing streptococcal infection in patients previously vaccinated with a multivalent pneumococcal vaccine. It is 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 Additional vaccines against Streptococcus pneumoniae serotypes to which the individual was previously vaccinated It is possible that the vaccine may provide substantial protection, or the patient may not have 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 S. pneumoniae polysaccharide-protein conjugate, wherein the polysaccharide-protein conjugate Conjugate is a Streptococcus pneumoniae antibody conjugated to a carrier protein. The patient had previously tested positive for 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 is administered to a subject previously treated with a multivalent pneumococcal vaccine. The multivalent immunogenic vaccine is administered to patients who have been exposed to 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: i.4, 6B, 9V, 14, 18C, 19F and 23F; ii.4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 6A, 7F and Yobi 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 blood strains within a group of serotypes selected from the group consisting of Previously, a multivalent pneumococcal vaccine was developed to prevent pneumococcal disease caused by the Streptococcus pneumoniae strain. 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. Contains polysaccharides derived from conjugated Streptococcus pneumoniae serovars. In embodiments, the multivalent pneumococcal vaccine is conjugated to a carrier protein. It contains multiple pneumococcal capsular polysaccharides that are not isolated.

[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 er, Inc., Philadelphia, PA, USA] had been previously treated.

[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 [Rixensart, Belgium] It was being done.

[0187] In embodiments of the method, the multivalent immunogenic compositions of the invention are administered to a medical professional, e.g. Patients received multivalent pneumococcal vaccine according to the 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. or 1 month to 1 year after the patient received the multivalent pneumococcal vaccine, 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 and up 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 patient is administered the drug 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, Approximately 9 months, approximately 10 months, approximately 11 months, approximately 1 year, approximately 1.25 years, approximately 1.5 years, approximately 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 other embodiments, the present invention provides a method for administering to a patient a multivalent immunogenic composition of the present invention. and administering a multivalent pneumococcal vaccine to the patient, 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 human patients. For example, a multivalent pneumococcal vaccine may be administered to a patient first, followed by administration of a multivalent pneumococcal vaccine. Alternatively, the patient may first be administered a multivalent immunogenic composition of the present invention. The composition is administered to the patient, and then a multivalent pneumococcal vaccine is administered to the patient. The vaccine is designed to treat pulmonary pulmonary infections caused by multiple serotypes of Streptococcus pneumoniae. It can 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 Yobi 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 and treatment with a multivalent pneumococcal vaccine indicated for the prevention of pneumococcal disease caused by the will be done.

[0190] In certain embodiments of the method, the multivalent pneumococcal vaccine comprises a Streptococcus pneumoniae vaccine. 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. Contains polysaccharides derived from conjugated Streptococcus pneumoniae serovars. In embodiments, the multivalent pneumococcal vaccine is conjugated to a carrier protein. Not containing 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 ia, PA, USA] in any order. The patient is first administered PREVNAR® 13, followed by the 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 other embodiments 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, followed by the administration of 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 receives a multivalent immunogenic composition of the invention. and SYNFLORIX™ [pneumococcal polysaccharide conjugate vaccine ( adsorption), GlaxoSmithKline Biologicals sa, Rix In one embodiment, the patient receives treatment with ensart, Belgium, in any order. In this case, SYNFLORIX™ is administered to the patient first, followed by the administration of the multivalent immunogen of the present invention. In an alternative embodiment, the patient is first administered a multivalent immunogenic composition of the present invention. 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 administered. As used herein, "co-administration" is not limited to simultaneous administration of two compositions. In some embodiments, the multivalent immunizations are administered sequentially in any order, including one after the other. The active ingredient composition and the multivalent pneumococcal vaccine are administered intramuscularly or subcutaneously to 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, the patient is first administered a multivalent pneumococcal vaccine, followed by 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 patients, including Also provided are methods of vaccinating or inducing a protective immune response. In this case, the multivalent immunogenic composition may be a Streptococcus neutrophil antibody as described herein. and any combination of Monnier polysaccharide protein conjugates, Cocci vaccines are used to treat infections caused by multiple serotypes of Streptococcus pneumoniae. The vaccine may be any vaccine indicated for the prevention of a disease.

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

[0200] In this method, the multivalent immunogenic composition comprises a streptococcal antibody as described herein. The composition may contain 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 capsular polysaccharides 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 the 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 polyvalent immunization therapy according to the present invention as described above. Those who have already received a multivalent pneumococcal vaccine before receiving the first dose of the immunogenic composition Alternatively, the patient may be given a steroid injection prior to receiving the multivalent immunogenic composition of the present invention. 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 Streptococcus pneumoniae. This vaccine is intended for patients who have received a multivalent pneumococcal vaccine indicated for the prevention of 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 administered using 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, the 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 older children, adolescents and adults (e.g., 18-45 years old). Also suitable for use in: 18-50 years, 18-55 years, 18-60 years or 18-65 years In other embodiments of any of the methods of the invention, the patient is between about 2 and about 18 years old. In other embodiments of any of the methods of the present 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 the multivalent immunogenic composition of the invention is administered to the infant. Administer one, two, or three doses of the composition to the infant. The length of time between administration of each dose can vary. However, an example dosing schedule is to administer a dose at 2 months of age, followed by a further dose at 4 months of age. The dosing schedule for infants includes administering the final dose at 6 months of age. Another example is administering a dose at 2 months of age, followed by another dose at 3 months of age. Another example of a dosing schedule for rhesus monkeys is to administer a dose at 2 months of age, followed by a dose at 3 months of age. and finally administering a final dose at 6 months of age. additional "booster" doses of the multivalent immunogenic composition of the present 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. 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 present invention, the immunogenic composition of the present invention is administered. 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 conjunction with a vaccine against influenza. The flu vaccine is a "senior flu vaccine," meaning it is for older people, e.g., those aged 6 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 the multivalent immunogenic composition of the present invention may be determined based on the appropriate immunogenicity in the subject. This can be confirmed by standard studies involving observation of the response. For example, in another embodiment, For these 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 present invention are useful for treating 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 administration. Administration may include one or more of oral / alimentary, 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 more effectively prevented and, therefore, the infection can be prevented at an early stage. In certain embodiments, the compositions of the present invention is administered to patients by intramuscular or subcutaneous injection.

[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. For purposes of describing and disclosing the materials, they are incorporated herein by reference.

[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 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 this can be done by

[0220] The following examples are intended to illustrate, but not limit, the present invention.

[0221] Example 1 Production of Streptococcus pneumoniae (S. pneumoniae) capsular polysaccharide Methods for culturing pneumococci are well known in the art. See, e.g., Chase, 1967, Me 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. In accordance with 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. The serotype 23B strain was obtained from the US Centers for Disease Control and Prevention ( 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) obtained from the City of Alabama Birmingham. The subtypes are distinguished based on the Quellung reaction using specific antisera. See, e.g., U.S. Patent No. 5,847,112. The resulting isolates were Animal-free, containing soy peptone, yeast extract, and glucose (hemin-free) Further cloning was performed by successively plating in two stages on agar plates consisting of a nutrient-rich medium. In the case of serotype 7F, the agar plates used also contained hemin. The clonal isolates of each serotype were cultured in a mixture of 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 (chemical inactivation) before 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. Growth was performed using shake flasks or culture bottles containing 100% ethanol. The mixture was placed in a sealed shake flask or bottle to minimize gas exchange while controlling the temperature. Cell growth cultures were subjected to growth. During cell growth of these serotypes, temperature, pH, pressure and The agitation was controlled. Sparging was not used, so the airflow was The overlay was also controlled. The specific After a suitable confluency is achieved, a portion of the cell growth culture is cultured in a medium containing soy peptone or soybean peptone. Ammonium chloride ultrafiltrate, yeast extract or yeast extract ultrafiltrate, sodium chloride, calcium phosphate A pre-sterilized, animal-component-free growth medium containing sodium and glucose is used. The temperature, pH, pressure and agitation were controlled. No sparging was used. This also controlled the airflow overlay.

[0224] Once the glucose is nearly consumed, the batch is deactivated by the addition of phenol, a chemical deactivator. Fermentation was terminated by adding pure phenol to a final concentration of 0.8–1.2% to dissolve the cells. The cells were inactivated and the capsular polysaccharides were released from the cell walls in a fermenter for a specified period of time. Perform primary inactivation, in which case temperature and agitation are kept under control. After primary inactivation, The mixture was transferred to another container and heated at controlled temperature and agitation with additional identified This was achieved by maintaining the bacteria for a period of time, resulting in complete inactivation. or by verification of phenol concentration and time specified. The inactivated broth was purified.

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

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

[0227] Further impurity removal 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 this process, sodium acetate and phenol (liquid) in sodium phosphate buffer were added. phenol (precipitated or solid phenol) is loaded onto the diafiltered retentate. The polysaccharides were then fractionated in two stages. Alcohol is added to the preparation to precipitate cell debris and other unwanted impurities, while preserving the crude The purified polysaccharide was left in solution. Impurities were removed by centrifugation and subsequent depth filtration steps. 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) Polyvalent Studies Using DMSO Conjugation Manufacturing of Jugate The polysaccharides are dissolved, sized to a target molecular weight, chemically activated, and purified by ultrafiltration into a buffer. The activated polysaccharide and purified CRM197 were separately lyophilized and then washed with dimethyl sulfoxide. The polysaccharide solution was then redissolved in DMSO. The resulting conjugate was dialyzed and then purified as described below. After further purification, the conjugate was subjected to a final 0.2 micron filtration. In order to obtain the desired results, several process parameters within each step, such as pH, temperature, and concentration, are controlled. and time were controlled.

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

[0230] The 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 of polysaccharides was minimized. The oxidation 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 through a 10 kDa NMWCO tangential flow ultrafiltration membrane in 10 mM phosphate buffer. Diafiltration was performed against potassium carbonate (pH 6.4), followed by diafiltration with 5 kDa N Diafiltration was performed against water using a MWCO tangential flow ultrafiltration membrane. Filtration was carried out at 2 to 8°C.

[0233] Polysaccharide conjugation to CRM197 Pseudomonas as previously described (WO 2012 / 173876 A1) Expression in Pseudomonas fluorescens The purified CRM197 obtained by the above procedure was filtered using a 5 kDa NMWCO tangential flow ultrafiltration membrane. Diafiltration 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 with a 1% w / v sucrose concentration. Formulated for freeze-drying.

[0235] The formulated Ps and CRM197 solutions were lyophilized separately. The CRM197 and CRM297 substances were separately redissolved in an equal volume of DMSO. The polysaccharide solution and CRM197 were mixed to obtain a polysaccharide concentration of 1.5 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 (per mole of polysaccharide repeating unit) (1 mol per 1000 mg of ATP) 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 approximately 4°C. The pH was neutralized by adding potassium phosphate buffer. Using a 150 mM sodium chloride solution, 0.05% (w / v) polysorbate 20 The batch was dialyzed against HCl at about 4° C. for 3 days.

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

[0238] Example 4 Serotype 1 Conjugates for PCV23 (DMSO+Aq) Multivalent Studies Using Aqueous Conjugation Conjugate manufacturing The polysaccharides are dissolved, sized, chemically activated, and buffer exchanged by ultrafiltration. The purified CRM197 was then converted into an activated polysaccharide using nickel chloride in the reaction mixture. 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] Polysaccharide size reduction and oxidation Purified pneumococcal capsular polysaccharide powder was dissolved in water and filtered at 0.45 microns. The resulting polysaccharide was homogenized to reduce its molecular weight. The pressure and number of passes through the homogenizer were controlled at 250 bar / 5 passes. Small polysaccharides were concentrated and filtered 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 of polysaccharides was minimized. The oxidation 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 through a 10 kDa NMWCO tangential flow ultrafiltration membrane in 10 mM phosphate buffer. The ultrafiltration was performed at 2-8°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 a buffered polysaccharide solution at a polysaccharide to CRM197 mass ratio of 0.5. The mass ratio was chosen so that the polysaccharide to CRM197 ratio in the granules was controlled. The concentrations of phosphate were 6.9 g / L and 100 mM, respectively. The polysaccharide concentration was chosen to control the size of the conjugates. Using the solution, nickel chloride was added 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 phosphate (1 mole per mole of polysaccharide repeating unit) was added. Potassium (pH 6.0) was added.

[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 batch was The mixture was filtered at 0.2 microns.

[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 ethanol (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 converted into an activated polysaccharide using nickel chloride in the reaction mixture. 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] Polysaccharide size reduction and oxidation Purified pneumococcal capsular polysaccharide powder was dissolved in water and filtered at 0.45 microns. The resulting polysaccharide was homogenized to reduce its molecular weight. The pressure and number of passes through the homogenizer were controlled at 250 bar / 5 passes. Small polysaccharides were concentrated and filtered 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 of polysaccharides was minimized. The oxidation 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 through a 10 kDa NMWCO tangential flow ultrafiltration membrane in 10 mM phosphate buffer. The ultrafiltration was performed at 2-8°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 a buffered polysaccharide solution at a polysaccharide to CRM197 mass ratio of 0.5. The mass ratio was chosen so that the polysaccharide to CRM197 ratio in the granules 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 was controlled. The solution was filtered through a 100 mM nickel chloride solution to dilute the nickel chloride to approximately 2 mM. 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 phosphate (1 mole per mole of polysaccharide repeating unit) was added. Potassium (pH 6.0) was added.

[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 batch was The mixture was filtered at 0.2 microns.

[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 ethanol (pH 7.0). The batch was divided into aliquots and frozen at -60°C or below.

[0254] Example 6 Serotype 3 Conjugate for PCV23(DMSO) Polyvalent Studies Using DMSO Conjugation Manufacturing of Jugate The polysaccharide is dissolved, sized to a target molecular weight, chemically activated, and purified by ultrafiltration. The activated polysaccharide and purified CRM197 were separately lyophilized and then washed with dimethyl sulfoxide. The polysaccharide solution was then redissolved in DMSO. 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, The concentration and time were controlled.

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

[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 of polysaccharides was minimized. The oxidation 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). and then dialysis against water using a 5 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was carried out at 2-8°C.

[0259] Polysaccharide conjugation to CRM197 Pseudomonas as previously described (WO 2012 / 173876 A1) Purified CRM197 obtained by expression in C. fluorescens was analyzed using a 5 kDa NMW CO2 was filtered using a tangential flow ultrafiltration membrane in 2 mM phosphate (pH 7.2) buffer. The solution was diafiltered and filtered to 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 with a 1% w / v sucrose concentration. Formulated for freeze-drying.

[0261] The formulated Ps and CRM197 solutions were lyophilized separately. The CRM197 material was separately redissolved in an equal volume of DMSO. The polysaccharide solution and CRM197 were mixed to obtain a polysaccharide concentration of 1.35 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 to control the amount of sodium cyanoborohydride (1 unit of polysaccharide repeating unit). (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 approximately 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% ethanol at 4°C.

[0264] The retentate batch was filtered to 0.2 microns (through a 0.5 micron prefilter) and then 150 mM sodium chloride (p) containing 0.015% (w / v) polysorbate 20 Dilute with additional 10 mM histidine in PBS (H7.0), dispense into aliquots, and store at -60°C. Frozen below.

[0265] Example 7 PCV22 and PCV23 (DMSO+Aq) multivalent synthesis 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 converted into an activated polysaccharide using nickel chloride in the reaction mixture. 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] Polysaccharide size reduction and oxidation Purified pneumococcal capsular polysaccharide powder was dissolved in water and filtered at 0.45 microns. The resulting polysaccharide was homogenized to reduce its molecular weight. The pressure and number of passes through the homogenizer were controlled at 380 bar / 5 passes. Small polysaccharides were concentrated and filtered 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 of polysaccharides was minimized. The oxidation 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 through a 10 kDa NMWCO tangential flow ultrafiltration membrane in 10 mM phosphate buffer. The ultrafiltration was performed at 2-8°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 Combine with 0.2 micron filtered buffered polysaccharide solution at a polysaccharide to CRM197 mass 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 that the size of the resulting conjugate could be controlled. 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 in a ratio of 0.2 to 0.2, with the aim of maximizing the consumption of polysaccharides and proteins. The nitrification was allowed to proceed at 10°C for 120 hours.

[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 phosphate (1 mole per mole of polysaccharide repeating unit) was added. Potassium (pH 6.0) was added.

[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 microns.

[0272] An additional 10 mM L-hydroxybenzoate 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 Conjugate for PCV23(DMSO) Polyvalent Studies Using DMSO Conjugation Manufacturing of Jugate The polysaccharide is dissolved, sized to a target molecular weight, chemically activated, and purified by ultrafiltration. 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 stirred 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] Polysaccharide size reduction and oxidation Purified pneumococcal Ps capsule powder was dissolved in water and filtered at 0.45 microns. The resulting polysaccharide was homogenized to reduce the molecular weight of Ps. The number of passes was controlled to 300 bar / 5 passes.

[0275] The 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. The sugars were partially deketalized, and 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 through a 10 kDa NMWCO tangential flow ultrafiltration membrane in 10 mM phosphate buffer. Diafiltration was performed against potassium carbonate (pH 6.4), followed by diafiltration with 5 kDa N Diafiltration was performed against water using a MWCO tangential flow ultrafiltration membrane. Filtration was carried out at 2 to 8°C.

[0278] Polysaccharide conjugation to CRM197 Pseudomonas as previously described (WO 2012 / 173876 A1) Purified CRM197 obtained by expression in C. fluorescens was analyzed using a 5 kDa NMW CO2 was filtered using a tangential flow ultrafiltration membrane in 2 mM phosphate (pH 7.2) buffer. The solution was diafiltered and filtered to 0.2 microns.

[0279] Activated polysaccharide formulated for lyophilization at 6 mg Ps / mL at 5% w / v sucrose concentration CRM197 was lyophilized at 1% w / v sucrose concentration and 6 mg Pr / mL. It was formulated for use.

[0280] The formulated Ps and CRM197 solutions were lyophilized separately. The CRM197 and CRM297 substances were separately redissolved in an equal volume of DMSO. The polysaccharide solution and CRM1 were mixed to obtain a polysaccharide concentration of 2.0 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 chosen so that sodium cyanoborohydride (per mole of polysaccharide repeating unit) (1 mol per 1000 mg of ATP) 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 approximately 4°C. The pH was neutralized by adding potassium phosphate buffer. Using a 150 mM sodium chloride solution, 0.05% (w / v) polysorbate 20 The batch was dialyzed against HCl at about 4° C. for 3 days.

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

[0283] Example 9 PCV23 (DMSO+Aq) and PCV22 multivalent lab experiments 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 converted into an activated polysaccharide using nickel chloride in the reaction mixture. 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] Polysaccharide size reduction and oxidation Purified pneumococcal capsular polysaccharide powder was dissolved in water and filtered at 0.45 microns. The resulting polysaccharide was homogenized to reduce its molecular weight. The pressure and number of passes through the homogenizer were controlled at 300 bar / 5 passes. Small polysaccharides were concentrated and filtered 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. The sugars were partially deketalized, and 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 through a 10 kDa NMWCO tangential flow ultrafiltration membrane in 10 mM phosphate buffer. The ultrafiltration was performed at 2-8°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 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 that the size of the resulting conjugates could be controlled. The solution was filtered at 0.2 microns. 100 mM nickel chloride solution was used to Nickel was added to approximately 2 mM. Sodium cyanoborohydride (per mole of polysaccharide repeating unit) The conjugate was added in a volume of 100 ml 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 phosphate (1 mole per mole of polysaccharide repeating unit) was added. Potassium (pH 6.0) was added.

[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 filtered at 0.2 microns.

[0290] An additional 10 mM L-hydroxybenzoate 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 Conjugate for PCV23(DMSO) Polyvalent Studies Using DMSO Conjugation Manufacturing of Jugate The polysaccharide is dissolved, sized to a target molecular weight, chemically activated, and purified by ultrafiltration. 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 stirred 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] Polysaccharide size reduction and oxidation Purified pneumococcal Ps capsule powder was dissolved in water and filtered at 0.45 microns. The resulting polysaccharide was homogenized to reduce the molecular weight of Ps. The number of passes was controlled to 600 bar / 5 passes.

[0293] The 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 obtain the active The polysaccharide size reduction due to activation was minimized. The oxidation 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 in 10 mM acetic acid using a 10 kDa NMWCO tangential flow ultrafiltration membrane. Diafiltration against sodium (pH 4.1) followed by 5 kDa N Diafiltration was performed against water using a MWCO tangential flow ultrafiltration membrane. Filtration was carried out at 2 to 8°C.

[0296] Polysaccharide conjugation to CRM197 Pseudomonas as previously described (WO 2012 / 173876 A1) Purified CRM197 obtained by expression in C. fluorescens was analyzed using a 5 kDa NMW CO2 was filtered using a tangential flow ultrafiltration membrane in 2 mM phosphate (pH 7.2) buffer. The solution was diafiltered and filtered to 0.2 microns.

[0297] Activated polysaccharide formulated for lyophilization at 6 mg Ps / mL at 5% w / v sucrose concentration CRM197 was lyophilized at 1% w / v sucrose concentration and 6 mg Pr / mL. It was formulated for use.

[0298] The formulated Ps and CRM197 solutions were lyophilized separately. The polysaccharide solution was redissolved 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 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. 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 in a 100 ml flask. Potassium phosphate buffer was then added to neutralize the pH. A 300 kDa NMWCO dialysis cassette was used to dialyze the lysate containing 150 mM sodium chloride, The batch was dialyzed 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 PCV22 and PCV23 (DMSO+Aq) multivalent synthesis 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 converted into an activated polysaccharide using nickel chloride in the reaction mixture. 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] Polysaccharide size reduction and oxidation Purified pneumococcal capsular polysaccharide powder was dissolved in water and filtered at 0.45 microns. The resulting polysaccharide was homogenized to reduce its molecular weight. The pressure and number of passes through the homogenizer were controlled at 600 bar / 5 passes. Small polysaccharides were concentrated and filtered 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 obtain the active The polysaccharide size reduction due to activation was minimized. The oxidation 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 through a 10 kDa NMWCO tangential flow ultrafiltration membrane in 10 mM acetic acid. 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 a buffered polysaccharide solution at a polysaccharide to CRM197 mass ratio of 0.4. The mass ratio was chosen so that the polysaccharide to CRM197 ratio in the granules was controlled. The concentrations of phosphate were 3.8 g / L and 150 mM, respectively. The polysaccharide concentration was chosen so that the size of the conjugates was controlled. The solution was filtered through a 100 mM nickel chloride solution to dilute the nickel chloride to approximately 2 mM. 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. Diaphragm the batch against 300 mM sodium bicarbonate (pH 9.3) at 2-8°C. The batch recovered in the retentate was then diluted with 1.5M potassium phosphate. The solution was neutralized with HCl (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 batch was The mixture was filtered at 0.2 microns.

[0308] 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 PBS (pH 7.0). The batch was divided into aliquots and frozen at -60°C or below.

[0309] Example 12 PCV23(DMSO) and PCV23(DMSO) 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 followed by a final 0.2 micron filtration. Several process parameters within each step are adjusted to obtain conjugates with desired properties. Parameters such as pH, temperature, concentration and time were controlled.

[0310] Polysaccharide size reduction and oxidation Purified pneumococcal Ps capsule powder was dissolved in water and filtered at 0.45 microns. The resulting 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. Diafiltration was performed 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 of polysaccharides was minimized. The oxidation 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). and then filtered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was carried out at 2-8°C.

[0313] Polysaccharide conjugation to CRM197 Pseudomonas as previously described (WO 2012 / 173876 A1) Purified CRM197 obtained by expression in C. fluorescens was analyzed using a 5 kDa NMW CO2 was filtered using a tangential flow ultrafiltration membrane in 2 mM phosphate (pH 7.2) buffer. The mixture was diafiltered.

[0314] Activated polysaccharide formulated for lyophilization at 6 mg Ps / mL at 5% w / v sucrose concentration CRM197 was lyophilized at 1% w / v sucrose concentration and 6 mg Pr / mL. It was formulated for use.

[0315] The formulated Ps and CRM197 solutions were lyophilized separately. The CRM197 and CRM297 substances were separately redissolved in an equal volume of DMSO. The polysaccharide solution and CRM197 were mixed to obtain a polysaccharide concentration of 1.4 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 chosen so that sodium cyanoborohydride (per mole of polysaccharide repeating unit) (1 mol per 1000 mg of ATP) 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 diluted with 150 mM sodium chloride containing phosphate 20 at approximately 4°C. The pH was neutralized by adding potassium carbonate buffer. The batch was concentrated to obtain 30 kDa NMWC. Using a tangential flow ultrafiltration membrane, filter the solution containing 150 mM sodium chloride, 25 mM potassium phosphate, The solution was diafiltered against 100 ml of 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% ethanol 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 solution was diluted with histidine, dispensed into aliquots, and frozen at -60°C or below.

[0319] Example 13 PCV23(DMSO) and PCV23(DMSO) using DMSO conjugation (O / Aq) Preparation of serotype 6B conjugates for multivalent studies The polysaccharide is dissolved, sized to a target molecular weight, chemically activated, and purified by ultrafiltration. 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 stirred 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] Polysaccharide size reduction and oxidation Purified pneumococcal Ps capsule powder was dissolved in water and filtered at 0.45 microns. The resulting polysaccharide was homogenized to reduce the molecular weight of Ps. The number of passes was controlled to 200 bar / 5 passes.

[0321] The 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 of polysaccharides was minimized. The oxidation 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). and then filtered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was carried out at 2-8°C.

[0324] Polysaccharide conjugation to CRM197 Pseudomonas as previously described (WO 2012 / 173876 A1) Purified CRM197 obtained by expression in C. fluorescens was analyzed using a 5 kDa NMW CO2 was filtered using a tangential flow ultrafiltration membrane in 2 mM phosphate (pH 7.2) buffer. The solution was diafiltered and filtered to 0.2 microns.

[0325] Activated polysaccharide formulated for lyophilization at 6 mg Ps / mL at 5% w / v sucrose concentration CRM197 was lyophilized at 1% w / v sucrose concentration and 6 mg Pr / mL. It was formulated for use.

[0326] The formulated Ps and CRM197 solutions were lyophilized separately. The CRM197 and CRM297 substances were separately redissolved in an equal volume of DMSO. The polysaccharide solution and CRM197 were mixed to obtain a polysaccharide concentration of 1.35 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 to control the amount of sodium cyanoborohydride (1 unit of polysaccharide repeating unit). (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 approximately 4°C. The pH was neutralized by adding potassium phosphate buffer. The batch was concentrated to a concentration of 30 kDa NMW. CO2 tangential flow ultrafiltration membrane was used to filter 150 mM sodium chloride, 25 mM potassium phosphate The solution was diafiltered against 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% ethanol 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 solution 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 Production of slate The polysaccharide is dissolved, sized to a target molecular weight, chemically activated, and purified by ultrafiltration. 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 stirred 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] Polysaccharide size reduction and oxidation Purified pneumococcal Ps capsule powder was dissolved in water and filtered at 0.45 microns. The resulting 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. Diafiltration was performed 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 of polysaccharides was minimized. The oxidation 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). and then filtered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was carried out at 2-8°C.

[0334] Polysaccharide conjugation to CRM197 Pseudomonas as previously described (WO 2012 / 173876 A1) Purified CRM197 obtained by expression in C. fluorescens was analyzed using a 5 kDa NMW CO2 was filtered using a tangential flow ultrafiltration membrane in 2 mM phosphate (pH 7.2) buffer. The solution was diafiltered and filtered to 0.2 microns.

[0335] Activated polysaccharide formulated for lyophilization at 6 mg Ps / mL at 5% w / v sucrose concentration CRM197 was lyophilized at 1% w / v sucrose concentration and 6 mg Pr / mL. It was formulated for use.

[0336] The formulated Ps and CRM197 solutions were lyophilized separately. The CRM197 and CRM297 substances were separately redissolved in an equal volume of DMSO. The polysaccharide solution and CRM197 were mixed to obtain a polysaccharide concentration of 1.35 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 to control the amount of sodium cyanoborohydride (1 unit of polysaccharide repeating unit). (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 approximately 4°C. The pH was neutralized by adding potassium phosphate buffer. The batch was concentrated to a concentration of 30 kDa NMW. CO2 tangential flow ultrafiltration membrane was used to filter 150 mM sodium chloride, 25 mM potassium phosphate The solution was diafiltered against 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% ethanol 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 solution was diluted with histidine, dispensed into aliquots, and frozen at -60°C or below.

[0340] Example 15 PCV23(DMSO) and PCV23(DMSO) using DMSO conjugation (O+Aq) Preparation of serotype 7F conjugates for multivalent studies The polysaccharide is dissolved, sized to a target molecular weight, chemically activated, and purified by ultrafiltration. 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 stirred 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] Polysaccharide size reduction and oxidation Purified pneumococcal Ps capsule powder was dissolved in water and filtered at 0.45 microns. The resulting 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. Diafiltration was performed 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 sodium solution and allowed to proceed for 4 hours at 4°C.

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

[0344] Polysaccharide conjugation to CRM197 Pseudomonas as previously described (WO 2012 / 173876 A1) Purified CRM197 obtained by expression in C. fluorescens was analyzed using a 5 kDa NMW CO2 was filtered using a tangential flow ultrafiltration membrane in 2 mM phosphate (pH 7.2) buffer. The solution was diafiltered and filtered to 0.2 microns.

[0345] Activated polysaccharide formulated for lyophilization at 6 mg Ps / mL at 5% w / v sucrose concentration CRM197 was lyophilized at 1% w / v sucrose concentration and 6 mg Pr / mL. It was formulated for use.

[0346] The formulated Ps and CRM197 solutions were lyophilized separately. The CRM197 and CRM297 substances were separately redissolved in an equal volume of DMSO. The polysaccharide solution and CRM197 were mixed to obtain a polysaccharide concentration of 1.5 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 (per mole of polysaccharide repeating unit) (1 mol per 1000 mg of ATP) 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 approximately 4°C. The pH was neutralized by adding potassium phosphate buffer. The batch was concentrated to a concentration of 30 kDa NMW. CO2 tangential flow ultrafiltration membrane was used to filter 150 mM sodium chloride, 25 mM potassium phosphate The solution was diafiltered against 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% ethanol 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 solution 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 Production of slate The polysaccharide is dissolved, sized to a target molecular weight, chemically activated, and purified by ultrafiltration. 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 stirred 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] Polysaccharide size reduction and oxidation Purified pneumococcal Ps capsule powder was dissolved in water and filtered at 0.45 microns. The resulting 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. Diafiltration was performed 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 sodium solution and allowed to proceed for 4 hours at 4°C.

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

[0354] Polysaccharide conjugation to CRM197 Pseudomonas as previously described (WO 2012 / 173876 A1) Purified CRM197 obtained by expression in C. fluorescens was analyzed using a 5 kDa NMW CO2 was filtered using a tangential flow ultrafiltration membrane in 2 mM phosphate (pH 7.2) buffer. The solution was diafiltered and filtered to 0.2 microns.

[0355] Activated polysaccharide formulated for lyophilization at 6 mg Ps / mL at 5% w / v sucrose concentration CRM197 was lyophilized at 1% w / v sucrose concentration and 6 mg Pr / mL. It was formulated for use.

[0356] The formulated Ps and CRM197 solutions were lyophilized separately. The CRM197 material was 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 mol of polysaccharide repeating unit) (1 mole per 1000 mg of ATP) 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 approximately 4°C. The pH was neutralized by adding potassium phosphate buffer. The batch was concentrated to a concentration of 30 kDa NMW. CO2 tangential flow ultrafiltration membrane was used to filter 150 mM sodium chloride, 25 mM potassium phosphate The solution was diafiltered against 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% ethanol 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 solution was diluted with histidine, dispensed into aliquots, and frozen at -60°C or below.

[0360] Example 17 PCV23(DMSO) and PCV23(DMSO) using DMSO conjugation (O+Aq) Preparation of serotype 8 conjugates for multivalent studies The polysaccharide is dissolved, sized to a target molecular weight, chemically activated, and purified by ultrafiltration. 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 stirred 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] Polysaccharide size reduction and oxidation Purified pneumococcal Ps capsule powder was dissolved in water and filtered at 0.45 microns. The resulting 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 of polysaccharides was minimized. The oxidation 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). and then dialysis against water using a 5 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was carried out at 2-8°C.

[0364] Polysaccharide conjugation to CRM197 Pseudomonas as previously described (WO 2012 / 173876 A1) Purified CRM197 obtained by expression in C. fluorescens was analyzed using a 5 kDa NMW CO2 was filtered using a tangential flow ultrafiltration membrane in 2 mM phosphate (pH 7.2) buffer. The solution was diafiltered and filtered to 0.2 microns.

[0365] Activated polysaccharide formulated for lyophilization at 6 mg Ps / mL at 5% w / v sucrose concentration CRM197 was lyophilized at 1% w / v sucrose concentration and 6 mg Pr / mL. It was formulated for use.

[0366] The formulated Ps and CRM197 solutions were lyophilized separately. The CRM197 and CRM297 substances were separately redissolved in an equal volume of DMSO. The polysaccharide solution and CRM197 were mixed to obtain a polysaccharide concentration of 1.5 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 approximately 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% ethanol at 4°C.

[0369] The retentate batch was filtered to 0.2 microns (through a 0.5 micron prefilter) and then 150 mM sodium chloride (p) containing 0.015% (w / v) polysorbate 20 Dilute with additional 10 mM histidine in PBS (H7.0), dispense into aliquots, and store at -60°C. Frozen below.

[0370] Example 18 Serotype 9V Conjugate for PCV23(DMSO) Polyvalent Studies Using DMSO Conjugation Conjugate manufacturing The polysaccharide is dissolved, sized to a target molecular weight, chemically activated, and purified by ultrafiltration. 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 stirred 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] Polysaccharide size reduction and oxidation Purified pneumococcal Ps capsule powder was dissolved in water and filtered at 0.45 microns. The resulting 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 10 kDa Diafiltration was performed 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 of polysaccharides was minimized. The oxidation 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). and then filtered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was carried out at 2-8°C.

[0374] Polysaccharide conjugation to CRM197 Pseudomonas as previously described (WO 2012 / 173876 A1) Purified CRM197 obtained by expression in C. fluorescens was analyzed using a 5 kDa NMW CO2 was filtered using a tangential flow ultrafiltration membrane in 2 mM phosphate (pH 7.2) buffer. The solution was diafiltered and filtered to 0.2 microns.

[0375] Activated polysaccharide formulated for lyophilization at 6 mg Ps / mL at 5% w / v sucrose concentration CRM197 was lyophilized at 1% w / v sucrose concentration and 6 mg Pr / mL. It was formulated for use.

[0376] The formulated Ps and CRM197 solutions were lyophilized separately. The CRM197 and CRM297 substances were separately redissolved in an equal volume of DMSO. The polysaccharide solution and CRM197 were mixed to obtain a polysaccharide concentration of 1.3 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 chosen so that sodium cyanoborohydride (per mole of polysaccharide repeating unit) (1 mol per 1000 mg of ATP) 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 approximately 4°C. The pH was neutralized by adding potassium phosphate buffer. The batch was concentrated to a concentration of 30 kDa NMW. CO2 tangential flow ultrafiltration membrane was used to filter 150 mM sodium chloride, 25 mM potassium phosphate The solution was diafiltered against 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% ethanol at 4°C.

[0379] The retentate batch was filtered to 0.2 microns (through a 0.5 micron prefilter) and 0.01 150 mM sodium chloride (pH 7.0) containing 5% (w / v) polysorbate 20 ) with additional 10 mM histidine, dispense into aliquots, and freeze at ≤60°C. 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 converted into an activated polysaccharide using nickel chloride in the reaction mixture. 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] Polysaccharide size reduction and oxidation Purified pneumococcal capsular polysaccharide powder was dissolved in water and filtered at 0.45 microns. The resulting polysaccharide was homogenized to reduce its molecular weight. The pressure and number of passes through the homogenizer were controlled at 100 bar / 5 passes. Small polysaccharides were concentrated and filtered 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 of polysaccharides was minimized. The oxidation 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 through a 10 kDa NMWCO tangential flow ultrafiltration membrane in 10 mM phosphate buffer. The ultrafiltration was performed at 2-8°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 Combine with 0.2 micron filtered buffered polysaccharide solution at a polysaccharide to CRM197 mass 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 selected so that the size of the resulting conjugates was controlled. The solution was then 0.2 micron filtered. The salt was removed using a 100 mM nickel chloride solution. Nickel chloride was added to approximately 2 mM. Sodium cyanoborohydride (1 mole of polysaccharide repeating unit) The conjugates were added in a ratio of 0.2 to 0.2, with the aim of maximizing the consumption of polysaccharides and proteins. 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 phosphate (1 mole per mole of polysaccharide repeating unit) was added. Potassium (pH 6.0) was added.

[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 batch was The mixture was filtered at 0.2 microns.

[0387] 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 PBS (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 converted into an activated polysaccharide using nickel chloride in the reaction mixture. 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] Polysaccharide size reduction and oxidation Purified pneumococcal capsular polysaccharide powder was dissolved in water and filtered at 0.45 microns. The resulting polysaccharide was homogenized to reduce its molecular weight. The pressure and number of passes through the homogenizer were controlled at 100 bar / 5 passes. Small polysaccharides were concentrated and filtered 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 of polysaccharides was minimized. The oxidation 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 through a 10 kDa NMWCO tangential flow ultrafiltration membrane in 10 mM phosphate buffer. The ultrafiltration was performed at 2-8°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 Combine with 0.2 micron filtered buffered polysaccharide solution at a polysaccharide to CRM197 mass 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 selected so that the size of the resulting conjugates was controlled. The solution was then 0.2 micron filtered. The salt was removed using a 100 mM nickel chloride solution. Nickel chloride was added to approximately 2 mM. Sodium cyanoborohydride (1 mole of polysaccharide repeating unit) The conjugates were added in a ratio of 0.2 to 0.2, with the aim of maximizing the consumption of polysaccharides and proteins. 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 phosphate (1 mole per mole of polysaccharide repeating unit) was added. Potassium (pH 6.0) was added.

[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 batch was The mixture was filtered at 0.2 microns.

[0395] 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 PBS (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 Manufacturing The polysaccharide is dissolved, sized to a target molecular weight, chemically activated, and purified by ultrafiltration. 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 stirred 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] Polysaccharide size reduction and oxidation Purified pneumococcal Ps capsule powder was dissolved in water and filtered at 0.45 microns. The resulting polysaccharide was homogenized to reduce the molecular weight of Ps. The number of passes was controlled to 615 bar / 5 passes.

[0398] The 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 of polysaccharides was minimized. The oxidation 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). and then filtered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was carried out at 2-8°C.

[0401] Polysaccharide conjugation to CRM197 Pseudomonas as previously described (WO 2012 / 173876 A1) Purified CRM197 obtained by expression in C. fluorescens was analyzed using a 5 kDa NMW CO2 was filtered using a tangential flow ultrafiltration membrane in 2 mM phosphate (pH 7.2) buffer. The solution was diafiltered and filtered to 0.2 microns.

[0402] Activated polysaccharide formulated for lyophilization at 6 mg Ps / mL at 5% w / v sucrose concentration CRM197 was lyophilized at 1% w / v sucrose concentration and 6 mg Pr / mL. It was formulated for use.

[0403] The formulated Ps and CRM197 solutions were lyophilized separately. The CRM197 and CRM297 substances were separately redissolved in an equal volume of DMSO. The polysaccharide solution and CRM197 were mixed to obtain a polysaccharide concentration of 1.6 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 chosen so that sodium cyanoborohydride (per mole of polysaccharide repeating unit) (1 mol per 1000 mg of ATP) 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 approximately 4°C. Potassium phosphate buffer was added to neutralize the pH.

[0405] Final Filtration and Product Storage Concentrate the batch to 0.015 ml 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 10 ml of PBS at 4°C.

[0406] The retentate batch was filtered to 0.2 microns (through a 0.5 micron prefilter) and then 150 mM sodium chloride (p) containing 0.015% (w / v) polysorbate 20 Dilute with additional 10 mM histidine in PBS (H7.0), dispense into aliquots, and store at -60°C. Frozen below.

[0407] Example 22 Serotypes for PCV23 (DMSO+Aq) multivalent studies using DMSO conjugation Preparation of 10A conjugate The polysaccharide is dissolved, sized to a target molecular weight, chemically activated, and purified by ultrafiltration. 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 stirred 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] Polysaccharide size reduction and oxidation Purified pneumococcal Ps capsule powder was dissolved in water and filtered at 0.45 microns. The resulting 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. Diafiltration was performed 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 of polysaccharides was minimized. The oxidation 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). and then filtered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was carried out at 2-8°C.

[0411] Polysaccharide conjugation to CRM197 Pseudomonas as previously described (WO 2012 / 173876 A1) Purified CRM197 obtained by expression in C. fluorescens was analyzed using a 5 kDa NMW CO2 was filtered using a tangential flow ultrafiltration membrane in 2 mM phosphate (pH 7.2) buffer. The solution was diafiltered and filtered to 0.2 microns.

[0412] Activated polysaccharide formulated for lyophilization at 6 mg Ps / mL at 5% w / v sucrose concentration CRM197 was lyophilized at 1% w / v sucrose concentration and 6 mg Pr / mL. It was formulated for use.

[0413] The formulated Ps and CRM197 solutions were lyophilized separately. The CRM197 and CRM297 substances were separately redissolved in an equal volume of DMSO. The polysaccharide solution and CRM197 were mixed to obtain a polysaccharide concentration of 1.6 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 chosen so that sodium cyanoborohydride (per mole of polysaccharide repeating unit) (1 mol per 1000 mg of ATP) 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 approximately 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 a 300 kDa NMWCO tangential flow Using an ultrafiltration membrane, 150 ml of ... Dialysis at 4°C against 10 mM histidine in 10 mM sodium chloride (pH 7.0) It was regulated.

[0416] The retentate batch was filtered to 0.2 microns (through a 0.5 micron prefilter) and then 150 mM sodium chloride (p) containing 0.015% (w / v) polysorbate 20 Dilute with additional 10 mM histidine in PBS (H7.0), dispense into aliquots, and store at -60°C. Frozen below.

[0417] Example 23 Serotype 10A conjugates for PCV22 multivalent studies using DMSO conjugation Manufacturing The polysaccharide is dissolved, sized to a target molecular weight, chemically activated, and purified by ultrafiltration. 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 stirred 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] Polysaccharide size reduction and oxidation Purified pneumococcal Ps capsule powder was dissolved in water and filtered at 0.45 microns. The resulting 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. Diafiltration was performed 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 of polysaccharides was minimized. The oxidation 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). and then filtered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was carried out at 2-8°C.

[0421] Polysaccharide conjugation to CRM197 Pseudomonas as previously described (WO 2012 / 173876 A1) Purified CRM197 obtained by expression in C. fluorescens was analyzed using a 5 kDa NMW CO2 was filtered using a tangential flow ultrafiltration membrane in 2 mM phosphate (pH 7.2) buffer. The solution was diafiltered and filtered to 0.2 microns.

[0422] Activated polysaccharide formulated for lyophilization at 6 mg Ps / mL at 5% w / v sucrose concentration CRM197 was lyophilized at 1% w / v sucrose concentration and 6 mg Pr / mL. It was formulated for use.

[0423] The formulated Ps and CRM197 solutions were lyophilized separately. The CRM197 and CRM297 substances were separately redissolved in an equal volume of DMSO. The polysaccharide solution and CRM197 were mixed to obtain a polysaccharide concentration of 1.75 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 mol of polysaccharide repeating unit) (1 mole per 1000 mg of ATP) 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 approximately 4°C. The pH was neutralized by adding potassium phosphate buffer. The batch was concentrated to a concentration of 30 kDa NMW. CO2 tangential flow ultrafiltration membrane was used to filter 150 mM sodium chloride, 25 mM potassium phosphate The solution was diafiltered against 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% ethanol at 4°C.

[0426] The retentate batch was filtered to 0.2 microns (through a 0.5 micron prefilter) and then 150 mM sodium chloride (p) containing 0.015% (w / v) polysorbate 20 Dilute with additional 10 mM histidine in PBS (H7.0), dispense into aliquots, and store at -60°C. Frozen below.

[0427] Example X Serotype 11A conjugates for PCV24 multivalent studies using DMSO conjugation Manufacturing The polysaccharide is dissolved, sized to a target molecular weight, chemically activated, and purified by ultrafiltration. 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 stirred 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] Polysaccharide size reduction and oxidation Purified pneumococcal Ps capsule powder was dissolved in water and filtered at 0.45 microns. The resulting 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 of polysaccharides was minimized. The oxidation 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). and then dialysis 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 obtained by expression in C. fluorescens was analyzed using a 5 kDa NMW CO2 was filtered using a tangential flow ultrafiltration membrane in 2 mM phosphate (pH 7.2) buffer. The solution was diafiltered and filtered to 0.2 microns.

[0432] Activated polysaccharide formulated for lyophilization at 6 mg Ps / mL at 5% w / v sucrose concentration CRM197 was lyophilized at 1% w / v sucrose concentration and 6 mg Pr / mL. It was formulated for use.

[0433] The formulated Ps and CRM197 solutions were lyophilized separately. The CRM197 and CRM297 substances were separately redissolved in an equal volume of DMSO. The polysaccharide solution and CRM197 were mixed to obtain a polysaccharide concentration of 1.5 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 (per mole of polysaccharide repeating unit) (1 mol per 1000 mg of ATP) 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 diluted with 150 mM sodium chloride containing phosphate 20 at approximately 4°C. The pH was neutralized by adding potassium carbonate 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% ethanol 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 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 below.

[0437] Example 24 PCV23(DMSO) and PCV23(DMSO) using DMSO conjugation (O+Aq) Preparation of serotype 12F conjugates for multivalent studies The polysaccharide is dissolved, sized to a target molecular weight, chemically activated, and purified by ultrafiltration. 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 stirred 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] Polysaccharide size reduction and oxidation Purified pneumococcal Ps capsule powder was dissolved in water and filtered at 0.45 microns. The resulting polysaccharide was reduced in size by acid hydrolysis. The acid hydrolysis was carried out by adding acetic acid to a concentration of 200 mM. , and incubated at 80°C for 155 minutes, followed by cold potassium phosphate (pH 7) buffer. This was neutralized by adding HCl to a concentration of 400 mM.

[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 of polysaccharides was minimized. The oxidation 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). and then dialysis against water using a 5 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was carried out at 2-8°C.

[0442] Polysaccharide conjugation to CRM197 Pseudomonas as previously described (WO 2012 / 173876 A1) Purified CRM197 obtained by expression in C. fluorescens was analyzed using a 5 kDa NMW CO2 was filtered using a tangential flow ultrafiltration membrane in 2 mM phosphate (pH 7.2) buffer. The solution was diafiltered and filtered to 0.2 microns.

[0443] Activated polysaccharide formulated for lyophilization at 6 mg Ps / mL at 5% w / v sucrose concentration CRM197 was lyophilized at 1% w / v sucrose concentration and 6 mg Pr / mL. It was formulated for use.

[0444] The formulated Ps and CRM197 solutions were lyophilized separately. The CRM197 and CRM297 substances were separately redissolved in an equal volume of DMSO. The polysaccharide solution and CRM197 were mixed to obtain a polysaccharide concentration of 1.8 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 chosen so that sodium cyanoborohydride (per mole of polysaccharide repeating unit) (1 mol per 1000 mg of ATP) 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 approximately 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% ethanol at 4°C.

[0447] The retentate batch was filtered to 0.2 microns (through a 0.5 micron prefilter) and then 150 mM sodium chloride (p) containing 0.015% (w / v) polysorbate 20 Dilute with additional 10 mM histidine in PBS (H7.0), dispense into aliquots, and store at -60°C. Frozen below.

[0448] Example 25 PCV23(DMSO) and PCV23(DMSO) using DMSO conjugation (O+Aq) Preparation of serotype 12F conjugates for multivalent studies The polysaccharide is dissolved, sized to a target molecular weight, chemically activated, and purified by ultrafiltration. 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 stirred 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] Polysaccharide size reduction and oxidation Purified pneumococcal Ps capsule powder was dissolved in water and filtered at 0.45 microns. The resulting polysaccharide was reduced in size by acid hydrolysis. The acid hydrolysis was carried out by adding acetic acid to a concentration of 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 HCl 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 of polysaccharides was minimized. The oxidation 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). and then dialysis against water using a 5 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was carried out at 2-8°C.

[0453] Polysaccharide conjugation to CRM197 Pseudomonas as previously described (WO 2012 / 173876 A1) Purified CRM197 obtained by expression in C. fluorescens was analyzed using a 5 kDa NMW CO2 was filtered using a tangential flow ultrafiltration membrane in 2 mM phosphate (pH 7.2) buffer. The solution was diafiltered and filtered to 0.2 microns.

[0454] Activated polysaccharide formulated for lyophilization at 6 mg Ps / mL at 5% w / v sucrose concentration CRM197 was lyophilized at 1% w / v sucrose concentration and 6 mg Pr / mL. It was formulated for use.

[0455] The formulated Ps and CRM197 solutions were lyophilized separately. The CRM197 and CRM297 substances were separately redissolved in an equal volume of DMSO. The polysaccharide solution and CRM197 were mixed to obtain a polysaccharide concentration of 1.5 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 (per mole of polysaccharide repeating unit) (1 mol per 1000 mg of ATP) 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 approximately 4°C. The pH was neutralized by adding potassium phosphate buffer. The batch was concentrated to a concentration of 30 kDa NMW. CO2 tangential flow ultrafiltration membrane was used to filter 150 mM sodium chloride, 25 mM potassium phosphate The solution was diafiltered against 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% ethanol at 4°C.

[0458] The retentate batch was filtered to 0.2 microns (through a 0.5 micron prefilter) and then 150 mM sodium chloride (p) containing 0.015% (w / v) polysorbate 20 Dilute with additional 10 mM histidine in PBS (H7.0), dispense into aliquots, and store at -60°C. Frozen below.

[0459] Example 26 Serotype 14 for PCV23(DMSO) polyvalent studies using DMSO conjugation Conjugate manufacturing The polysaccharide is dissolved, sized to a target molecular weight, chemically activated, and purified by ultrafiltration. 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 stirred 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] Polysaccharide size reduction and oxidation Purified pneumococcal Ps capsule powder was dissolved in water and filtered at 0.45 microns. The resulting polysaccharide was homogenized to reduce the molecular weight of Ps. The number of passes was controlled to 200 bar / 6 passes.

[0461] The 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 of polysaccharides was minimized. The oxidation 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). and then filtered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was carried out at 2-8°C.

[0464] Polysaccharide conjugation to CRM197 Pseudomonas as previously described (WO 2012 / 173876 A1) Purified CRM197 obtained by expression in C. fluorescens was analyzed using a 5 kDa NMW CO2 was filtered using a tangential flow ultrafiltration membrane in 2 mM phosphate (pH 7.2) buffer. The solution was diafiltered and filtered to 0.2 microns.

[0465] Activated polysaccharide formulated for lyophilization at 6 mg Ps / mL at 5% w / v sucrose concentration CRM197 was lyophilized at 1% w / v sucrose concentration and 6 mg Pr / mL. It was formulated for use.

[0466] The formulated Ps and CRM197 solutions were lyophilized separately. The CRM197 and CRM297 substances were separately redissolved in an equal volume of DMSO. The polysaccharide solution and CRM197 were mixed to obtain a polysaccharide concentration of 1.5 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 (per mole of polysaccharide repeating unit) (1 mol per 1000 mg of ATP) 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 approximately 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 dialyzed 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 PCV22 and PCV23 (DMSO+Aq) multivalent synthesis 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 converted into an activated polysaccharide using nickel chloride in the reaction mixture. 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] Polysaccharide size reduction and oxidation Purified pneumococcal capsular polysaccharide powder was dissolved in water and filtered at 0.45 microns. The resulting polysaccharide was homogenized to reduce its molecular weight. The pressure and number of passes through the homogenizer were controlled at 200 bar / 6 passes. Small polysaccharides were concentrated and filtered 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 of polysaccharides was minimized. The oxidation 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 through a 10 kDa NMWCO tangential flow ultrafiltration membrane in 10 mM phosphate buffer. The ultrafiltration was performed at 2-8°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 Combine with 0.2 micron filtered buffered polysaccharide solution at a polysaccharide to CRM197 mass 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 that the size of the resulting conjugate could be controlled. 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 in a ratio of 0.2 to 0.2, with the aim of maximizing the consumption of polysaccharides and proteins. The 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 phosphate (1 mole per mole of polysaccharide repeating unit) was added. Potassium (pH 6.0) was added.

[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 filtered at 0.2 microns.

[0476] An additional 10 mM L-hydroxybenzoate 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(DMSO) using DMSO conjugation (O+Aq) Preparation of serotype 15A conjugates for multivalent studies The polysaccharide is dissolved, sized to a target molecular weight, chemically activated, and purified by ultrafiltration. 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 stirred 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] Polysaccharide size reduction and oxidation Purified pneumococcal Ps capsule powder was dissolved in water and filtered at 0.45 microns. The resulting polysaccharide was homogenized to reduce the molecular weight of Ps. The number of passes was controlled to 210 bar / 5 passes.

[0479] The 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 of polysaccharides was minimized. The oxidation 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). and then filtered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was carried out at 2-8°C.

[0482] Polysaccharide conjugation to CRM197 Pseudomonas as previously described (WO 2012 / 173876 A1) Purified CRM197 obtained by expression in C. fluorescens was analyzed using a 5 kDa NMW CO2 was filtered using a tangential flow ultrafiltration membrane in 2 mM phosphate (pH 7.2) buffer. The solution was diafiltered and filtered to 0.2 microns.

[0483] Activated polysaccharide formulated for lyophilization at 6 mg Ps / mL at 5% w / v sucrose concentration CRM197 was lyophilized at 1% w / v sucrose concentration and 6 mg Pr / mL. It was formulated for use.

[0484] The formulated Ps and CRM197 solutions were lyophilized separately. The CRM197 substance was separately 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 was 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 ratio was 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 approximately 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% ethanol at 4°C.

[0487] The retentate batch was filtered to 0.2 microns (through a 0.5 micron prefilter) and then 150 mM sodium chloride (p) containing 0.015% (w / v) polysorbate 20 Dilute with additional 10 mM histidine in PBS (H7.0), dispense into aliquots, and store at -60°C. Frozen below.

[0488] Example 29 Serotype 15A conjugates for PCV22 multivalent studies using DMSO conjugation Manufacturing The polysaccharide is dissolved, sized to a target molecular weight, chemically activated, and purified by ultrafiltration. 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 stirred 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] Polysaccharide size reduction and oxidation Purified pneumococcal Ps capsule powder was dissolved in water and filtered at 0.45 microns. The resulting polysaccharide was homogenized to reduce the molecular weight of Ps. The number of passes was controlled to 200 bar / 5 passes.

[0490] The 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 of polysaccharides was minimized. The oxidation 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). and then filtered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was carried out at 2-8°C.

[0493] Polysaccharide conjugation to CRM197 Pseudomonas as previously described (WO 2012 / 173876 A1) Purified CRM197 obtained by expression in C. fluorescens was analyzed using a 5 kDa NMW CO2 was filtered using a tangential flow ultrafiltration membrane in 2 mM phosphate (pH 7.2) buffer. The solution was diafiltered and filtered to 0.2 microns.

[0494] Activated polysaccharide formulated for lyophilization at 6 mg Ps / mL at 5% w / v sucrose concentration CRM197 was lyophilized at 1% w / v sucrose concentration and 6 mg Pr / mL. It was formulated for use.

[0495] The formulated Ps and CRM197 solutions were lyophilized separately. The CRM197 substance was separately 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 was 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 ratio was 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 approximately 4°C. The pH was neutralized by adding potassium phosphate buffer. The batch was concentrated to a concentration of 30 kDa NMW. CO2 tangential flow ultrafiltration membrane was used to filter 150 mM sodium chloride, 25 mM potassium phosphate The solution was diafiltered against 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% ethanol at 4°C.

[0498] The retentate batch was filtered to 0.2 microns (through a 0.5 micron prefilter) and then 150 mM sodium chloride (p) containing 0.015% (w / v) polysorbate 20 Dilute with additional 10 mM histidine in PBS (H7.0), dispense into aliquots, and store at -60°C. Frozen below.

[0499] Example 30 PCV23(DMSO) and PCV23(DMSO) using DMSO conjugation (O+Aq) Preparation of serotype 15C conjugates for 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 basic hydrolysis to release the groups, chemically activated, and purified by ultrafiltration. The activated polysaccharide and purified CRM197 were separately lyophilized and diluted with DMSO. The redissolved polysaccharide solution was then combined with the CRM197 solution and the mixture was stirred for 1 hour at 20°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] Polysaccharide size reduction, base hydrolysis, and oxidation 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 filter was controlled at 300 bar / 5 passes.

[0501] The 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 mg of HCl and the solution was cooled to ambient temperature. Diafiltration was performed 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 allow for 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 hours at 22°C.

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

[0505] Polysaccharide conjugation to CRM197 Pseudomonas as previously described (WO 2012 / 173876 A1) Purified CRM197 obtained by expression in C. fluorescens was analyzed using a 5 kDa NMW CO2 was filtered using a tangential flow ultrafiltration membrane in 2 mM phosphate (pH 7.2) buffer. The solution was diafiltered and filtered to 0.2 microns.

[0506] Activated polysaccharide formulated for lyophilization at 6 mg Ps / mL at 5% w / v sucrose concentration CRM197 was lyophilized at 1% w / v sucrose concentration and 6 mg Pr / mL. It was formulated for use.

[0507] The formulated Ps and CRM197 solutions were lyophilized separately. The CRM197 and CRM297 substances were separately redissolved in an equal volume of DMSO. The polysaccharide solution and CRM197 were mixed to obtain a polysaccharide concentration of 1.75 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 mol of polysaccharide repeating unit) (1 mole per 1000 mg of ATP) 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 approximately 4°C. The pH was neutralized by adding potassium phosphate buffer. The batch was concentrated to a concentration of 30 kDa NMW. CO2 tangential flow ultrafiltration membrane was used to filter 150 mM sodium chloride, 25 mM potassium phosphate The solution was diafiltered against 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% ethanol at 4°C.

[0510] The retentate batch was filtered to 0.2 microns (through a 0.5 micron prefilter) and then 150 mM sodium chloride (p) containing 0.015% (w / v) polysorbate 20 Dilute with additional 10 mM histidine in PBS (H7.0), dispense into aliquots, and store at -60°C. Frozen below.

[0511] Example 31 Serotype 15C conjugates for PCV22 multivalent studies using DMSO conjugation Manufacturing Streptococcus pneumoniae ae) Polysaccharide derived from serotype 15B is dissolved, sized to a target molecular weight, and O-acetylated. It is subjected to mild basic hydrolysis to release the groups, chemically activated, and purified by ultrafiltration. The activated polysaccharide and purified CRM197 were separately lyophilized and diluted with DMSO. The redissolved polysaccharide solution was then combined with the CRM197 solution and the mixture was stirred for 1 hour at 20°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] Polysaccharide size reduction, base hydrolysis, and oxidation 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, 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 adjust the pH. The solution was then concentrated and cooled to ambient temperature. 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 allow for 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 hours at 22°C.

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

[0515] Polysaccharide conjugation to CRM197 Pseudomonas as previously described (WO 2012 / 173876 A1) Purified CRM197 obtained by expression in C. fluorescens was analyzed using a 5 kDa NMW CO2 was filtered using a tangential flow ultrafiltration membrane in 2 mM phosphate (pH 7.2) buffer. The solution was diafiltered and filtered to 0.2 microns.

[0516] Activated polysaccharide formulated for lyophilization at 6 mg Ps / mL at 5% w / v sucrose concentration CRM197 was lyophilized at 1% w / v sucrose concentration and 6 mg Pr / mL. It was formulated for use.

[0517] The formulated Ps and CRM197 solutions were lyophilized separately. The CRM197 and CRM297 substances were separately redissolved in an equal volume of DMSO. The polysaccharide solution and CRM197 were mixed to obtain a polysaccharide concentration of 1.75 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 mol of polysaccharide repeating unit) (1 mole per 1000 mg of ATP) 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 approximately 4°C. The pH was neutralized by adding potassium phosphate buffer. The batch was concentrated to a concentration of 30 kDa NMW. CO2 tangential flow ultrafiltration membrane was used to filter 150 mM sodium chloride, 25 mM potassium phosphate The solution was diafiltered against 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% ethanol at 4°C.

[0520] The retentate batch was filtered to 0.2 microns (through a 0.5 micron prefilter) and then 150 mM sodium chloride (p) containing 0.015% (w / v) polysorbate 20 Dilute with additional 10 mM histidine in PBS (H7.0), dispense into aliquots, and store at -60°C. Frozen below.

[0521] Example 32 PCV23(DMSO) and PCV23(DMSO) using DMSO conjugation (O+Aq) Preparation of serotype 18C conjugates for multivalent studies The polysaccharide is dissolved, sized to a target molecular weight, chemically activated, and purified by ultrafiltration. 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 stirred 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] Polysaccharide size reduction and oxidation Purified pneumococcal Ps capsule powder was dissolved in water and filtered at 0.45 microns. The resulting polysaccharide was reduced in size by acid hydrolysis. The acid hydrolysis was carried out by adding acetic acid to a concentration of 200 mM. , and incubated at 90°C for 160 minutes, followed by cold potassium phosphate (pH 7) buffer. This was neutralized by adding HCl to a concentration of 400 mM.

[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 of polysaccharides was minimized. The oxidation 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). and then filtered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was carried out at 2-8°C.

[0526] Polysaccharide conjugation to CRM197 Pseudomonas as previously described (WO 2012 / 173876 A1) Purified CRM197 obtained by expression in C. fluorescens was analyzed using a 5 kDa NMW CO2 was filtered using a tangential flow ultrafiltration membrane in 2 mM phosphate (pH 7.2) buffer. The solution was diafiltered and filtered to 0.2 microns.

[0527] Activated polysaccharide formulated for lyophilization at 6 mg Ps / mL at 5% w / v sucrose concentration CRM197 was lyophilized at 1% w / v sucrose concentration and 6 mg Pr / mL. It was formulated for use.

[0528] The formulated Ps and CRM197 solutions were lyophilized separately. The CRM197 material was 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 mol of polysaccharide repeating unit) (1 mole per 1000 mg of ATP) 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 approximately 4°C. The pH was neutralized by adding potassium phosphate buffer. The batch was concentrated to a concentration of 30 kDa NMW. CO2 tangential flow ultrafiltration membrane was used to filter 150 mM sodium chloride, 25 mM potassium phosphate The solution was diafiltered against 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% ethanol at 4°C.

[0531] The retentate batch was filtered to 0.2 microns (through a 0.5 micron prefilter) and then 150 mM sodium chloride (p) containing 0.015% (w / v) polysorbate 20 Dilute with additional 10 mM histidine in PBS (H7.0), dispense into aliquots, and store at -60°C. Frozen below.

[0532] Example 33 Serotype 18C conjugates for PCV22 multivalent studies using DMSO conjugation Manufacturing The polysaccharide is dissolved, sized to a target molecular weight, chemically activated, and purified by ultrafiltration. 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 stirred 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] Polysaccharide size reduction and oxidation Purified pneumococcal Ps capsule powder was dissolved in water and filtered at 0.45 microns. The resulting polysaccharide was reduced in size by acid hydrolysis. The acid hydrolysis was carried out by adding acetic acid to a concentration of 200 mM. , and incubated at 90°C for 160 minutes, followed by cold potassium phosphate (pH 7) buffer. This was neutralized by adding HCl to a concentration of 400 mM.

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

[0535] 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 o...

Claims

1. 1. A multivalent immunogenic composition for the prevention of acute otitis media and sinusitis comprising 24 different S. pneumoniae polysaccharide carrier protein conjugates, each of the conjugates comprising the polysaccharide of a particular Streptococcus pneumoniae serotype conjugated to a carrier protein, wherein the Streptococcus pneumoniae serotypes are 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, de-O-acetylated 15B, and 16A. 15B), 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B, wherein the carrier protein is CRM197, and the composition does not comprise a polysaccharide carrier protein conjugate containing a polysaccharide of any other Streptococcus pneumoniae serotype.

2. The multivalent immunogenic composition of claim 1 , comprising an adjuvant.

3. The multivalent immunogenic composition of claim 2, wherein the adjuvant is an aluminum phosphate adjuvant.

4. 1. A multivalent immunogenic composition for the prevention of acute otitis media and sinusitis comprising Streptococcus pneumoniae (S. pneumoniae) polysaccharide carrier protein conjugates, each of the conjugates comprising a polysaccharide of a particular Streptococcus pneumoniae serotype conjugated to a carrier protein, wherein the Streptococcus pneumoniae serotypes consist of 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, de-O-acetylated 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B, and wherein the carrier protein is CRM197.

5. The multivalent immunogenic composition of claim 4, comprising an adjuvant.

6. The multivalent immunogenic composition of claim 5, wherein the adjuvant is an aluminum phosphate adjuvant.

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