Multivalent pneumococcal polysaccharide-protein conjugate composition

Abstract

An immunogenic composition having 13 distinct polysaccharide-protein conjugates and optionally, an aluminum-based adjuvant, is described. Each conjugate contains a capsular polysaccharide prepared from a different serotype of Streptococcus pneumoniae (1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F) conjugated to a carrier protein. The immunogenic composition, formulated as a vaccine, increases coverage against pneumococcal disease in infants and young children globally, and provides coverage for serotypes 6A and 19A that is not dependent on the limitations of serogroup cross-protection. Methods for making an immunogenic conjugate comprising Streptococcus pneumoniae serotype 19A polysaccharide are also provided in which the serotype 19A polysaccharide is co-lyophilized with a carrier protein and conjugation is carried out in dimethyl sulfoxide (DMSO) via a reductive amination mechanism.

1 - 15 . (canceled) 
     
     
         16 . A multivalent immunogenic composition, comprising polysaccharide-protein conjugates, together with a physiologically acceptable vehicle, wherein each of the conjugates comprises a capsular polysaccharide from a different serotype of  Streptococcus pneumoniae  conjugated to a carrier protein, and the capsular polysaccharides are prepared from serotype 19A and at least one additional serotype, wherein each capsular polysaccharide is separately conjugated to a carrier protein, and wherein said serotype 19A polysaccharide is covalently linked to a carrier protein according to a method comprising:
 (a) reacting purified serotype 19A polysaccharide with an oxidizing agent resulting in an activated serotype 19A polysaccharide;   (b) compounding the activated serotype 19A polysaccharide with a carrier protein;   (c) co-lyophilizing the compounded activated serotype 19A polysaccharide and carrier protein;   (d) re-suspending the compounded activated serotype 19A polysaccharide and carrier protein in dimethyl sulfoxide (DMSO);   (e) reacting the compounded, activated serotype 19A polysaccharide and carrier protein with a reducing agent resulting in a serotype 19A polysaccharide:carrier protein conjugate; and   (f) capping unreacted aldehydes in the serotype 19A polysaccharide:carrier protein conjugate resulting in an immunogenic conjugate comprising  Streptococcus pneumoniae  serotype 19A polysaccharide covalently linked to a carrier protein;   wherein step (c) is performed after step (b) and before steps (d) and (e).   
     
     
         17 . The multivalent immunogenic composition of  claim 16 ; wherein each capsular polysaccharide is conjugated to the same carrier protein. 
     
     
         18 . The multivalent immunogenic composition of  claim 17 , wherein the carrier protein is CRM 197 . 
     
     
         19 . The multivalent immunogenic composition of  claim 18 , wherein the activated serotype 19A polysaccharide and CRM 197  are compounded at a ratio of 0.8:1. 
     
     
         20 . The multivalent immunogenic composition of  claim 16 , wherein the pH of the activated serotype 19A polysaccharide is adjusted to 6.5±0.2 prior to compounding with the carrier protein. 
     
     
         21 . The multivalent immunogenic composition of  claim 16 , wherein the activated serotype 19A polysaccharide is compounded with sucrose prior to compounding with the carrier protein. 
     
     
         22 . The multivalent immunogenic composition of  claim 16 , further comprising purifying the immunogenic conjugate. 
     
     
         23 . The multivalent immunogenic composition of  claim 16 , wherein the oxidizing agent is sodium periodate. 
     
     
         24 . The multivalent immunogenic composition of  claim 16 , wherein the reducing agent is sodium cyanoborohydride. 
     
     
         25 . The multivalent immunogenic composition of  claim 16 , wherein capping unreacted aldehydes comprises reacting the serotype 19A polysaccharide:carrier protein conjugate with sodium borohydride. 
     
     
         26 . A multivalent immunogenic composition, comprising polysaccharide-protein conjugates, together with a physiologically acceptable vehicle, wherein each of the conjugates comprises a capsular polysaccharide from a different serotype of  Streptococcus pneumoniae  conjugated to a carrier protein, and the capsular polysaccharides are prepared from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F, wherein each capsular polysaccharide is separately conjugated to a carrier protein, and wherein said serotype 19A polysaccharide is covalently linked to a carrier protein according to a method comprising:
 (a) reacting purified serotype 19A polysaccharide with an oxidizing agent resulting in an activated serotype 19A polysaccharide;   (b) compounding the activated serotype 19A polysaccharide with a carrier protein;   (c) co-lyophilizing the compounded activated serotype 19A polysaccharide and carrier protein;   (d) re-suspending the compounded activated serotype 19A polysaccharide and carrier protein in dimethyl sulfoxide (DMSO);   (e) reacting the compounded, activated serotype 19A polysaccharide and carrier protein with a reducing agent resulting in a serotype 19A polysaccharide:carrier protein conjugate; and   (f) capping unreacted aldehydes in the serotype 19A polysaccharide:carrier protein conjugate resulting in an immunogenic conjugate comprising  Streptococcus pneumoniae  serotype 19A polysaccharide covalently linked to a carrier protein;   wherein step (c) is performed after step (b) and before steps (d) and (e).   
     
     
         27 . The multivalent immunogenic composition of  claim 26 , wherein each capsular polysaccharide is conjugated to the same carrier protein. 
     
     
         28 . The multivalent immunogenic composition of  claim 27 , wherein the carrier protein is CRM 197 . 
     
     
         29 . The multivalent immunogenic composition of  claim 28 , wherein the activated serotype 19A polysaccharide and CRM 197  are compounded at a ratio of 0.8:1. 
     
     
         30 . The multivalent immunogenic composition of  claim 26 , wherein the pH of the activated serotype 19A polysaccharide is adjusted to 6.5±0.2 prior to compounding with the carrier protein. 
     
     
         31 . The multivalent immunogenic composition of  claim 26 , wherein the activated serotype 19A polysaccharide is compounded with sucrose prior to compounding with the carrier protein. 
     
     
         32 . The multivalent immunogenic composition of  claim 26 , further comprising purifying the immunogenic conjugate. 
     
     
         33 . The multivalent immunogenic composition of  claim 26 , wherein the oxidizing agent is sodium periodate. 
     
     
         34 . The multivalent immunogenic composition of  claim 26 , wherein the reducing agent is sodium cyanoborohydride. 
     
     
         35 . The multivalent immunogenic composition of  claim 26 , wherein capping unreacted aldehydes comprises reacting the serotype 19A polysaccharide:carrier protein conjugate with sodium borohydride. 
     
     
         36 . A multivalent immunogenic composition, comprising polysaccharide-protein conjugates, together with a physiologically acceptable vehicle, wherein each of the conjugates comprises a capsular polysaccharide front a different serotype  Streptococcus pneumoniae  conjugated to a carrier protein, and the capsular polysaccharides are prepared from a capsular polysaccharide that comprises a phosphodiester linkage between repeat units and at least one additional serotype, wherein each capsular polysaccharide is separately conjugated to a carrier protein, and wherein said capsular polysaccharide that comprises a phosphodiester linkage between repeat units is covalently linked to a carrier protein according to a method comprising:
 (a) reacting said polysaccharide with an oxidizing agent resulting in an activated polysaccharide;   (b) compounding the activated polysaccharide with a carrier protein;   (c) co-lyophilizing the compounded activated polysaccharide and carrier protein;   (d) re-suspending the compounded activated polysaccharide and carrier protein in dimethyl sulfoxide (DMSO);   (e) reacting the compounded, activated polysaccharide and carrier protein with a reducing agent resulting in a polysaccharide:carrier protein conjugate; and   (f) capping unreacted aldehydes in the polysaccharide:carrier protein conjugate resulting in an immunogenic conjugate comprising  Streptococcus pneumoniae  polysaccharide covalently linked to a carrier protein;   wherein step (c) is performed after step (b) and before steps (d) and (e).   
     
     
         37 . The multivalent immunogenic composition of  claim 36 , wherein said capsular polysaccharide comprising a phosphodiester linkage between repeat units is  Streptococcus pneumoniae  polysaccharide serotype 19A, 19F, 6A or 6B. 
     
     
         38 . The multivalent immunogenic composition of  claim 36 , wherein each capsular polysaccharide is conjugated to the same carrier protein. 
     
     
         39 . The multivalent immunogenic composition of  claim 38 , wherein the carrier protein is CRM 197 . 
     
     
         40 . An immunogenic conjugate comprising  Streptococcus pneumoniae  serotype 19A polysaccharide covalently linked to a carrier protein, wherein said serotype 19A polysaccharide is covalently linked to a carrier protein according to a method comprising:
 (a) reacting purified serotype 19A polysaccharide with an oxidizing agent resulting in an activated serotype 19A polysaccharide;   (b) compounding the activated serotype 19A polysaccharide with a carrier protein;   (c) co-lyophilizing the compounded activated serotype 19A polysaccharide and carrier protein;   (d) re-suspending the compounded activated serotype 19A polysaccharide and carrier protein in dimethyl sulfoxide (DMSO);   (e) reacting the compounded, activated serotype 19A polysaccharide and carrier protein with a reducing agent resulting in a serotype 19A polysaccharide:carrier protein conjugate; and   (f) capping unreacted aldehydes in the serotype 19A polysaccharide:carrier protein conjugate resulting in an immunogenic conjugate comprising  Streptococcus pneumoniae  serotype 19A polysaccharide covalently linked to a carrier protein;   wherein step (c) is performed after step (b) and before steps (d) and (e).   
     
     
         41 . The immunogenic conjugate of  claim 40 , wherein the carrier protein is CRM 197 . 
     
     
         42 . The immunogenic conjugate of  claim 41 , wherein the activated serotype 19A polysaccharide and CRM 197  are compounded at a ratio of 0.8:1. 
     
     
         43 . The immunogenic conjugate of  claim 40 , wherein the pH of the activated serotype 19A polysaccharide is adjusted to 6.5±0.2 prior to compounding with the carrier protein. 
     
     
         44 . The immunogenic conjugate of  claim 40 , wherein the activated serotype 19A polysaccharide is compounded with sucrose prior to compounding with the carrier protein. 
     
     
         45 . The immunogenic conjugate of  claim 40 , further comprising purifying the immunogenic conjugate. 
     
     
         46 . The immunogenic conjugate of  claim 40 , wherein the oxidizing agent is sodium periodate. 
     
     
         47 . The immunogenic conjugate of  claim 40 , wherein the reducing agent is sodium cyanoborohydride. 
     
     
         48 . The immunogenic conjugate of  claim 40 , wherein capping unreacted aldehydes comprises reacting the serotype 19A polysaccharide:carrier protein conjugate with sodium borohydride. 
     
     
         49 . An immunogenic conjugate comprising a  Streptococcus pneumoniae  polysaccharide covalently linked to a carrier protein, wherein said polysaccharide comprises a phosphodiester linkage between repeat units, and wherein said capsular polysaccharide that comprises a phosphodiester linkage between repeat units is covalently linked to a carrier protein according to a method comprising:
 (a) reacting said polysaccharide with art oxidizing agent resulting in an activated polysaccharide;   (b) compounding the activated polysaccharide with a carrier protein;   (c) co-lyophilizing the compounded activated polysaccharide and carrier protein;   (d) re-suspending the compounded activated polysaccharide and carrier protein in dimethyl suboxide (DMSO);   (e) reacting the compounded, activated polysaccharide and carrier protein with a reducing agent resulting in a polysaccharide:carrier protein conjugate; and   (f) capping unreacted aldehydes in the polysaccharide carrier protein conjugate resulting in an immunogenic conjugate comprising  Streptococcus pneumoniae  polysaccharide covalently linked to a carrier protein;   wherein step (c) is performed after step (b) and before steps (d) and (e).   
     
     
         50 . The immunogenic conjugate of  claim 49 , wherein said capsular polysaccharide comprising a phosphodiester linkage between repeat units is  Streptococcus pneumoniae  polysaccharide serotype 19A, 19F, 6A or 6B. 
     
     
         51 . The immunogenic conjugate of  claim 49 , wherein the carrier protein is CRM 197 . 
     
     
         52 . A method for making a multivalent immunogenic composition comprising polysaccharide-protein conjugates, wherein each of the conjugates comprises a capsular polysaccharide from a different serotype of  Streptococcus pneumoniae  conjugated to a carrier protein, and the capsular polysaccharides are prepared from serotype 19A and at least one additional serotype, the method comprising:
 (a) reacting purified serotype 19A polysaccharide with an oxidizing agent resulting in an activated serotype 19A polysaccharide;   (b) compounding the activated serotype 19A polysaccharide with a carrier protein;   (c) co-lyophilizing the compounded activated serotype 19A polysaccharide and carrier protein;   (d) re-suspending the compounded activated serotype 19A polysaccharide and carrier protein in dimethyl sulfoxide (DMSO);   (e) reacting the compounded, activated serotype 19A polysaccharide and carrier protein with a reducing agent resulting in a serotype 19A polysaccharide:carrier protein conjugate;   (f) capping unreacted aldehydes in the serotype 19A polysaccharide:carrier protein conjugate resulting in an immunogenic conjugate comprising  Streptococcus pneumoniae  serotype 19A polysaccharide covalently linked to a carrier protein; and   (g) compounding the immunogenic conjugate comprising  Streptococcus pneumoniae  serotype 19A polysaccharide and at least one immunogenic conjugate comprising an additional  Streptococcus pneumoniae  serotype polysaccharide resulting in a multivalent immunogenic composition;   wherein step (c) is performed after step (h) and before steps (d) and (e).   
     
     
         53 . The method of  claim 52 , wherein the capsular polysaccharides are prepared from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F. 
     
     
         54 . The method of  claim 52 , wherein each capsular polysaccharide is separately conjugated to a carrier protein. 
     
     
         55 . The method of  claim 54 , wherein each capsular polysaccharide is conjugated to the same carrier protein. 
     
     
         56 . The method of  claim 55 , wherein the carrier protein is CRM 197 . 
     
     
         57 . The method of  claim 56 , wherein the activated serotype 19A polysaccharide and CRM 197  are compounded at a ratio of 0.8:1. 
     
     
         58 . The method of  claim 52 , wherein the pH of the activated serotype 19A polysaccharide is adjusted to 6.5±0.2 prior to compounding with the carrier protein. 
     
     
         59 . The method of  claim 52 , wherein the activated serotype 19A polysaccharide is compounded with sucrose prior to compounding with the carrier protein. 
     
     
         60 . The method of  claim 52 , further comprising purifying the immunogenic conjugate. 
     
     
         61 . The method of  claim 52 , wherein the oxidizing agent is sodium periodate. 
     
     
         62 . The method of  claim 52 , wherein the reducing agent is sodium cyanoborohydride. 
     
     
         63 . The method of  claim 52 , wherein capping unreacted aldehydes comprises reacting the serotype 19A polysaccharide:carrier protein conjugate with sodium borohydride.