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.

1 . A method for making an immunogenic conjugate comprising  Streptococcus pneumoniae  serotype 1 polysaccharide covalently linked to a carrier protein, the method comprising:
 (a) reacting purified serotype 1 polysaccharide with an alkaline pH buffer resulting in a partially de-O-acetylated serotype 1 polysaccharide;   (b) reacting the partially de-O-acetylated serotype 1 polysaccharide with a mild acid resulting in a neutralized, partially de-O-acetylated serotype 1 polysaccharide;   (c) reacting the neutralized, partially de-O-acetylated serotype 1 polysaccharide with an oxidizing agent resulting in an activated serotype 1 polysaccharide;   (d) compounding the activated serotype 1 polysaccharide with a carrier protein;   (e) reacting the compounded, activated serotype 1 polysaccharide and carrier protein with a reducing agent resulting in a serotype 1 polysaccharide:carrier protein conjugate; and   (f) capping unreacted aldehydes in the serotype 1 polysaccharide:carrier protein conjugate resulting in an immunogenic conjugate comprising  Streptococcus pneumoniae  serotype 1 polysaccharide covalently linked to a carrier protein.   
     
     
         2 . The method of  claim 1 , further comprising purifying the activated serotype 1 polysaccharide prior to compounding with the carrier protein. 
     
     
         3 . The method of  claim 1 , further comprising co-lyophilizing the compounded, activated serotype 1 polysaccharide and carrier protein prior to reacting with the reducing agent. 
     
     
         4 . The method of  claim 1 , further comprising purifying the immunogenic conjugate. 
     
     
         5 . The method of  claim 1 , wherein the alkaline pH buffer is a bicarbonate/carbonate buffer. 
     
     
         6 . The method of  claim 1 , wherein the mild acid is acetic acid. 
     
     
         7 . The method of  claim 1 , wherein the mild acid is a low ionic strength mineral acid. 
     
     
         8 . The method of  claim 7 , wherein the low ionic strength mineral acid is hydrochloric acid. 
     
     
         9 . The method of  claim 1 , wherein the oxidizing agent is sodium periodate. 
     
     
         10 . The method of  claim 1 , wherein the carrier protein is CRM 197 . 
     
     
         11 . The method of  claim 1 , wherein the reducing agent is sodium cyanoborohydride. 
     
     
         12 . The method of  claim 1 , wherein capping unreacted aldehydes comprises reacting the serotype 1 polysaccharide:carrier protein conjugate with sodium borohydride. 
     
     
         13 . A method for making an immunogenic conjugate comprising  Streptococcus pneumoniae  serotype 1 polysaccharide covalently linked to a carrier protein, the method comprising:
 (a) reacting purified serotype 1 polysaccharide with a bicarbonate/carbonate buffer resulting in a partially de-O-acetylated serotype 1 polysaccharide;   (b) reacting the partially de-O-acetylated serotype 1 polysaccharide with acetic acid resulting in a neutralized, partially de-O-acetylated serotype 1 polysaccharide;   (c) reacting the neutralized, partially de-O-acetylated serotype 1 polysaccharide with sodium periodate resulting in an activated serotype 1 polysaccharide;   (d) purifying the activated serotype 1 polysaccharide;   (e) compounding the activated serotype 1 polysaccharide with a carrier protein;   (f) co-lyophilizing the compounded, activated serotype 1 polysaccharide and carrier protein;   (g) reacting the co-lyophilized, activated serotype 1 polysaccharide and carrier protein with sodium cyanoborohydride resulting in a serotype 1 polysaccharide:carrier protein conjugate; and   (h) capping unreacted aldehydes in the serotype 1 polysaccharide:carrier protein conjugate with sodium borohydride resulting in an immunogenic conjugate comprising  Streptococcus pneumoniae  serotype 1 polysaccharide covalently linked to a carrier protein.   
     
     
         14 . The method of  claim 13 , further comprising purifying the immunogenic conjugate. 
     
     
         15 . The method of  claim 13 , wherein the carrier protein is CRM 197 . 
     
     
         16 . A method for making an activated  Streptococcus pneumoniae  serotype 1 polysaccharide, the method comprising:
 (a) reacting purified serotype 1 polysaccharide with an alkaline pH buffer resulting in a partially de-O-acetylated serotype 1 polysaccharide;   (b) reacting the partially de-O-acetylated serotype 1 polysaccharide with a mild acid resulting in a neutralized, partially de-O-acetylated serotype 1 polysaccharide; and   (c) reacting the neutralized, partially de-O-acetylated serotype 1 polysaccharide with an oxidizing agent resulting in an activated serotype 1 polysaccharide.   
     
     
         17 . The method of  claim 16 , further comprising purifying the activated serotype 1 polysaccharide following oxidation. 
     
     
         18 . The method of  claim 16 , wherein the alkaline pH buffer is a bicarbonate/carbonate buffer. 
     
     
         19 . The method of  claim 16 , wherein the mild acid is acetic acid. 
     
     
         20 . The method of  claim 16 , wherein the oxidizing agent is sodium periodate. 
     
     
         21 . A purified  Streptococcus pneumoniae  serotype 1 polysaccharide that is de-β-acetylated to approximately 40 mole percent. 
     
     
         22 . The purified  Streptococcus pneumoniae  serotype 1 polysaccharide of  claim 21 , wherein said polysaccharide is conjugated to a carrier protein. 
     
     
         23 . The purified  Streptococcus pneumoniae  serotype 1 polysaccharide of  claim 21 , wherein said polysaccharide is obtained by reacting purified  Streptococcus pneumoniae  serotype 1 polysaccharide with an alkaline pH buffer. 
     
     
         24 . The purified  Streptococcus pneumoniae  serotype 1 polysaccharide of  claim 23 , wherein said polysaccharide is conjugated to a carrier protein. 
     
     
         25 . The purified  Streptococcus pneumoniae  serotype 1 polysaccharide of  claim 23 , wherein said alkaline pH buffer is a sodium bicarbonate/carbonate buffer at pH 9.0. 
     
     
         26 . The purified  Streptococcus pneumoniae  serotype 1 polysaccharide of  claim 25 , wherein said buffer concentration is 30 mM.