Multivalent Pneumococcal Polysaccharide-Protein Conjugate Composition
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, 66, 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 . A method for making an immunogenic conjugate comprising Streptococcus pneumoniae serotype 19A polysaccharide covalently linked to a carrier protein, 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; 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.
2 . The method of claim 1 , wherein the carrier protein is CRM 197 .
3 . The method of claim 2 , wherein the activated serotype 19A polysaccharide and CRM 197 are compounded at a ratio of 0.8:1.
4 . The method of claim 1 , wherein the pH of the activated serotype 19A polysaccharide is adjusted to 6.5±0.2 prior to compounding with the carrier protein.
5 . The method of claim 1 , wherein the activated serotype 19A polysaccharide is compounded with sucrose prior to compounding with the carrier protein.
6 . The method of claim 1 , further comprising purifying the immunogenic conjugate.
7 . The method of claim 1 , wherein the oxidizing agent is sodium periodate.
8 . The method of claim 1 , wherein the reducing agent is sodium cyanoborohydride.
9 . The method of claim 1 , wherein capping unreacted aldehydes comprises reacting the serotype 19A polysaccharide:carrier protein conjugate with sodium borohydride.
10 . A method for making an immunogenic conjugate comprising Streptococcus pneumoniae serotype 19A polysaccharide covalently linked to a carrier protein, the method comprising:
(a) reacting purified serotype 19A polysaccharide with sodium periodate resulting in an activated serotype 19A polysaccharide;
(b) adjusting the pH of the activated serotype 19A polysaccharide to 6.5±0.2;
(c) compounding the activated serotype 19A polysaccharide with sucrose;
(d) compounding the activated serotype 19A polysaccharide with a carrier protein at a ratio of 0.8:1;
(e) co-lyophilizing the compounded activated serotype 19A polysaccharide and carrier protein;
(f) re-suspending the compounded activated serotype 19A polysaccharide and carrier protein in dimethyl sulfoxide (DMSO);
(g) reacting the compounded, activated serotype 19A polysaccharide and carrier protein with sodium cyanoborohydride resulting in a serotype 19A polysaccharide:carrier protein conjugate; and
(h) capping unreacted aldehydes in the serotype 19A polysaccharide:carrier protein conjugate with sodium borohydride resulting in an immunogenic conjugate comprising Streptococcus pneumoniae serotype 19A polysaccharide covalently linked to a carrier protein.
11 . The method of claim 1 , wherein the carrier protein is CRM 197 .
12 . The method of claim 1 , further comprising purifying the immunogenic conjugate.
13 . A method for making an immunogenic conjugate comprising a Streptococcus pneumoniae polysaccharide covalently linked to a carrier protein wherein said polysaccharide comprises a phosphodiester linkage between repeat units, the 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.
14 . The method of claim 13 , wherein said polysaccharide comprising a phosphodiester linkage between repeat units is Streptococcus pneumoniae polysaccharide serotype 19A, 19F, 6A, or 68.
15 . The method of claim 13 , wherein the carrier protein is CRM 197 .