Methods for producing streptococcus pneumoniae capsular polysaccharide carrier protein conjugates

Abstract

A method is described for producing a pneumococcal capsular polysaccharide protein conjugate in which one or more activated pneumococcal polysaccharides of particular pneumococcal serotypes and carrier protein are separately lyophilized, the separately lyophilized polysaccharides and carrier protein are separately reconstituted in an organic solvent, and the reconstituted polysaccharide and carrier protein are then combined together by Tee-mixing and conjugated together to produce polysaccharide carrier protein conjugates. A plurality of conjugates, each comprising polysaccharides of a particular serotype, may be used to produce multivalent pneumococcal immunogenic compositions having a combination of conjugates for use in vaccines.

What is claimed: 
     
         1 . A method for making a composition comprising a  Streptococcus pneumoniae  polysaccharide covalently linked to a carrier protein, the method comprising:
 (a) providing a first dried composition comprising activated  Streptococcus pneumoniae  polysaccharides from one or more  Streptococcus pneumoniae  serotypes and a second dried composition comprising carrier protein;   (b) separately reconstituting the first dried composition and the second dried composition in an organic solvent and mixing to provide a first homogenous solution comprising the one or more activated polysaccharides and a second homogenous solution comprising the carrier protein;   (c) combining the first homogenous solution with the second homogenous solution by Tee-mixing to produce a mixture; and   (d) adding a reducing agent to the mixture to produce a conjugate solution comprising a carrier protein conjugated to one or more polysaccharides of the  Streptococcus pneumoniae  serotype.   
     
     
         2 . The method of  claim 1 , wherein the first and second dried compositions are prepared by a sublimative drying process selected from lyophilization and radiant energy vacuum (REV) dehydration. 
     
     
         3 . The method of  claim 2 , wherein the sublimative drying process comprises freezing the first and second aqueous solutions in the form of cakes or lyosphere beads. 
     
     
         4 . The method of  claim 2 , wherein the sublimative drying is bulk drying performed in a container selected from the group consisting of metal tray, plastic tray, plastic bag, and class I tubing vials. 
     
     
         5 . The method of  claim 1 , wherein the first and second dried compositions are prepared by a sublimative drying process comprising lyophilization or radiant energy vacuum (REV) dehydration comprising providing a first aqueous solution comprising activated  Streptococcus pneumoniae  polysaccharides from one or more  Streptococcus pneumoniae  serotypes and a second aqueous solution comprising a carrier protein and a buffer, wherein the first and second aqueous solutions comprise about 0.5% (w/v) or more sucrose, and subjecting the first and second aqueous solutions to the sublimative drying process to produce the first and second dried compositions. 
     
     
         6 . The method of  claim 5 , wherein the buffer is a Histidine, Succinate, MES, MOPS, HEPES, or Acetate buffer in a pH range of 5.0-7.0. 
     
     
         7 . The method of  claim 5 , wherein the buffer is a Phosphate or Citrate buffer in a pH range of 5.0-7.0. 
     
     
         8 . The method of  claim 1 , wherein the first and second dried compositions have a moisture content of less than 6%. 
     
     
         9 . The method of  claim 1 , wherein the organic solvent is dimethylsulfoxide (DMSO). 
     
     
         10 . The method of  claim 1 , wherein the reconstituting comprises eight minutes or less and the mixing comprises 120 minutes or less. 
     
     
         11 . The method of  claim 1 , wherein each conjugate solution comprises polysaccharide conjugated to the carrier protein at a ratio from about 0.6 to about 1.3 polysaccharide to carrier protein on a weight to weight basis. 
     
     
         12 . The method of  claim 1 , wherein the conjugate solution comprises a free polysaccharide amount that is less than about 15% of the total polysaccharide in the solution. 
     
     
         13 . The method of  claim 1 , wherein the one or more polysaccharides are obtained from a  Streptococcus pneumoniae  serotype selected from the group consisting of serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 6E, 6G, 6H, 7F, 7A, 7B, 7C, 8, 9A, 9L, 9N, 9V, 10F, 10A, 10B, 10C, 11F, 11A, 11B, 11C, 11D, 11E, 12F, 12A, 12B, 13, 14, 15F, 15A, 15B, 15C, 16F, 16A, 17F, 17A, 18F, 18A, 18B, 18C, 19F, 19A, 19B, 19C, 20A, 20B, 21, 22F, 22A, 23F, 23A, 23B, 24F, 24A, 24B, 25F, 25A, 27, 28F, 28A, 29, 31, 32F, 32A, 33F, 33A, 33B, 33C, 33D, 33E, 34, 35F, 35A, 35B, 35C, 36, 37, 38, 39, 40, 41F, 41A, 42, 43, 44, 45, 46, 47F, 47A, 48, CWPS1, CWPS2, and CWPS3. 
     
     
         14 . The method of  claim 1 , wherein the carrier protein is an inactivated bacterial toxoid selected from the group consisting of tetanus toxoid, diphtheria toxoid, pertussis toxoid, bacterial cytolysins or pneumolysin. 
     
     
         15 . The method of  claim 12 , wherein the inactivated bacterial toxoid is CRM 197 . 
     
     
         16 . The method of  claim 1 , wherein the conjugate solution is sterile filtered. 
     
     
         17 . The method  claim 1 , wherein the  Streptococcus pneumoniae  polysaccharide is activated by reacting with an oxidizing agent. 
     
     
         18 . A method for making a composition comprising two or more conjugates, each conjugate having  Streptococcus pneumoniae  polysaccharides from one or more serotypes covalently linked to a carrier protein, the method comprising:
 (a) providing
 (i) two or more first aqueous solutions, each first aqueous solution comprising activated polysaccharides of a specific  Streptococcus pneumoniae  serotype, wherein the polysaccharides have been reacted with an oxidizing agent to provide the activated polysaccharides and wherein the two or more first aqueous solutions are not the same; or, 
 (ii) two or more first aqueous solutions, each first aqueous solution comprising activated polysaccharides of two or more  Streptococcus pneumoniae  serotypes wherein the polysaccharides have been reacted with an oxidizing agent to provide the activated polysaccharide and wherein the two or more first aqueous solutions are not the same; 
   (b) providing two or more second aqueous solutions, each comprising a carrier protein and a buffer, wherein the quantity of two or more second aqueous solutions corresponds to at least the quantity of two or more first aqueous solutions;   (c) separately drying the two or more first aqueous solutions and the two or more second aqueous solutions in a sublimative drying process to produce two or more first dried compositions, each comprising dried polysaccharides and two or more second dried compositions, each comprising dried carrier protein;   (d) reconstituting separately each of the two or more first dried compositions and each of the two or more second dried compositions in an organic solvent and mixing to provide two or more first homogenous solutions, each independently comprising either (i) polysaccharides of a specific  Streptococcus pneumoniae  serotype or (ii) polysaccharides of two or more specific  Streptococcus pneumoniae  serotypes, and two or more second homogenous solutions, each comprising the carrier protein;   (e) separately combining each first homogenous solution with a second homogenous solution by Tee-mixing to produce a plurality of mixtures;   (f) adding a reducing agent to the plurality of mixtures to produce a plurality conjugate solutions; and   (g) combining two or more of the plurality of conjugate solutions to produce the composition comprising two or more conjugates, each conjugate having  Streptococcus pneumoniae  polysaccharides from one or more serotypes covalently linked to a carrier protein.   
     
     
         19 . The method of  claim 18 , wherein at least one conjugate solution is prepared by separately Tee-mixing a first homogenous solution with a second homogenous solution to produce the plurality of conjugate solutions; or,
 wherein at least one conjugate solution is prepared by separately Tee-mixing a first aqueous solution with a second homogenous solution to produce the plurality of conjugate solutions; or,   wherein each conjugate solution is prepared by separately Tee-mixing a first homogenous solution with a second homogenous solution to produce the plurality of conjugate solutions; or,   wherein each conjugate solution is prepared by separately Tee-mixing a first aqueous solution with a second homogenous solution to produce the plurality of conjugate solutions.   
     
     
         20 . The method of  claim 18 , wherein the sublimative drying process comprises lyophilization or radiant energy vacuum (REV) dehydration. 
     
     
         21 . The method of  claim 18 , wherein the sublimative drying process comprises freezing the first and second aqueous solutions in the form of cakes or lyosphere beads. 
     
     
         22 . The method of  claim 18 , wherein the sublimative drying is bulk drying performed in a container selected from the group consisting of metal tray, plastic tray, plastic bag, and class I tubing vials. 
     
     
         23 . The method of  claim 18 , wherein the first and second aqueous solutions comprise about 0.5% (w/v) or more sucrose. 
     
     
         24 . The method of  claim 18 , wherein the organic solvent is dimethylsulfoxide (DMSO). 
     
     
         25 . The method of  claim 18 , wherein the reconstituting comprises eight minutes or less and the mixing comprises 120 minutes or less. 
     
     
         26 . The method of  claim 18 , wherein each conjugate solution comprises polysaccharide conjugated to the carrier protein at a ratio from about 0.6 to about 1.3 polysaccharide to carrier protein on a weight to weight basis. 
     
     
         27 . The method of  claim 18 , wherein each conjugate solution comprises a free polysaccharide amount that is less than about 15% of the total polysaccharide in the solution. 
     
     
         28 . The method of  claim 18 , wherein the buffer is a Histidine, Succinate, MOPS, HEPES, or Acetate buffer in a pH range of 5.0-7.0. 
     
     
         29 . The method of  claim 18 , wherein the buffer is a Phosphate or Citrate buffer in a pH range of 5.0-7.0. 
     
     
         30 . The method of  claim 18 , wherein the one or more polysaccharides are obtained from one or more  Streptococcus pneumoniae  serotypes selected from the group consisting of at least one of serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 6E, 6G, 6H, 7F, 7A, 7B, 7C, 8, 9A, 9L, 9N, 9V, 10F, 10A, 10B, 10C, 11F, 11A, 11B, 11C, 11D, 11E, 12F, 12A, 12B, 13, 14, 15F, 15A, 15B, 15C, 16F, 16A, 17F, 17A, 18F, 18A, 18B, 18C, 19F, 19A, 19B, 19C, 20A, 20B, 21, 22F, 22A, 23F, 23A, 23B, 24F, 24A, 24B, 25F, 25A, 27, 28F, 28A, 29, 31, 32F, 32A, 33F, 33A, 33B, 33C, 33D, 33E, 34, 35F, 35A, 35B, 35C, 36, 37, 38, 39, 40, 41F, 41A, 42, 43, 44, 45, 46, 47F, 47A, 48, CWPS1, CWPS2, and CWPS3. 
     
     
         31 . The method of  claim 18 , wherein the carrier protein is an inactivated bacterial toxoid selected from the group consisting of tetanus toxoid, diphtheria toxoid, pertussis toxoid, bacterial cytolysins or pneumolysin. 
     
     
         32 . The method of  claim 31 , wherein the inactivated bacterial toxoid is CRM 197 . 
     
     
         33 . The method of  claim 18 , wherein the conjugate solutions are sterile filtered. 
     
     
         34 . A method for making a multivalent pneumococcal conjugate vaccine containing  Streptococcus pneumoniae  serotype polysaccharides 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F conjugated to carrier proteins, the method comprising:
 (a) providing 23 dried carrier protein compositions, and 23 dried activated polysaccharide compositions, each dried activated polysaccharide composition comprising dried activated polysaccharides from a  Streptococcus pneumoniae  serotype selected from 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F, wherein none of the 23 dried activated polysaccharide compositions comprises activated polysaccharides from the same  Streptococcus pneumoniae  serotype;   (b) separately reconstituting the 23 dried carrier protein compositions and the 23 dried activated polysaccharide compositions in an organic solvent and mixing to provide 23 homogenous carrier protein solutions, each comprising the carrier protein, and 23 homogenous activated polysaccharide solutions, each comprising activated polysaccharides of a specific  Streptococcus pneumoniae  serotype, wherein none of the 23 homogenous solutions comprise activated polysaccharides from the same  Streptococcus pneumoniae  serotype;   (c) combining each of the 23 homogenous carrier protein solutions with one of the 23 activated polysaccharide homogenous solutions by Tee-mixing to produce 23 mixtures, each comprising carrier protein and activated polysaccharides of a specific  Streptococcus pneumoniae  serotype, wherein none of the 23 mixtures comprise activated polysaccharides from the same  Streptococcus pneumoniae  serotype;   (d) adding a reducing agent to each of the 23 mixtures to produce 23 conjugate solutions, each comprising a carrier protein conjugated to polysaccharides of a specific  Streptococcus pneumoniae  serotype, wherein none of the 23 conjugate solutions comprise activated polysaccharides from the same  Streptococcus pneumoniae  serotype; and   (e) combining the 23 conjugate solutions to provide the multivalent pneumococcal conjugate vaccine containing  Streptococcus pneumoniae  serotype polysaccharides 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F conjugated to carrier proteins.   
     
     
         35 . A method for making a multivalent pneumococcal conjugate vaccine containing  Streptococcus pneumoniae  serotype polysaccharides 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, and 33F conjugated to carrier proteins, the method comprising:
 (a) providing 15 dried carrier protein compositions and 15 dried activated polysaccharide compositions, each comprising dried activated polysaccharides from a  Streptococcus pneumoniae  serotype selected from 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, and 33F, wherein none of the 15 dried activated polysaccharide compositions comprises activated polysaccharides from the same  Streptococcus pneumoniae  serotype;   (b) separately reconstituting the 15 dried carrier protein compositions and the 15 dried activated polysaccharide compositions in an organic solvent and mixing to provide 15 homogenous carrier protein solutions, each comprising the carrier protein, and 15 homogenous activated polysaccharide solutions, each comprising activated polysaccharides of a specific  Streptococcus pneumoniae  serotype, wherein none of the 15 homogenous solutions comprise activated polysaccharides from the same  Streptococcus pneumoniae  serotype;   (c) combining each of the 15 homogenous carrier protein solutions with one of the 15 activated polysaccharide homogenous solutions by Tee-mixing to produce 15 mixtures, each comprising carrier protein and activated polysaccharides of a specific  Streptococcus pneumoniae  serotype, wherein none of the 15 mixtures comprise activated polysaccharides from the same  Streptococcus pneumoniae  serotype;   (d) adding a reducing agent to each of the 15 mixtures to produce 15 conjugate solutions, each comprising a carrier protein conjugated to polysaccharides of a specific  Streptococcus pneumoniae  serotype, wherein none of the 15 conjugate solutions comprise activated polysaccharides from the same  Streptococcus pneumoniae  serotype; and   (e) combining the 15 conjugate solutions to provide the multivalent pneumococcal conjugate vaccine containing  Streptococcus pneumoniae  serotype polysaccharides 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, and 33F conjugated to carrier proteins.   
     
     
         36 . A method for making a multivalent pneumococcal conjugate vaccine containing  Streptococcus pneumoniae  serotype polysaccharides 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F conjugated to carrier proteins, the method comprising:
 (a) providing 13 dried carrier protein compositions, and 13 dried activated polysaccharide compositions, each dried activated polysaccharide composition comprising dried activated polysaccharides from a  Streptococcus pneumoniae  serotype selected from 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F, wherein none of the 13 dried activated polysaccharide compositions comprises activated polysaccharides from the same  Streptococcus pneumoniae  serotype;   (b) separately reconstituting the 13 dried carrier protein compositions and the 13 dried activated polysaccharide compositions in an organic solvent and mixing to provide 13 homogenous carrier protein solutions, each comprising the carrier protein, and 13 homogenous activated polysaccharide solutions, each comprising activated polysaccharides of a specific  Streptococcus pneumoniae  serotype, wherein none of the 13 homogenous solutions comprise activated polysaccharides from the same  Streptococcus pneumoniae  serotype;   (c) combining each of the 13 homogenous carrier protein solutions with one of the 13 activated polysaccharide homogenous solutions by Tee-mixing to produce 13 mixtures, each comprising carrier protein and activated polysaccharides of a specific  Streptococcus pneumoniae  serotype, wherein none of the 13 mixtures comprise activated polysaccharides from the same  Streptococcus pneumoniae  serotype;   (d) adding a reducing agent to each of the 13 mixtures to produce 13 conjugate solutions, each comprising a carrier protein conjugated to polysaccharides of a specific  Streptococcus pneumoniae  serotype, wherein none of the 13 conjugate solutions comprise activated polysaccharides from the same  Streptococcus pneumoniae  serotype; and   (e) combining the 13 conjugate solutions to provide the multivalent pneumococcal conjugate vaccine containing  Streptococcus pneumoniae  serotype polysaccharides 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F conjugated to carrier proteins.   
     
     
         37 . A method for making a multivalent pneumococcal conjugate vaccine containing  Streptococcus pneumoniae  serotype polysaccharides 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, and 23F conjugated to carrier proteins, the method comprising:
 (a) providing 10 dried carrier protein compositions, and 10 dried activated polysaccharide compositions, each dried activated polysaccharide composition comprising dried activated polysaccharides from a  Streptococcus pneumoniae  serotype selected from 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, and 23F, wherein none of the 10 dried activated polysaccharide compositions comprises activated polysaccharides from the same  Streptococcus pneumoniae  serotype;   (b) separately reconstituting the 10 dried carrier protein compositions and the 10 dried activated polysaccharide compositions in an organic solvent and mixing to provide 10 homogenous carrier protein solutions, each comprising the carrier protein, and 10 homogenous activated polysaccharide solutions, each comprising activated polysaccharides of a specific  Streptococcus pneumoniae  serotype, wherein none of the 10 homogenous solutions comprise activated polysaccharides from the same  Streptococcus pneumoniae  serotype;   (c) combining each of the 10 homogenous carrier protein solutions with one of the 10 activated polysaccharide homogenous solutions by Tee-mixing to produce 10 mixtures, each comprising carrier protein and activated polysaccharides of a specific  Streptococcus pneumoniae  serotype, wherein none of the 10 mixtures comprise activated polysaccharides from the same  Streptococcus pneumoniae  serotype;   (d) adding a reducing agent to each of the 10 mixtures to produce 10 conjugate solutions, each comprising a carrier protein conjugated to polysaccharides of a specific  Streptococcus pneumoniae  serotype, wherein none of the 10 conjugate solutions comprise activated polysaccharides from the same  Streptococcus pneumoniae  serotype; and   (e) combining the 10 conjugate solutions to provide the multivalent pneumococcal conjugate vaccine containing  Streptococcus pneumoniae  serotype polysaccharides 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, and 23F conjugated to carrier proteins.