Method for producing streptococcus pneumoniae capsular polysaccharide carrier protein conjugates

Abstract

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

Description

[0001] This application is a divisional application of the patent application with application number 201980029273.X, application date April 25, 2019, and name “Method for producing pneumococcal capsular polysaccharide carrier protein conjugate”. Background Art (1) Field of the Invention

[0002] The present invention relates to a method for producing a pneumococcal capsular polysaccharide-protein conjugate, wherein one or more activated pneumococcal polysaccharides of a specific pneumococcal serotype and a carrier protein are separately lyophilized, wherein the separately lyophilized polysaccharides and carrier proteins are separately reconstituted in an organic solvent, and then the reconstituted polysaccharides and carrier proteins are combined and conjugated together by Tee mixing to produce a polysaccharide-carrier-protein conjugate. Multiple conjugates, each containing a polysaccharide of a specific serotype, can be used to produce a multivalent pneumococcal immunogenic composition containing a combination of conjugates for use in a vaccine.

[0003] (2) Description of related fields

[0004] Streptococcus pneumoniae is a capsular bacterium and an important cause of serious diseases worldwide. In 1997, the Centers for Disease Control and Prevention (CDC) estimated that the United States had 3,000 cases of pneumococcal meningitis, 50,000 cases of pneumococcal bacteremia, 7,000,000 cases of pneumococcal otitis media, and 500,000 cases of pneumococcal pneumonia each year. Referring to Centers for Disease Control and Prevention, MMWR Morb Mortal Wkly Rep 1997, 46 (RR-8): 1-13. In addition, the complications of these diseases may be serious. Some studies report that the mortality rate of pneumococcal meningitis is as high as 8%, and the neurological sequelae are as high as 25%. Referring to Arditi et al., 1998, Pediatrics 102: 1087-97.

[0005] The multivalent pneumococcal polysaccharide vaccine, which has been licensed for many years, has proven invaluable in preventing pneumococcal disease in adults, especially the elderly and high-risk groups. However, infants and young children respond poorly to unconjugated pneumococcal polysaccharides. Bacterial polysaccharides are T-cell-independent immunogens that elicit weak or no responses in infants. Chemical conjugation of bacterial polysaccharide immunogens to carrier proteins can convert the infant's immune response to a T-cell-dependent one. Diphtheria toxoid (DTx, a chemically detoxified version of DT) and CRM 197 They are described as carrier proteins for bacterial polysaccharide immunogens due to the presence of T-cell stimulating epitopes in their amino acid sequences.

[0006] Thus, polysaccharide-protein conjugate vaccines comprising 15 polysaccharides of bacterial capsules conjugated to carrier proteins have been developed, and others are being developed. Examples of conjugate vaccines that have been developed include Haemophilus influenzae type B (Hib) conjugate vaccines (e.g., ) and against Streptococcus pneumoniae (e.g. and PREVNAR ) and Neisseria meningitidis (e.g., ) conjugate vaccine.

[0007] After the polysaccharide antigen is conjugated to the carrier protein, the reaction mixture can be purified to remove the free polysaccharide without protein conjugated thereto, the free carrier protein without polysaccharide antigen conjugated thereto, and the low molecular weight polysaccharide protein conjugate. Various methods for purifying free polysaccharides, free proteins, and low molecular weight conjugates are well known in the art, including, for example, hydrophobic chromatography, tangential ultrafiltration, diafiltration, etc. See, for example, International Patent Application Publication No. WO00 / 38711, U.S. Patent No. 6,146,902, and Lei et al., 2000, Dev.Biol.103:259-264. The method for reducing the amount of free polysaccharides also includes lyophilizing the carrier protein and polysaccharide together, as disclosed in U.S. Patent No. 7,709,001 and U.S. Patent Application Publication No. 20110201791, which also shows that lyophilizing the carrier protein and polysaccharide together is better than lyophilizing the carrier protein and polysaccharide discretely, particularly for capsular polysaccharide 19A. However, co-lyophilization does not offer the ability to optimize individual formulation and cycle parameters without inconvenient processing and the ability to produce carrier protein and polysaccharide serotype supplies at the desired solution specifications.

[0008] Therefore, there is a continuing need for improved methods of producing stable polysaccharide-protein conjugates that are free of impurities such as free polysaccharide and low molecular weight conjugates. Summary of the Invention

[0009] The present invention provides a method for producing a multivalent pneumococcal polysaccharide-protein conjugate for use in a pneumococcal vaccine, wherein discrete lyophilized pneumococcal polysaccharide and carrier protein are prepared using various sublimation methods and then used in a conjugation process to produce a multivalent pneumococcal polysaccharide-polysaccharide-protein conjugate for use in a pneumococcal vaccine. The discrete drying method offers various advantages over co-lyophilization of the polysaccharide and carrier protein, including but not limited to the ability to optimize individual formulation and cycle parameters, ease of handling, and the ability to produce individual polysaccharide and carrier protein formulations at desired solution specifications.

[0010] The present invention provides (A) a method for preparing a composition comprising a polysaccharide from one serotype of Streptococcus pneumoniae covalently linked to a carrier protein, the method comprising:

[0011] (a) providing a first dried composition comprising an activated Streptococcus pneumoniae polysaccharide from a Streptococcus pneumoniae serotype and a second dried composition comprising a carrier protein;

[0012] (b) reconstituting the first dried composition and the second dried composition in an organic solvent, respectively, and mixing them to provide a first homogenous solution comprising the activated polysaccharide of the S. pneumoniae serotype and a second homogenous solution comprising the carrier protein;

[0013] (c) combining the first homogeneous solution with the second homogeneous solution by Tee mixing to produce a mixture; and

[0014] (d) adding a reducing agent to the mixture to produce a conjugate solution comprising a carrier protein conjugated to the polysaccharide of one of the S. pneumoniae serotypes; (B) a method of preparing a composition comprising one or more S. pneumoniae polysaccharides covalently linked to a carrier protein, the method comprising:

[0015] (a) providing a first dried composition comprising activated Streptococcus pneumoniae polysaccharide from one or more Streptococcus pneumoniae serotypes and a second dried composition comprising a carrier protein;

[0016] (b) reconstituting the first dried composition and the second dried composition in an organic solvent, respectively, and mixing them to provide a first homogenous solution comprising one or more activated polysaccharides and a second homogenous solution comprising the carrier protein;

[0017] (c) combining the first homogeneous solution with the second homogeneous solution by Tee mixing to produce a mixture; and

[0018] (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 S. pneumoniae serotype; and (c) a method of preparing a composition comprising two or more S. pneumoniae polysaccharides covalently linked to a carrier protein, the method comprising:

[0019] (a) providing a first dried composition comprising activated Streptococcus pneumoniae polysaccharides from two or more Streptococcus pneumoniae serotypes and a second dried composition comprising a carrier protein;

[0020] (b) reconstituting the first dried composition and the second dried composition in an organic solvent, respectively, and mixing to provide a first homogenous solution comprising two or more activated polysaccharides and a second homogenous solution comprising the carrier protein;

[0021] (c) combining the first homogeneous solution with the second homogeneous solution by Tee mixing to produce a mixture; and

[0022] (d) adding a reducing agent to the mixture to produce a conjugate solution comprising the carrier protein conjugated to two or more polysaccharides of the S. pneumoniae serotype.

[0023] In a specific embodiment of the method, the first dried composition and the second dried composition are prepared by a sublimation drying method selected from freeze drying and radiant energy vacuum (REV) dehydration. In a further embodiment, the sublimation drying method comprises freezing the first aqueous solution and the second aqueous solution in the form of a cake or lyophilized pellets. In another embodiment, the sublimation drying is performed in batches in a container selected from the group consisting of: a metal tray, a plastic tray, a plastic bag, and a Type I vial.

[0024] In certain embodiments, the first dried composition and the second dried composition each have a final moisture content of about 6% or less. In certain embodiments, the first dried composition and the second dried composition each have a final moisture content of about 5% or less. In certain embodiments, the first dried composition and the second dried composition each have a final moisture content of about 4% or less. In certain embodiments, the first dried composition and the second dried composition each have a final moisture content of about 3% or less. In certain embodiments, the first dried composition and the second dried composition each have a final moisture content of about 2% or less.

[0025] In a specific embodiment, the first dried composition and the second dried composition are prepared by sublimation drying a first aqueous solution comprising activated pneumococcal polysaccharide from one, two or more pneumococcal serotypes and a second aqueous solution comprising a carrier protein and a buffer to produce the first dried composition and the second dried composition, wherein the first aqueous solution and the second aqueous solution comprise about 0.5% (w / v) or more sucrose, and wherein the sublimation drying is selected from freeze drying and radiant energy vacuum (REV) dehydration. In a specific embodiment, the first aqueous solution comprises about 4% to 6% (w / v) sucrose and the second aqueous solution comprises about 4% to 8% (w / v) sucrose.

[0026] In a specific embodiment, the first aqueous solution comprises the polysaccharide at a concentration of about 6 to 9 mg / mL and the second aqueous solution comprises the carrier protein at a concentration of about 6 to 12 mg / mL. In a specific embodiment, the first aqueous solution comprises the polysaccharide at a concentration of about 6 or 9 mg / mL and the second aqueous solution comprises the carrier protein at a concentration of about 6, 9, 10 or 12 mg / mL.

[0027] In a specific embodiment, the organic solvent is an aprotic solvent. In a specific embodiment, the aprotic solvent is dichloromethane, tetrahydrofuran, ethyl acetate, acetonitrile, dimethylformamide, dimethyl sulfoxide (DMSO), acetone or hexamethylphosphoric acid triamide. In a specific embodiment, the organic solvent is DMSO.

[0028] In a specific embodiment of the method, the reconstructing in step (b) is performed in eight minutes or less. In a specific embodiment, the reconstructing in step (b) is performed in six minutes or less. In a specific embodiment, the reconstructing in step (b) is performed in four minutes or less. In a specific embodiment, the reconstructing in step (b) is performed in two minutes or less. In a specific embodiment, the reconstructing in step (b) is performed in about two minutes. In a specific embodiment, the reconstructing in step (b) is performed in one minute or less.

[0029] In a specific embodiment, the mixing in step (b) is carried out in 120 minutes or less to prepare the first homogeneous solution and the second homogeneous solution. In a specific embodiment, the mixing in step (b) is carried out in 90 minutes or less. In a specific embodiment, the mixing in step (b) is carried out in 60 minutes or less. In a specific embodiment, the mixing in step (b) is carried out in 30 minutes or less. In a specific embodiment, the mixing in step (b) is carried out in 15 minutes or less. In a specific embodiment, the mixing in step (b) is carried out in 10 minutes or less. In a specific embodiment, the second homogeneous solution comprising the carrier protein is placed in a DMSO solution for about six hours or less before being combined with the first homogeneous solution comprising the polysaccharide by Tee mixing.

[0030] In a specific embodiment, each conjugate solution comprises polysaccharide conjugated to the carrier protein at a polysaccharide to carrier protein ratio of about 0.6 to about 1.3 on a weight-to-weight basis. In a specific embodiment, each conjugate solution comprises polysaccharide conjugated to the carrier protein at a polysaccharide to carrier protein ratio of about 0.9 to about 1.5 on a weight-to-weight basis. In a specific embodiment, each conjugate solution comprises polysaccharide conjugated to the carrier protein at a polysaccharide to carrier protein ratio of about 0.6 to about 1.5 on a weight-to-weight basis.

[0031] In a specific embodiment, the conjugate solution comprises a free polysaccharide concentration of less than about 15% of the total polysaccharides in the solution. In a specific embodiment, the conjugate solution comprises a free polysaccharide concentration of less than about 10% of the total polysaccharides in the solution.

[0032] In certain embodiments, the conjugate has a carrier protein lysine loss value greater than 5 (mol / mol).

[0033] In a specific embodiment, each conjugate solution comprises polysaccharide conjugated to the carrier protein at a polysaccharide to carrier protein ratio of about 0.6 to about 1.3 on a weight to weight basis, a free polysaccharide concentration of less than about 15% of the total polysaccharide in the solution, and the conjugate has a carrier protein lysine loss value greater than 5 (mol / mol).

[0034] In specific embodiments, the buffer is a histidine, succinate, MES, MOPS, HEPES, or acetate buffer in the pH range of 5.0-7.0.

[0035] In certain embodiments, the buffer is a phosphate or citrate buffer having a pH range of 5.0-7.0.

[0036] In a specific embodiment, the polysaccharide is obtained from a serotype of S. pneumoniae 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, 19V, F, 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, 33 B, 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.

[0037] In a specific embodiment, the S. pneumoniae polysaccharide is activated by reaction with an oxidizing agent.

[0038] In a specific embodiment, the carrier protein is an inactivated bacterial toxoid selected from the group consisting of tetanus toxoid, diphtheria toxoid, pertussis toxoid, bacterial cytolysin, or pneumolysin. In a specific embodiment, the inactivated bacterial toxoid is CRM 197 .

[0039] In certain embodiments, the conjugate solution is sterile filtered.

[0040] The present invention provides a method for preparing a composition comprising one or more Streptococcus pneumoniae polysaccharides covalently linked to a carrier protein, the method comprising

[0041] (a) providing (i) a first aqueous solution comprising activated Streptococcus pneumoniae polysaccharide from one or more Streptococcus pneumoniae serotypes, and (ii) a second aqueous solution comprising a carrier protein and a buffer;

[0042] (b) drying the first aqueous solution and the second aqueous solution separately in a sublimation drying process to produce a first dried composition comprising dried one or more activated polysaccharides and a second dried composition comprising dried carrier protein;

[0043] (c) reconstituting the first dried composition and the second dried composition, respectively, by adding the dried composition to 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;

[0044] (d) combining the first homogeneous solution with the second homogeneous solution by Tee mixing to produce a mixture; and

[0045] (e) adding a reducing agent to the mixture to produce a conjugate solution comprising the carrier protein conjugated to the one or more activated polysaccharides of the S. pneumoniae serotype.

[0046] In a specific embodiment of the method, the sublimation drying method is selected from freeze drying and radiant energy vacuum (REV) dehydration. In a further embodiment, the sublimation drying method comprises freezing the first aqueous solution and the second aqueous solution in the form of a cake or lyophilized pellets. In another embodiment, the sublimation drying is performed in batches in a container selected from the group consisting of: a metal tray, a plastic tray, a plastic bag, and a Type I vial.

[0047] In certain embodiments, the first dried composition and the second dried composition each have a final moisture content of about 6% or less. In certain embodiments, the first dried composition and the second dried composition each have a final moisture content of about 5% or less. In certain embodiments, the first dried composition and the second dried composition each have a final moisture content of about 4% or less. In certain embodiments, the first dried composition and the second dried composition each have a final moisture content of about 3% or less. In certain embodiments, the first dried composition and the second dried composition each have a final moisture content of about 2% or less.

[0048] In certain embodiments, the first aqueous solution and the second aqueous solution comprise about 0.5% (w / v) or more sucrose. In certain embodiments, the first aqueous solution comprises about 4% to 6% (w / v) sucrose and the second aqueous solution comprises about 4% to 8% (w / v) sucrose.

[0049] In a specific embodiment, the first aqueous solution comprises the polysaccharide at a concentration of about 6 to 9 mg / mL and the second aqueous solution comprises the carrier protein at a concentration of about 6 to 12 mg / mL. In a specific embodiment, the first aqueous solution comprises the polysaccharide at a concentration of about 6 or 9 mg / mL and the second aqueous solution comprises the carrier protein at a concentration of about 6, 9, 10 or 12 mg / mL.

[0050] In a specific embodiment, the organic solvent is an aprotic solvent. In a specific embodiment, the aprotic solvent is dichloromethane, tetrahydrofuran, ethyl acetate, acetonitrile, dimethylformamide, dimethyl sulfoxide (DMSO), acetone or hexamethylphosphoric acid triamide. In a specific embodiment, the organic solvent is DMSO.

[0051] In a specific embodiment of the method, the remodeling in step (c) is performed in eight minutes or less. In a specific embodiment, the remodeling in step (c) is performed in six minutes or less. In a specific embodiment, the remodeling in step (c) is performed in four minutes or less. In a specific embodiment, the remodeling in step (c) is performed in two minutes or less. In a specific embodiment, the remodeling comprises about two minutes. In a specific embodiment, the remodeling in step (c) is performed in one minute or less.

[0052] In a specific embodiment, the mixing in step (c) is carried out in 120 minutes or less to prepare the first homogeneous solution and the second homogeneous solution. In a specific embodiment, the mixing in step (c) is carried out in 90 minutes or less. In a specific embodiment, the mixing in step (c) is carried out in 60 minutes or less. In a specific embodiment, the mixing in step (c) is carried out in 30 minutes or less. In a specific embodiment, the mixing in step (c) is carried out in 15 minutes or less. In a specific embodiment, the mixing in step (c) is carried out in 10 minutes or less. In a specific embodiment, the second homogeneous solution comprising the carrier protein is placed in a DMSO solution for about six hours or less before being combined with the first homogeneous solution comprising the polysaccharide by Tee mixing.

[0053] In a specific embodiment, each conjugate solution comprises polysaccharide conjugated to the carrier protein at a polysaccharide to carrier protein ratio of about 0.6 to about 1.3 on a weight-to-weight basis. In a specific embodiment, each conjugate solution comprises polysaccharide conjugated to the carrier protein at a polysaccharide to carrier protein ratio of about 0.9 to about 1.5 on a weight-to-weight basis. In a specific embodiment, each conjugate solution comprises polysaccharide conjugated to the carrier protein at a polysaccharide to carrier protein ratio of about 0.6 to about 1.5 on a weight-to-weight basis.

[0054] In a specific embodiment, the conjugate solution comprises a free polysaccharide concentration of less than about 15% of the total polysaccharides in the solution. In a specific embodiment, the conjugate solution comprises a free polysaccharide concentration of less than about 10% of the total polysaccharides in the solution.

[0055] In certain embodiments, the conjugate has a carrier protein lysine loss value greater than 5 (mol / mol).

[0056] In a specific embodiment, each conjugate solution comprises polysaccharide conjugated to the carrier protein at a polysaccharide to carrier protein ratio of about 0.6 to about 1.3 on a weight to weight basis, a free polysaccharide concentration of less than about 15% of the total polysaccharide in the solution, and the conjugate has a carrier protein lysine loss value greater than 5 (mol / mol).

[0057] In specific embodiments, the buffer is a histidine, succinate, MES, MOPS, HEPES, or acetate buffer in the pH range of 5.0-7.0.

[0058] In certain embodiments, the buffer is a phosphate or citrate buffer having a pH range of 5.0-7.0.

[0059] In a specific embodiment, the one or more S. pneumoniae serotypes are obtained from a S. 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, 18D, 18E ...C, 18D, 18E, 18F, 18A, 18B, 18C, 18C, 18 43, 44, 45, 46, 47F, 47A, 48, CWPS1, CWPS2 and CWPS3.

[0060] In a specific embodiment, the S. pneumoniae polysaccharide is activated by reaction with an oxidizing agent.

[0061] In a specific embodiment, the carrier protein is an inactivated bacterial toxoid selected from the group consisting of tetanus toxoid, diphtheria toxoid, pertussis toxoid, bacterial cytolysin, or pneumolysin. In a specific embodiment, the inactivated bacterial toxoid is CRM 197 .

[0062] In certain embodiments, the conjugate solution is sterile filtered.

[0063] The present invention also provides a method for preparing a composition comprising two or more conjugates, each conjugate comprising a polysaccharide from one or more serotypes of Streptococcus pneumoniae covalently linked to a carrier protein, the method comprising:

[0064] (a) Provide

[0065] (i) two or more first aqueous solutions, each first aqueous solution comprising an activated polysaccharide of a specific S. pneumoniae serotype, wherein the polysaccharide has been reacted with an oxidizing agent to provide the activated polysaccharide, and wherein the two or more first aqueous solutions are different; or

[0066] (ii) two or more first aqueous solutions, each first aqueous solution comprising activated polysaccharides of two or more serotypes of S. pneumoniae, 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 different;

[0067] (b) providing two or more second aqueous solutions, each comprising a carrier protein and a buffer, wherein the amount of the two or more second aqueous solutions corresponds to the amount of at least the two or more first aqueous solutions;

[0068] (c) separately drying the two or more first aqueous solutions and the two or more second aqueous solutions in a sublimation drying process to produce two or more first dried compositions and two or more second dried compositions, each of the first dried compositions comprising a dried polysaccharide and each of the second dried compositions comprising a dried carrier protein;

[0069] (d) reconstituting each of the two or more first dried compositions and each of the two or more second dried compositions separately in an organic solvent and mixing to provide two or more first homogenous solutions and two or more second homogenous solutions, each of the first homogenous solutions independently comprising (i) a polysaccharide of a specific S. pneumoniae serotype or (ii) polysaccharides of two or more specific S. pneumoniae serotypes, and each of the second homogenous solutions comprising the carrier protein;

[0070] (e) combining each of the first homogeneous solutions with the second homogeneous solution separately by Tee mixing to produce a plurality of mixtures;

[0071] (f) adding a reducing agent to the plurality of mixtures to produce a plurality of conjugate solutions; and

[0072] (g) combining two or more of the plurality of conjugate solutions to produce a composition comprising two or more conjugates, each conjugate comprising a polysaccharide from one or more serotypes of Streptococcus pneumoniae covalently linked to a carrier protein.

[0073] In a specific embodiment of the method, at least one conjugate solution is prepared by Tee mixing a first homogeneous solution with a second homogeneous solution separately to produce a plurality of conjugate solutions; or, at least one conjugate solution is prepared by Tee mixing a first aqueous solution with a second homogeneous solution separately to produce a plurality of conjugate solutions; or, each conjugate solution is prepared by Tee mixing a first homogeneous solution with a second homogeneous solution separately to produce a plurality of conjugate solutions; or, each conjugate solution is prepared by Tee mixing a first aqueous solution with a second homogeneous solution separately to produce a plurality of conjugate solutions.

[0074] In a specific embodiment of the method, the sublimation drying method is selected from freeze drying and radiant energy vacuum (REV) dehydration. In a further embodiment, the sublimation drying method comprises freezing the first aqueous solution and the second aqueous solution in the form of a cake or lyophilized pellets. In another embodiment, the sublimation drying is performed in batches in a container selected from the group consisting of: a metal tray, a plastic tray, a plastic bag, and a Type I vial.

[0075] In certain embodiments, the first dried composition and the second dried composition each have a final moisture content of about 6% or less. In certain embodiments, the first dried composition and the second dried composition each have a final moisture content of about 5% or less. In certain embodiments, the first dried composition and the second dried composition each have a final moisture content of about 4% or less. In certain embodiments, the first dried composition and the second dried composition each have a final moisture content of about 3% or less. In certain embodiments, the first dried composition and the second dried composition each have a final moisture content of about 2% or less.

[0076] In certain embodiments, the first aqueous solution and the second aqueous solution comprise about 0.5% (w / v) or more sucrose. In certain embodiments, the first aqueous solution comprises about 4% to 6% (w / v) sucrose and the second aqueous solution comprises about 4% to 8% (w / v) sucrose.

[0077] In a specific embodiment, the first aqueous solution comprises the polysaccharide at a concentration of about 6 to 9 mg / mL and the second aqueous solution comprises the carrier protein at a concentration of about 6 to 12 mg / mL. In a specific embodiment, the first aqueous solution comprises the polysaccharide at a concentration of about 6 or 9 mg / mL and the second aqueous solution comprises the carrier protein at a concentration of about 6, 9, 10 or 12 mg / mL.

[0078] In a specific embodiment, the organic solvent is an aprotic solvent. In a specific embodiment, the aprotic solvent is dichloromethane, tetrahydrofuran, ethyl acetate, acetonitrile, dimethylformamide, dimethyl sulfoxide (DMSO), acetone or hexamethylphosphoric acid triamide. In a specific embodiment, the organic solvent is DMSO.

[0079] In a specific embodiment of the method, the remodeling in step (d) is performed in eight minutes or less. In a specific embodiment, the remodeling in step (d) is performed in six minutes or less. In a specific embodiment, the remodeling in step (d) is performed in four minutes or less. In a specific embodiment, the remodeling in step (d) is performed in two minutes or less. In a specific embodiment, the remodeling comprises about two minutes. In a specific embodiment, the remodeling in step (d) is performed in one minute or less.

[0080] In a specific embodiment, the mixing in step (d) is performed in 120 minutes or less to prepare the first homogeneous solution and the second homogeneous solution. In a specific embodiment, the mixing in step (d) is performed in 90 minutes or less. In a specific embodiment, the mixing in step (d) is performed in 60 minutes or less. In a specific embodiment, the mixing in step (d) is performed in 30 minutes or less. In a specific embodiment, the mixing in step (d) is performed in 15 minutes or less. In a specific embodiment, the mixing in step (d) is performed in 10 minutes or less. In a specific embodiment, the second homogeneous solution comprising the carrier protein is placed in a DMSO solution for about six hours or less before being combined with the first homogeneous solution comprising the polysaccharide by Tee mixing.

[0081] In a specific embodiment, each conjugate solution comprises polysaccharide conjugated to the carrier protein at a polysaccharide to carrier protein ratio of about 0.6 to about 1.3 on a weight-to-weight basis. In a specific embodiment, each conjugate solution comprises polysaccharide conjugated to the carrier protein at a polysaccharide to carrier protein ratio of about 0.9 to about 1.5 on a weight-to-weight basis. In a specific embodiment, each conjugate solution comprises polysaccharide conjugated to the carrier protein at a polysaccharide to carrier protein ratio of about 0.6 to about 1.5 on a weight-to-weight basis.

[0082] In a specific embodiment, the conjugate solution comprises a free polysaccharide concentration of less than about 15% of the total polysaccharides in the solution. In a specific embodiment, the conjugate solution comprises a free polysaccharide concentration of less than about 10% of the total polysaccharides in the solution.

[0083] In certain embodiments, the conjugate has a carrier protein lysine loss value greater than 5 (mol / mol).

[0084] In a specific embodiment, each conjugate solution comprises polysaccharide conjugated to the carrier protein at a polysaccharide to carrier protein ratio of about 0.6 to about 1.3 on a weight to weight basis, a free polysaccharide concentration of less than about 15% of the total polysaccharide in the solution, and the conjugate has a carrier protein lysine loss value greater than 5 (mol / mol).

[0085] In specific embodiments, the buffer is a histidine, succinate, MES, MOPS, HEPES, or acetate buffer in the pH range of 5.0-7.0.

[0086] In certain embodiments, the buffer is a phosphate or citrate buffer having a pH range of 5.0-7.0.

[0087] In a specific embodiment, the polysaccharide is obtained from a serotype of S. pneumoniae 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, 19V, F, 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, 33 B, 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.

[0088] In a specific embodiment, the S. pneumoniae polysaccharide is activated by reaction with an oxidizing agent.

[0089] In a specific embodiment, the carrier protein is an inactivated bacterial toxoid selected from the group consisting of tetanus toxoid, diphtheria toxoid, pertussis toxoid, bacterial cytolysin, or pneumolysin. In a specific embodiment, the inactivated bacterial toxoid is CRM 197 .

[0090] In certain embodiments, the conjugate solution is sterile filtered.

[0091] The present invention also includes a method for preparing a multivalent pneumococcal conjugate vaccine comprising polysaccharides of Streptococcus pneumoniae serotypes 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 a carrier protein, the method comprising:

[0092] (a) providing 23 dried carrier protein compositions and 23 dried activated polysaccharide compositions, each dried activated polysaccharide composition comprising a dried activated polysaccharide from a serotype of Streptococcus pneumoniae 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 an activated polysaccharide from the same serotype of Streptococcus pneumoniae;

[0093] (b) reconstituting the 23 dried carrier protein compositions and the 23 dried activated polysaccharide compositions with an organic solvent, respectively, and mixing the resulting mixture to provide 23 homogeneous carrier protein solutions and 23 homogeneous activated polysaccharide solutions, each homogeneous carrier protein solution comprising the carrier protein, and each homogeneous activated polysaccharide solution comprising an activated polysaccharide of a specific S. pneumoniae serotype, wherein none of the 23 homogeneous solutions comprises an activated polysaccharide from the same S. pneumoniae serotype;

[0094] (c) combining each of the 23 homogenous carrier protein solutions with one of the 23 homogenous activated polysaccharide solutions by Tee mixing to produce 23 mixtures, each mixture comprising a carrier protein and an activated polysaccharide of a specific S. pneumoniae serotype, wherein none of the 23 mixtures comprises an activated polysaccharide from the same S. pneumoniae serotype;

[0095] (d) adding a reducing agent to each of the 23 mixtures to produce 23 conjugate solutions, each conjugate solution comprising a carrier protein conjugated to a polysaccharide of a specific S. pneumoniae serotype, wherein none of the 23 conjugate solutions comprises an activated polysaccharide from the same S. pneumoniae serotype; and

[0096] (e) combining the 23 conjugate solutions to provide the multivalent immunogenic complex or vaccine against S. pneumoniae serotypes 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F.

[0097] The present invention also includes a method for preparing a multivalent immunogenic complex or vaccine against polysaccharides of Streptococcus pneumoniae serotypes 1, 4, 3, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F, the method comprising:

[0098] (a) providing 15 dried carrier protein compositions and 15 dried activated polysaccharide compositions, each dried activated polysaccharide composition comprising a dried activated polysaccharide from a serotype of Streptococcus pneumoniae 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 an activated polysaccharide from the same serotype of Streptococcus pneumoniae;

[0099] (b) reconstituting the 15 dried carrier protein compositions and the 15 dried activated polysaccharide compositions with an organic solvent, respectively, and mixing the resulting 15 homogeneous carrier protein solutions and 15 homogeneous activated polysaccharide solutions, each homogeneous carrier protein solution comprising the carrier protein, and each homogeneous activated polysaccharide solution comprising an activated polysaccharide of a specific Streptococcus pneumoniae serotype, wherein none of the 15 homogeneous solutions comprises an activated polysaccharide from the same Streptococcus pneumoniae serotype;

[0100] (c) combining each of the 15 homogenous carrier protein solutions with one of the 15 homogenous activated polysaccharide solutions by Tee mixing to produce 15 mixtures, each mixture comprising a carrier protein and an activated polysaccharide of a specific S. pneumoniae serotype, wherein none of the 15 mixtures comprises an activated polysaccharide from the same S. pneumoniae serotype;

[0101] (d) adding a reducing agent to each of the 15 mixtures to produce 15 conjugate solutions, each conjugate solution comprising a carrier protein conjugated to a polysaccharide of a specific S. pneumoniae serotype, wherein none of the 15 conjugate solutions comprises an activated polysaccharide from the same S. pneumoniae serotype; and

[0102] (e) combining the 15 conjugate solutions to provide the multivalent pneumococcal conjugate vaccine containing S. pneumoniae serotype polysaccharides 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F conjugated to a carrier protein.

[0103] The present invention also provides a method for preparing a multivalent pneumococcal conjugate vaccine, wherein the multivalent pneumococcal conjugate vaccine contains polysaccharides of Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F conjugated to a carrier protein, the method comprising:

[0104] (a) providing 13 dried carrier protein compositions and 13 dried activated polysaccharide compositions, each dried activated polysaccharide composition comprising a dried activated polysaccharide from a serotype of Streptococcus pneumoniae 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 an activated polysaccharide from the same serotype of Streptococcus pneumoniae;

[0105] (b) reconstituting the 13 dried carrier protein compositions and the 13 dried activated polysaccharide compositions with an organic solvent, respectively, and mixing the resulting mixture to provide 13 homogeneous carrier protein solutions and 13 homogeneous activated polysaccharide solutions, each homogeneous carrier protein solution comprising the carrier protein, and each homogeneous activated polysaccharide solution comprising an activated polysaccharide of a specific Streptococcus pneumoniae serotype, wherein none of the 13 homogeneous solutions comprises an activated polysaccharide from the same Streptococcus pneumoniae serotype;

[0106] (c) combining each of the 13 homogenous carrier protein solutions with one of the 13 homogenous activated polysaccharide solutions by Tee mixing to produce 13 mixtures, each mixture comprising a carrier protein and an activated polysaccharide of a specific S. pneumoniae serotype, wherein none of the 13 mixtures comprises an activated polysaccharide from the same S. pneumoniae serotype;

[0107] (d) adding a reducing agent to each of the 13 mixtures to produce 13 conjugate solutions, each conjugate solution comprising a carrier protein conjugated to a polysaccharide of a specific S. pneumoniae serotype, wherein none of the 13 conjugate solutions comprises an activated polysaccharide from the same S. pneumoniae serotype; and

[0108] (e) combining the 13 conjugate solutions to provide the multivalent pneumococcal conjugate vaccine containing S. pneumoniae serotype polysaccharides 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F conjugated to a carrier protein.

[0109] The present invention also provides a method for preparing a multivalent pneumococcal conjugate vaccine, wherein the multivalent pneumococcal conjugate vaccine contains Streptococcus pneumoniae serotype polysaccharides 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F conjugated to a carrier protein, the method comprising:

[0110] (a) providing 10 dried carrier protein compositions and 10 dried activated polysaccharide compositions, each dried activated polysaccharide composition comprising a dried activated polysaccharide from a serotype of Streptococcus pneumoniae selected from 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F, wherein none of the 10 dried activated polysaccharide compositions comprises an activated polysaccharide from the same serotype of Streptococcus pneumoniae;

[0111] (b) reconstituting the 10 dried carrier protein compositions and the 10 dried activated polysaccharide compositions with an organic solvent, respectively, and mixing the reconstitution compositions to provide 10 homogeneous carrier protein solutions and 10 homogeneous activated polysaccharide solutions, each homogeneous carrier protein solution comprising the carrier protein, and each homogeneous activated polysaccharide solution comprising an activated polysaccharide of a specific S. pneumoniae serotype, wherein none of the 10 homogeneous solutions comprises an activated polysaccharide from the same S. pneumoniae serotype;

[0112] (c) combining each of the 10 homogenous carrier protein solutions with one of the 10 homogenous activated polysaccharide solutions by Tee mixing to produce 10 mixtures, each mixture comprising a carrier protein and an activated polysaccharide of a specific S. pneumoniae serotype, wherein none of the 10 mixtures comprises an activated polysaccharide from the same S. pneumoniae serotype;

[0113] (d) adding a reducing agent to each of the 10 mixtures to produce 10 conjugate solutions, each conjugate solution comprising a carrier protein conjugated to a polysaccharide of a specific S. pneumoniae serotype, wherein none of the 10 conjugate solutions comprises an activated polysaccharide from the same S. pneumoniae serotype; and

[0114] (e) combining the 10 conjugate solutions to provide the multivalent pneumococcal conjugate vaccine containing S. pneumoniae serotype polysaccharides 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F conjugated to a carrier protein.

[0115] In a specific embodiment, each of said dried carrier protein compositions has a final moisture content of 6% or less and each of said dried polysaccharide compositions has a final moisture content of about 6% or less.

[0116] In a specific embodiment of the method, each of the dried carrier protein and dried polysaccharide composition is prepared by a sublimation drying method, which includes freeze drying or radiant energy vacuum (REV) dehydration. In another embodiment, the sublimation drying is performed in batches in a container selected from the group consisting of: a metal tray, a plastic tray, a plastic bag, and a Type I vial. In a further embodiment, the sublimation drying method includes freezing each aqueous solution comprising the carrier protein and each aqueous solution comprising the polysaccharide in the form of a cake or lyophilized pellets, respectively.

[0117] In a specific embodiment, a composition of dried carrier and dried polysaccharide is prepared by sublimation drying a plurality of separate aqueous polysaccharide solutions and a plurality of aqueous carrier protein solutions, each aqueous polysaccharide solution comprising an activated Streptococcus pneumoniae polysaccharide from a specific listed Streptococcus pneumoniae serotype as indicated above, and each carrier protein solution comprising a carrier protein and a buffer, to produce a dried polysaccharide and carrier protein composition, wherein the amount of aqueous carrier protein solution at least corresponds to the amount of aqueous polysaccharide solution, and wherein the aqueous polysaccharide and carrier protein solutions each comprise about 0.5% (w / v) or more sucrose, and wherein the sublimation drying is selected from freeze drying and radiant energy vacuum (REV) dehydration.

[0118] In a specific embodiment, the aqueous solutions of the polysaccharide and the carrier protein each contain about 0.5% (w / v) or more sucrose. In a specific embodiment, the aqueous polysaccharide solution each contains about 4% to 6% (w / v) sucrose and the aqueous carrier protein solution each contains about 4% to 8% (w / v) sucrose.

[0119] In a specific embodiment, the polysaccharide aqueous solution comprises the polysaccharide at a concentration of about 6 to 9 mg / mL, and the carrier protein aqueous solution comprises the carrier protein at a concentration of about 6 to 12 mg / mL, respectively. In a specific embodiment, the polysaccharide aqueous solution comprises the polysaccharide at a concentration of about 6 or 9 mg / mL, respectively, and the carrier protein aqueous solution comprises the carrier protein at a concentration of about 6, 9, 10, or 12 mg / mL, respectively.

[0120] In a specific embodiment, the organic solvent is an aprotic solvent. In a specific embodiment, the aprotic solvent is dichloromethane, tetrahydrofuran, ethyl acetate, acetonitrile, dimethylformamide, dimethyl sulfoxide (DMSO), acetone or hexamethylphosphoric acid triamide. In a specific embodiment, the organic solvent is DMSO.

[0121] In a specific embodiment of the method, the remodeling in step (b) is performed in eight minutes or less. In a specific embodiment, the remodeling in step (b) is performed in six minutes or less. In a specific embodiment, the remodeling in step (b) is performed in four minutes or less. In a specific embodiment, the remodeling in step (b) is performed in two minutes or less. In a specific embodiment, the remodeling comprises about two minutes. In a specific embodiment, the remodeling in step (b) is performed in one minute or less.

[0122] In a specific embodiment, the mixing in step (b) is carried out in 120 minutes or less to prepare the first homogeneous solution and the second homogeneous solution. In a specific embodiment, the mixing in step (b) is carried out in 90 minutes or less. In a specific embodiment, the mixing in step (b) is carried out in 60 minutes or less. In a specific embodiment, the mixing in step (b) is carried out in 30 minutes or less. In a specific embodiment, the mixing in step (b) is carried out in 15 minutes or less. In a specific embodiment, the mixing in step (b) is carried out in 10 minutes or less. In a specific embodiment, the second homogeneous solution comprising the carrier protein is placed in a DMSO solution for about six hours or less before being combined with the first homogeneous solution comprising the polysaccharide by Tee mixing.

[0123] In a specific embodiment, each conjugate solution comprises polysaccharide conjugated to the carrier protein at a polysaccharide to carrier protein ratio of about 0.6 to about 1.3 on a weight-to-weight basis. In a specific embodiment, each conjugate solution comprises polysaccharide conjugated to the carrier protein at a polysaccharide to carrier protein ratio of about 0.9 to about 1.5 on a weight-to-weight basis. In a specific embodiment, each conjugate solution comprises polysaccharide conjugated to the carrier protein at a polysaccharide to carrier protein ratio of about 0.6 to about 1.5 on a weight-to-weight basis.

[0124] In a specific embodiment, the conjugate solution comprises a free polysaccharide concentration of less than about 15% of the total polysaccharides in the solution. In a specific embodiment, the conjugate solution comprises a free polysaccharide concentration of less than about 10% of the total polysaccharides in the solution.

[0125] In certain embodiments, the conjugate has a carrier protein lysine loss value greater than 5 (mol / mol).

[0126] In a specific embodiment, each conjugate solution comprises polysaccharide conjugated to the carrier protein at a polysaccharide to carrier protein ratio of about 0.6 to about 1.3 on a weight to weight basis, a free polysaccharide concentration of less than about 15% of the total polysaccharide in the solution, and the conjugate has a carrier protein lysine loss value greater than 5 (mol / mol).

[0127] In a specific embodiment, the S. pneumoniae polysaccharide is activated by reaction with an oxidizing agent.

[0128] In certain embodiments, the conjugate solution is sterile filtered. BRIEF DESCRIPTION OF THE DRAWINGS

[0129] Figure 1The results show that the combination of components and the effect of component concentration, polysaccharide (Ps) concentration, CRM 197 Effect of (Pr) concentration and ratio of Ps to Pr (Ps:Pr) (components were initially dissolved in anhydrous DMSO at 6 mg / mL and combined to a final Ps:Pr ratio of 1:1).

[0130] Figure 2 The results show that the combination of components and the effect of component concentration, polysaccharide (Ps) concentration, CRM 197 Effect of (Pr) concentration and ratio of Ps to Pr (Ps:Pr) (components were initially dissolved in anhydrous DMSO at 6 mg / mL and combined to a final Ps:Pr ratio of 1:1).

[0131] Figure 3 The results show that the binding of components and the effect of component concentration, polysaccharide (Ps) concentration, CRM 197 Effect of (Pr) concentration and the ratio of Ps to Pr (Ps:Pr) (Pr was initially dissolved in anhydrous DMSO at 3 mg / mL and used to dissolve lyophilized Ps).

[0132] Figure 4 The results show that the binding of components and the effect of component concentration, polysaccharide (Ps) concentration, CRM 197 Effect of (Pr) concentration and ratio of Ps to Pr (Ps:Pr) (components were initially dissolved in anhydrous DMSO at 6 mg / mL and the volumetric addition rates of the components were the same).

[0133] Figure 5 Shown are the rapid (two minutes) versus slow (eight minutes) reconstitution of CRMs in anhydrous DMSO. 197 FTIR spectrum of .

[0134] Figure 6 Shown are CRMs with intermediate reconstitution (four minutes) followed by increasing hold times (30 minutes; 60 minutes; 120 minutes; 180 minutes; 240 minutes; 300 minutes) in anhydrous DMSO. 197 FTIR spectrum of .

[0135] Figure 7 The CRM was rapidly reconstituted (two minutes) in anhydrous DMSO at various concentrations (10 mg / mL; 30 mg / mL; 50 mg / mL). 197 FTIR spectrum of .

[0136] Figure 8A and 8B Shown at 10 mg / mL ( Figure 8A ) and 30 mg / mL ( Figure 8B ) CRM that was rapidly reconstituted (two minutes) in anhydrous DMSO 197 Dynamic light scattering (DLS) spectra of the samples. Three measurements (three tracks) were collected for each sample.

[0137] Figure 9 Shown are CRMs that were rapidly reconstituted (two minutes) in DMSO containing varying levels of water (anhydrous DMSO; 99% DMSO / water; 97% DMSO / water; 95% DMSO / water). 197 FTIR spectrum of .

[0138] Figure 10 Shows rapid (two minutes) versus slow (eight minutes) reconstitution of extremely dry lyophilized CRM in anhydrous DMSO 197 FTIR spectra (fast (two minutes); slow (eight minutes)).

[0139] Figure 11 Shows CRM 197 : Polysaccharide serotype 6B (CRM 197 -6B) FTIR spectrum of the conjugate, where before polysaccharide 6B conjugation, CRM 197 The CRM was reconstituted either rapidly (two minutes) or slowly (eight minutes). Also shown was the CRM conjugated to polysaccharide 6B under aqueous conditions. 197 .

[0140] Figure 12 A general scheme for the preparation of carrier protein (Pr) polysaccharide (Ps) conjugates is shown.

[0141] Figure 13 The sizes of the conjugates produced from reactions using polysaccharides of increasing size are shown. The figure shows that slow addition of anhydrous DMSO can yield even larger conjugates from polysaccharides of the same size compared to rapid addition of anhydrous DMSO. DETAILED DESCRIPTION

[0142] I. Definition

[0143] As used herein, the term "polysaccharide" (Ps) is intended to include any antigenic carbohydrate element (or antigenic unit) commonly used in the field of immunization and bacterial vaccines, including but not limited to "saccharide", "oligosaccharide", "polysaccharide", "liposaccharide", "lipooligosaccharide (LOS)", "lipopolysaccharide (LPS)", "glycosylation", "glycoconjugate", etc. Depending on the context, Ps can be singular or plural.

[0144] As used herein, when used with the immunogenic compositions of the present invention, the term "comprising" refers to the inclusion of any other components (subject to the limitation of the term "consisting of" for antigenic mixtures), such as adjuvants and excipients. When used with a multivalent polysaccharide-protein conjugate mixture, the term "consisting of" refers to a mixture having those specific Streptococcus pneumoniae polysaccharide-protein conjugates and not having other Streptococcus pneumoniae polysaccharide-protein conjugates from different serotypes.

[0145] As defined herein, the terms "precipitation" or "precipitate," "particle formation," "turbidity," and "aggregation" are used interchangeably and refer to any physical or chemical interaction that leads to aggregation of polysaccharide-protein conjugates. Aggregation processes (e.g., protein aggregation) can be induced by a variety of physicochemical stresses, including heat, pressure, pH, agitation, shear force, freeze-thaw, dehydration, heavy metals, phenolic compounds, silicone oils, denaturants, and the like.

[0146] As used herein, the term "reconstituting" or "reconstitution" refers to the addition of a liquid to a dried substance to dissolve the dried substance to provide a solution of the substance dissolved therein. However, the solution provided by reconstitution may have a concentration gradient or layer of the substance dissolved therein. Therefore, after reconstitution, the solution is physically stirred to provide a homogeneous solution of the reconstituted substance.

[0147] As used herein, the term "mixing" is used to refer to the physical agitation of a solution by shaking, stirring, rocking, rotating, or the like.

[0148] As used herein, the term "homogeneous solution" refers to a solution in which all components are thoroughly mixed such that there are no concentration gradients or layers between the components in the solution.

[0149] As used herein, "lyophilized spheres" are discrete particles of a freeze-dried material, for example in the form of beads or spheres or other shapes. Lyophilized spheres may also be referred to as freeze-dried granular spheres or freeze-dried beads. In some embodiments, the diameter of the freeze-dried spheres is from about 2 to about 12 mm, preferably from 2 to 8 mm, such as from 2.5 to 6 mm or 2.5 to 5 mm. In some embodiments, the volume of the freeze-dried spheres is from about 20 to 550 μL, preferably from 20 to 100 μL, such as from 20 to 50 μL. In embodiments in which the freeze-dried spheres are not substantially spherical, the size of the freeze-dried spheres may be described with respect to their aspect ratio, which is the ratio of the major diameter to the minor diameter. The aspect ratio of the freeze-dried spheres may be from 0.5 to 2.5, preferably from 0.75 to 2, such as from 1 to 1.5.

[0150] As used herein, an "immunogenic composition" can be a multivalent composition containing one or more antigens conjugated to one or more carrier proteins. In certain embodiments of the invention, the antigen is a saccharide from an encapsulated bacterium. In such compositions, the saccharide is composed of long chains of sugar molecules similar to those found on the surface of certain types of bacteria. Encapsulated bacteria include, but are not limited to, Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae type b. The antigens can be from the same organism or from different organisms. In a preferred embodiment of the invention, the antigen is a Streptococcus pneumoniae capsular polysaccharide.

[0151] As used herein, the term "radiant energy vacuum (REV) dehydration" is also known as microwave vacuum drying (MVD).

[0152] II. Process

[0153] The present invention provides a method or process for producing a multivalent pneumococcal polysaccharide-protein conjugate useful as an anti-pneumococcal vaccine, wherein pneumococcal polysaccharide and a carrier protein are discretely (or separately) lyophilized using various sublimation methods, and the discrete lyophilized polysaccharide and carrier protein are then mixed under conditions that result in the formation of a conjugate composition having a low level of free polysaccharide (e.g., less than 15% free polysaccharide).

[0154] The present invention is an improvement over prior art methods for producing multivalent pneumococcal polysaccharide-protein conjugates with lower levels of free polysaccharide. Prior art methods such as those disclosed in U.S. Patent No. 7,709,001 and U.S. Patent Application Publication No. 20110201791 use co-lyophilization of a carrier protein and polysaccharide because it has been demonstrated that co-lyophilizing the polysaccharide and carrier protein together results in a conjugate composition with less free polysaccharide (less than 18%), whereas, as shown in the results shown in Table 1 of U.S. Patent Application Publication No. 20110201791, a conjugate composition formulated by combining discrete lyophilized polysaccharides and carrier proteins results in a composition with approximately 31% free polysaccharide.

[0155]

[0156] However, the inventors of the present invention have discovered that under certain conditions, discrete (or separate) lyophilized carrier proteins and activated polysaccharides can be conjugated to produce compositions comprising high molecular weight conjugates, but which, unlike those described in U.S. Patent Application Publication No. 20110201791, have lower levels of free polysaccharides. These specific conditions include providing a solution of the carrier protein in an organic solvent and a solution comprising one or more polysaccharides in the organic solvent, wherein in a specific embodiment, the organic solvent is dimethyl sulfoxide (DMSO), and combining or combining the two solutions simultaneously by Tee mixing. Figure 4As shown in , by simultaneously mixing two solutions together via Tee mixing, the concentration gradient that typically occurs when two solutions are combined by adding one solution to another or dissolving a dry material into a solution can be significantly reduced or eliminated (see Figure 1 、 2 or 3), and as exemplified by serotype 19F polysaccharide in Example 10, which can reduce or eliminate gel formation for polysaccharides obtained from various serotypes. The present invention has various advantages over co-lyophilization of polysaccharide and carrier protein in a single composition, including but not limited to the ability to optimize individual formulations and cycle parameters, ease of handling, and the ability to produce individual polysaccharide and carrier protein formulations at desired solution specifications.

[0157] The inventors also found that using CRM 197 As a model, the length of time that an organic solvent (such as DMSO) is added to a dried carrier protein during reconstitution of the dried carrier protein and the length of time the reconstituted carrier protein is subsequently stored prior to conjugation affect the secondary structure of the carrier protein and therefore the yield of conjugates having the desired molecular weight and ratio of carrier protein to polysaccharide conjugated thereto. Figure 5-11 As shown, the time for reconstitution of a dried carrier protein in an organic solvent (such as anhydrous DMSO) under rapid addition conditions (e.g., adding the organic solvent to the dried carrier protein in less than eight minutes or in about two minutes or less) provides a reconstituted carrier protein solution in which the carrier protein is fully expanded and has no detectable β-sheet formation, as determined by Fourier transform infrared spectroscopy (FTIR) or dynamic light scattering (DLS). It has further been found that the presence of any detectable water in the organic solvent and / or a carrier protein having a final concentration of carrier protein greater than 12 mg / mL results in β-sheet-mediated aggregation. Therefore, in a preferred embodiment of the present invention, the organic solvent comprises 100% of the organic solvent and does not contain detectable water (e.g., an anhydrous organic solvent); the organic solvent is added to the dried carrier protein over a period of less than eight minutes; preferably less than five minutes, and more preferably two minutes or less; and the carrier protein is maintained at a concentration of 10 mg / mL or less or 12 mg / mL or less, and in a specific embodiment, at about 6 mg / mL.

[0158] In a specific embodiment, to ensure complete dissolution of the dried carrier protein and polysaccharide, the first homogenous solution and the second homogenous solution can be mixed for 120 minutes or less. In a specific embodiment, the mixing can be for 90 minutes or less. In a specific embodiment, the mixing can be for 60 minutes or less. In a specific embodiment, the mixing can be for 30 minutes or less. In a specific embodiment, the mixing can be for 10 minutes or less. In a specific embodiment, the second homogenous solution containing the carrier protein is maintained for about six hours or less before being combined with the first homogenous solution containing the polysaccharide by Tee mixing. The inventors have found that maintaining the carrier protein in an anhydrous organic solvent for more than six hours may result in the formation of detectable beta-sheet-mediated carrier protein aggregation. Therefore, in a specific embodiment, the carrier protein is maintained in DMSO for six hours or less.

[0159] Figure 12 Shown is a general flow chart for preparing carrier protein polysaccharide conjugates, wherein a homogenous solution of carrier protein is prepared according to the teachings herein, and a homogenous solution of carrier protein and polysaccharide is prepared by Tee mixing according to the teachings herein.

[0160] Typically, purified pneumococcal capsular polysaccharide (Ps) powders were dissolved in water separately and all serotypes except serotype 19A were filtered through 0.45 micron. All serotypes except serotype 19A were homogenized to reduce the molecular weight of Ps. Serotype 18C was size-reduced by acid hydrolysis at 90°C or higher. Serotype 19A was not size-reduced due to its relatively low initial size. The homogenization pressure and number of passes through the homogenizer were controlled to achieve serotype-specific molecular weights for serotype-specific targets. The polysaccharides were separately filtered through 0.22-micron and then concentrated in ultrafiltration step 1 using a 10 kDa ultrafiltration membrane (type 5) with an open channel and diafiltered with water to produce diafiltrate 1.

[0161] The leachate 1 can then be adjusted to a serotype-specific temperature (between 4–22°C) and pH (4-5) using a buffer (e.g., sodium acetate) to minimize polysaccharide size reduction during the activation step. Polysaccharide activation is performed by periodate oxidation. For serotype 4, the solution is incubated at approximately 50°C and pH 4 prior to activation to partially deketalize the polysaccharide. Polysaccharide activation is initiated by the addition of a sodium metaperiodate solution. The amount of sodium metaperiodate added is serotype-specific, ranging from approximately 0.1 to 0.5 moles of sodium metaperiodate per mole of polysaccharide repeating unit. The serotype-specific charge of sodium metaperiodate is selected to achieve a target level of polysaccharide activation (moles of aldehyde per mole of polysaccharide repeating unit).

[0162] In ultrafiltration step 2, the activated polysaccharide can be diafiltered for all serotypes and then concentrated by tangential flow ultrafiltration using a 10 kDa ultrafiltration membrane with an open channel to produce diafiltrate 2. At the end of the diafiltration, diafiltrate 2 can be concentrated to a target of 15 g Ps / L. Preferably, ultrafiltration for all serotypes is performed at 2-8°C.

[0163] The filtrate 2 is then diluted in water containing sucrose to a final concentration of about 4 to 12 mg / mL polysaccharide and 0.5% to 6% (w / v) sucrose, and subjected to a sublimation drying process to produce a dried polysaccharide, preferably with a final moisture content of 6% or less. In a specific embodiment, the final moisture content of the composition is about 5% or less. In a specific embodiment, the final moisture content of the composition is about 4% or less. In a specific embodiment, the final moisture content of the composition is about 3% or less. In a specific embodiment, the final moisture content of the composition is about 2% or less. The amount of sucrose added to the solution before lyophilization is serotype-specific and can range from 3.0 to 5.0% (w / v) before lyophilization. In a specific embodiment, two or more polysaccharides can be dried together to produce a dried polysaccharide mixture.

[0164] Use 5kDa tangential flow ultrafiltration membrane, with 2mM phosphate buffer (pH 7.0), the carrier protein of purification is carried out diafiltration, then filter through 0.22 micron filter.In the water containing sucrose, dilute the filtered solution so that final concentration is about 6 to 12mg / mL carrier protein and 4 to 10% (w / v) sucrose, and carry out sublimation drying in the process, to produce the dry carrier protein that final water content is 6% or lower.In a specific embodiment, the final water content of composition is about 5% or lower.In a specific embodiment, the final water content of composition is about 4% or lower.In a specific embodiment, the final water content of composition is about 3% or lower.In a specific embodiment, the final water content of composition is about 2% or lower.Sublimation drying method can comprise freeze-drying or radiation energy vacuum (REV) dehydration.

[0165] The dried polysaccharide or dried polysaccharide mixture and the dried carrier protein are reconstituted or redissolved in an organic solvent (e.g., dimethyl sulfoxide (DMSO)). In a specific embodiment, the carrier protein is reconstituted at a concentration of about 10 to 20 mg / mL or about 10 mg / mL or less. The inventors have found that the presence of 1% (v / v) or more water in DMSO will lead to β-sheet mediated aggregation (see Figure 9). Thus, the present invention provides embodiments wherein the organic solvent has a moisture content of less than 1% (v / v) or wherein the organic solvent contains no detectable moisture (e.g., an anhydrous organic solvent or a 100% (v / v) organic solvent). The dried carrier protein is reconstituted by adding the anhydrous organic solvent to the dried carrier protein over a period of less than eight minutes, preferably less than six minutes or less than four minutes, or more preferably two minutes or less, or one minute or less.

[0166] The inventors also found that using CRM 197 As a model, based on Fourier transform infrared spectroscopy (FTIR) and dynamic light scattering (DLS) analysis, the shorter the addition time for adding anhydrous organic solvent to a dried carrier protein, the less irreversible β-sheet-mediated aggregation is formed. Figure 5 As shown in , the addition time of anhydrous DMSO is two minutes so that detectable β sheet-mediated aggregation is not formed. Therefore, in a specific embodiment, the invention provides an organic solvent (for example, DMSO) containing less than 1% moisture or an organic solvent (for example, anhydrous or 100% (v / v) organic solvent) containing no detectable moisture in a reconstructed carrier protein, wherein the organic solvent added is added to the dry carrier protein within a period of two minutes or less than two minutes to provide a concentration of 20 mg / mL or less or about 12 mg / mL, or about 10 mg / mL or less of the reconstructed carrier protein. In a specific embodiment, after reconstruction, the mixture can be mixed for up to 30 minutes to provide a homogeneous solution of the carrier protein and a homogeneous solution of the polysaccharide. However, in a specific embodiment, mixing can be carried out in 120 minutes or less, 90 minutes or less, 60 minutes or less, 30 minutes or less, or 10 minutes or less. In a specific embodiment, 500 mM sodium phosphate buffer (pH 7.2) is added to the homogenous solution containing the carrier protein to a final concentration of 1.0 mM sodium phosphate.

[0167] The homogenous solution of the carrier protein and the homogenous solution of the polysaccharide or two or more polysaccharides are combined by Tee mixing to provide a conjugated solution comprising both the carrier protein and the polysaccharide or two or more polysaccharides. The rate of Tee mixing can be varied, but is typically set to a rate that allows complete mixing in 10 minutes or less. Tee mixing can be performed at any temperature, such as room temperature or 4°C or between 18°C ​​and 23°C. After reconstitution or redissolution in an anhydrous organic solvent, the polysaccharide and carrier protein homogenous solutions are combined in a manner that results in a serotype-specific final polysaccharide concentration and ratio of polysaccharide to carrier protein. Typically, the carrier protein and polysaccharide are mixed together in amounts that will provide a final conjugated polysaccharide:carrier protein ratio of from about 0.6 to 1.3 (w / w).

[0168] To perform the conjugation reaction, an aqueous solution of sodium cyanoborohydride was prepared and 1.0 meq of sodium cyanoborohydride (1.0 mole of sodium cyanoborohydride per mole of polysaccharide repeating unit) was added to the conjugation solution. The molar concentration of the sodium cyanoborohydride solution was based on a target amount of approximately 0.5% of the total water content of the solution during the conjugation process. The conjugation solution was reacted at a serotype-specific temperature for a serotype-specific duration to produce the carrier protein:polysaccharide conjugate intermediate.

[0169] Next, an aqueous solution of sodium borohydride was prepared and 2.0 meq of sodium borohydride (relative to the polysaccharide repeating units) was added to the conjugation solution. The molar concentration of the sodium borohydride solution was based on a target total water content of approximately 1.0% in the conjugation solution after addition of the sodium borohydride. The conjugation solution was allowed to react at room temperature for three hours (except for certain serotypes, such as serotype 7F, which were allowed to react for two hours) to produce the carrier protein:polysaccharide conjugate.

[0170] To quench the conjugation reaction, for conjugates containing polysaccharides from specific serotypes, the conjugated solution is diluted to 20% (v / v) or less anhydrous DMSO in a dilution step by slowly adding the conjugated solution to a solution containing 150 mM sodium chloride (150 mM sodium chloride containing 0.025% (w / v) polysorbate 20) to produce a quenched conjugate solution. In a specific embodiment, the temperature is maintained at 15°C or less during the dilution step. After about 1 hour, a 1.5 M potassium phosphate (pH 6.0) solution can be added to the solution to a final concentration of 25 mM potassium phosphate. Conjugation performance can be assessed by the consumption of total polysaccharide and carrier protein, the ratio of conjugate polysaccharide to carrier protein, and the molecular weight of the conjugate.

[0171] In ultrafiltration step 3, the quenched conjugate solution can be concentrated to about 2.5 g / L and diafiltered using a 30 kDa tangential flow ultrafiltration membrane at 2-8° C. using up to about 10 diafiltration volumes of 150 mM sodium chloride or 150 mM sodium chloride containing 25 mM potassium phosphate to produce diafiltrate 3. The polysaccharide concentration in diafiltrate 3 from ultrafiltration step 3 can be determined by high performance size exclusion chromatography (HPSEC) ultraviolet multi-angle light scattering refractive index (UV-MALS-RI). For conjugates containing polysaccharides from certain serotypes (e.g., serotype 19F), diafiltrate 3 can be filtered through a 0.22 micron filter to produce a filtrate, which can then be incubated at 22° C. for about 120 hours.

[0172] In ultrafiltration step 4, the diafiltrate 3 or filtrate from ultrafiltration step 3 can be concentrated to a polysaccharide concentration of about 2.5 g / L and diafiltered using a 300 kDa tangential flow ultrafiltration membrane at 2-8°C using 20 diafiltration volumes of 150 mM sodium chloride, pH 7.0, containing 10 mM L-histidine to produce diafiltrate 4. For conjugates comprising polysaccharides from certain serotypes (e.g., serotype 7F), the conjugate can be diafiltered using a 100 kDa tangential flow ultrafiltration membrane; for example, conjugates comprising serotypes 6A, 6B, and 18C polysaccharides can be concentrated to about 3.5 g / L and diafiltered using a 300 kDa tangential flow ultrafiltration membrane at 2-8°C using a buffer solution containing 150 mM sodium chloride and 0.03% (w / v) polysorbate 20, pH 7.0, to produce diafiltrate 4. The conjugate comprising serotype 7F, 19A, 19F and 23F polysaccharides can be concentrated to about 2.0 g / L and diafiltered using a 300 kDa regenerated cellulose tangential flow ultrafiltration membrane at 2-8°C using a buffer (pH 7.0) containing 150 mM sodium chloride and 0.015% (w / v) polysorbate 20 to produce diafiltrate 4. The polysaccharide concentration in diafiltrate 2 can be determined by HPSEC UV-MALS-RI. The buffer can be 10-50 mM acetate, phosphate, TRIS, HEPES or an amino acid buffer (such as histidine). In a specific embodiment, the buffer is 10 mM L-histidine.

[0173] The diafiltrate 4 from the ultrafiltration step 4 can be filtered through a 0.22 micron filter to produce a second filtrate. The polysaccharide concentration of the second filtrate can be determined by HPSEC UV-MALS-RI. If the polysaccharide concentration of the second filtrate is greater than 1.0 g / L, the second filtrate can be diluted to a polysaccharide concentration of 1.0 g / L using additional 150 mM sodium chloride (pH 7.0) containing 10 mM L-histidine. This provides a monovalent bulk conjugate intermediate (MBC) or a monovalent drug substance. The MBC can be dispensed into aliquots and frozen at -60°C to -80°C.

[0174] For conjugates containing serotype 6A, 6B, and 18C polysaccharides, the diafiltrate 4 from ultrafiltration step 4 can be filtered through a double membrane 0.5 / 0.2 micron filter to produce a second filtrate. The polysaccharide concentration of the second filtrate can be determined by HPSEC UV-MALS-RI. If the polysaccharide concentration of the second filtrate is greater than 1.0 g / L, the second filtrate can be diluted to a polysaccharide concentration of 1.0 g / L using additional 10 mM L-histidine, 0.03% (w / v) polysorbate 20 in 150 mM sodium chloride, pH 7.0. This provides the MBC or monovalent drug substance. The MBC can be dispensed into aliquots and frozen at -60°C to -80°C.

[0175] For conjugates containing 7F, 19A, 19F and 23F polysaccharides, the diafiltrate 4 can be filtered through a 0.22 micron filter to produce a second filtrate. The polysaccharide concentration of the second filtrate can be determined by HPSEC UV-MALS-RI. If the polysaccharide concentration of the second filtrate is greater than 1.0 g / L, the second filtrate can be diluted to a polysaccharide concentration of 1.0 g / L using additional 10 mM L-histidine, 0.015% (w / v) polysorbate 20, pH 7.0 in 150 mM sodium chloride. This provides an MBC or monovalent drug substance. The MBC can be distributed into aliquots and frozen at -60°C to -80°C.

[0176] III. Polysaccharides

[0177] Capsular polysaccharides from Streptococcus pneumoniae can be prepared by standard techniques known to those skilled in the art. For example, polysaccharides can be isolated from bacteria and can be sized to a certain extent by known methods (see, for example, European Patent Nos. EP497524 and EP497525); and in specific embodiments, by microfluidization using a homogenizer or chemical hydrolysis. In one embodiment, the Streptococcus pneumoniae strain is grown in a soy-based culture medium. The various polysaccharides are then purified by standard procedures (including centrifugation, precipitation, and ultrafiltration). See, for example, U.S. Patent Application Publication No. 2008 / 0286838 and U.S. Patent No. 5,847,112. The polysaccharide can be sized to reduce viscosity and / or improve the filterability of subsequent conjugated products. In the present invention, capsular polysaccharide is prepared from one or more of serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 7B, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 21, 22A, 22F, 23A, 23B, 23F, 24F, 27, 28A, 31, 33F, 34, 35A, 35B, 35F and 38.

[0178] IV. Carrier Protein

[0179] In a specific embodiment of the present invention, CRM 197 Used as a carrier protein. CRM 197 It is a non-toxic variant of diphtheria toxin (i.e., a toxoid). In one embodiment, the CRM is isolated from a culture of Corynebacterium diphtheriae strain C7 (β197) grown in a casamino acid and yeast extract-based medium. 197In another embodiment, the CRM is recombinantly prepared according to the method described in U.S. Patent No. 5,614,382. 197 Typically, CRMs are purified by a combination of ultrafiltration, ammonium sulfate precipitation, and ion exchange chromatography. 197 In some embodiments, CRMs in Pseudomonas fluorescens are prepared using PFENEX EXPRESSION TECHNOLOGY (Pfenex Inc., San Diego, CA). 197 .

[0180] Other suitable carrier proteins include additional inactivated bacterial toxins, such as DT (diphtheria toxoid), TT (tetanus toxoid) or fragment C of TT, pertussis toxoid, cholera toxoid (e.g., as described in International Patent Application Publication No. WO 2004 / 083251), E. coli LT, E. coli ST, and exotoxin A of Pseudomonas aeruginosa. Bacterial outer membrane proteins, such as outer membrane complex c (OMPC), porins, transferrin binding proteins, pneumococcal surface protein A (PspA; see International Patent Application Publication No. WO 02 / 091998), pneumococcal adhesin protein (PsaA), C5a peptidase from group A or group B streptococci, or Haemophilus influenzae protein D, pneumolysin (Kuo et al., 1995, Infect Immun 63:2706-13), including ply that is detoxified in some way, such as dPLY-GMBS (see International Patent Application Publication No. WO 04 / 081515) or dPLY-formol, PhtX, including fusions of PhtA, PhtB, PhtD, PhtE and Pht proteins, such as PhtDE fusions, PhtBE fusions (see International Patent Application Publication Nos. WO 01 / 98334 and WO 03 / 54007), can also be used. Other proteins, such as ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or purified protein derivative of tuberculin (PPD), PorB (from Neisseria meningitidis), PD (Haemophilus influenzae protein D; see, e.g., European Patent No. EP 0 594 610B) or immunologically functional equivalents thereof, synthetic peptides (see European Patent Nos. EP 0 378 881 and EP 0 427 347), heat shock proteins (see International Patent Application Publication Nos. WO 93 / 17712 and WO 94 / 03208), pertussis proteins (see International Patent Application Publication No. WO 98 / 58668 and European Patent No. EP 0 471 177), cytokines, lymphokines, growth factors or hormones (see International Patent Application Publication No. WO 91 / 01146), artificial proteins comprising multiple human CD4+ T cell epitopes from antigens derived from various pathogens (see Falugi et al., 2001, Eur J Immunol 31:3816-3824), such as N19 protein (see Baraldoi et al., 2004, Infect Immun 72:4884-7), iron uptake protein (see International Patent Application Publication No. WO 01 / 72337), toxin A or B of Clostridium difficile (C. difficile) (see International Patent Publication No. WO 00 / 61761) and flagellin (see Ben-Yedidia et al., 1998, Immunol Lett 64:9) can also be used as carrier proteins.

[0181] Other DT mutants can also be used as carrier proteins, such as CRM 176 , CRM 228 , CRM 45 (Uchida et al., 1973, JBiol Chem 218:3838-3844), CRM9, CRM 45 , CRM 102 , CRM 103 and CRM 107 , and other mutations described by Nicholls and Youle in Genetically Engineered Toxins, Ed: Frankel, Maecel Dekker Inc, 1992; deletion or mutation of Glu-148 to Asp, Gln or Ser and / or mutation of Ala 158 to Gly and other mutations described in U.S. Patent No. 4,709,017 or U.S. Patent No. 4,950,740; mutation of at least one or more residues Lys 516, Lys 526, Phe 530 and / or Lys 534 and other mutations described in U.S. Patent No. 5,917,017 or U.S. Patent No. 6,455,673; or fragments described in U.S. Patent No. 5,843,711.

[0182] V. Sublimation drying

[0183] The composition containing the polysaccharide of a specific S. pneumoniae serotype and the composition containing the carrier protein can be dried separately using a sublimation method to provide a composition comprising a dried carrier protein having a final moisture content of 6% or less and a composition comprising a dried polysaccharide of a specific S. pneumoniae serotype having a final moisture content of about 6% or less. For example, the dried composition can be prepared by freeze drying or radiant energy vacuum (REV) dehydration (microwave vacuum drying). See, for example, Encyclopedia of Agriculture, Food, and Biological Engineering. Marcel Dekker, Inc or Xu & Sunada, Chem Pharm Bull (Tokyo) 55 (11): 1545-50 (2007). The freeze-dried formulation components can be in the form of cakes or particles, for example, pellets, beads or spheres of the freeze-dried material, such as freeze-dried balls. See, for example, AS Mujumdar (2007). Handbook of Industrial Drying. CRC Press.

[0184] For example, lyophilized pellets can be prepared by loading aliquots of an aqueous solution comprising one or more polysaccharide serotypes onto a solid surface in the form of droplets (e.g., about 20, 50, 100, or 250 microliters) so that the droplets remain intact. In one embodiment of the invention, the surface is a plate, such as a metal plate, for example, at a temperature of about -180°C to about -196°C or about -180°C to about -273°C. For example, in one embodiment of the invention, the aqueous solution is loaded onto the surface by a dispensing tip. In one embodiment of the invention, the aqueous solution is dispensed at a dispensing rate of about 3 ml / min to about 75 ml / min, about 5 ml / min to about 75 ml / min, about 3 ml / min to about 60 ml / min, about 20 ml / min to about 75 ml / min, and about 20 ml / min to about 60 ml / min. In one embodiment of the invention, the aqueous solution distributed is 250 microliters, and the distribution speed is between about 5mL / min and about 75mL / min, or wherein the aliquot is 100 microliters, and the distribution speed is between about 3mL / min and about 60mL / min. In one embodiment of the invention, the gap between the distribution tip and the surface of the distribution aqueous solution is about 0.1cm or larger (for example, about 0.5cm or between 0.1cm and 1cm or between 0.1cm and 0.75cm). Once on the surface, the aqueous solution is frozen and then sublimated and dried by freeze drying. Methods for preparing freeze-dried balls are well known in the art. See, for example, US5656597; WO2013066769; WO2014093206; WO2015057540; WO2015057541 or WO2015057548.

[0185] In one embodiment of the invention, the aqueous solution is freeze-dried by radiant energy vacuum (REV) dehydration (microwave vacuum drying). REV dehydration is a drying method performed under reduced pressure conditions in which the boiling point of water and the oxygen content of the atmosphere are reduced. For example, in one embodiment of the invention, an aliquot of the aqueous solution is deposited on a solid surface in the form of individual droplets, wherein the surface temperature is about -90°C or lower, and the droplets are maintained as individual droplets when contacted and frozen on the surface as frozen pellets; microwave radiation is applied to the frozen pellets at a pressure below atmospheric pressure to produce dried pellets, such as spheres. In another example, the aqueous solution is provided on a tray and subjected to microwave radiation at a pressure below atmospheric pressure to produce a dried cake. See, for example, U.S. Patent Nos. 4,389,794; 4,664,924; 4,809,596; 4,882,851.

[0186] VI. Polysaccharide-Protein Conjugation

[0187] The purified polysaccharides are chemically activated to introduce functional groups capable of reacting with carrier proteins. Once activated, each capsular polysaccharide is conjugated individually to a carrier protein to form a glycoconjugate according to the method of the present invention.

[0188] In one embodiment, chemical activation of polysaccharides can be achieved by the methods described in U.S. Patent Nos. 4,365,170; 4,673,574; and 4,902,506. Briefly, pneumococcal polysaccharides are reacted with an oxidizing agent (e.g., a periodate-based oxidizing agent such as sodium periodate, potassium periodate, or periodic acid) to result in random oxidative cleavage of vicinal hydroxyl groups to generate reactive aldehyde groups. Direct amine coupling of the oxidized polysaccharide with primary amine groups (primarily lysine residues) on a protein carrier can be achieved by reductive amination. For example, conjugation can be performed in aqueous solution or in an organic solvent such as dimethyl sulfoxide (DMSO). See, for example, US2015 / 0231270A1, EP 0471 177B1, US2011 / 0195086A1. At the end of the conjugation reaction, the unreacted aldehyde is capped by adding a strong reducing agent (e.g., sodium borohydride).

[0189] In one embodiment, chemical activation of polysaccharides and subsequent conjugation to carrier proteins are achieved by the method described in U.S. Patent Nos. 4,365,170, 4,673,574 and 4,902,506. In short, polysaccharides are reacted with periodate-based oxidants such as sodium periodate, potassium periodate or periodic acid, resulting in random oxidative cleavage of ortho-hydroxyl groups to generate reactive aldehyde groups. Then, direct amine coupling of the primary amine groups (mainly lysine residues) on the polysaccharide and protein carrier can be achieved by reductive amination. For example, in the presence of nickel, conjugation is carried out by reacting a mixture of activated polysaccharides and carrier proteins with a reducing agent such as sodium cyanoborohydride. Conjugation reaction can be carried out in aqueous solution or in an organic solvent such as dimethyl sulfoxide (DMSO). See, for example, US2015 / 0231270A1, EP 0471 177B1, US2011 / 0195086A1. At the end of the conjugation reaction, unreacted aldehyde is optionally reduced by adding a strong reducing agent such as sodium borohydride.

[0190] In one embodiment, prior to formulation, each pneumococcal capsular polysaccharide is purified separately from Streptococcus pneumoniae, activated to form a reactive aldehyde, and then covalently conjugated to a carrier protein by reductive amination using sodium cyanoborohydride in the presence of nickel. Nickel forms a complex with residual, interfering cyanide from the sodium cyanoborohydride reducing agent used for reductive amination. Therefore, nickel can be used in the methods herein to improve the efficiency of the conjugation reaction and facilitate the removal of free cyanide.

[0191] Transition metals are known to form stable complexes with cyanide and are known to improve the reductive methylation of protein amino groups and formaldehyde with sodium cyanoborohydride. See Gidley et al., Biochem J. 1982, 203:331-334; Jentoft et al., Anal Biochem. 1980, 106:186-190. However, the applicants surprisingly discovered that the addition of nickel, by complexing residual, interfering cyanide, increased protein consumption during the conjugation process and resulted in the formation of larger, potentially more immunogenic conjugates.

[0192] Variations in free cyanide levels among batches of commercially available sodium cyanoborohydride reagent can lead to inconsistent conjugation performance, resulting in variations in conjugate properties, including molecular weight and polysaccharide-to-protein ratio. Adding nickel to the conjugation reaction reduces free cyanide levels, thereby improving the degree of batch-to-batch consistency of the conjugate.

[0193] In another embodiment, the conjugation method can employ activation of the polysaccharide using 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) to form a cyanate ester. The activated sugar can be coupled directly to amino groups on the carrier protein.

[0194] In an alternative embodiment, reactive homobifunctional or heterobifunctional groups can be introduced onto activated polysaccharides by reacting cyanate with any of several available methods. For example, cystamine or cysteamine can be used to prepare thiolated polysaccharides, which can be coupled to a carrier via a thioether bond obtained after reaction with a maleimide-activated carrier protein (e.g., using GMBS) or a haloacetylated carrier protein (e.g., using iodoacetimidate [e.g., ethyl iodoacetimidate HCl] or N-succinimidyl bromoacetate or SIAB, or SIA, or SBAP). Such conjugates are described in International Patent Application Publication Nos. WO 93 / 15760, WO 95 / 08348, and WO 96 / 29094; and Chu et al., 1983, Infect. Immunity 40: 245-256.

[0195] Other suitable conjugation methods use carbodiimides, hydrazides, active esters, norbornane, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC and / or TSTU. Many are described in International Patent Application Publication No. WO98 / 42721. Conjugation can involve a carbonyl linker, which can be formed by reacting the free hydroxyl groups of the sugar with CDI (see, Bethell et al., 1979, J. Biol. Chem. 254: 2572-4; Hearn et al., 1981, J. Chromatogr. 218: 509-18), followed by reaction with a carrier protein to form a carbamate bond. The chemical reaction involves reducing the terminal end of the carbohydrate to form a primary hydroxyl group, which is then reacted with CDI to form a carbamate intermediate, which is then coupled to the amino group of the protein carrier. The reaction may require optional protection / deprotection of other primary hydroxyl groups on the sugar.

[0196] The following examples are intended to facilitate further understanding of the present invention.

[0197] Example 1

[0198] Preparation of Streptococcus pneumoniae capsular polysaccharides 6A, 6B, 7F, 18C, 19A, 19F and 23F.

[0199] Methods for culturing pneumococci are well known in the art. See, for example, Chase, 1967, Methods of Immunology and Immunochemistry 1:52. Methods for preparing pneumococcal capsular polysaccharides are well known in the art. See, for example, European Patent No. EP 0 497 524. Isolates of pneumococcal subtypes are available from the American Type Culture Collection (Manassas, VA). The bacteria are identified as encapsulated, non-motile, Gram-positive, lancet-shaped diplococci that are α-hemolytic on blood agar. Subtypes can be distinguished using specific antisera based on the Quelling reaction. See, for example, U.S. Patent No. 5,847,112.

[0200] Cell banks representing each S. pneumoniae serotype present were obtained from frozen vials from Merck Culture Collection (Rahway, NJ). The thawed seed culture was transferred to a seed fermentor containing pre-sterilized growth medium suitable for S. pneumoniae. The culture was grown in a seed fermentor with temperature and pH control. The entire volume of the seed fermentor was transferred to a production fermentor containing pre-sterilized growth medium. The production fermentation is the final cell growth stage of the process. Temperature, pH, and agitation rate were controlled.

[0201] The fermentation process is terminated by adding an inactivator. Following inactivation, the solution is transferred to an inactivation tank, where it is maintained at a controlled temperature and under agitation. Cell debris is removed using a combination of centrifugation and filtration. The solution is then subjected to ultrafiltration and diafiltration. The solution is then subjected to solvent-based fractionation to remove impurities and recover the polysaccharides.

[0202] Example 2

[0203] Polysaccharide size reduction and activation were performed as follows.

[0204] Approximately 6 g of purified pneumococcal capsular polysaccharide (Ps) powder was dissolved in water for injection (WFI) at room temperature to a target concentration of approximately 4 g / L. The solution was then filtered through a 0.45 μm filter to reduce bioburden. The Ps concentration of the filtered Ps solution was determined by HPSEC UV-MALS-RI.

[0205] For all serotypes disclosed herein except serotypes 18C and 19A, the solution was diluted to about 2.5 g / L and then homogenized using a GEA-Niro Soavi Panda 2K homogenizer to reduce the molecular weight of Ps. All serotypes except serotype 19A were homogenized to reduce the molecular weight of the polysaccharide. Due to its relatively low initial size, the size of serotype 19A was not reduced. The homogenization pressure and the number of passes through the homogenizer (150-1000 bar; 4-7 times) were controlled for serotype-specific targets to achieve serotype-specific molecular weights. The polysaccharides with reduced size were filtered at 0.2 microns and then concentrated and diafiltered with water using a 10 kDa NMWCO tangential flow ultrafiltration membrane. During the homogenization process, the temperature was controlled using cooling water supplied to the heat exchanger at the homogenizer outlet.

[0206] Acid hydrolysis was used instead of homogenization to reduce the molecular weight of serotype 18C Ps. The temperature of the filtered serotype 18C Ps solution was increased to approximately 96°C and 90°C for batches A and B, respectively. The solution was then adjusted with glacial acetic acid (17.4 M) to a final concentration of 0.2 M and maintained for approximately 180 minutes for batches A and 160 minutes for batches B, respectively. 1.5 M potassium phosphate (pH 7.0) was added to a final concentration of 0.46 M to terminate the acid hydrolysis by increasing the pH of the solution, and the solution was then cooled to room temperature.

[0207] Serotype 19A was not subjected to 0.45 micron filtration and size reduction. Due to its relatively low initial size, size reduction was not required. After dissolution, serotype 19A was filtered through 0.22 micron as described in the next paragraph.

[0208] Each solution was then filtered using a 0.22 micron filter to reduce the bioburden before proceeding to the ultrafiltration 1 step. The filtered Ps was concentrated to approximately 10 g / L using a 10 kDa NMWCO tangential flow ultrafiltration membrane and then diafiltered using 6 diafiltration volumes of WFI at room temperature to obtain an ultrafiltration 1 process intermediate (UF1-FR). Serotype 18C used a 5 kDa NMWCO membrane instead of a 10 kDa NMWCO membrane to improve Ps recovery by retaining the lower molecular weight Ps produced by acid hydrolysis. The Ps concentration of UF1-FR was determined by HPSEC UV-MALS-RI. Before activating the Ps, WFI was added to UF1-FR to achieve a Ps concentration of approximately 10 g / L.

[0209] Then, 2 M sodium acetate buffer was added to control the pH of the activation reaction step. The sodium acetate concentration and pH as well as the temperature during the Ps activation reaction were controlled to specific values ​​for each serotype (Table 2).

[0210] Periodate activation was initiated by adding a 100 mM sodium metaperiodate solution to the solution based on the number of moles of periodate per mole of PnPs repeating units (RU). During activation, the vicinal diol (vicinaldiol) was oxidized to a reactive aldehyde within a serotype-specific reaction time. This reaction produced an activated product (AP) process intermediate. The amount of sodium metaperiodate added and the reaction time were controlled to specific values ​​for each serotype (Table 1).

[0211]

[0212] After Ps activation, the solution was diafiltered with 6 diafiltration volumes of 10 mM potassium phosphate (pH 6.4), followed by another 6 diafiltration volumes of WFI using a 10 kDa NMWCO tangential flow ultrafiltration membrane at 2-8 ° C. Serotype 18C uses a 5 kDa NMWCO membrane instead of a 10 kDa NMWCO membrane to improve Ps recovery by retaining the lower molecular weight Ps produced by acid hydrolysis. The solution is then concentrated to produce an ultrafiltration 2 process intermediate (UF2-FR). The Ps concentration of UF2-FR is determined by HPSEC UV-MALS-RI. The degree of activation is determined by derivatizing the UF2-FR sample with thiosemicarbazide and then detecting thiosemicarbazone by HPSEC with UV detection.

[0213] Example 3

[0214] CRM can be performed as follows 197 Preparation of carrier protein.

[0215] Frozen, purified CRM197, expressed in Pseudomonas fluorescens as previously described (see International Patent Application Publication No. WO 2012 / 173876), was diafiltered using a 5 kDa NMWCO tangential flow ultrafiltration membrane with 10 diafiltration volumes of 2 mM phosphate buffer, pH 7.2, and filtered at 0.22 microns. In a specific embodiment, 5 mM potassium phosphate, pH 6.4 (5 mM sodium phosphate, pH 7.0) is used for serotype 18C. The diafiltered solution was diluted in water containing sucrose to a final concentration of between 1.0 and 5.3% (w / v) and lyophilized to produce a dried CRM having a final moisture content of 6% or less. 197 Table 2 below provides the CRMs conjugated to polysaccharides of specific serotypes. 197 Representative sucrose concentrations.

[0216] Example 4

[0217] Lyophilize polysaccharide (Ps) and CRM as shown below. 197 (Pr).

[0218] Prior to lyophilization, dilute CRM as follows 197 and Ps solution.

[0219] In some embodiments, CRMs are prepared using WFI, 5 mM sodium phosphate, pH 6.4 (pH 7.0 for serotype 18C) and a freshly prepared 30% (w / v) sucrose solution in WFI. 197 The solution was diluted to a protein concentration of 6.0 mg / mL. The UF2-FR Ps solution was diluted to a Ps concentration of 6.0 mg / mL using WFI and a freshly prepared 30% w / v sucrose solution in WFI.

[0220] In some embodiments, CRM is prepared using WFI, 2 mM sodium phosphate (pH 7.2), and a freshly prepared 50% (w / v) sucrose solution in WFI. 197 The solution was diluted to a protein concentration of 6.0 mg / mL.The UF2-FR Ps solution was diluted to a Ps concentration of 6.0 mg / mL using WFI and a freshly prepared 50% (w / v) sucrose solution in WFI.

[0221] Serotype-specific sucrose and phosphate concentrations were used (see, e.g., Table 2). The diluted CRM was lyophilized using a Virtis Genesis freeze dryer. 197 and UF2-FR solution was lyophilized.

[0222]

[0223] Example 5

[0224] This example shows that CRM 197 Reconstitution time in anhydrous DMSO affects the secondary structure.

[0225] Lyophilized CRM that had been lyophilized in 6 mg / mL, 5% (w / v) sucrose, 1.25 mM sodium phosphate, pH 7.2 was reconstituted in DMSO (anhydrous DMSO from Sigma-Aldrich, 27655-100 mL) using either slow (eight minutes) or fast reconstitution (two minutes). 197 The solution was stirred at 400 nm and then mixed to a final concentration of 10 mg / mL. Reconstitution was performed by adding an appropriate volume of DMSO every 30 seconds until the time point was reached. FTIR spectra were collected with a BioTools PROTA-3S at a controlled temperature (25°C) using transmission mode with a 50 mm CaF2 window. -1 Fifty scans were collected at a resolution of 100 nm, averaged, buffer subtracted, and corrected for water vapor.

[0226] Reconstitute lyophilized CRM in anhydrous DMSO using rapid addition (two minutes) 197 Shown at 1660cm -1 The expected free carbonyl amide I peak was found at 197 Completely developed in anhydrous DMSO ( Figure 5 When reconstituted in anhydrous DMSO using a slow addition time (eight minutes) 197 When the second peak (1626 cm -1 ). This second peak is attributed to the formation of intermolecular β-sheets.

[0227] Example 6

[0228] This example demonstrates that reconstitution of CRM197 at an intermediate reconstitution time (four minutes) and holding for up to 300 minutes results in an increase in the proportion of intermolecular β-sheets with increasing hold time.

[0229] Lyophilized CRM that had been lyophilized in 6 mg / mL, 5% (w / v) sucrose, 1.25 mM sodium phosphate, pH 7.2 was reconstituted in DMSO (anhydrous DMSO can be obtained from Sigma-Aldrich, 27655-100 mL) within 4 minutes. 197To a final concentration of 10 mg / mL. Reconstitution was performed by adding an appropriate volume of DMSO every 30 seconds until the time point was reached. After the initial time point (T0) was collected, the sample was kept in the FTIR and further time points (30, 60, 120, 180, 240, 300 minutes) were collected. FTIR spectra were collected with a BioTools PROTA-3S at a controlled temperature (25 ° C) using a transmission mode with a 50 mm CaF2 window. The 4 cm -1 Fifty scans were collected at a resolution of 100 nm, averaged, buffer subtracted, and corrected for water vapor.

[0230] As shown by the presence of intermolecular β-sheet amide I peak ( Figure 6 ), CRM was reconstituted in anhydrous DMSO at an intermediate time (4 min) 197 Increasing the retention time (up to 300 min) resulted in a proportional increase in the amount of intermolecular β-sheets and a decrease in the amount of free carbonylamide I peak ( Figure 6 ).

[0231] Example 7

[0232] This example demonstrates that the formation of intermolecular β-sheets is concentration dependent.

[0233] Lyophilized CRM that had been lyophilized in 6 mg / mL, 5% (w / v) sucrose, 1.25 mM sodium phosphate, pH 7.2 was reconstituted in DMSO (anhydrous DMSO can be obtained from Sigma-Aldrich, 27655-100 mL) within two minutes. 197 To a final concentration of 10, 30 or 50 mg / mL. Reconstitution was performed by adding an appropriate volume of DMSO every 30 seconds until the time point was reached. FTIR spectra were collected with a BioTools PROTA-3S using transmission mode with a 50 mm CaF2 window at a controlled temperature (25°C). -1 Fifty scans were collected at a resolution of 500 nm, averaged, buffer subtracted, and corrected for water vapor. Dynamic light scattering (DLS) measurements were collected using a Malvern Zetasizer ZS at a controlled temperature (25° C.) and a viscosity of 1.996 cP at the prepared concentration.

[0234] When rapidly reconstituted with 10 mg / mL CRM 197 ( Figure 7 , two minutes), there was no evidence of intermolecular β-sheet formation, confirming earlier observations ( Figure 5At higher concentrations (30 mg / mL, 50 mg / mL), intermolecular β-sheets were formed even at a fast reconstitution time (two minutes). This supports the hypothesis that the increased CRM present during the slow reconstitution process 197 concentration is one of the reasons for the formation of intermolecular β-sheets. This concentration hypothesis is also supported by DLS data, which show that with lower concentrations ( Figure 8A ) compared to higher concentrations ( Figure 8B ) Reconstructed CRM 197 The proportion of larger particles increased, which is consistent with protein aggregation.

[0235] Example 8

[0236] For reconstructing CRM 197 Increasing the water concentration in DMSO leads to increased formation of intermolecular β-sheets.

[0237] Lyophilized CRM that had been lyophilized in 6 mg / mL, 5% (w / v) sucrose, 1.25 mM sodium phosphate, pH 7.2 was reconstituted in DMSO (anhydrous DMSO can be obtained from Sigma-Aldrich, 27655-100 mL) within two minutes. 197 To a final concentration of 10 mg / mL, the DMSO is 95%, 97%, 99% (v / v) DMSO / water or anhydrous DMSO. Anhydrous DMSO can be Sigma-Aldrich D2438-50 mL. Reconstitution is performed by adding an appropriate volume of DMSO every 30 seconds until the time point is reached. FTIR spectra were collected using a BioTools PROTA-3S in transmission mode with a 50 mm CaF2 window at a controlled temperature (25°C). Spectra can be taken at 4 cm -1 Fifty scans were collected at a resolution of 100 nm, averaged, buffer subtracted, and corrected for water vapor.

[0238] When CRM is rapidly reconstituted in anhydrous DMSO at 10 mg / mL 197 hour( Figure 9 ), there was no evidence of intermolecular β-sheet formation, confirming earlier observations ( Figure 5 , Figure 7 A very small amount of water (99% DMSO / water) showed a small shoulder of intermolecular β-sheets, which increased with increasing amounts of water (97%, 95% DMSO / water). This supports the hypothesis that increasing the amount of water during reconstitution leads to an increase in the amount of intermolecular β-sheets.

[0239] Example 9

[0240] Reduce the CRM of freeze-dried 197 The water content in the medium does not reduce the sensitivity to slow reconstitution.

[0241] CRM 197 The lyophilized CMR was lyophilized in a manner that reduced the amount of water present in the filter cake (longer drying cycles, lower fill volumes) (approximately 6 mg / mL in 1.25 mM phosphate (pH 7.2) containing 1% (w / v) sucrose). The lyophilized CMR was dried in DMSO (e.g., anhydrous DMSO from Sigma-Aldrich, D2438-50 mL) over a two-minute or eight-minute period. 197 Reconstitute to a final concentration of 10 mg / mL. Reconstitute by adding an appropriate volume of DMSO every 30 seconds until the time point is reached. FTIR spectra were collected using a BioTools PROTA-3S in transmission mode with a 50 mm CaF2 window at a controlled temperature (25°C). -1 Fifty scans were collected at a resolution of 100 nm, averaged, buffer subtracted, and corrected for water vapor.

[0242] CRM 197 The increase of water in the DMSO reconstitution leads to an increase in the proportion of intermolecular β-sheet formation ( Figure 9 CRM that had been lyophilized to reduce water content was reconstituted in anhydrous DMSO at two different rates, fast (two minutes) and slow (eight minutes). 197 . Reduce the CRM of freeze-dried 197 The water content in the medium does not reduce the sensitivity of the intermolecular β-sheet to the remodeling time ( Figure 10 ).

[0243] Example 10

[0244] In preparation for conjugation, freeze-dried polysaccharide (Ps) and freeze-dried CRM 197 The protein (Pr) is dissolved separately in anhydrous DMSO as described above. The two solutions are then combined to achieve a specific Ps to Pr (Ps:Pr) ratio and final Ps concentration. Sodium cyanoborohydride is added to the Ps:Pr conjugate solution and the solution is incubated at the desired conjugation temperature for an appropriate reaction time, both of which are specific for the Ps serotype to which the Pr is conjugated. It has been found that the size of the resulting conjugate is significantly affected by the Ps concentration.

[0245] There are several ways to bring the conjugated components (Ps and Pr) together. Figure 1-4 Several combination strategies are detailed, along with their effects on component concentrations and Ps:Pr when combined. Figure 1 ) or add Ps to Pr solution ( Figure 2), the concentration of the component added to the solution containing the other components and the concentration of the other components change over time, especially at the interface between the two solutions. The change in concentration over time during the addition process causes the change in Ps concentration and the change in the Ps:Pr ratio, which has an impact on the conjugate size. When lyophilized Ps is dissolved in the Pr solution ( Figure 3 ), the Ps concentration and Ps:Pr ratio at the powder-liquid interface started out high (at the solubility limit of Ps) and then decreased to the target value as the polysaccharide dissolved.

[0246] However, the conjugation was carried out by Tee mixing to a separate conjugation vessel ( Figure 4 ) Simultaneous addition of the two components eliminates this concentration gradient and maintains a constant ratio of the two components during addition, significantly reducing potential effects on conjugate size. Compared to other modes of simultaneous transfer, using a pump to combine the two components in a chamber (Tee mixing) provides a high level of control and scalability.

[0247] In several solutions developed for serotype 19F, as summarized in Table 3, viscous gels formed during the conjugation process: gel formation indicates the formation of larger, highly networked conjugates. In these solutions, higher Ps concentrations for conjugation were achieved. In the gelled solutions, dry Ps was dissolved in a Pr solution or a Ps solution was added to a Pr solution. Both combination strategies exhibited a Ps concentration gradient during the addition process, with the initial Ps concentration and the Ps:Pr high ( Figure 1 and Figure 3 ). Both combination strategies showed a Ps concentration gradient during the addition process. No concentration gradient ( Figure 4 ) or a concentration gradient with a lower initial Ps concentration and Ps:Pr ratio ( Figure 2 This supports the hypothesis that high Ps concentrations and Ps:Pr ratios can produce very large conjugates, leading to the formation of viscous gels.

[0248]

[0249] Example 11

[0250] Lyophilized Ps and lyophilized CRM were mixed with equal volumes of anhydrous DMSO at room temperature. 197 (Pr) was redissolved to prepare a homogeneous solution of Ps and Pr. CRM was redissolved at a faster rate of 2 minutes 197 In some embodiments, 500 mM sodium phosphate buffer (pH 7.2) is spiked into the DMSO-reconstituted protein solution to a final concentration of 1.0 mM sodium phosphate. After reconstitution in DMSO, Ps and CRM are separated by Tee mixing as described above. 197Solutions were combined to achieve serotype-specific final Ps concentrations and Ps:CRMs. 197 ratio (Table 4).

[0251] A solution of sodium cyanoborohydride in WFI was prepared, and 1.0 meq of sodium cyanoborohydride (1.0 mole of sodium cyanoborohydride per mole of Ps repeating unit) was added to the solution. The molar concentration of the sodium cyanoborohydride solution (Table 4) was based on a target of approximately 0.5% total water content of the solution during conjugation. The solution was reacted at a serotype-specific temperature for a serotype-specific duration (Table 4) to produce the conjugated product intermediate (CP).

[0252] A solution of sodium borohydride in WFI was prepared and 2.0 meq of sodium borohydride (relative to the Ps repeating unit) was added to the solution. The molar concentration of the sodium borohydride solution (Table 4) was based on a target of approximately 1.0% total water content of the solution after addition of the borohydride. The solution was allowed to react at room temperature for 2-3 hours to produce the conjugate product quench intermediate (CPQ).

[0253]

[0254] The conjugate solution is then diluted to 20% or less (v / v) anhydrous DMSO by slowly adding the solution to 150 mM sodium chloride (or, for the conjugate in some embodiments, 150 mM sodium chloride plus 0.025% w / v polysorbate 20). The solution temperature is maintained below 15°C during the dilution step. After approximately one hour, 1.5 M potassium phosphate (pH 6.0) is added to the solution to a final concentration of 25 mM potassium phosphate. The total Ps and CRM can be determined by 197 Consumption of conjugate Ps with CRM 197 The conjugation performance was evaluated by the ratio of and the molecular weight of the conjugate.

[0255] The conjugated solution was concentrated to approximately 2.5 g / L and diafiltered using a 30 kDa NMWCO tangential flow ultrafiltration membrane at 2-8°C using 10 diafiltration volumes of 150 mM sodium chloride or 150 mM sodium chloride containing 25 mM potassium phosphate. This produced an ultrafiltration 3 process intermediate (UF3-FR). The Ps concentration of UF3-FR was determined by HPSEC UV-MALS-RI.

[0256] For 19F in some embodiments, UF3-FR is 0.22 micron filtered and then incubated at 22°C for about 120 hours.

[0257] Then, the UF3-FR solution is processed in an ultrafiltration 4 step. During the ultrafiltration 4 step in some embodiments, the solution is concentrated to a Ps concentration of about 2.5 g / L and diafiltered at 2-8°C using 20 diafiltration volumes of 150 mM sodium chloride (pH 7.0) containing 10 mM L-histidine. Serotype 7F is subjected to an ultrafiltration 4 step using a 100 kDa NMWCO membrane. For serotypes 6A, 6B, and 18C, the UF3-FR solution is concentrated to about 3.5 g / L and diafiltered at 2-8°C using 10 mM L-histidine, 0.03% (w / v) PS-20, pH 7.0 in 150 mM sodium chloride using a 300 kDa NMWCO Biomax PES tangential flow ultrafiltration membrane. In some embodiments, serotype 7F, 19A, 19F and 23F UF3-FR solutions are concentrated to about 2.0 g / L and diafiltered using a 300 kDa NMWCO UltraCel regenerated cellulose tangential flow ultrafiltration membrane at 2-8°C using 10 mM L-histidine, 0.015% (w / v) PS-20, pH 7.0 in 150 mM sodium chloride. This produces an ultrafiltration 4 process intermediate (UF4-FR). The Ps concentration of UF4-FR is determined by HPSEC UV-MALS-RI.

[0258] UF4-FR was filtered through a PVDF filter at 0.22 microns. The Ps concentration of the filtrate was determined by HPSEC UV-MALS-RI. If the Ps concentration of the filtrate was greater than 1.0 g / L, the filtrate was diluted to a Ps concentration of 1.0 g / L using additional 150 mM sodium chloride (pH 7.0) containing 10 mM L-histidine. This produced a monovalent bulk conjugate intermediate (MBC). The MBC was distributed into aliquots and frozen at -60°C to -80°C.

[0259] For serotype 6A, 6B and 18C, UF4-FR is carried out 0.5 / 0.2 micron filtration by double membrane PES filter. Determine the Ps concentration of the filtrate by HPSEC UV-MALS-RI. If the Ps concentration of the filtrate is greater than 1.0g / L, the filtrate is diluted to a Ps concentration of 1.0g / L using another 10mM L-histidine, 0.03% w / v PS-20, pH 7.0 in 150mM sodium chloride. This has produced a monovalent batch conjugate intermediate (MBC). MBC is dispensed into aliquots and frozen at -60°C to -80°C.

[0260] For serotype 7F, 19A, 19F and 23F, UF4-FR is carried out to 0.22 micron filtration through PVDF filter.The PS concentration of filtrate is determined by HPSEC UV-MALS-RI.If the Ps concentration of filtrate is greater than 1.0g / L, the filtrate is diluted to Ps concentration 1.0g / L using another 10mM L histidine in 150mM sodium chloride, 0.015% w / v PS-20, pH 7.0. This has produced unit price batch conjugate intermediate (MBC).MBC is distributed into aliquots and frozen at -60 ℃ to -80 ℃.

[0261] Example 12

[0262] When CRM 197 Upon slow reconstitution (eight minutes) in anhydrous DMSO and subsequent conjugation to 6B polysaccharide, intermolecular β-sheets were present after conjugation.

[0263] CRM was reconstituted in DMSO either rapidly (two minutes) or slowly (eight minutes) as previously discussed. 197 The 6B polysaccharide was then conjugated to the 6B polysaccharide, purified as previously described, and diafiltered in (10 mM histidine, 150 mM NaCl pH 7) to a concentration of approximately 1 mg / mL. The sample was then concentrated using an Amicon Ultra 11K MWCO filter to enable FTIR measurements. FTIR spectra were collected using a BioTools PROTA-3S at a controlled temperature (25° C.) using transmission mode with a 50 mm CaF2 window. -1 Fifty scans were collected at a resolution of 100 nm, averaged, buffer subtracted, and corrected for water vapor. Data were then analyzed using Omic software (ThermoFisher).

[0264] Use CRM that has been slowly reconstituted in DMSO 197 Prepared CRM 197 -6B conjugate ( Figure 11 ) showed evidence for intermolecular β-sheet formation, indicating that the CRM persists even after remodeling 197 Aggregate. Use CRM that has been rapidly refactored 197 Prepared CRM 197 -6B( Figure 11 ) shows the expected free C=O amide and no β-sheet formation.

[0265] Example 13

[0266] This example shows the use of reductive amination of Ps from serotype 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 7B, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 21, 22A, 22F, 23A, 23B, 23F, 24F, 27, 28A, 31, 33F, 34, 35A, 35B, 35F, or 38 with a CRM in anhydrous DMSO using reductive amination. 197 (Pr) conjugation method. Different serotype polysaccharides were conjugated to purified CRM using a common process. 197 Carrier protein.

[0267] By rapidly adding anhydrous DMSO to the dry CRM within two minutes 197 The dried CRM prepared as described previously was reconstituted in anhydrous DMSO. 197 , and Ps was reconstituted separately in anhydrous DMSO to prepare Pr and Ps homogeneous solutions. Ps and Pr were reconstituted with half of the total conjugation reaction volume, respectively. Therefore, during the conjugation process, the concentration of Ps after DMSO reconstitution was 2x Ps concentration, which was calculated to be in the range of 2.2 to 7.6 g / L according to Table 3. After DMSO reconstitution, the Pr concentration was 2x (Ps concentration / Ps:Pr ratio during conjugation), which was in the range of 1.5 to 5.7 g / L. The Pr homogeneous solution was then combined with the Ps homogeneous solution by Tee mixing. Sodium cyanoborohydride (1 mole per mole of polysaccharide repeating unit) was then added to the mixture, and conjugation was performed with a serum-specific duration (1 to 48 hours) to achieve the target conjugated size.

[0268] Reduction with sodium borohydride

[0269] After the conjugation reaction, sodium borohydride (2 moles per mole of polysaccharide repeating unit) was added and the solution was incubated at 22°C for 1 hour. The solution was diluted to 150 mM sodium chloride, 0.025% (w / v) polysorbate 20 at approximately 4°C. Phosphate buffer was then added to neutralize the pH. The solution was concentrated and diafiltered using a 10 kDa NMWCO tangential flow ultrafiltration membrane at approximately 4°C using 150 mM sodium chloride.

[0270] Final filtration and product storage

[0271] Each solution was then concentrated and diafiltered using a 300 kDa NMWCO tangential flow ultrafiltration membrane in 150 mM sodium chloride (pH 7.0) containing 10 mM histidine at 4° C. The retentate solution was 0.22 μm filtered.

[0272] Serotype 19F was incubated for approximately 5 days and diafiltered using a 300 kDa NMWCO tangential flow ultrafiltration membrane at approximately 4°C in 150 mM sodium chloride, pH 7.0, containing 10 mM histidine and filtered at 0.22 micron.

[0273] Serotype 18C was diafiltered using a 300 kDa NMWCO tangential flow ultrafiltration membrane at approximately 4°C in 150 mM sodium chloride, pH 7.0, containing 10 mM histidine and filtered at 0.22 micron.

[0274] Additional dilution solution of 10 mM L-histidine in 150 mM sodium chloride (pH 7.0) was used, dispensed into aliquots and frozen at ≤ -60°C.

[0275] Example 14

[0276] This example shows the formulation of a 15-valent pneumococcal conjugate vaccine with different surfactants and stabilizers.

[0277] Pneumococcal polysaccharide-protein conjugates prepared as described above were used to formulate a 15-valent pneumococcal conjugate vaccine (PCV15) with serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F.

[0278] As previously discussed, pneumococcal polysaccharide-CRM produced by reductive amination in DMSO was used. 197 The formulations were prepared by combining the conjugates. The bulk conjugate volume required to achieve the target final concentration for each serotype was calculated based on the solution volume and bulk polysaccharide concentration. The 15 conjugates were combined with excipients selected from sodium chloride, L-histidine, and pH 5.8 buffer containing polysorbate (PS)-20, PS-80, or poloxamer (P) 188.

[0279] During this time, the aseptically formulated materials were gently mixed and subsequently mixed with bulk aluminum phosphate adjuvant (APA), which may or may not contain propylene glycol (PG) and polyethylene glycol 400 (PEG400). Two concentrations of conjugate and APA were studied in various formulations. One contained 8 μg / mL serotype 6B polysaccharide, 4 μg / mL polysaccharide for all other serotypes, and 250 μg / mL APA. The other contained 16 μg / mL serotype 6B polysaccharide, 8 μg / mL polysaccharide for all other serotypes, and 500 μg / mL APA. The formulated vaccines were stored at 2–8°C.

[0280] APA is an aqueous suspension of aluminum hydroxyphosphate. It is prepared by mixing aluminum chloride and sodium phosphate in a 1:1 volume ratio to precipitate the aluminum hydroxyphosphate. Following the mixing process, the material is size-reduced using a high-shear mixer to achieve a monodisperse particle size distribution. The product is then diafiltered with saline and steam sterilized.

[0281] Example 15

[0282] In this example, 6B polysaccharide (Ps) and CRM prepared as discussed above were discretely lyophilized by REV 197 (Pr) to form a dry cake. The dry cake was reconstituted in DMSO and conjugated as discussed herein.

[0283] The target Ps:Pr ratio (w / w) was 0.9 to 1.5, and the target molecular weight (MW) of the conjugate was 1500 to 3500 kDa. The target free Ps was 15% or less, and the loss of free lysine was greater than 5 mol / mol. The results are shown in Table 5. The REV dried material prepared according to the present invention had a conjugate MW within the target range, a Ps:Pr ratio within the target range, free Ps within the target range, and free lysine within the target range.

[0284]

[0285] Determination of conjugate Mw, conjugate Mn, and conjugate Ps:Pr in Table 5.

[0286] The molecular weight and concentration analysis of the conjugate were determined using HPSEC / UV / MALS / RI. The conjugate sample was injected and separated by high performance size exclusion chromatography (HPSEC). Detection was completed using ultraviolet (UV), multi-angle light scattering (MALS) and refractive index (RI) detectors in series. Protein concentration was calculated using the extinction coefficient by UV280. The polysaccharide concentration was deconvoluted using the dn / dc factor from the RI signal (contributed by both protein and polysaccharide). The dn / dc factor is the change in the refractive index of the solution and the change in solute concentration (in mL / g). The molecular weight average of the sample was calculated using the concentration and light scattering information measured on the entire sample peak by Astra software (Wyatt Technology Corporation, Santa Barbara, CA). The determination of lysine loss in Table 5.

[0287] The lysine consumption in the conjugated protein was determined by the number of covalent linkages between the polysaccharide and the carrier protein.

[0288] Waters AccQ-Tag amino acid analysis (AAAaa) is used to measure the degree of conjugation in the conjugate sample. In the Eldex workstation, gas phase acid hydrolysis is used to hydrolyze the sample to break down the carrier protein into its constituent amino acids. Free amino acids are derivatized using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC). The derivatized sample is then analyzed using UPLC with UV detection on a C18 column. Average protein concentration is obtained using representative amino acids other than lysine. The consumption of lysine (i.e., lysine loss) during the conjugation process is determined by the difference between the average measured value of lysine in the conjugate and the amount of lysine expected in the initial protein.

[0289] Determination of free Ps in Table 5

[0290] Free polysaccharide (not conjugated to CRM) was measured by first precipitating free protein and conjugates using deoxycholate (DOC) and hydrochloric acid. 197 The precipitate was then filtered off and the concentration of free polysaccharide in the filtrate was analyzed by HPSEC / UV / MALS / RI. Free polysaccharide was calculated based on the percentage of total polysaccharide measured by HPSEC / UV / MALS / RI.

[0291] Determination of free Pr in Table 5

[0292] Separation of free polysaccharides and polysaccharide CRMs in conjugate samples by capillary electrophoresis in micellar electrokinetic chromatography (MEKC) mode 197 Conjugate and free CRM 197 Briefly, samples were mixed with MEKC running buffer containing 25 mM borate, 100 mM SDS, pH 9.3 and separated in pretreated bare fused-20 silica capillaries. Separation was monitored at 200 nm and the CRM 197 Standard curve for free CRM 197 Quantitation was performed. Free protein results were reported as a percentage of the total protein content determined by the HPSEC / UV / MALS / RI procedure.

[0293] Example 16

[0294] In this example, 6A polysaccharide (Ps) and CRM 197 (Pr) Samples were prepared separately (discretely) and lyophilized or pooled and lyophilized according to the present invention. The lyophilized samples were then reconstituted in DMSO and conjugated as discussed herein.

[0295] Two different MVD cycles were tested to dry the beads (Table 6). For both cycles, the pressure was maintained in the range of 50 to 60 mTorr. The temperature was maintained in the range of 25 to 30°C, depending on the amount of power applied.

[0296]

[0297] The preliminary drying cycle (Lyo1) took 18 hours, as shown in Table 7.

[0298]

[0299] The drying cycle (Lyo 2) was modified by adding a secondary drying cycle and increasing the primary drying time, as shown in Table 8.

[0300]

[0301] The target Ps:Pr ratio (w / w) was 0.9 to 1.5, and the target molecular weight (MW) of the conjugate was 1500 to 3500 kDa. The results are shown in Table 9.

[0302]

[0303] Example 17

[0304] In this example, 23F polysaccharide (Ps) and CRM 197 (Pr) Samples were prepared separately (discretely) and lyophilized or pooled and lyophilized according to the present invention. The lyophilized samples were then reconstituted in DMSO and conjugated as discussed previously herein.

[0305] The target Ps:Pr ratio (w / w) was 0.9 to 1.5, and the target molecular weight (MW) of the conjugate was 1500 to 3500 kDa. The results are shown in Table 10, which shows that Ps and Pr were within the expected targets for the separately lyophilized samples, except for sample B2, which had a ratio of 2.4.

[0306]

[0307] Example 18

[0308] This example shows the addition of DMSO to the lyophilized carrier protein CRM 197 Effect of time of (Pr) on conjugate size using 6A polysaccharide (Ps). As previously discussed, DMSO was added rapidly (two minutes) and slowly (eight minutes) to lyophilized Pr, mixed, and then conjugated to activated polysaccharide reconstituted in DMSO.

[0309] Figure 13The size of the conjugates produced from the conjugation reaction using polysaccharides of increasing size is shown. The general relationship is that the larger the polysaccharide used in the conjugation reaction (UF2 size), the larger the resulting conjugate. For each polysaccharide size, slow and fast addition of DMSO to the lyophilized Pr were compared. The figure shows that slow addition of DMSO can produce even larger conjugates of the same size polysaccharide compared to fast addition of DMSO.

[0310] Example 19

[0311] This example shows the methods for producing polysaccharides (Ps) and CRMs. 197 Development of lyophilizer conditions for (Pr;CRM) lyophilized pellets.

[0312] Discrete solutions of CRM and activated polysaccharides from serotypes 6A and 23F were prepared as described in Examples 3 and 4 and had the Ps, CRM and sucrose concentrations as indicated in the table in this example.

[0313] A modified Biomek FX pipetting robot (Cryomek) is used to dispense 50 μL aliquots of the solution onto the flat freezing surface of the Cryomek. The beads can be dispensed into small cold containers using a shovel mechanism without causing any breakage. After completing the cycle of each different solution, the beads are transferred to an intermediate storage container and kept at -70°C until sublimation drying in a freeze dryer or by microwave vacuum drying. For freeze dryer drying, a monolayer of beads is dispensed into a drying tray. The cabinet pressure, shelf temperature, and cycle time are set. After drying the beads, the freeze-dried balls are stored at 2-8°C. (See Example 5 for specific parameters)

[0314] The initial drying cycle (Lyo1) took 18 hours and is shown in Table 19.1. The residual moisture content of the lyophilized pellets was determined by Karl Fisher titration and is shown in Table 19.2.

[0315]

[0316]

[0317] The results showed that the moisture content in the lyophilized pellets was high. Furthermore, the pellets were very fragile and hygroscopic. The solids content was increased by increasing the polysaccharide, protein, and sucrose concentrations, as shown in Table 20.2. The drying cycle (Lyo 2) was modified by adding a secondary drying cycle and increasing the primary drying time, as shown in Table 20.1.

[0318]

[0319]

[0320] Due to the improved drying cycle, the residual moisture content of Lyo2 is significantly lower than that of Lyo1. Even after all the ice has sublimated, secondary drying can improve the removal of bound moisture that is still present in the product. Secondary drying requires a higher temperature (30°C) than primary drying (15°C).

[0321] At this point, the total drying cycle time in the freeze dryer was 45 hours. Some parameters, such as pressure and temperature, were varied to further reduce the drying cycle time (Method: Lyo 3), as shown in Tables 21.1 and 21.2.

[0322]

[0323]

[0324] Example 20

[0325] This example shows the methods for producing polysaccharides (Ps) and CRMs. 197 Development of Radiant Energy Vacuum (REV) Dehydration (Microwave Vacuum Drying (MVD)) Conditions for (Pr;CRM) Freeze-Dried Spheres.

[0326] Solutions of CRM and activated polysaccharides from serotypes 6A and 23F were prepared as described in Examples 3 and 4 and had the Ps, CRM and sucrose concentrations shown in Tables 22.1 and 22.2.

[0327] A modified Biomek FX pipetting robot (Cryomek) is used to dispense 50 μL aliquots of the solution onto the flat freezing surface of the Cryomek. The beads can be dispensed into small cold containers using a shovel mechanism without causing any rupture. After completing the cycle of each different solution, the beads are transferred to an intermediate storage container and kept at -70°C until sublimation drying is performed in a freeze dryer or by microwave vacuum drying. For microwave drying, a monolayer of beads is dispensed into the container. Power, pressure, and cycle time are set. After drying the beads, the freeze-dried balls are stored at 2-8°C (see Example 6 for specific parameters).

[0328] Two different MVD cycles were tested to dry the beads. For both cycles, the pressure was maintained in the range of 50 to 60 mTorr. The temperature was maintained in the range of 25 to 30°C, depending on the applied power.

[0329]

[0330]

[0331] The MVD1 cycle took 4 hours and 30 minutes, but was insufficient to reduce the residual moisture content to at most 2%. The MVD2 cycle took longer and used higher power, therefore providing a significantly lower residual moisture content.

[0332] Although the present invention has been described herein with reference to the embodiments shown, it should be understood that the present invention is not limited thereto. Those of ordinary skill in the art and those who have access to the teachings herein will recognize other modifications and embodiments within the scope thereof. Therefore, the present invention is limited only by the claims appended hereto.

[0333] The present invention also includes the following items.

[0334] 1. A method for preparing a composition comprising a Streptococcus pneumoniae polysaccharide covalently linked to a carrier protein, the method comprising:

[0335] (a) providing a first dried composition comprising activated Streptococcus pneumoniae polysaccharide from one or more Streptococcus pneumoniae serotypes and a second dried composition comprising a carrier protein;

[0336] (b) reconstituting the first dried composition and the second dried composition in an organic solvent, respectively, and mixing them to provide a first homogenous solution comprising one or more activated polysaccharides and a second homogenous solution comprising the carrier protein;

[0337] (c) combining the first homogeneous solution with the second homogeneous solution by Tee mixing to produce a mixture; and

[0338] (d) adding a reducing agent to the mixture to produce a conjugate solution comprising the carrier protein conjugated to one or more polysaccharides of the S. pneumoniae serotype.

[0339] 2. The method according to item 1, wherein the first dried composition and the second dried composition are prepared by a sublimation drying method selected from freeze drying and radiant energy vacuum (REV) dehydration.

[0340] 3. The method according to item 2, wherein the sublimation drying method comprises freezing the first aqueous solution and the second aqueous solution in the form of cakes or freeze-dried beads.

[0341] 4. The method according to item 2, wherein the sublimation drying is performed in batches in a container selected from the group consisting of a metal tray, a plastic tray, a plastic bag, and a type I vial.

[0342] 5. The method of claim 1 , wherein the first dried composition and the second dried composition are prepared by a sublimation drying process including freeze drying or radiant energy vacuum (REV) dehydration, comprising providing a first aqueous solution and a second aqueous solution, the first aqueous solution comprising activated Streptococcus pneumoniae polysaccharide from one or more serotypes of Streptococcus pneumoniae, the second aqueous solution comprising a carrier protein and a buffer, wherein the first aqueous solution and the second aqueous solution comprise about 0.5% (w / v) or more sucrose, and subjecting the first aqueous solution and the second aqueous solution to a sublimation drying process to produce the first dried composition and the second dried composition.

[0343] 6. The method according to item 5, wherein the buffer is histidine, succinate, MES, MOPS, HEPES or acetate buffer with a pH range of 5.0-7.0.

[0344] 7. The method according to item 5, wherein the buffer is a phosphate or citrate buffer having a pH range of 5.0-7.0.

[0345] 8. The method according to item 1, wherein the first dried composition and the second dried composition have a moisture content of less than 6%.

[0346] 9. The method according to item 1, wherein the organic solvent is dimethyl sulfoxide (DMSO).

[0347] 10. The method of claim 1, wherein the reconstitution comprises eight minutes or less and the mixing comprises 120 minutes or less.

[0348] 11. The method of item 1, wherein each conjugate solution comprises polysaccharide conjugated to the carrier protein at a polysaccharide to carrier protein ratio of about 0.6 to about 1.3 on a weight to weight basis.

[0349] 12. The method of item 1, wherein the conjugate solution comprises an amount of free polysaccharide that is less than about 15% of the total polysaccharides in the solution.

[0350] 13. The method of claim 1, wherein the one or more polysaccharides are obtained from a serotype of Streptococcus pneumoniae selected from the group consisting of: serotype 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, 43, 44, 45, 46, 47F, 47A, 48, CWPS1, CWPS2 and CWPS3.

[0351] 14. The method according to item 1, wherein the carrier protein is an inactivated bacterial toxoid selected from the group consisting of tetanus toxoid, diphtheria toxoid, pertussis toxoid, bacterial cytolysin or pneumolysin.

[0352] 15. The method according to item 12, wherein the inactivated bacterial toxin is CRM 197 .

[0353] 16. The method according to item 1, wherein the conjugate solution is sterile filtered.

[0354] 17. The method according to item 1, wherein the Streptococcus pneumoniae polysaccharide is activated by reacting with an oxidizing agent.

[0355] 18. A method of preparing a composition comprising two or more conjugates, each conjugate comprising a polysaccharide from one or more serotypes of Streptococcus pneumoniae covalently linked to a carrier protein, the method comprising:

[0356] (a) Provide

[0357] (i) two or more first aqueous solutions, each first aqueous solution comprising an activated polysaccharide of a specific S. pneumoniae serotype, wherein the polysaccharide has been reacted with an oxidizing agent to provide the activated polysaccharide, and wherein the two or more first aqueous solutions are different; or

[0358] (ii) two or more first aqueous solutions, each first aqueous solution comprising activated polysaccharides of two or more serotypes of S. pneumoniae, 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 different;

[0359] (b) providing two or more second aqueous solutions, each comprising a carrier protein and a buffer, wherein the amount of the two or more second aqueous solutions corresponds to the amount of at least the two or more first aqueous solutions;

[0360] (c) separately drying the two or more first aqueous solutions and the two or more second aqueous solutions in a sublimation drying process to produce two or more first dried compositions and two or more second dried compositions, each of the first dried compositions comprising a dried polysaccharide and each of the second dried compositions comprising a dried carrier protein;

[0361] (d) reconstituting each of the two or more first dried compositions and each of the two or more second dried compositions separately in an organic solvent and mixing to provide two or more first homogenous solutions and two or more second homogenous solutions, each of the first homogenous solutions independently comprising (i) a polysaccharide of a specific S. pneumoniae serotype or (ii) polysaccharides of two or more specific S. pneumoniae serotypes, and each of the second homogenous solutions comprising the carrier protein;

[0362] (e) combining each of the first homogeneous solutions with the second homogeneous solution separately by Tee mixing to produce a plurality of mixtures;

[0363] (f) adding a reducing agent to the plurality of mixtures to produce a plurality of conjugate solutions; and

[0364] (g) combining two or more of the plurality of conjugate solutions to produce a composition comprising two or more conjugates, each conjugate comprising a polysaccharide from one or more serotypes of Streptococcus pneumoniae covalently linked to a carrier protein.

[0365] 19. The method according to item 18, wherein at least one conjugate solution is prepared by separately mixing a first homogeneous solution with a second homogeneous solution Tee to produce the plurality of conjugate solutions; or

[0366] wherein at least one conjugate solution is prepared by separately mixing a first aqueous solution with a second homogeneous solution Tee to produce the plurality of conjugate solutions; or

[0367] wherein each conjugate solution is prepared by separately mixing the first homogeneous solution with the second homogeneous solution Tee to produce the plurality of conjugate solutions; or

[0368] Each conjugate solution is prepared by mixing the first aqueous solution with the second homogeneous solution Tee, respectively, to produce the plurality of conjugate solutions.

[0369] 20. The method of item 18, wherein the sublimation drying method comprises freeze drying or radiant energy vacuum (REV) dehydration.

[0370] 21. The method according to item 18, wherein the sublimation drying method comprises freezing the first aqueous solution and the second aqueous solution in the form of cakes or freeze-dried beads.

[0371] 22. The method according to item 18, wherein the sublimation drying is performed in batches in a container selected from the group consisting of a metal tray, a plastic tray, a plastic bag, and a type I vial.

[0372] 23. The method according to item 18, wherein the first aqueous solution and the second aqueous solution comprise about 0.5% (w / v) or more sucrose.

[0373] 24. The method according to item 18, wherein the organic solvent is dimethyl sulfoxide (DMSO).

[0374] 25. The method of claim 18, wherein the reconstitution comprises eight minutes or less and the mixing comprises 120 minutes or less.

[0375] 26. The method of item 18, wherein each conjugate solution comprises polysaccharide conjugated to the carrier protein at a polysaccharide to carrier protein ratio of about 0.6 to about 1.3 on a weight to weight basis.

[0376] 27. The method of item 18, wherein each conjugate solution comprises an amount of free polysaccharide that is less than about 15% of the total polysaccharide in the solution.

[0377] 28. The method according to item 18, wherein the buffer is histidine, succinate, MES, MOPS, HEPES or acetate buffer in the pH range of 5.0-7.0.

[0378] 29. The method according to item 18, wherein the buffer is a phosphate or citrate buffer having a pH range of 5.0-7.0.

[0379] 30. The method of item 18, wherein the one or more polysaccharides are obtained from one or more serotypes of Streptococcus pneumoniae 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, 43, 44, 45, 46, 47F, 47A, 48, at least one of CWPS1, CWPS2 and CWPS3.

[0380] 31. The method according to item 18, wherein the carrier protein is an inactivated bacterial toxoid selected from the group consisting of tetanus toxoid, diphtheria toxoid, pertussis toxoid, bacterial cytolysin or pneumolysin.

[0381] 32. The method according to item 31, wherein the inactivated bacterial toxin is CRM 197 .

[0382] 33. The method of claim 18, wherein the conjugate solution is sterile filtered.

[0383] 34. A method for preparing a multivalent pneumococcal conjugate vaccine comprising polysaccharides of Streptococcus pneumoniae serotypes 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 a carrier protein, the method comprising:

[0384] (a) providing 23 dried carrier protein compositions and 23 dried activated polysaccharide compositions, each dried activated polysaccharide composition comprising a dried activated polysaccharide from a serotype of Streptococcus pneumoniae 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 an activated polysaccharide from the same serotype of Streptococcus pneumoniae;

[0385] (b) reconstituting the 23 dried carrier protein compositions and the 23 dried activated polysaccharide compositions with an organic solvent, respectively, and mixing the resulting mixture to provide 23 homogeneous carrier protein solutions and 23 homogeneous activated polysaccharide solutions, each homogeneous carrier protein solution comprising the carrier protein, and each homogeneous activated polysaccharide solution comprising an activated polysaccharide of a specific S. pneumoniae serotype, wherein none of the 23 homogeneous solutions comprises an activated polysaccharide from the same S. pneumoniae serotype;

[0386] (c) combining each of the 23 homogenous carrier protein solutions with one of the 23 homogenous activated polysaccharide solutions by Tee mixing to produce 23 mixtures, each mixture comprising a carrier protein and an activated polysaccharide of a specific S. pneumoniae serotype, wherein none of the 23 mixtures comprises an activated polysaccharide from the same S. pneumoniae serotype;

[0387] (d) adding a reducing agent to each of the 23 mixtures to produce 23 conjugate solutions, each conjugate solution comprising a carrier protein conjugated to a polysaccharide of a specific S. pneumoniae serotype, wherein none of the 23 conjugate solutions comprises an activated polysaccharide from the same S. pneumoniae serotype; and

[0388] (e) combining the 23 conjugate solutions to provide the multivalent pneumococcal conjugate vaccine containing S. pneumoniae serotype polysaccharide 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 a carrier protein.

[0389] 35. A method for preparing a multivalent pneumococcal conjugate vaccine comprising polysaccharides of Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F conjugated to a carrier protein, the method comprising:

[0390] (a) providing 15 dried carrier protein compositions and 15 dried activated polysaccharide compositions, each dried activated polysaccharide composition comprising a dried activated polysaccharide from a serotype of Streptococcus pneumoniae 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 an activated polysaccharide from the same serotype of Streptococcus pneumoniae;

[0391] (b) reconstituting the 15 dried carrier protein compositions and the 15 dried activated polysaccharide compositions with an organic solvent, respectively, and mixing the resulting 15 homogeneous carrier protein solutions and 15 homogeneous activated polysaccharide solutions, each homogeneous carrier protein solution comprising the carrier protein, and each homogeneous activated polysaccharide solution comprising an activated polysaccharide of a specific Streptococcus pneumoniae serotype, wherein none of the 15 homogeneous solutions comprises an activated polysaccharide from the same Streptococcus pneumoniae serotype;

[0392] (c) combining each of the 15 homogenous carrier protein solutions with one of the 15 homogenous activated polysaccharide solutions by Tee mixing to produce 15 mixtures, each mixture comprising a carrier protein and an activated polysaccharide of a specific S. pneumoniae serotype, wherein none of the 15 mixtures comprises an activated polysaccharide from the same S. pneumoniae serotype;

[0393] (d) adding a reducing agent to each of the 15 mixtures to produce 15 conjugate solutions, each conjugate solution comprising a carrier protein conjugated to a polysaccharide of a specific S. pneumoniae serotype, wherein none of the 15 conjugate solutions comprises an activated polysaccharide from the same S. pneumoniae serotype; and

[0394] (e) combining the 15 conjugate solutions to provide the multivalent pneumococcal conjugate vaccine containing S. pneumoniae serotype polysaccharides 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F conjugated to a carrier protein.

[0395] 36. A method for preparing a multivalent pneumococcal conjugate vaccine comprising polysaccharides of Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F conjugated to a carrier protein, the method comprising:

[0396] (a) providing 13 dried carrier protein compositions and 13 dried activated polysaccharide compositions, each dried activated polysaccharide composition comprising a dried activated polysaccharide from a serotype of Streptococcus pneumoniae 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 an activated polysaccharide from the same serotype of Streptococcus pneumoniae;

[0397] (b) reconstituting the 13 dried carrier protein compositions and the 13 dried activated polysaccharide compositions with an organic solvent, respectively, and mixing the resulting mixture to provide 13 homogeneous carrier protein solutions and 13 homogeneous activated polysaccharide solutions, each homogeneous carrier protein solution comprising the carrier protein, and each homogeneous activated polysaccharide solution comprising an activated polysaccharide of a specific Streptococcus pneumoniae serotype, wherein none of the 13 homogeneous solutions comprises an activated polysaccharide from the same Streptococcus pneumoniae serotype;

[0398] (c) combining each of the 13 homogenous carrier protein solutions with one of the 13 homogenous activated polysaccharide solutions by Tee mixing to produce 13 mixtures, each mixture comprising a carrier protein and an activated polysaccharide of a specific S. pneumoniae serotype, wherein none of the 13 mixtures comprises an activated polysaccharide from the same S. pneumoniae serotype;

[0399] (d) adding a reducing agent to each of the 13 mixtures to produce 13 conjugate solutions, each conjugate solution comprising a carrier protein conjugated to a polysaccharide of a specific S. pneumoniae serotype, wherein none of the 13 conjugate solutions comprises an activated polysaccharide from the same S. pneumoniae serotype; and

[0400] (e) combining the 13 conjugate solutions to provide the multivalent pneumococcal conjugate vaccine containing S. pneumoniae serotype polysaccharides 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F conjugated to a carrier protein.

[0401] 37. A method for preparing a multivalent pneumococcal conjugate vaccine comprising polysaccharides of Streptococcus pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F conjugated to a carrier protein, the method comprising:

[0402] (a) providing 10 dried carrier protein compositions and 10 dried activated polysaccharide compositions, each dried activated polysaccharide composition comprising a dried activated polysaccharide from a serotype of Streptococcus pneumoniae selected from 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F, wherein none of the 10 dried activated polysaccharide compositions comprises an activated polysaccharide from the same serotype of Streptococcus pneumoniae;

[0403] (b) reconstituting the 10 dried carrier protein compositions and the 10 dried activated polysaccharide compositions with an organic solvent, respectively, and mixing the reconstitution compositions to provide 10 homogeneous carrier protein solutions and 10 homogeneous activated polysaccharide solutions, each homogeneous carrier protein solution comprising the carrier protein, and each homogeneous activated polysaccharide solution comprising an activated polysaccharide of a specific S. pneumoniae serotype, wherein none of the 10 homogeneous solutions comprises an activated polysaccharide from the same S. pneumoniae serotype;

[0404] (c) combining each of the ten homogenous carrier protein solutions with one of the ten homogenous activated polysaccharide solutions by Tee mixing to produce ten mixtures, each mixture comprising a carrier protein and an activated polysaccharide of a specific S. pneumoniae serotype, wherein none of the ten mixtures comprises an activated polysaccharide from the same S. pneumoniae serotype;

[0405] (d) adding a reducing agent to each of the 10 mixtures to produce 10 conjugate solutions, each conjugate solution comprising a carrier protein conjugated to a polysaccharide of a specific S. pneumoniae serotype, wherein none of the 10 conjugate solutions comprises an activated polysaccharide from the same S. pneumoniae serotype; and

[0406] (e) combining the 10 conjugate solutions to provide the multivalent pneumococcal conjugate vaccine containing S. pneumoniae serotype polysaccharides 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F conjugated to a carrier protein.

Claims

1. A method for preparing a composition comprising two or more conjugates, each conjugate comprising a polysaccharide from one or more serotypes of Streptococcus pneumoniae covalently linked to a carrier protein, the method comprising: (a) Provide (i) two or more first aqueous solutions, each first aqueous solution comprising an activated polysaccharide of a particular S. pneumoniae serotype, wherein the polysaccharide has been reacted with an oxidizing agent to provide the activated polysaccharide, and wherein the two or more first aqueous solutions do not contain the same S. pneumoniae serotype polysaccharide; or (ii) two or more first aqueous solutions, each first aqueous solution comprising activated polysaccharides of two or more serotypes of S. pneumoniae, wherein the polysaccharides have been reacted with an oxidizing agent to provide the activated polysaccharides, and wherein the two or more first aqueous solutions do not contain the same polysaccharide of the S. pneumoniae serotype; (b) providing two or more second aqueous solutions, each comprising a carrier protein and a buffer, wherein the amount of the two or more second aqueous solutions corresponds to the amount of at least the 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 sublimation drying process to produce two or more first dried compositions and two or more second dried compositions, each of the first dried compositions comprising a dried polysaccharide and each of the second dried compositions comprising a dried carrier protein; (d) reconstitute each of the two or more first dried compositions and each of the two or more second dried compositions, respectively, in an organic solvent, wherein reconstitution takes eight minutes or less; and mixing to provide two or more first homogenous solutions and two or more second homogenous solutions, each of the first homogenous solutions independently comprising (i) a polysaccharide of a specific S. pneumoniae serotype or (ii) polysaccharides of two or more specific S. pneumoniae serotypes, and each of the second homogenous solutions comprising the carrier protein, wherein the mixing comprises 120 minutes or less; (e) combining each of the first homogeneous solutions with the second homogeneous solution by Tee mixing to produce a plurality of mixtures, each mixture comprising the first homogeneous solution and the second homogeneous solution; (f) adding a reducing agent to each mixture comprising the plurality of mixtures to produce a plurality of conjugate solutions, each conjugate solution independently comprising (i) a polysaccharide of a specific S. pneumoniae serotype or (ii) polysaccharides of two or more specific S. pneumoniae serotypes conjugated to the carrier protein; and (g) combining two or more of the plurality of conjugate solutions to produce a composition comprising two or more conjugates, each conjugate comprising a polysaccharide from one or more serotypes of Streptococcus pneumoniae covalently linked to a carrier protein.

2. The method according to claim 1, wherein at least one conjugate solution is prepared by separately mixing the first homogeneous solution with the second homogeneous solution Tee and adding a reducing agent to produce the plurality of conjugate solutions; or wherein at least one conjugate solution is prepared by separately mixing the first aqueous solution with the second homogeneous solution Tee and adding a reducing agent to produce the plurality of conjugate solutions; or wherein each conjugate solution is prepared by separately mixing the first homogeneous solution with the second homogeneous solution Tee and adding a reducing agent to produce the plurality of conjugate solutions; or Each conjugate solution is prepared by separately mixing the first aqueous solution with the second homogeneous solution Tee and adding a reducing agent to produce the plurality of conjugate solutions.

3. The method of claim 1, wherein the sublimation drying method comprises freeze drying or radiant energy vacuum (REV) dehydration.

4. The method of claim 1, wherein the sublimation drying process comprises freezing the first aqueous solution and the second aqueous solution in the form of cakes or lyophilized pellets prior to the sublimation drying process.

5. The method of claim 1, wherein the sublimation drying comprises batch drying in a container selected from the group consisting of a metal tray, a plastic tray, a plastic bag, and a Type I vial.

6. The method of claim 1, wherein the first aqueous solution and the second aqueous solution comprise about 0.5% (w / v) or more sucrose. The method according to claim 1 , wherein the organic solvent is dimethyl sulfoxide (DMSO).

8. The method of claim 1, wherein each conjugate solution comprises polysaccharide conjugated to the carrier protein at a polysaccharide to carrier protein ratio of about 0.6 to about 1.3 on a weight to weight basis.

9. The method of claim 1, wherein each conjugate solution comprises an amount of free polysaccharide that is less than about 15% of the total polysaccharide in the solution.

10. The method of claim 1, wherein the buffer is histidine, succinate, MES, MOPS, HEPES or acetate buffer in the pH range of 5.0-7.

0.

11. The method of claim 1, wherein the buffer is a phosphate or citrate buffer having a pH range of 5.0-7.

0.

12. The method of claim 18, wherein the one or more and two or more S. pneumoniae serotypes are selected from the group consisting of the following S. pneumoniae serotypes: serotype 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, at least one of CWPS1, CWPS2 and CWPS3.

13. 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 cytolysin, or pneumolysin.

14. The method of claim 13, wherein the diphtheria toxoid is CRM 197 .

15. The method of claim 1, wherein the conjugate solution is sterile filtered.

Images