Method for producing a pneumococcal capsular polysaccharide carrier protein conjugate

Abstract

Describes a method for producing a pneumococcal capsular polysaccharide-protein conjugate, in which one or more activated pneumococcal polysaccharides of a specific pneumococcal serotype and a carrier protein are freeze-dried separately, the separately freeze-dried polysaccharides and carrier proteins are reconstituted separately 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. A plurality of 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.

Description

BACKGROUND OF THE INVENTION

[0001] (1) Field of the Invention

[0002] The present invention relates to a method for producing a pneumococcal capsular polysaccharide - protein conjugate, in which one or more activated pneumococcal polysaccharides of a specific pneumococcal serotype and a carrier protein are separately lyophilized, the separately lyophilized polysaccharide and carrier protein are separately reconstituted in an organic solvent, and then the reconstituted polysaccharide and carrier protein are combined and conjugated together by Tee mixing to produce a polysaccharide - carrier protein conjugate. A plurality of 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 a vaccine.

[0003] (2) Description of the Related Art

[0004] Streptococcus pneumoniae is a capsulated bacterium and is an important cause of serious diseases worldwide. In 1997, the Centers for Disease Control and Prevention (CDC) in the United States estimated that there were 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 in the United States each year. See Centers for Disease Control and Prevention, MMWR Morb Mortal Wkly Rep 1997, 46(RR - 8):1 - 13. In addition, the complications of these diseases can be severe. Some studies have reported that the mortality rate of pneumococcal meningitis is as high as 8% and the neurological sequelae are as high as 25%. See Arditi et al., 1998, Pediatrics 102:1087 - 97.

[0005] The multivalent pneumococcal polysaccharide vaccines that have been licensed for many years have proven to be of inestimable value in preventing pneumococcal diseases in adults, especially the elderly and high - risk populations. However, infants respond poorly to unconjugated pneumococcal polysaccharides. Bacterial polysaccharides are T - cell - independent immunogens that elicit a weak or no response in infants. Chemical conjugation of a bacterial polysaccharide immunogen with a carrier protein can convert the immune response in infants into a T - cell - dependent immune response. Diphtheria toxoid (DTx, a chemically detoxified version of DT) and CRM 197 are described as carrier proteins for bacterial polysaccharide immunogens because of the presence of T - cell - stimulating epitopes in their amino acid sequences.

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

[0007] After the polysaccharide antigen is conjugated to the carrier protein, the reaction mixture can be purified to remove free polysaccharides that do not have the protein conjugated thereto, free carrier proteins that do not have the polysaccharide antigen conjugated thereto, and low molecular weight polysaccharide-protein conjugates. 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. Methods for reducing the amount of free polysaccharides also include co-lyophilizing the carrier protein and the polysaccharide, as disclosed in U.S. Patent No. 7,709,001 and U.S. Patent Application Publication No. 20110201791, which also shows that co-lyophilization of the carrier protein and the polysaccharide is superior to discrete lyophilization of the carrier protein and the polysaccharide, especially for capsular polysaccharide 19A. However, co-lyophilization does not have the ability to optimize individual formulation and cycle parameters in situations where handling is inconvenient and the ability to produce carrier protein and polysaccharide serotype supplies in the desired solution specifications.

[0008] Accordingly, there is a continuing need for improved methods for producing stable polysaccharide-protein conjugates that are free of impurities such as free polysaccharides 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 a pneumococcal vaccine, wherein discrete lyophilized pneumococcal polysaccharides and carrier proteins are prepared using various sublimation methods and then used in a conjugation process to produce a multivalent pneumococcal polysaccharide polysaccharide-protein conjugate for a pneumococcal vaccine. The method of discrete drying provides various advantages over co-lyophilization of the polysaccharide and the 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 in the desired solution specifications.

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

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

[0012] (b) reconstituting the first dried composition and the second dried composition separately in an organic solvent and mixing them to provide a first homogeneous solution and a second homogeneous solution, the first homogeneous solution comprising the activated polysaccharide of the pneumococcal serotype, and the second homogeneous 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, the conjugate solution comprising a carrier protein conjugated to the polysaccharide of one of the pneumococcal serotypes; (B) a method for preparing a composition comprising one or more pneumococcal polysaccharides covalently linked to a carrier protein, the method comprising:

[0015] (a) providing a first dried composition and a second dried composition, the first dried composition comprising an activated pneumococcal polysaccharide from one or more pneumococcal serotypes, and the second dried composition comprising a carrier protein;

[0016] (b) reconstituting the first dried composition and the second dried composition separately in an organic solvent and mixing them to provide a first homogeneous solution and a second homogeneous solution, the first homogeneous solution comprising one or more activated polysaccharides, and the second homogeneous 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, the conjugate solution comprising a carrier protein conjugated to one or more polysaccharides of the pneumococcal serotype; and (C) a method for preparing a composition comprising two or more pneumococcal polysaccharides covalently linked to a carrier protein, the method comprising:

[0019] (a) Provide a first dried composition and a second dried composition, wherein the first dried composition comprises activated pneumococcal polysaccharides from two or more pneumococcal serotypes, and the second dried composition comprises a carrier protein;

[0020] (b) Reconstitute the first dried composition and the second dried composition separately in an organic solvent and mix them to provide a first homogeneous solution and a second homogeneous solution, wherein the first homogeneous solution comprises two or more activated polysaccharides, and the second homogeneous solution comprises the carrier protein;

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

[0022] (d) Add a reducing agent to the mixture to produce a conjugate solution, wherein the conjugate solution comprises a carrier protein conjugated to two or more polysaccharides of pneumococcal serotypes.

[0023] In a particular 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 a first aqueous solution and a second aqueous solution in the form of cakes or lyophilized beads. In another embodiment, the sublimation drying is carried out in a batch drying in a container selected from: metal trays, plastic trays, plastic bags, and type I vials.

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

[0025] In certain embodiments, the first and second dried compositions are prepared by lyophilizing a first aqueous solution and a second aqueous solution to produce a first dried composition and a second dried composition, the first aqueous solution comprising activated pneumococcal polysaccharides from one, two, or more pneumococcal serotypes, the second aqueous solution comprising a carrier protein and a buffer, wherein the first and second aqueous solutions comprise about 0.5% (w / v) or more sucrose, and wherein the lyophilization is selected from freeze-drying and radiant energy vacuum (REV) dehydration. 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.

[0026] In certain embodiments, 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 certain embodiments, 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 certain embodiments, the organic solvent is an aprotic solvent. In certain embodiments, the aprotic solvent is dichloromethane, tetrahydrofuran, ethyl acetate, acetonitrile, dimethylformamide, dimethyl sulfoxide (DMSO), acetone, or hexamethylphosphoric triamide. In certain embodiments, the organic solvent is DMSO.

[0028] In certain embodiments of the method, the reconstitution in step (b) is carried out in eight minutes or less. In certain embodiments, the reconstitution in step (b) is carried out in six minutes or less. In certain embodiments, the reconstitution in step (b) is carried out in four minutes or less. In certain embodiments, the reconstitution in step (b) is carried out in two minutes or less. In certain embodiments, the reconstitution is carried out in about two minutes. In certain embodiments, the reconstitution in step (b) is carried out in one minute or less.

[0029] In certain embodiments, 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 certain embodiments, the mixing in step (b) is carried out in 90 minutes or less. In certain embodiments, the mixing in step (b) is carried out in 60 minutes or less. In certain embodiments, the mixing in step (b) is carried out in 30 minutes or less. In certain embodiments, the mixing in step (b) is carried out in 15 minutes or less. In certain embodiments, the mixing in step (b) is carried out in 10 minutes or less. In certain embodiments, the second homogeneous solution containing the carrier protein is placed in the DMSO solution for about six hours or less before being combined with the first homogeneous solution containing the polysaccharide through a Tee mixer.

[0030] In certain embodiments, each conjugate solution contains a polysaccharide conjugated to the carrier protein at a polysaccharide to carrier protein ratio of about 0.6 to about 1.3 weight to weight. In certain embodiments, each conjugate solution contains a polysaccharide conjugated to the carrier protein at a polysaccharide to carrier protein ratio of about 0.9 to about 1.5 weight to weight. In certain embodiments, each conjugate solution contains a polysaccharide conjugated to the carrier protein at a polysaccharide to carrier protein ratio of about 0.6 to about 1.5 weight to weight.

[0031] In certain embodiments, the conjugate solution contains a free polysaccharide concentration of less than about 15% of the total polysaccharide in the solution. In certain embodiments, the conjugate solution contains a free polysaccharide concentration of less than about 10% of the total polysaccharide in the solution.

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

[0033] In certain embodiments, each conjugate solution contains a polysaccharide conjugated to the carrier protein at a polysaccharide to carrier protein ratio of about 0.6 to about 1.3 weight to weight, 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 (moles / mole).

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

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

[0036] In certain embodiments, the polysaccharide is obtained from Streptococcus pneumoniae serotypes 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, 18C, 19F, 19A, 19B, 19C, 20A, 20B, 21, 22F, 22A, 23F, 23A, 23B, 24F, 24A, 24B, 25F, 25A, 27, 28F, 28A, 29, 31, 32F, 32A, 33F, 33A, 33B, 33C, 33D, 33E, 34, 35F, 35A, 35B, 35C, 36, 37, 38, 39, 40, 41F, 41A, 42, 43, 44, 45, 46, 47F, 47A, 48, CWPS1, CWPS2, and CWPS3.

[0037] In certain embodiments, the Streptococcus pneumoniae polysaccharide is activated by reaction with an oxidizing agent.

[0038] In certain embodiments, the carrier protein is an inactivated bacterial toxoid selected from the group consisting of: tetanus toxoid, diphtheria toxoid, pertussis toxoid, bacteriolysin, or pneumolysin. In certain embodiments, 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 polysaccharides from one or more Streptococcus pneumoniae serotypes, and (ii) a second aqueous solution comprising a carrier protein and a buffer;

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

[0043] (c) Reconstituting the first dried composition and the second dried composition by adding the dried composition to an organic solvent and mixing separately to provide a first homogeneous solution and a second homogeneous solution, wherein the first homogeneous solution comprises the one or more activated polysaccharides and the second homogeneous solution comprises 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, the conjugate solution comprising the carrier protein conjugated to the one or more activated polysaccharides of the Streptococcus pneumoniae serotype.

[0046] In a particular 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 cakes or lyophilized beads. In another embodiment, the sublimation drying is carried out in batch in a container selected from: metal trays, plastic trays, plastic bags and type I glass vials.

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

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

[0049] In a particular 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 particular 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 certain embodiments, the organic solvent is an aprotic solvent. In certain embodiments, the aprotic solvent is dichloromethane, tetrahydrofuran, ethyl acetate, acetonitrile, dimethylformamide, dimethyl sulfoxide (DMSO), acetone, or hexamethylphosphoric triamide. In certain embodiments, the organic solvent is DMSO.

[0051] In certain embodiments of the method, the reconstitution in step (c) is carried out in eight minutes or less. In certain embodiments, the reconstitution in step (c) is carried out in six minutes or less. In certain embodiments, the reconstitution in step (c) is carried out in four minutes or less. In certain embodiments, the reconstitution in step (c) is carried out in two minutes or less. In certain embodiments, the reconstitution comprises about two minutes. In certain embodiments, the reconstitution in step (c) is carried out in one minute or less.

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

[0053] In certain embodiments, each conjugate solution contains a polysaccharide conjugated to the carrier protein at a polysaccharide to carrier protein ratio of about 0.6 to about 1.3 weight to weight. In certain embodiments, each conjugate solution contains a polysaccharide conjugated to the carrier protein at a polysaccharide to carrier protein ratio of about 0.9 to about 1.5 weight to weight. In certain embodiments, each conjugate solution contains a polysaccharide conjugated to the carrier protein at a polysaccharide to carrier protein ratio of about 0.6 to about 1.5 weight to weight.

[0054] In certain embodiments, the conjugate solution contains a free polysaccharide concentration of less than about 15% of the total polysaccharide in the solution. In certain embodiments, the conjugate solution contains a free polysaccharide concentration of less than about 10% of the total polysaccharide in the solution.

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

[0056] In certain embodiments, each conjugate solution comprises a polysaccharide conjugated to the carrier protein at a polysaccharide to carrier protein ratio of from about 0.6 to about 1.3 weight to weight, 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 (moles / mole).

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

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

[0059] In certain embodiments, the one or more Streptococcus pneumoniae serotypes are obtained from Streptococcus pneumoniae serotypes selected from: 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, CWPS1, CWPS2 and CWPS3.

[0060] In certain embodiments, the Streptococcus pneumoniae polysaccharide is activated by reaction with an oxidizing agent.

[0061] In certain embodiments, the carrier protein is an inactivated bacterial toxoid selected from: tetanus toxoid, diphtheria toxoid, pertussis toxoid, bacteriolysin or pneumolysin. In certain embodiments, 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 that comprises two or more conjugates, each conjugate containing a Streptococcus pneumoniae polysaccharide from one or more serotypes covalently linked to a carrier protein, the method comprising:

[0064] (a) providing

[0065] (i) two or more first aqueous solutions, each first aqueous solution comprising an activated polysaccharide of a specific Streptococcus 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 Streptococcus pneumoniae serotypes, 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 amounts of the two or more second aqueous solutions correspond to at least the amounts of 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 lyophilization process to produce two or more first dried compositions and two or more second dried compositions, the first dried compositions each comprising a dried polysaccharide and the second dried compositions each comprising a dried carrier protein;

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

[0070] (e) combining each first homogeneous solution with a second homogeneous solution respectively 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 said multiple conjugate solutions to produce a composition comprising two or more conjugates, each conjugate comprising a Streptococcus pneumoniae polysaccharide from one or more serotypes covalently linked to a carrier protein.

[0073] In certain embodiments of the method, at least one conjugate solution is prepared by separately Tee mixing a first homogeneous solution and a second homogeneous solution to produce multiple conjugate solutions; or, wherein at least one conjugate solution is prepared by separately Tee mixing a first aqueous solution and a second homogeneous solution to produce multiple conjugate solutions; or, wherein each conjugate solution is prepared by separately Tee mixing a first homogeneous solution and a second homogeneous solution to produce multiple conjugate solutions; or, wherein each conjugate solution is prepared by separately Tee mixing a first aqueous solution and a second homogeneous solution to produce multiple conjugate solutions.

[0074] In certain embodiments of the method, the lyophilization method is selected from freeze drying and radiant energy vacuum (REV) dehydration. In a further embodiment, the lyophilization method comprises freezing the first aqueous solution and the second aqueous solution in the form of cakes or lyophilized beadlets. In another embodiment, the lyophilization is carried out in a batch drying in a container selected from: metal trays, plastic trays, plastic bags, and type I vials.

[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 of sucrose. In certain embodiments, the first aqueous solution comprises about 4% to 6% (w / v) of sucrose and the second aqueous solution comprises about 4% to 8% (w / v) of sucrose.

[0077] In certain embodiments, 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 certain embodiments, 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 certain embodiments, the organic solvent is an aprotic solvent. In certain embodiments, the aprotic solvent is dichloromethane, tetrahydrofuran, ethyl acetate, acetonitrile, dimethylformamide, dimethyl sulfoxide (DMSO), acetone, or hexamethylphosphoric triamide. In certain embodiments, the organic solvent is DMSO.

[0079] In certain embodiments of the method, the reconstitution in step (d) is carried out in eight minutes or less. In certain embodiments, the reconstitution in step (d) is carried out in six minutes or less. In certain embodiments, the reconstitution in step (d) is carried out in four minutes or less. In certain embodiments, the reconstitution in step (d) is carried out in two minutes or less. In certain embodiments, the reconstitution comprises about two minutes. In certain embodiments, the reconstitution in step (d) is carried out in one minute or less.

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

[0081] In certain embodiments, each conjugate solution comprises a polysaccharide conjugated to the carrier protein at a polysaccharide to carrier protein ratio of from about 0.6 to about 1.3 weight to weight. In certain embodiments, each conjugate solution comprises a polysaccharide conjugated to the carrier protein at a polysaccharide to carrier protein ratio of from about 0.9 to about 1.5 weight to weight. In certain embodiments, each conjugate solution comprises a polysaccharide conjugated to the carrier protein at a polysaccharide to carrier protein ratio of from about 0.6 to about 1.5 weight to weight.

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

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

[0084] In certain embodiments, each conjugate solution comprises a polysaccharide conjugated to the carrier protein at a polysaccharide to carrier protein ratio of from about 0.6 to about 1.3 weight to weight, 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 (moles / mole).

[0085] In certain embodiments, the buffer is a histidine, succinate, MES, MOPS, HEPES or acetate buffer having a 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 certain embodiments, the polysaccharide is obtained from Streptococcus pneumoniae serotypes selected from: 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, CWPS1, CWPS2, and CWPS3.

[0088] In certain embodiments, the Streptococcus pneumoniae polysaccharide is activated by reaction with an oxidizing agent.

[0089] In certain embodiments, the carrier protein is an inactivated bacterial toxoid selected from: tetanus toxoid, diphtheria toxoid, pertussis toxoid, bacteriolysin, or pneumolysin. In certain embodiments, 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 containing Streptococcus pneumoniae serotype polysaccharides 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F conjugated to a carrier protein, the method comprising:

[0092] (a) Provide 23 dry carrier protein compositions and 23 dry activated polysaccharide compositions, each dry activated polysaccharide composition comprising dry activated polysaccharide from a Streptococcus pneumoniae serotype selected from 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F, wherein none of the 23 dry activated polysaccharide compositions comprises activated polysaccharide from the same Streptococcus pneumoniae serotype;

[0093] (b) Reconstitute the 23 dry carrier protein compositions and the 23 dry activated polysaccharide compositions separately with an organic solvent and mix them 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 activated polysaccharide of a specific Streptococcus pneumoniae serotype, wherein none of the 23 homogeneous solutions comprises activated polysaccharide from the same Streptococcus pneumoniae serotype;

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

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

[0096] (e) Combine the 23 conjugate solutions to provide the multivalent immunogenic complex or vaccine against 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.

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

[0098] (a) Provide 15 dry carrier protein compositions and 15 dry activated polysaccharide compositions, each dry activated polysaccharide composition comprising dry activated polysaccharide from a Streptococcus pneumoniae serotype selected from 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, and 33F, wherein none of the 15 dry activated polysaccharide compositions comprises activated polysaccharide from the same Streptococcus pneumoniae serotype;

[0099] (b) Reconstitute the 15 dry carrier protein compositions and the 15 dry activated polysaccharide compositions separately with an organic solvent and mix them to provide 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 activated polysaccharide of a specific Streptococcus pneumoniae serotype, wherein none of the 15 homogeneous solutions comprises activated polysaccharide from the same Streptococcus pneumoniae serotype;

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

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

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

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

[0104] (a) Provide 13 dry carrier protein compositions and 13 dry activated polysaccharide compositions, each dry activated polysaccharide composition containing dry activated polysaccharide from Streptococcus pneumoniae serotypes selected from 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F, wherein none of the 13 dry activated polysaccharide compositions contain activated polysaccharide from the same Streptococcus pneumoniae serotype;

[0105] (b) Reconstitute the 13 dry carrier protein compositions and the 13 dry activated polysaccharide compositions separately with an organic solvent and mix them to provide 13 homogeneous carrier protein solutions and 13 homogeneous activated polysaccharide solutions, each homogeneous carrier protein solution containing the carrier protein and each homogeneous activated polysaccharide solution containing activated polysaccharide of a specific Streptococcus pneumoniae serotype, wherein none of the 13 homogeneous solutions contain activated polysaccharide from the same Streptococcus pneumoniae serotype;

[0106] (c) Combine each of the 13 homogeneous carrier protein solutions with one of the 13 activated polysaccharide homogeneous solutions by Tee mixing to produce 13 mixtures, each mixture containing a carrier protein and activated polysaccharide of a specific Streptococcus pneumoniae serotype, wherein none of the 13 mixtures contain activated polysaccharide from the same Streptococcus pneumoniae serotype;

[0107] (d) Add a reducing agent to each of the 13 mixtures to produce 13 conjugate solutions, each conjugate solution containing a carrier protein conjugated to polysaccharide of a specific Streptococcus pneumoniae serotype, wherein none of the 13 conjugate solutions contain activated polysaccharide from the same Streptococcus pneumoniae serotype; and

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

[0109] The present invention also provides a method for preparing a multivalent pneumococcal conjugate vaccine containing 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) Provide 10 dry carrier protein compositions and 10 dry activated polysaccharide compositions, each dry activated polysaccharide composition containing dry activated polysaccharide from Streptococcus pneumoniae serotypes selected from 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, and 23F, wherein none of the 10 dry activated polysaccharide compositions contain activated polysaccharide from the same Streptococcus pneumoniae serotype;

[0111] (b) Reconstitute each of the 10 dried carrier protein compositions and the 10 dried activated polysaccharide compositions with an organic solvent and mix them to provide 10 homogeneous carrier protein solutions and 10 homogeneous activated polysaccharide solutions, each homogeneous carrier protein solution containing the carrier protein and each homogeneous activated polysaccharide solution containing the activated polysaccharide of a specific Streptococcus pneumoniae serotype, wherein none of the 10 homogeneous solutions contains the activated polysaccharide from the same Streptococcus pneumoniae serotype;

[0112] (c) Combine each of the 10 homogeneous carrier protein solutions with one of the 10 homogeneous activated polysaccharide solutions by Tee mixing to produce 10 mixtures, each mixture containing the carrier protein and the activated polysaccharide of a specific Streptococcus pneumoniae serotype, wherein none of the 10 mixtures contains the activated polysaccharide from the same Streptococcus pneumoniae serotype;

[0113] (d) Add a reducing agent to each of the 10 mixtures to produce 10 conjugate solutions, each conjugate solution containing the carrier protein conjugated to the polysaccharide of a specific Streptococcus pneumoniae serotype, wherein none of the 10 conjugate solutions contains the activated polysaccharide from the same Streptococcus pneumoniae serotype; and

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

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

[0116] In certain embodiments of the method, each of the dried carrier protein and dried polysaccharide compositions is prepared by a sublimation drying method, the sublimation drying method including freeze-drying or radiant energy vacuum (REV) dehydration. In another embodiment, the sublimation drying is carried out in a batch drying in a container selected from: metal trays, plastic trays, plastic bags, and type I vials. In a further embodiment, the sublimation drying method includes freezing each aqueous solution containing the carrier protein and each aqueous solution containing the polysaccharide in the form of cakes or lyophilized beadlets, respectively.

[0117] In certain embodiments, a composition of a dried carrier and a dried polysaccharide is prepared by lyophilizing a plurality of separate aqueous polysaccharide solutions and a plurality of aqueous carrier protein solutions, each aqueous polysaccharide solution comprising an activated pneumococcal polysaccharide from a specific enumerated pneumococcal serotype as shown above, each carrier protein solution comprising a carrier protein and a buffer, to produce a dried polysaccharide and carrier protein composition, wherein the number of aqueous carrier protein solutions corresponds at least to the number of aqueous polysaccharide solutions, and wherein the aqueous solutions of the polysaccharide and the carrier protein each comprise about 0.5% (w / v) or more sucrose, and wherein the lyophilization is selected from freeze drying and radiant energy vacuum (REV) dehydration.

[0118] In certain embodiments, the aqueous solutions of the polysaccharide and the carrier protein each comprise about 0.5% (w / v) or more sucrose. In certain embodiments, the aqueous polysaccharide solutions each comprise about 4% to 6% (w / v) sucrose and the aqueous carrier protein solutions each comprise about 4% to 8% (w / v) sucrose.

[0119] In certain embodiments, the aqueous polysaccharide solutions each comprise the polysaccharide at a concentration of about 6 to 9 mg / mL and the aqueous carrier protein solutions each comprise the carrier protein at a concentration of about 6 to 12 mg / mL. In certain embodiments, the aqueous polysaccharide solutions each comprise the polysaccharide at a concentration of about 6 or 9 mg / mL and the aqueous carrier protein solutions each comprise the carrier protein at a concentration of about 6, 9, 10, or 12 mg / mL.

[0120] In certain embodiments, the organic solvent is an aprotic solvent. In certain embodiments, the aprotic solvent is dichloromethane, tetrahydrofuran, ethyl acetate, acetonitrile, dimethylformamide, dimethyl sulfoxide (DMSO), acetone, or hexamethylphosphoramide. In certain embodiments, the organic solvent is DMSO.

[0121] In certain embodiments of the method, the reconstitution in step (b) is carried out in eight minutes or less. In certain embodiments, the reconstitution in step (b) is carried out in six minutes or less. In certain embodiments, the reconstitution in step (b) is carried out in four minutes or less. In certain embodiments, the reconstitution in step (b) is carried out in two minutes or less. In certain embodiments, the reconstitution comprises about two minutes. In certain embodiments, the reconstitution in step (b) is carried out in one minute or less.

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

[0123] In certain embodiments, each conjugate solution contains a polysaccharide conjugated to the carrier protein at a polysaccharide to carrier protein ratio of about 0.6 to about 1.3 weight to weight. In certain embodiments, each conjugate solution contains a polysaccharide conjugated to the carrier protein at a polysaccharide to carrier protein ratio of about 0.9 to about 1.5 weight to weight. In certain embodiments, each conjugate solution contains a polysaccharide conjugated to the carrier protein at a polysaccharide to carrier protein ratio of about 0.6 to about 1.5 weight to weight.

[0124] In certain embodiments, the conjugate solution contains a free polysaccharide concentration of less than about 15% of the total polysaccharide in the solution. In certain embodiments, the conjugate solution contains a free polysaccharide concentration of less than about 10% of the total polysaccharide in the solution.

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

[0126] In certain embodiments, each conjugate solution contains a polysaccharide conjugated to the carrier protein at a polysaccharide to carrier protein ratio of about 0.6 to about 1.3 weight to weight, 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 (moles / mole).

[0127] In certain embodiments, the Streptococcus 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 1Shows the binding of components and the effects on component concentrations, polysaccharide (Ps) concentration, CRM 197 (Pr) concentration, and the ratio of Ps to Pr (Ps:Pr) during the addition of Pr solution to Ps solution (initially dissolving the components in anhydrous DMSO at 6 mg / mL and combining to a final Ps:Pr ratio of 1:1).

[0130] Figure 2 Shows the binding of components and the effects on component concentrations, polysaccharide (Ps) concentration, CRM 197 (Pr) concentration, and the ratio of Ps to Pr (Ps:Pr) during the addition of Ps solution to Pr solution (initially dissolving the components in anhydrous DMSO at 6 mg / mL and combining to a final Ps:Pr ratio of 1:1).

[0131] Figure 3 Shows the binding of components and the effects on component concentrations, polysaccharide (Ps) concentration, CRM 197 (Pr) concentration, and the ratio of Ps to Pr (Ps:Pr) during the addition of lyophilized Ps to Pr solution (initially dissolving Pr in anhydrous DMSO at 3 mg / mL and using it to dissolve lyophilized Ps).

[0132] Figure 4 Shows the binding of components and the effects on component concentrations, polysaccharide (Ps) concentration, CRM 197 (Pr) concentration, and the ratio of Ps to Pr (Ps:Pr) during the simultaneous addition of Pr solution and Ps solution to a separate conjugation container (initially dissolving the components in anhydrous DMSO at 6 mg / mL and with the same volumetric addition rate of the components).

[0133] Figure 5 Shows the FTIR spectra of CRM 197 reconstituted rapidly (two minutes) compared to slowly (eight minutes) in anhydrous DMSO.

[0134] Figure 6 Shows the FTIR spectra of CRM 197 reconstituted at an intermediate rate (four minutes) in anhydrous DMSO, followed by increasing the holding time (30 minutes; 60 minutes; 120 minutes; 180 minutes; 240 minutes; 300 minutes).

[0135] Figure 7 Shows the FTIR spectra of CRM 197 reconstituted rapidly (two minutes) in anhydrous DMSO at different concentrations (10 mg / mL; 30 mg / mL; 50 mg / mL).

[0136] Figure 8A and 8B shows the dynamic light scattering (DLS) profiles of CRM Figure 8A ) and 30 mg / mL ( Figure 8B ) rapidly reconstituted (two minutes) in anhydrous DMSO. Three measurements (three traces) were taken for each sample. 197

[0137] Figure 9 shows the FTIR spectra of CRM 197 rapidly reconstituted (two minutes) in DMSO containing different levels of water (anhydrous DMSO; 99% DMSO / water; 97% DMSO / water; 95% DMSO / water).

[0138] Figure 10 shows the FTIR spectra (rapid (two minutes); slow (eight minutes)) of very dry lyophilized CRM 197 rapidly reconstituted (two minutes) compared to slowly reconstituted (eight minutes) in anhydrous DMSO.

[0139] Figure 11 shows CRM 197 : the FTIR spectra of the polysaccharide serotype 6B (CRM 197 -6B) conjugate, where CRM 197 has been rapidly (two minutes) or slowly (eight minutes) reconstituted prior to conjugation to polysaccharide 6B. Also shown is CRM 197 conjugated to polysaccharide 6B under aqueous conditions.

[0140] Figure 12 shows a general flow chart for preparing carrier protein (Pr)-polysaccharide (Ps) conjugates.

[0141] Figure 13 shows the size of the conjugate produced from the reaction using polysaccharides of increasing size. Shown in the figure is that slower addition of anhydrous DMSO can result in even larger conjugates from the same size polysaccharides compared to rapid addition of anhydrous DMSO. DETAILED DESCRIPTION

[0142] I. Definitions

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

[0144] As used herein, when used in connection with an immunogenic composition of the invention, the term "comprising" means including any other components (subject to the limitation of the term "consisting of" for antigen mixtures), such as adjuvants and excipients. When used in connection with a mixture of multivalent polysaccharide-protein conjugates, the term "consisting of" means a mixture having those specific pneumococcal polysaccharide-protein conjugates and no other pneumococcal polysaccharide-protein conjugates from different serotypes.

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

[0146] As used herein, the term "reconstituting / reconstitution" refers to adding a liquid to a dry substance to dissolve the dry 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 agitated 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, swirling, rotating, etc.

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

[0149] As used herein, a "lyophilized bead" is a discrete particle of a lyophilized substance, e.g., in the form of a bead or sphere or other shape. A lyophilized bead may also be referred to as a lyophilized particle bead or a lyophilized bead. In some embodiments, the diameter of the lyophilized bead 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 lyophilized bead is from about 20 to 550 μL, preferably from 20 to 100 μL, such as from 20 to 50 μL. In embodiments where the lyophilized bead is substantially non-spherical, the dimensions of the lyophilized bead can be described in terms of its aspect ratio, which is the ratio of the major axis to the minor axis. The aspect ratio of the lyophilized bead can 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 that contains one or more antigens conjugated to one or more carrier proteins. In certain embodiments of the invention, the antigen is a sugar from an encapsulated bacterium. In such compositions, the sugar consists of long chains of sugar molecules similar to those 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 can be from different organisms. In a preferred embodiment of the invention, the antigen is Streptococcus pneumoniae capsular polysaccharide.

[0151] As used herein, the term "radiant energy vacuum (REV) dehydration" is also referred to 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 that can be used as an anti-pneumococcal vaccine, wherein the pneumococcal polysaccharide and the carrier protein are discretely (or separately) lyophilized using various sublimation methods, and then the discretely lyophilized polysaccharide and carrier protein are mixed under conditions that result in the formation of a conjugate composition having a lower 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 having a lower level 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 used co-lyophilization of the carrier protein and the polysaccharide because it has been shown that co-lyophilization of the polysaccharide and the carrier protein together results in the production of a conjugate composition having less free polysaccharide (less than 18%), while as shown by the results in Table 1 of U.S. Patent Application Publication No. 20110201791, a conjugate composition produced by combining discretely lyophilized polysaccharide and carrier protein results in a composition having approximately 31% free polysaccharide.

[0155]

[0156] However, the inventors of the present invention have found that under certain conditions, discretely (or separately) lyophilized carrier protein and activated polysaccharide can be conjugated to produce a composition containing high molecular weight conjugates, but different from those shown in U.S. Patent Application Publication No. 20110201791, the composition has a lower level of free polysaccharide. These specific conditions include providing a solution of the carrier protein in an organic solvent and a solution containing one or more polysaccharides in an organic solvent, wherein in a particular embodiment, the organic solvent is dimethyl sulfoxide (DMSO), and the two solutions are combined or merged together simultaneously by Tee mixing. As Figure 4As shown, the two solutions are simultaneously mixed together by Tee mixing, which can significantly reduce or eliminate the concentration gradient that usually occurs when two solutions are combined by adding one solution to another or dissolving dry material into a solution (see Figure 1 , 2 or 3), and as exemplified by the serotype 19F polysaccharide in Example 10, which can reduce or eliminate the gel formation of polysaccharides obtained from various serotypes. Compared with the co-lyophilization of polysaccharide and carrier protein in a single composition, the present invention has various advantages, including but not limited to the ability to optimize individual formulations and cycling parameters, ease of handling, and the ability to produce individual polysaccharide and carrier protein formulations with the required solution specifications.

[0157] The inventors also found that using CRM 197 as a model, the length of time for adding an organic solvent (such as DMSO) to the dry carrier protein during the reconstitution of the dry carrier protein and the length of time for storing the reconstituted carrier protein subsequent to conjugation before conjugation affect the secondary structure of the carrier protein and thus affect the yield of the conjugate having the desired molecular weight and the ratio of the carrier protein to the polysaccharide conjugated thereto. As Figures 5 - 11 shown, as determined by Fourier transform infrared spectroscopy (FTIR) or dynamic light scattering (DLS), the time for reconstituting the dry carrier protein in an organic solvent (such as anhydrous DMSO) under rapid addition conditions (for example, adding the organic solvent to the dry 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 unfolded and no detectable β-sheet formation occurs. It was further found that the presence of any detectable water in the organic solvent and / or a carrier protein with a final concentration of the carrier protein greater than 12 mg / mL would result in β-sheet-mediated aggregation. Therefore, in a preferred embodiment of the present invention, the organic solvent is 100% of the organic solvent and contains no detectable water (for example, anhydrous organic solvent); the organic solvent is added to the dry carrier protein within 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 at or less than 10 mg / mL or 12 mg / mL or lower, and in a particular embodiment, at about 6 mg / mL.

[0158] In certain embodiments, to ensure complete dissolution of the dried carrier protein and polysaccharide, the first homogenized solution and the second homogenized solution can be mixed for 120 minutes or less. In certain embodiments, the mixing can be for 90 minutes or less. In certain embodiments, the mixing can be for 60 minutes or less. In certain embodiments, the mixing can be for 30 minutes or less. In certain embodiments, the mixing can be for 10 minutes or less. In certain embodiments, the second homogenized solution containing the carrier protein is held for about six hours or less before being combined with the first homogenized solution containing the polysaccharide by Tee mixing. The inventors have found that holding the carrier protein in an anhydrous organic solvent for more than six hours can lead to the formation of detectable β-sheet mediated carrier protein aggregates. Thus, in certain embodiments, the carrier protein is held in DMSO for six hours or less.

[0159] Figure 12 A general flowchart for preparing a carrier protein-polysaccharide conjugate is shown, where a homogenized solution of the carrier protein is prepared according to the teachings herein, and a homogenized solution of the carrier protein and polysaccharide is prepared by Tee mixing according to the teachings herein.

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

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

[0162] In ultrafiltration step 2, diafiltration of the activated polysaccharide can be performed for all serotypes, followed by concentration by tangential flow ultrafiltration using a 10 kDa ultrafiltration membrane with open channels to produce diafiltrate 2. At the end of 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] Then, diafiltrate 2 is diluted in water containing sucrose such that the final concentration is about 4 to 12 mg / mL polysaccharide and 0.5% to 6% (w / v) sucrose, and it is subjected to a sublimation drying method to produce dried polysaccharide, preferably with a final water content of 6% or less. In a particular embodiment, the final water content of the composition is about 5% or less. In a particular embodiment, the final water content of the composition is about 4% or less. In a particular embodiment, the final water content of the composition is about 3% or less. In a particular embodiment, the final water content of the composition is about 2% or less. The amount of sucrose added to the solution prior to lyophilization is serotype - specific and can range from 3.0 to 5.0% (w / v) prior to lyophilization. In a particular embodiment, two or more polysaccharides can be dried together to produce a dried polysaccharide mixture.

[0164] The purified carrier protein is diafiltered using a 5 kDa tangential flow ultrafiltration membrane with 2 mM phosphate buffer (pH 7.0), and then filtered through a 0.22 micron filter. The filtered solution is diluted in water containing sucrose such that the final concentration is about 6 to 12 mg / mL carrier protein and 4 to 10% (w / v) sucrose, and is subjected to sublimation drying during the process to produce dried carrier protein with a final water content of 6% or less. In a particular embodiment, the final water content of the composition is about 5% or less. In a particular embodiment, the final water content of the composition is about 4% or less. In a particular embodiment, the final water content of the composition is about 3% or less. In a particular embodiment, the final water content of the composition is about 2% or less. The sublimation drying method can include lyophilization or radiant energy vacuum (REV) dehydration.

[0165] The dried polysaccharide or dried polysaccharide mixture and the dried carrier protein are separately reconstituted or redissolved in an organic solvent (e.g., dimethyl sulfoxide (DMSO)). In a particular 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 1% (v / v) or more water content in DMSO will result in β - sheet mediated aggregation (see Figure 9)。Therefore, the present invention provides embodiments in which the water content of the organic solvent is less than 1% (v / v) or in which the organic solvent does not contain detectable water (e.g., anhydrous organic solvent or 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 in 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 of the anhydrous organic solvent to the dried carrier protein, the less formation of irreversible β-sheet-mediated aggregation. As Figure 5 shown, an addition time of two minutes for anhydrous DMSO resulted in no detectable β-sheet-mediated aggregation. Thus, in certain embodiments, the present invention provides for reconstituting the carrier protein in an organic solvent containing less than 1% water (e.g., DMSO) or an organic solvent without detectable water (e.g., anhydrous or 100% (v / v) organic solvent), wherein the added organic solvent is added to the dried carrier protein over a period of two minutes or less to provide a reconstituted carrier protein having a concentration of 20 mg / mL or less or about 12 mg / mL, or about 10 mg / mL or less. In certain embodiments, after reconstitution, 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 certain embodiments, the 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 certain embodiments, 500 mM sodium phosphate buffer (pH 7.2) is added to the homogeneous solution containing the carrier protein such that the final concentration is 1.0 mM sodium phosphate.

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

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

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

[0170] To quench the conjugation reaction, for conjugates containing polysaccharides from a specific serotype, the conjugation solution is diluted to 20% (v / v) or less anhydrous DMSO in a dilution step by slowly adding the conjugation 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 certain embodiments, the temperature is maintained at 15 °C or lower 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. The conjugation performance can be evaluated by the consumption of total polysaccharide and carrier protein, the ratio of conjugate polysaccharide to carrier protein, and the conjugate molecular weight.

[0171] In ultrafiltration step 3, the quenched conjugate solution can be concentrated to approximately 2.5 g / L and diafiltered using a 30 kDa tangential flow ultrafiltration membrane with 150 mM sodium chloride or 150 mM sodium chloride containing 25 mM potassium phosphate at 2 - 8 °C using up to approximately 10 diafiltration volumes 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 (such as serotype 19F), diafiltrate 3 can be filtered through a 0.22 micron filter to produce a filtrate, which is then incubated at 22 °C for approximately 120 hours.

[0172] In ultrafiltration step 4, the retentate 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 with 20 diafiltration volumes of 150 mM sodium chloride containing 10 mM L-histidine (pH 7.0) at 2 - 8 °C to produce retentate 4. For conjugates containing polysaccharides from certain serotypes (e.g., serotype 7F), the conjugate can be diafiltered using a 100 kDa tangential flow ultrafiltration membrane; for example, a conjugate containing serotype 6A, 6B, and 18C polysaccharides can be concentrated to about 3.5 g / L and diafiltered using a 300 kDa tangential flow ultrafiltration membrane with a buffer containing 150 mM sodium chloride and 0.03% (w / v) polysorbate 20 (pH 7.0) at 2 - 8 °C to produce retentate 4. Conjugates containing 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 with a buffer containing 150 mM sodium chloride and 0.015% (w / v) polysorbate 20 (pH 7.0) at 2 - 8 °C to produce retentate 4. The polysaccharide concentration in retentate 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 retentate 4 from 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 containing 10 mM L-histidine (pH 7.0). This provides a monovalent bulk conjugate intermediate (MBC) or monovalent drug substance. The MBC can be aliquoted and frozen at -60 °C to -80 °C.

[0174] For conjugates containing serotype 6A, 6B, and 18C polysaccharides, the retentate 4 from ultrafiltration step 4 can be filtered through a dual 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 in 150 mM sodium chloride, 0.03% (w / v) polysorbate 20, pH 7.0. This provides the MBC or monovalent drug substance. The MBC can be aliquoted and frozen at -60 °C to -80 °C.

[0175] For conjugates containing polysaccharides 7F, 19A, 19F, and 23F, the filtrate 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 in 150 mM sodium chloride, 0.015% (w / v) polysorbate 20, pH 7.0. This provides the MBC or the monovalent API. The MBC can be aliquoted 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, the polysaccharides can be isolated from the bacteria and sized to a certain extent by known methods (see, e.g., European Patent Nos. EP497524 and EP497525); and in certain embodiments, microfluidization accomplished using a homogenizer or chemical hydrolysis. In one embodiment, a Streptococcus pneumoniae strain is grown in a soy-based medium. The various polysaccharides are then purified by standard procedures including centrifugation, precipitation, and ultrafiltration. See, e.g., U.S. Patent Application Publication No. 2008 / 0286838 and U.S. Patent No. 5,847,112. The polysaccharides can be sized to reduce viscosity and / or improve the filterability of the subsequent conjugate product. In the present invention, capsular polysaccharides are 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 is used as the carrier protein. CRM 197 is a non-toxic variant of diphtheria toxin (i.e., a toxoid). In one embodiment, CRM is isolated from a culture of Corynebacterium diphtheriae strain C7(β197) grown in a casein amino acid and yeast extract-based medium. 197Carrier protein. In another embodiment, CRM is recombinantly prepared according to the method described in U.S. Patent No. 5,614,382 197 . Typically, CRM is purified by a combination of ultrafiltration, ammonium sulfate precipitation, and ion exchange chromatography 197 . In some embodiments, PFENEX EXPRESSION TECHNOLOGY (Pfenex Inc., San Diego, CA) is used to prepare CRM in Pseudomonas fluorescens 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. WO2004 / 083251), Escherichia coli LT, Escherichia coli ST, and Pseudomonas aeruginosa exotoxin A. Bacterial outer membrane proteins can also be used, 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 B streptococci, or Haemophilus influenzae protein D, pneumolysin (Kuo et al., 1995, Infect Immun 63:2706-13), including ply 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 fusion, PhtBE fusion (see International Patent Application Publication Nos. WO 01 / 98334 and WO 03 / 54007). 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; e.g., see European Patent No. EP 0 594 610B) or its immunologically equivalent, synthetic peptides (see European Patent Nos. EP0378881 and EP0427347), heat shock proteins (see International Patent Application Publication Nos. WO 93 / 17712 and WO 94 / 03208), pertussis proteins (see International Patent Application Publication Nos. WO98 / 58668 and European Patent No. EP0471177), cytokines, lymphokines, growth factors or hormones (see International Patent Application Publication No. WO 91 / 01146), artificial proteins containing multiple human CD4+ T cell epitopes derived from various pathogen antigens (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 proteins (see International Patent Application Publication No. WO01 / 72337), toxin A or B of Clostridium 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, J Biol 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; Glu-148 deletion or mutation to Asp, Gln or Ser and / or Ala 158 mutation 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 of the 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. Lyophilization

[0183] Compositions containing polysaccharides of specific Streptococcus pneumoniae serotypes and compositions containing carrier proteins can be dried separately using lyophilization methods to provide a composition containing a dried carrier protein with a final moisture content of 6% or less and a composition containing a dried polysaccharide of a specific Streptococcus pneumoniae serotype with a final moisture content of about 6% or less. For example, the dried compositions 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 lyophilized formulation components can be in the form of cakes or granules, e.g., pellets, beads or spheres of the lyophilized material, such as lyophilized balls. See, for example, A.S. Mujumdar (2007). Handbook of Industrial Drying. CRC Press.

[0184] For example, lyophilized beads can be prepared by loading an aliquot of an aqueous solution containing one or more polysaccharide serotypes in the form of droplets (e.g., about 20, 50, 100, or 250 microliters) onto a solid plane in such a way 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 means of 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 dispensed aqueous solution is 250 microliters and the dispensing rate is between about 5 mL / min and about 75 mL / min, or wherein the aliquot is 100 microliters and the dispensing rate is between about 3 mL / min and about 60 mL / min. In one embodiment of the invention, the gap between the dispensing tip and the surface on which the aqueous solution is dispensed is about 0.1 cm or greater (e.g., about 0.5 cm or between 0.1 cm and 1 cm or between 0.1 cm and 0.75 cm). Once on the surface, the aqueous solution is frozen and then lyophilized by sublimation drying. Methods for preparing lyophilized beads 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 lyophilized by radiation energy vacuum (REV) dehydration (microwave vacuum drying). REV dehydration is a drying method carried out under reduced pressure conditions in which the boiling point of water and the oxygen content in 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 a single droplet, wherein the surface temperature is about -90°C or lower, and the droplet is maintained as a single droplet when it contacts and freezes on the surface as a frozen bead; microwave radiation is applied to the frozen bead at a pressure below atmospheric pressure to produce a dried bead, such as spherical. In another example, the aqueous solution is provided on a tray and microwave radiation is carried out 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 a carrier protein. Once activated, each capsular polysaccharide is conjugated to the carrier protein separately to form a glycoconjugate according to the method of the present invention.

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

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

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

[0191] It is known that transition metals can form stable complexes with cyanides, and it is known that the reductive methylation of protein amino groups and formaldehyde with sodium cyanoborohydride can be improved. See Gidley et al., Biochem J. 1982, 203: 331-334; Jentoft et al., Anal Biochem. 1980, 106: 186-190. However, the applicant has surprisingly found that by complexing residual interfering cyanides, the addition of nickel increases the consumption of protein during the conjugation process and results in the formation of larger and possibly more immunogenic conjugates.

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

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

[0194] In an alternative embodiment, reactive homobifunctional or heterobifunctional groups can be introduced onto the activated polysaccharide by reacting the cyanate ester in any of several available ways. For example, cystamine or cysteamine can be used to prepare thiolated polysaccharides, which can be coupled to the carrier via thioether bonds obtained after reaction with maleimide-activated carrier proteins (such as using GMBS) or haloacetylated carrier proteins (such as using iodoacetimide [e.g., iodoacetimide ethyl ester 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. Immun. 40: 245-256.

[0195] Other suitable conjugation methods use carbodiimide, hydrazide, active ester, 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 reaction of the free hydroxyl group 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, then reacting the primary hydroxyl group with CDI to form a carbamate intermediate, and then coupling 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, e.g., Chase, 1967, Methods of Immunology and Immunochemistry 1:52. Methods for preparing Streptococcus pneumoniae capsular polysaccharides are well known in the art. See, e.g., European Patent No. EP0497524. Isolates of pneumococcal serotypes are available from the American Type Culture Collection (Manassas, VA). The bacteria are identified as encapsulated, non-motile, Gram-positive, lancet-shaped diplococci with α-hemolysis on blood agar. Subtypes can be distinguished using specific antisera according to the Quelling reaction. See, e.g., U.S. Patent No. 5,847,112.

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

[0201] The fermentation process is terminated by adding an inactivating agent. After inactivation, the solution is transferred to an inactivation tank where it is maintained at a controlled temperature with stirring. Cell debris is removed using a combination of centrifugation and filtration. The solution is ultrafiltered and diafiltered. Then, the solution is fractionated based on solvents to remove impurities and recover the polysaccharide.

[0202] Example 2

[0203] Polysaccharide size reduction and activation are carried out as follows.

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

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

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

[0207] Serotype 19A is not filtered through a 0.45 μm filter and its size is not reduced. Since it has a relatively low initial size, size reduction is not required. After dissolution, serotype 19A is filtered through a 0.22 μm filter as described in the following paragraph.

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

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

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

[0211]

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

[0213] Example 3

[0214] CRM can be carried out as shown below 197 Preparation of carrier protein.

[0215] Using a 5 kDa NMWCO tangential flow ultrafiltration membrane, diafilter the frozen, purified CRM197 obtained by expression in Pseudomonas fluorescens as previously described (see International Patent Application Publication No. WO 2012 / 173876) with 10 diafiltration volumes of 2 mM phosphate buffer (pH 7.2), and perform 0.22 micron filtration. In certain embodiments, 5 mM potassium phosphate pH 6.4 (5 mM sodium phosphate, pH 7.0) is used for serotype 18C. Dilute the diafiltered solution in water containing sucrose to a final concentration between 1.0 and 5.3% (w / v), and lyophilize to produce a dry CRM with a final moisture content of 6% or less. 197 Table 2 below provides the representative sucrose concentrations of CRM conjugated to polysaccharides of specific serotypes. 197

[0216] Example 4

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

[0218] Before lyophilization, dilute the CRM 197 and Ps solutions as shown below.

[0219] In some embodiments, use 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 to dilute the CRM 197 solution to a protein concentration of 6.0 mg / mL. Use WFI and a freshly prepared 30% w / v sucrose solution in WFI to dilute the UF2-FR Ps solution to a Ps concentration of 6.0 mg / mL.

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

[0221] Use serotype-specific sucrose and phosphate concentrations (see, for example, Table 2). Use a Virtis Genesis freeze dryer to lyophilize the diluted CRM 197 and UF2-FR solutions.

[0222]

[0223] Example 5

[0224] This example shows the CRM as measured by Fourier transform infrared spectroscopy (FTIR) 197 The reconstitution time of the CRM in anhydrous DMSO affects the secondary structure.

[0225] Reconstitute the lyophilized CRM that has been lyophilized in 6 mg / mL, 5% (w / v) sucrose, 1.25 mM sodium phosphate, pH 7.2 in DMSO (anhydrous DMSO from Sigma-Aldrich, 27655-100 mL) using slow (eight minutes) or rapid reconstitution (two minutes) 197 to a final concentration of 10 mg / mL, and then mix to produce a homogeneous solution. Reconstitution is carried out by adding appropriate volumes of DMSO every 30 seconds until the time point is reached. FTIR spectra are acquired using a BioTools PROTA-3S in transmission mode with a 50 mm CaF2 window at a controlled temperature (25 °C). Fifty scans can be acquired at a resolution of 4 cm -1 and averaged, subtracting the buffer and correcting for water vapor.

[0226] Reconstitute the lyophilized CRM in anhydrous DMSO using rapid addition (two minutes) 197 shows the expected free carbonyl amide I peak at 1660 cm -1 , which indicates that the CRM 197 is fully unfolded in anhydrous DMSO ( Figure 5 ). When the CRM is reconstituted in anhydrous DMSO using a slow addition time (eight minutes) 197 , a second peak (1626 cm -1 ) appears in the amide I region. This second peak is attributed to the formation of intermolecular β-sheets.

[0227] Example 6

[0228] This example shows that reconstituting 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 as the holding time increases.

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

[0230] As shown by the presence of the intermolecular β-sheet amide I peak ( Figure 6 ), reconstitution of CRM in anhydrous DMSO at an intermediate time (4 minutes) 197 led to protein aggregation. Increasing the holding time (up to 300 minutes) led to a proportional increase in the amount of intermolecular β-sheet, while the amount of the free carbonyl amide I peak decreased ( Figure 6 ).

[0231] Example 7

[0232] This example shows 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 from Sigma-Aldrich, 27655-100 mL) within two minutes 197 to a final concentration of 10, 30 or 50 mg / mL. Reconstitution was carried out by adding appropriate volumes of DMSO every 30 seconds until the time point was reached. Using the transmission mode with a 50 mm CaF2 window, FTIR spectra were acquired with BioTools PROTA-3S at a controlled temperature (25 °C). Fifty scans could be acquired at a resolution of 4 cm -1 and averaged, the buffer was subtracted and water vapor was corrected. Dynamic light scattering (DLS) measurements were collected at the prepared concentrations at a controlled temperature (25 °C) with a viscosity of 1.996 cP using a Malvern Zetasizer ZS.

[0234] When CRM was rapidly reconstituted at 10 mg / mL 197 ( Figure 7 , two minutes), there was no evidence of the formation of intermolecular β-sheets, confirming earlier observations ( Figure 5)。At higher concentrations (30 mg / mL, 50 mg / mL), intermolecular β-sheets form even at rapid refolding times (two minutes). This supports the hypothesis that the increased CRM 197 concentration present during slow refolding is one of the causes of intermolecular β-sheet formation. DLS data also support this concentration hypothesis, which shows that compared to lower concentrations ( Figure 8A ), higher concentrations ( Figure 8B ) of refolded CRM 197 have an increased proportion of larger particles, which is consistent with protein aggregation.

[0235] Example 8

[0236] Increasing the water concentration in DMSO used for refolding CRM 197 results in increased formation of intermolecular β-sheets.

[0237] Refold lyophilized CRM 197 previously lyophilized in 6 mg / mL, 5% (w / v) sucrose, 1.25 mM sodium phosphate, pH 7.2 to a final concentration of 10 mg / mL in two minutes in DMSO (anhydrous DMSO can be from Sigma-Aldrich, 27655 - 100 mL), where the DMSO is 95%, 97%, 99% (v / v) DMSO / water or anhydrous DMSO. Anhydrous DMSO can be Sigma-Aldrich D2438 - 50 mL. Refolding is carried out by adding appropriate volumes of DMSO every 30 seconds until the time point is reached. FTIR spectra are acquired using a transmission mode with a 50 mm CaF2 window at a controlled temperature (25 °C) with a BioTools PROTA-3S. Fifty scans can be acquired at a resolution of 4 cm -1 and averaged, subtracting the buffer and correcting for water vapor.

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

[0239] Example 9

[0240] Reducing the water content in lyophilized CRM 197 does not reduce the sensitivity to slow refolding.

[0241] CRM 197 was lyophilized in a manner that reduces the water present in the cake (longer drying cycles, lower fill volumes) (approx. 6 mg / mL in 1.25 mM phosphate (pH 7.2) containing 1% (w / v) sucrose). In DMSO (e.g., anhydrous DMSO from Sigma-Aldrich, D2438 - 50 mL), the lyophilized CMR was reconstituted to a final concentration of 10 mg / mL within a two-minute or eight-minute period. Reconstitution was carried out by adding appropriate volumes of DMSO every 30 seconds until the time point was reached. FTIR spectra were acquired using a transmission mode with a 50 mm CaF2 window at a controlled temperature (25 °C) with a BioTools PROTA-3S. Fifty scans could be acquired at a resolution of 4 cm 197 and averaged, subtracting the buffer and correcting for water vapor. -1

[0242] CRM 197 An increase in water during DMSO reconstitution of CRM led to an increase in the proportion of intermolecular β-sheet formation ( Figure 9 ). CRM that had been lyophilized in a manner to reduce the water content was reconstituted in anhydrous DMSO at two different rates, fast (two minutes) and slow (eight minutes). Reducing the water content in the lyophilized CRM 197 did not reduce the sensitivity of intermolecular β-sheets to the reconstitution time ( 197 ). Figure 10 )

[0243] Example 10

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

[0245] There are several ways to combine the conjugation components (Ps and Pr). Figures 1 - 4 Several combination strategies are detailed, as well as their effects on the component concentrations and Ps:Pr when combined. When Pr is added to the Ps solution ( Figure 1 ) or when Ps is added to the Pr solution ( Figure 2) When added, the concentrations of the component added to the solution containing other components and the other components change with time, especially at the interface between the two solutions. The changes in the Ps concentration and the Ps:Pr ratio caused by the change in concentration with time during the addition process have an impact on the conjugate size. When dissolving lyophilized Ps in the Pr solution ( Figure 3 ), the Ps concentration and the Ps:Pr ratio at the powder-liquid interface start high (at the solubility limit of Ps), and then decrease to the target value as the polysaccharide dissolves.

[0246] However, adding the two components simultaneously to a separate conjugation vessel ( Figure 4 ) by Tee mixing eliminates this concentration gradient and keeps the ratio of the two components constant during the addition, which significantly reduces the potential impact on the conjugate size. Compared with 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, a viscous gel was formed during conjugation: the formation of the gel indicates the formation of larger, highly networked conjugates. In these solutions, a higher Ps concentration for conjugation was achieved. In the gelling solution, the dried Ps was dissolved in the Pr solution or the Ps solution was added to the Pr solution. Both of these combination strategies showed a Ps concentration gradient during the addition process, with an initial high Ps concentration and Ps:Pr ( Figure 1 and Figure 3 ). Both of these combination strategies showed a Ps concentration gradient during the addition process. Batches without a concentration gradient ( Figure 4 ) or with a concentration gradient having a lower initial Ps concentration and Ps:Pr ratio ( Figure 2 ) did not show gel formation. This supports the hypothesis that high Ps concentration and Ps:Pr ratio can produce very large conjugates, leading to the formation of a viscous gel.

[0248]

[0249] Example 11

[0250] At room temperature, lyophilized Ps and lyophilized CRM 197 (Pr) were redissolved using an equal volume of anhydrous DMSO to prepare a homogeneous solution of Ps and Pr. CRM 197 was redissolved at a relatively fast rate of 2 minutes. In some embodiments, 500 mM sodium phosphate buffer (pH 7.2) was spiked into the protein solution redissolved in DMSO to a final concentration of 1.0 mM sodium phosphate. After redissolving in DMSO, as described above, Ps and CRM 197Solutions were combined to achieve serotype-specific final Ps concentrations and Ps:CRM 197 ratios (Table 4).

[0251] Prepare a solution of sodium cyanoborohydride in WFI and add 1.0 meq of sodium cyanoborohydride (1.0 mole of sodium cyanoborohydride per mole of Ps repeat unit) to the solution. The molar concentration of the sodium cyanoborohydride solution (Table 4) is based on a target value of approximately 0.5% total water content of the solution during conjugation. React the solution at the serotype-specific temperature for the serotype-specific duration (Table 4) to generate a conjugate product intermediate (CP).

[0252] Prepare a solution of sodium borohydride in WFI and add 2.0 meq of sodium borohydride (relative to the Ps repeat unit) to the solution. The molar concentration of the sodium borohydride solution (Table 4) is based on a target value of approximately 1.0% total water content of the solution after addition of the borohydride. React the solution at room temperature for 2 - 3 hours to produce a conjugate product quenching intermediate (CPQ).

[0253]

[0254] Then, dilute the conjugation solution to 20% or less (v / v) anhydrous DMSO by slowly adding the solution to 150 mM sodium chloride (150 mM sodium chloride plus 0.025% w / v polysorbate 20 for conjugates in some embodiments). During the dilution step, maintain the solution temperature below 15°C. After about one hour, add 1.5 M potassium phosphate (pH 6.0) to the solution such that the final concentration is 25 mM potassium phosphate. Conjugation performance can be evaluated by the consumption of total Ps and CRM 197 and the ratio of conjugate Ps to CRM 197 and the conjugate molecular weight.

[0255] Concentrate the conjugation solution to approximately 2.5 g / L and use a 30 kDa NMWCO tangential flow ultrafiltration membrane to diafilter at 2 - 8°C with 10 diafiltration volumes of 150 mM sodium chloride or 150 mM sodium chloride containing 25 mM potassium phosphate. This produces an ultrafiltration 3 process intermediate (UF3-FR). Determine the Ps concentration of UF3-FR by HPSEC UV-MALS-RI.

[0256] For 19F in some embodiments, filter UF3-FR through a 0.22 micron filter and then incubate at 22°C for approximately 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 with 20 volumes of diafiltration of 150 mM sodium chloride containing 10 mM L-histidine (pH 7.0) using a 300 kDa NMWCO Biomax PES tangential flow ultrafiltration membrane. Serotype 7F uses a 100 kDa NMWCO membrane for the ultrafiltration 4 step. For serotypes 6A, 6B, and 18C, the UF3-FR solution is concentrated to about 3.5 g / L and diafiltered at 2 - 8°C with 10 mM L-histidine, 0.03% (w / v) PS-20 in 150 mM sodium chloride, pH 7.0 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 at 2 - 8°C with 10 mM L-histidine, 0.015% (w / v) PS-20 in 150 mM sodium chloride, pH 7.0 using a 300 kDa NMWCO UltraCel regenerated cellulose tangential flow ultrafiltration membrane. This produces an ultrafiltration 4 process intermediate (UF4-FR). The Ps concentration of UF4-FR is determined by HPSEC UV-MALS-RI.

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

[0259] For serotypes 6A, 6B, and 18C, the UF4-FR is filtered through a dual membrane PES filter of 0.5 / 0.2 microns. The Ps concentration of the filtrate is determined by HPSEC UV-MALS-RI. If the Ps concentration of the filtrate is greater than 1.0 g / L, the filtrate is diluted to a Ps concentration of 1.0 g / L with additional 10 mM L-histidine, 0.03% w / v PS-20 in 150 mM sodium chloride, pH 7.0. This produces a monovalent bulk conjugate intermediate (MBC). The MBC is aliquoted and frozen at -60°C to -80°C.

[0260] For serotypes 7F, 19A, 19F, and 23F, 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 10 mM L-histidine in 150 mM sodium chloride, 0.015% w / v PS-20, pH 7.0. This produced the monovalent batch conjugate intermediate (MBC). The MBC was aliquoted and frozen at -60 °C to -80 °C.

[0261] Example 12

[0262] When CRM 197 was slowly reconstituted (eight minutes) in anhydrous DMSO and subsequently conjugated to 6B polysaccharide, intermolecular β-sheets were present after conjugation.

[0263] As previously discussed, CRM was rapidly (two minutes) or slowly (eight minutes) reconstituted in DMSO 197 and subsequently conjugated to 6B polysaccharide, purified as previously described, and dialyzed to a concentration of approximately 1 mg / mL in (10 mM histidine, 150 mM NaCl pH 7). The sample was then concentrated using an Amicon Ultra 11K MWCO filter to enable FTIR measurements. FTIR spectra were acquired using a transmission mode with a 50 mm CaF2 window at a controlled temperature (25 °C) using a BioTools PROTA-3S. Fifty scans could be acquired at a resolution of 4 cm -1 and averaged, the buffer subtracted, and water vapor corrected. The data was subsequently analyzed using Omic software (ThermoFisher).

[0264] The CRM 197 prepared using CRM 197 slowly reconstituted in DMSO Figure 11 ) showed evidence of intermolecular β-sheet formation, indicating that CRM 197 aggregates remained even after reconstitution. The CRM 197 prepared using 197 rapidly reconstituted CRM Figure 11 ) showed the expected free C=O amide and did not show β-sheet formation.

[0265] Example 13

[0266] This example shows the use of reductive amination in anhydrous DMSO to react 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. 197 (Pr) conjugation method. Use common process to conjugate different serotype polysaccharides to purified CRM 197 Carrier protein.

[0267] By rapidly adding anhydrous DMSO to the dried CRM within two minutes 197 The dried CRM prepared as described previously was reconstituted in anhydrous DMSO. 197 , and Ps was reconstructed separately in anhydrous DMSO to prepare Pr and Ps homogeneous solutions. Ps and Pr were reconstructed with half of the total conjugation reaction volume, respectively. Therefore, during the conjugation process, the concentration of Ps after DMSO reconstruction was 2x Ps concentration, and its range was 2.2 to 7.6 g / L calculated according to Table 3. After DMSO reconstruction, the Pr concentration was 2x (Ps concentration / Ps:Pr ratio during conjugation), which ranged from 1.5 to 5.7 g / L. Then, the Pr homogeneous solution was combined with the Ps homogeneous solution by Tee mixing. Then sodium cyanoborohydride (1 mole per mole of polysaccharide repeating unit) was added to the mixture, and conjugation was performed with a serum-specific duration (1 to 48 hours) to achieve the target conjugation 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. At about 4°C, the solution was diluted to 150 mM sodium chloride, 0.025% (w / v) polysorbate 20. 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 about 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 micron filtered.

[0272] Incubate serotype 19F for about 5 days, diafilter at about 4 °C in 150 mM sodium chloride containing 10 mM histidine (pH 7.0) using a 300 kDa NMWCO tangential flow ultrafiltration membrane, and perform 0.22 micron filtration.

[0273] Diafilter serotype 18C at about 4 °C in 150 mM sodium chloride containing 10 mM histidine (pH 7.0) using a 300 kDa NMWCO tangential flow ultrafiltration membrane, and perform 0.22 micron filtration.

[0274] Dilute with an additional solution of 150 mM sodium chloride containing 10 mM L-histidine (pH 7.0), aliquot it, and freeze 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] Use the pneumococcal polysaccharide-protein conjugate prepared as described above 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, use the pneumococcal polysaccharide-CRM conjugate produced by reductive amination in DMSO to prepare the formulation. Calculate the volume of the batch conjugate required to obtain the target final concentration for a single serotype based on the solution volume and the batch polysaccharide concentration. Combine the 15 conjugates with excipients selected from sodium chloride, L-histidine, pH 5.8 buffer containing polysorbate (PS)-20, PS-80, or poloxamer (P) 188. 197 Gently mix the aseptically formulated materials during this period, and then mix them with the batch aluminum phosphate adjuvant (APA), which may or may not contain propylene glycol (PG) and polyethylene glycol 400 (PEG400). Two concentrations of the conjugate and APA were studied in various formulations. One contains 8 μg / mL serotype 6B polysaccharide, 4 μg / mL polysaccharide for all other serotypes, and 250 μg / mL APA. The other contains 16 μg / mL serotype 6B polysaccharide, 8 μg / mL polysaccharide for all other serotypes, and 500 μg / mL APA. Store the formulated vaccine at 2 - 8 °C.

[0279]

[0280] ​APA is an aqueous suspension of aluminum hydroxyphosphate. APA is prepared by mixing aluminum chloride and sodium phosphate in a 1:1 volume ratio to precipitate aluminum hydroxyphosphate. After the mixing process, a high-shear mixer is used to reduce the size of the material to achieve a monodisperse particle size distribution. Then, the product is diafiltered with physiological saline and steam sterilized.

[0281] Example 15

[0282] In this example, the 6B polysaccharide (Ps) and CRM 197 (Pr) prepared as discussed above are lyophilized discretely by REV to form dry cakes. As discussed herein, the dry cakes are reconstituted in DMSO and conjugated.

[0283] The target Ps:Pr ratio (w / w) is 0.9 to 1.5, and the target molecular weight (MW) of the conjugate is 1500 to 3500 kDa. The target free Ps is 15% or less, and the free lysine loss is greater than 5 moles / mole. The results are shown in Table 5. The REV-dried material prepared according to the present invention has 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] Molecular weight and concentration analysis of the conjugate is performed using HPSEC / UV / MALS / RI determination. The conjugate sample is injected and separated by high-performance size-exclusion chromatography (HPSEC). Detection is completed using a tandem of ultraviolet (UV), multi-angle light scattering (MALS), and refractive index (RI) detectors. The protein concentration is calculated from UV280 using the extinction coefficient. The polysaccharide concentration is deconvoluted from the RI signal (contributed by both protein and polysaccharide) using the dn / dc factor, which is the change in the refractive index of the solution and the change in solute concentration (in mL / g). The average molecular weight of the sample is calculated using Astra software (Wyatt Technology Corporation, Santa Barbara, CA) using the concentration and light scattering information measured over the entire sample peak.

[0287] Determination of lysine loss in Table 5.

[0288] The lysine consumption in the conjugated protein is determined by the number of covalent linkages between the polysaccharide and the carrier protein.

[0289] Waters AccQ-Tag amino acid analysis (AAAaa) was used to measure the degree of conjugation in the conjugate samples. The samples were hydrolyzed using gas-phase acid hydrolysis in an Eldex workstation to break down the carrier protein into its constituent amino acids. The free amino acids were derivatized using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC). The derivatized samples were then analyzed by UPLC with UV detection on a C18 column. The average protein concentration was obtained using a representative amino acid other than lysine. The consumption of lysine (i.e., lysine loss) during the conjugation process was determined by the difference between the average measured lysine in the conjugate and the amount of lysine expected in the initial protein.

[0290] Determination of free Ps in Table 5

[0291] Free polysaccharides (not conjugated to CRM 197 conjugated polysaccharides) were measured by first precipitating the free protein and conjugate using deoxycholate (DOC) and hydrochloric acid. The precipitate was then filtered out, and the concentration of free polysaccharides in the filtrate was analyzed by HPSEC / UV / MALS / RI. The free polysaccharides were calculated as a percentage of the total polysaccharides measured by HPSEC / UV / MALS / RI.

[0292] Determination of free Pr in Table 5

[0293] Free polysaccharides, polysaccharide CRM 197 in the conjugate samples, and free CRM 197 were separated by capillary electrophoresis in micellar electrokinetic chromatography (MEKC) mode. Briefly, the samples were mixed with an MEKC running buffer containing 25 mM borate, 100 mM SDS, pH 9.3, and separated in a pre-treated bare fused 20 quartz capillary. The separation was monitored at 200 nm, and the free CRM 197 was quantified using a CRM 197 standard curve. The free protein results were reported as a percentage of the total protein content determined by the HPSEC / UV / MALS / RI procedure.

[0294] Example 16

[0295] In this example, 6A polysaccharide (Ps) and CRM 197 (Pr) samples were prepared and lyophilized separately (discretely) or combined and lyophilized according to the present invention. As discussed herein, the lyophilized samples were subsequently reconstituted in DMSO and conjugated.

[0296] Two different MVD cycles were examined for drying 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 power applied.

[0297]

[0298] The initial (preliminary) drying cycle (Lyo1) took 18 hours, as shown in Table 7.

[0299]

[0300] The drying cycle (Lyo 2) was improved by adding a secondary drying cycle and increasing the primary drying time, as shown in Table 8.

[0301]

[0302] 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.

[0303]

[0304] Example 17

[0305] In this example, 23F polysaccharide (Ps) and CRM 197 (Pr) samples were prepared and lyophilized separately (discretely) or combined and lyophilized according to the present invention. As discussed previously herein, the lyophilized samples were then reconstituted in DMSO and conjugated.

[0306] 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 for the separately lyophilized samples, both Ps and Pr were within the expected targets, except for sample B2, which had a ratio of 2.4.

[0307]

[0308] Example 18

[0309] This example shows the effect of the time of adding DMSO to the lyophilized carrier protein CRM 197 (Pr) on the conjugate size using 6A polysaccharide (Ps). As discussed previously, DMSO was added to the lyophilized Pr either rapidly (two minutes) or slowly (eight minutes), mixed, and then conjugated to the activated polysaccharide reconstituted in DMSO.

[0310] Figure 13Shows the size of the conjugate produced from the conjugation reaction using polysaccharides of increasing size. The general relationship is that the larger the polysaccharide used in the conjugation reaction (UF2 size), the larger the conjugate obtained. For each polysaccharide size, the slow and rapid addition of DMSO to Pr after lyophilization was compared. The figure shows that slow addition of DMSO results in even larger conjugates from polysaccharides of the same size compared to rapid addition of DMSO.

[0311] Example 19

[0312] This example shows the development of lyophilizer conditions for producing lyophilized beads of polysaccharide (Ps) and CRM 197 (Pr; CRM).

[0313] Discrete solutions of CRM and activated polysaccharides from serotypes 6A and 23F were prepared as described in Examples 3 and 4 and had Ps, CRM, and sucrose concentrations as shown in the table in this example.

[0314] A modified Biomek FX pipetting robot (Cryomek) was used to dispense 50 μL aliquots of the solutions onto the flat freezing surface of the Cryomek. The beads can be dispensed into small cold containers using a shoveling mechanism without causing any breakage. After completing the cycle for each different solution, the beads were transferred to an intermediate storage container and held at -70 °C until sublimation drying in a lyophilizer or by microwave vacuum drying. For lyophilizer drying, the beads were dispensed in a single layer onto drying trays. The cabinet pressure, shelf temperature, and cycle time were set. After drying the beads, the lyophilized beads were stored at 2 - 8 °C. (See Example 5 for specific parameters)

[0315] The initial drying cycle (Lyo1) took 18 hours as shown in Table 19.1. The residual moisture content of the lyophilized beads determined by Karl Fisher titration is shown in Table 19.2.

[0316]

[0317]

[0318] The results showed a high moisture content in the lyophilized beads. In addition, the lyophilized beads were very fragile and hygroscopic. The solid content was increased by increasing the polysaccharide, protein, and sucrose concentrations as shown in Table 20.2. The drying cycle (Lyo 2) was improved by adding a secondary drying cycle and increasing the primary drying time as shown in Table 20.1.

[0319]

[0320]

[0321] 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 the bound moisture still present in the product. Secondary drying requires a higher temperature (30 °C) than primary drying (15 °C).

[0322] At this time, the total drying cycle time of the freeze dryer is 45 hours. Some parameters, such as pressure and temperature, are changed to further shorten the drying cycle time (method: Lyo 3), as shown in Tables 21.1 and 21.2.

[0323]

[0324]

[0325] Example 20

[0326] This example shows the development of radiant energy vacuum (REV) dehydration (microwave vacuum drying (MVD)) conditions for the production of freeze-dried beads of polysaccharide (Ps) and CRM 197 (Pr; CRM).

[0327] Solutions of CRM and activated polysaccharides from serotypes 6A and 23F were prepared as described in Examples 3 and 4 and had Ps, CRM, and sucrose concentrations as shown in Tables 22.1 and 22.2.

[0328] A modified Biomek FX pipetting robot (Cryomek) was 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 shoveling mechanism without causing any breakage. After completing the cycle for 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 microwave drying, the beads are dispensed in a single layer into the container. The power, pressure, and cycle time are set. After drying the beads, the freeze-dried beads are stored at 2 - 8 °C (specific parameters see Example 6).

[0329] 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 amount of power applied.

[0330]

[0331]

[0332] The MVD1 cycle takes 4 hours and 30 minutes, but it is not sufficient to reduce the residual moisture content to at most 2%. The MVD2 cycle takes longer and consumes more power, and thus, it provides a significantly reduced residual moisture content.

[0333] Although the present invention has been described with reference to the embodiments shown herein, it should be understood that the present invention is not limited thereto. Other modifications and embodiments within its scope will be recognized by those of ordinary skill in the art and those who have obtained the teachings herein. Therefore, the present invention is defined only by the appended claims herein.

Claims

1. A method for preparing a composition, the composition comprising a Streptococcus pneumoniae polysaccharide covalently linked to a carrier protein, the method comprising: (a) providing a first dried composition and a second dried composition, the first dried composition comprising an activated Streptococcus pneumoniae polysaccharide from one or more Streptococcus pneumoniae serotypes, and the second dried composition comprising a carrier protein; (b) reconstituting the first dried composition and the second dried composition separately in an organic solvent and mixing them to provide a first homogeneous solution and a second homogeneous solution, the first homogeneous solution comprising one or more activated polysaccharides and the second homogeneous solution comprising the carrier protein; (c) combining the first homogeneous solution with the second homogeneous solution by Tee mixing to produce a mixture; and (d) adding a reducing agent to the mixture to produce a conjugate solution, the conjugate solution comprising a carrier protein conjugated to one or more polysaccharides of the Streptococcus pneumoniae serotype; wherein the one or more polysaccharides are obtained from Streptococcus pneumoniae serotypes selected from: 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, CWPS1, CWPS2 and CWPS3; wherein the water content of the organic solvent is less than 1% (v / v); wherein the reconstitution comprises eight minutes or less and the mixing comprises 120 minutes or less; wherein the carrier protein is CRM 197 ; wherein the concentration of the carrier protein in the second homogeneous solution is 12 mg / mL or lower; wherein the second homogeneous solution containing the carrier protein is held for six hours or less before being combined by Tee mixing with the first homogeneous solution containing the polysaccharide, and wherein each conjugate solution comprises a polysaccharide conjugated to the carrier protein at a polysaccharide to carrier protein ratio of 0.6 to 1.3 by weight to weight.

2. The method according to claim 1, wherein the one or more polysaccharides are obtained from Streptococcus pneumoniae serotypes selected from: (i) 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; or (ii) Serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, and 33F; or (iii) Serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F; or (iv) Serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, and 23F.

3. The method according to claim 1 or claim 2, 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.

4. The method according to claim 3, wherein the sublimation drying method comprises freezing the first homogeneous solution and the second homogeneous solution in the form of cakes or freeze-dried beadlets.

5. The method according to claim 3, wherein the sublimation drying is carried out in a batch drying in a container selected from metal trays, plastic trays, plastic bags, and type I vials.

6. The method according to claim 1 or claim 2, wherein the first dried composition and the second dried composition are prepared by a sublimation drying method comprising freeze-drying or radiant energy vacuum (REV) dehydration, which comprises providing a first aqueous solution and a second aqueous solution, the first aqueous solution comprising activated pneumococcal polysaccharide from one or more pneumococcal serotypes, the second aqueous solution comprising a carrier protein and a buffer, wherein the first aqueous solution and the second aqueous solution comprise 0.5% (w / v) or more of sucrose, and subjecting the first aqueous solution and the second aqueous solution to the sublimation drying method to produce the first dried composition and the second dried composition.

7. The method according to claim 6, wherein the buffer is a histidine, succinate, MES, MOPS, HEPES, or acetate buffer having a pH range of 5.0 - 7.

0.

8. The method according to claim 6, wherein the buffer is a phosphate or citrate buffer having a pH range of 5.0 - 7.

0.

9. The method according to claim 1, wherein the first dried composition and the second dried composition have a moisture content of less than 6%.

10. The method according to claim 1, wherein the organic solvent is dimethyl sulfoxide (DMSO).

11. The method according to claim 1, wherein the reconstitution comprises less than five minutes and the mixing comprises 120 minutes or less.

12. The method according to claim 11, wherein the reconstitution comprises less than two minutes or less and the mixing comprises 120 minutes or less.

13. The method according to claim 1, wherein the conjugate solution contains a free polysaccharide amount of less than 15% of the total polysaccharide in the solution.

14. The method according to claim 1, wherein the conjugate solution is sterile filtered.

15. The method according to claim 1, wherein the Streptococcus pneumoniae polysaccharide is activated by reaction with an oxidizing agent.

16. A method for preparing a multivalent pneumococcal conjugate vaccine, the multivalent pneumococcal conjugate vaccine containing Streptococcus pneumoniae serotype polysaccharides 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F conjugated to a carrier protein, the method comprising: (a) providing 23 dry carrier protein compositions and 23 dry activated polysaccharide compositions, each dry activated polysaccharide composition containing a dry activated polysaccharide from a Streptococcus pneumoniae serotype selected from 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F, wherein none of the 23 dry activated polysaccharide compositions contains an activated polysaccharide from the same Streptococcus pneumoniae serotype; (b) separately reconstituting the 23 dry carrier protein compositions and the 23 dry activated polysaccharide compositions with an organic solvent and mixing them to provide 23 homogeneous carrier protein solutions and 23 homogeneous activated polysaccharide solutions, each homogeneous carrier protein solution containing the carrier protein and each homogeneous activated polysaccharide solution containing an activated polysaccharide of a specific Streptococcus pneumoniae serotype, wherein none of the 23 homogeneous solutions contains an activated polysaccharide from the same Streptococcus pneumoniae serotype; (c) combining each of the 23 homogeneous carrier protein solutions with one of the 23 activated polysaccharide homogeneous solutions by Tee mixing to produce 23 mixtures, each mixture containing a carrier protein and an activated polysaccharide of a specific Streptococcus pneumoniae serotype, wherein none of the 23 mixtures contains an activated polysaccharide from the same Streptococcus pneumoniae serotype; (d) adding a reducing agent to each of the 23 mixtures to produce 23 conjugate solutions, each conjugate solution containing a carrier protein conjugated to a polysaccharide of a specific Streptococcus pneumoniae serotype, wherein none of the 23 conjugate solutions contains an activated polysaccharide from the same Streptococcus pneumoniae serotype; and (e) combining the 23 conjugate solutions to provide the multivalent pneumococcal conjugate vaccine containing Streptococcus pneumoniae serotype polysaccharides 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F conjugated to a carrier protein; wherein the water content of the organic solvent is less than 1% (v / v); wherein the reconstitution includes a time of eight minutes or less and the mixing includes a time of 120 minutes or less; wherein the carrier protein is CRM 197 ; wherein the concentration of the carrier protein in the second homogeneous solution is 12 mg / mL or lower; wherein the second homogeneous solution containing the carrier protein is maintained for six hours or less before being combined, via Tee mixing, with the first homogeneous solution containing the polysaccharide, and wherein each conjugate solution contains a polysaccharide conjugated to the carrier protein at a polysaccharide to carrier protein ratio of 0.6 to 1.3 weight to weight.

17. A method for preparing a multivalent pneumococcal conjugate vaccine, the multivalent pneumococcal conjugate vaccine containing Streptococcus pneumoniae serotype polysaccharides 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F conjugated to a carrier protein, the method comprising: (a) providing 15 dry carrier protein compositions and 15 dry activated polysaccharide compositions, each dry activated polysaccharide composition containing a dry activated polysaccharide from a Streptococcus pneumoniae serotype selected from 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F, wherein none of the 15 dry activated polysaccharide compositions contain activated polysaccharides from the same Streptococcus pneumoniae serotype; (b) separately reconstituting the 15 dry carrier protein compositions and the 15 dry activated polysaccharide compositions with an organic solvent and mixing them to provide 15 homogeneous carrier protein solutions and 15 homogeneous activated polysaccharide solutions, each homogeneous carrier protein solution containing the carrier protein and each homogeneous activated polysaccharide solution containing an activated polysaccharide of a specific Streptococcus pneumoniae serotype, wherein none of the 15 homogeneous solutions contain activated polysaccharides from the same Streptococcus pneumoniae serotype; (c) combining, via Tee mixing, each of the 15 homogeneous carrier protein solutions with one of the 15 activated polysaccharide homogeneous solutions to produce 15 mixtures, each mixture containing the carrier protein and an activated polysaccharide of a specific Streptococcus pneumoniae serotype, wherein none of the 15 mixtures contain activated polysaccharides from the same Streptococcus pneumoniae serotype; (d) adding a reducing agent to each of the 15 mixtures to produce 15 conjugate solutions, each conjugate solution containing a carrier protein conjugated to a polysaccharide of a specific Streptococcus pneumoniae serotype, wherein none of the 15 conjugate solutions contain activated polysaccharides from the same Streptococcus pneumoniae serotype; and (e) combining the 15 conjugate solutions to provide the multivalent pneumococcal conjugate vaccine containing Streptococcus pneumoniae serotype polysaccharides 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F conjugated to the carrier protein; wherein the water content of the organic solvent is less than 1% (v / v); wherein the reconstitution comprises eight minutes or less and the mixing comprises 120 minutes or less; wherein the carrier protein is CRM 197 ; wherein the concentration of the carrier protein in the second homogeneous solution is 12 mg / mL or lower; wherein the second homogeneous solution containing the carrier protein is held for six hours or less before being combined, via Tee mixing, with the first homogeneous solution containing the polysaccharide, and wherein each conjugate solution contains a polysaccharide conjugated to the carrier protein at a polysaccharide to carrier protein ratio of 0.6 to 1.3 weight to weight.

18. A method for preparing a multivalent pneumococcal conjugate vaccine, the multivalent pneumococcal conjugate vaccine containing Streptococcus pneumoniae serotype polysaccharides 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F conjugated to a carrier protein, the method comprising: (a) providing 13 dry carrier protein compositions and 13 dry activated polysaccharide compositions, each dry activated polysaccharide composition containing a dry activated polysaccharide from a Streptococcus pneumoniae serotype selected from 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F, wherein none of the 13 dry activated polysaccharide compositions contain activated polysaccharides from the same Streptococcus pneumoniae serotype; (b) separately reconstituting the 13 dry carrier protein compositions and the 13 dry activated polysaccharide compositions with an organic solvent and mixing to provide 13 homogeneous carrier protein solutions and 13 homogeneous activated polysaccharide solutions, each homogeneous carrier protein solution containing the carrier protein and each homogeneous activated polysaccharide solution containing an activated polysaccharide of a specific Streptococcus pneumoniae serotype, wherein none of the 13 homogeneous solutions contain activated polysaccharides from the same Streptococcus pneumoniae serotype; (c) combining each of the 13 homogeneous carrier protein solutions with one of the 13 activated polysaccharide homogeneous solutions via Tee mixing to produce 13 mixtures, each mixture containing the carrier protein and an activated polysaccharide of a specific Streptococcus pneumoniae serotype, wherein none of the 13 mixtures contain activated polysaccharides from the same Streptococcus pneumoniae serotype; (d) adding a reducing agent to each of the 13 mixtures to produce 13 conjugate solutions, each conjugate solution containing a carrier protein conjugated to a polysaccharide of a specific Streptococcus pneumoniae serotype, wherein none of the 13 conjugate solutions contain activated polysaccharides from the same Streptococcus pneumoniae serotype; and (e) combining the 13 conjugate solutions to provide the multivalent pneumococcal conjugate vaccine containing Streptococcus pneumoniae serotype polysaccharides 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F conjugated to the carrier protein; wherein the organic solvent has a water content of less than 1% (v / v); wherein the reconstitution comprises eight minutes or less and the mixing comprises 120 minutes or less; wherein the carrier protein is CRM 197 ; wherein the concentration of the carrier protein in the second homogeneous solution is 12 mg / mL or less; wherein the second homogeneous solution containing the carrier protein is held for six hours or less before being combined, via Tee mixing, with the first homogeneous solution containing the polysaccharide, and Each conjugate solution contains a polysaccharide conjugated to the carrier protein at a polysaccharide to carrier protein ratio of 0.6 to 1.3 weight for weight.

19. A method for preparing a multivalent pneumococcal conjugate vaccine, the multivalent pneumococcal conjugate vaccine containing Streptococcus pneumoniae serotype polysaccharides 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, and 23F conjugated to a carrier protein, the method comprising: (a) providing 10 dry carrier protein compositions and 10 dry activated polysaccharide compositions, each dry activated polysaccharide composition containing a dry activated polysaccharide from a Streptococcus pneumoniae serotype selected from 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, and 23F, wherein none of the 10 dry activated polysaccharide compositions contains an activated polysaccharide from the same Streptococcus pneumoniae serotype; (b) separately reconstituting the 10 dry carrier protein compositions and the 10 dry activated polysaccharide compositions with an organic solvent and mixing to provide 10 homogeneous carrier protein solutions and 10 homogeneous activated polysaccharide solutions, each homogeneous carrier protein solution containing the carrier protein and each homogeneous activated polysaccharide solution containing an activated polysaccharide of a specific Streptococcus pneumoniae serotype, wherein none of the 10 homogeneous solutions contains an activated polysaccharide from the same Streptococcus pneumoniae serotype; (c) combining each of the 10 homogeneous carrier protein solutions with one of the 10 activated polysaccharide homogeneous solutions by Tee mixing to produce 10 mixtures, each mixture containing the carrier protein and an activated polysaccharide of a specific Streptococcus pneumoniae serotype, wherein none of the 10 mixtures contains an activated polysaccharide from the same Streptococcus pneumoniae serotype; (d) adding a reducing agent to each of the 10 mixtures to produce 10 conjugate solutions, each conjugate solution containing a carrier protein conjugated to a polysaccharide of a specific Streptococcus pneumoniae serotype, wherein none of the 10 conjugate solutions contains an activated polysaccharide from the same Streptococcus pneumoniae serotype; and (e) combining the 10 conjugate solutions to provide the multivalent pneumococcal conjugate vaccine containing Streptococcus pneumoniae serotype polysaccharides 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, and 23F conjugated to the carrier protein; wherein the water content of the organic solvent is less than 1% (v / v); wherein the reconstitution includes eight minutes or less and the mixing includes 120 minutes or less; wherein the carrier protein is CRM 197 ; wherein the concentration of the carrier protein in the second homogeneous solution is 12 mg / mL or less; wherein the second homogeneous solution containing the carrier protein is held for six hours or less before being combined by Tee mixing with the first homogeneous solution containing the polysaccharide, and wherein each conjugate solution contains a polysaccharide conjugated to the carrier protein at a polysaccharide to carrier protein ratio of 0.6 to 1.3 weight for weight.

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