Method for producing a pneumococcal capsular polysaccharide carrier protein conjugate from freeze-dried spheres

Abstract

A method for producing a pneumococcal capsular polysaccharide-protein conjugate is described, in which one or more activated pneumococcal polysaccharides of a specific pneumococcal serotype and a carrier protein are lyophilized separately in the form of carrier protein lyophilized beads and one or more polysaccharide lyophilized beads. A predetermined amount of the carrier protein lyophilized beads and the activated polysaccharide lyophilized beads are mixed together, and the mixture is reconstituted in an organic solvent to produce the polysaccharide-carrier protein conjugate. A multivalent pneumococcal immunogenic composition containing a combination of conjugates for use in a vaccine can be produced using a plurality of conjugates each containing a polysaccharide of a specific serotype.

Description

Background Art

[0001] (1) Field of the Invention

[0002] The present invention relates to a method for producing a pneumococcal capsular polysaccharide - carrier protein conjugate, in which one or more activated pneumococcal polysaccharides of a specific pneumococcal serotype and a carrier protein are lyophilized in the form of carrier protein lyophilized beads and one or more activated polysaccharide lyophilized beads, respectively. A predetermined amount of the carrier protein lyophilized beads and the activated polysaccharide lyophilized beads are mixed together and the mixture is reconstituted in an organic solvent to produce the polysaccharide - carrier protein conjugate. Multiple conjugates each containing polysaccharides 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) of 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 reported that the mortality rate of pneumococcal meningitis was as high as 8% and the neurological sequelae were as high as 25%. See Arditi et al., 1998, Pediatrics 102: 1087 - 97.

[0005] It has been proven that the multivalent pneumococcal polysaccharide vaccine that has been licensed for many years has invaluable value in preventing pneumococcal diseases in adults, especially the elderly and high - risk populations. However, infants and young children 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 bacterial polysaccharide immunogens with carrier proteins 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 that include bacterial capsular polysaccharides conjugated to carrier proteins 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 a protein conjugated thereto, free carrier proteins that do not have a 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 known in the art and include, 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, particularly 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 carrier protein conjugate for a pneumococcal vaccine, wherein the activated pneumococcal polysaccharide and the carrier protein are discretely or separately lyophilized into lyophilized spheres. The lyophilized spheres can be mixed at a predetermined ratio and then reconstituted in an organic solvent to provide a reconstituted mixture of the activated polysaccharide and the carrier protein, which is then conjugated together to produce a multivalent pneumococcal polysaccharide carrier protein conjugate for a pneumococcal vaccine. The method of discretely drying the polysaccharide and the carrier protein into lyophilized spheres 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 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 lyophilized bead composition and a second lyophilized bead composition, the first lyophilized bead composition comprising an activated pneumococcal polysaccharide from a pneumococcal serotype, and the second lyophilized bead composition comprising a carrier protein;

[0012] (b) combining a quantity of the first lyophilized bead composition with a quantity of the second lyophilized bead composition to provide a lyophilized bead mixture having a predetermined ratio of the activated polysaccharide to the carrier protein;

[0013] (c) reconstituting and mixing the lyophilized bead mixture in an organic solvent to provide a reconstituted mixture; and

[0014] (d) adding a reducing agent to the reconstituted mixture to produce a conjugate solution comprising the carrier protein conjugated to the polysaccharide of a pneumococcal serotype.

[0015] In another embodiment, the present invention provides a method for preparing a composition comprising one or more pneumococcal polysaccharides covalently linked to a carrier protein, the method comprising: the method comprising:

[0016] (a) providing a first lyophilized bead composition and a second lyophilized bead composition, the first lyophilized bead composition comprising one or more activated pneumococcal polysaccharides from a pneumococcal serotype, and the second lyophilized bead composition comprising a carrier protein;

[0017] (b) combining a quantity of the first lyophilized bead composition with a quantity of the second lyophilized bead composition to provide a lyophilized bead mixture having a predetermined ratio of the one or more activated polysaccharides to the carrier protein;

[0018] (c) reconstituting and mixing the lyophilized bead mixture in an organic solvent to provide a reconstituted mixture; and

[0019] (d) adding a reducing agent to the reconstituted mixture to produce a conjugate solution comprising the carrier protein conjugated to the one or more pneumococcal serotype polysaccharides.

[0020] In another embodiment, the present invention provides a method for preparing a composition comprising two or more pneumococcal polysaccharides covalently linked to a carrier protein, the method comprising:

[0021] (a) Provide a first lyophilized bead composition and a second lyophilized bead composition, wherein the first lyophilized bead composition comprises two or more activated pneumococcal polysaccharides from pneumococcal serotypes, and the second lyophilized bead composition comprises a carrier protein;

[0022] (b) Combine a quantity of the first lyophilized bead composition with a quantity of the second lyophilized bead composition to provide a lyophilized bead mixture having a predetermined ratio of the two or more activated polysaccharides to the carrier protein;

[0023] (c) Reconstitute and mix the lyophilized bead mixture in an organic solvent to provide a reconstituted mixture; and

[0024] (d) Add a reducing agent to the reconstituted mixture to produce a conjugate solution comprising a carrier protein conjugated to the polysaccharides of two or more pneumococcal serotypes.

[0025] In a specific embodiment of any of the methods of the present invention, the first lyophilized bead composition and the second lyophilized bead composition are prepared by a sublimation drying method selected from freeze drying and radiant energy vacuum (REV) dehydration (microwave vacuum drying (MDV)).

[0026] In a specific embodiment, the first lyophilized bead composition and the second lyophilized bead composition each have a final moisture content of about 6% or less. In a specific embodiment, the first lyophilized bead composition and the second lyophilized bead composition each have a final moisture content of about 5% or less. In a specific embodiment, the first lyophilized bead composition and the second lyophilized bead composition each have a final moisture content of about 4% or less. In a specific embodiment, the first lyophilized bead composition and the second lyophilized bead composition each have a final moisture content of about 3% or less. In a specific embodiment, the first lyophilized bead composition and the second lyophilized bead composition each have a final moisture content of about 2% or less.

[0027] In a specific embodiment, the first lyophilized bead composition and the second lyophilized bead composition are prepared by sublimation drying 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 aqueous solution and the second aqueous solution comprise about 0.5% or more sucrose, and wherein the sublimation drying is selected from freeze drying and radiant energy vacuum (REV) dehydration. In a specific embodiment, the first aqueous solution comprises about 4% to 6% sucrose and the second aqueous solution comprises about 4% to 8% sucrose.

[0028] 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, or 12 mg / mL.

[0029] 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.

[0030] In certain embodiments of the method, the reconstitution in step (c) is carried out in eight minutes or less. In certain embodiments, the reconstitution is carried out in six minutes or less. In certain embodiments, the reconstitution is carried out in four minutes or less. In certain embodiments, the reconstitution 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 is carried out in one minute or less.

[0031] In certain embodiments, the mixing in step (c) is carried out in 120 minutes or less. In certain embodiments, the mixing is carried out in 90 minutes or less. In certain embodiments, the mixing is carried out in 60 minutes or less. In certain embodiments, the mixing is carried out in 30 minutes or less. In certain embodiments, the mixing is carried out in 10 minutes or less.

[0032] In certain embodiments, the conjugate solution comprises 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, the conjugate solution comprises 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, the conjugate solution comprises 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.

[0033] 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.

[0034] In certain embodiments, the conjugate has a carrier protein lysine loss value greater than 5 (moles / mole).

[0035] In certain embodiments, the 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 for 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).

[0036] 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.

[0037] In certain embodiments, the buffer is a phosphate or citrate buffer having a pH in the range of 5.0 - 7.0.

[0038] 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.

[0039] In certain embodiments, the Streptococcus pneumoniae polysaccharide is activated by reaction with an oxidizing agent.

[0040] 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 。

[0041] In certain embodiments, the conjugate solution is sterile filtered.

[0042] The present invention provides a method for preparing a composition comprising one or more pneumococcal polysaccharides covalently linked to a carrier protein, the method comprising

[0043] (a) providing (i) a first aqueous solution comprising activated pneumococcal polysaccharides from one or more pneumococcal serotypes, and (ii) a second aqueous solution comprising a carrier protein and a buffer;

[0044] (b) separately drying the first aqueous solution and the second aqueous solution in a sublimation drying method for producing lyophilized beads to produce a first lyophilized bead composition and a second lyophilized bead composition, the first lyophilized bead composition comprising the dried one or more activated polysaccharides and the second lyophilized bead composition comprising the dried carrier protein;

[0045] (c) combining a quantity of the first lyophilized bead composition with a quantity of the second lyophilized bead composition to provide a lyophilized bead mixture having a predetermined ratio of the one or more activated polysaccharides to the carrier protein;

[0046] (d) reconstituting and mixing the lyophilized bead mixture in an organic solvent to provide a reconstituted mixture; and

[0047] (e) adding a reducing agent to the reconstituted mixture to produce a conjugate solution comprising a carrier protein conjugated to the one or more activated polysaccharides of the pneumococcal serotype.

[0048] In a particular embodiment of the method, the sublimation drying method is selected from lyophilization and radiant energy vacuum (REV) dehydration.

[0049] In a particular embodiment, the first lyophilized bead composition and the second lyophilized bead composition each have a final moisture content of about 6% or less. In a particular embodiment, the first lyophilized bead composition and the second lyophilized bead composition each have a final moisture content of about 5% or less. In a particular embodiment, the first lyophilized bead composition and the second lyophilized bead composition each have a final moisture content of about 4% or less. In a particular embodiment, the first lyophilized bead composition and the second lyophilized bead composition each have a final moisture content of about 3% or less. In a particular embodiment, the first lyophilized bead composition and the second lyophilized bead composition each have a final moisture content of about 2% or less.

[0050] In a particular embodiment, the first aqueous solution and the second aqueous solution comprise about 0.5% or more sucrose. In a particular embodiment, the first aqueous solution comprises about 4% to 6% sucrose and the second aqueous solution comprises about 4% to 8% sucrose.

[0051] 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 or 12 mg / mL.

[0052] 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.

[0053] In certain embodiments of the method, the reconstitution in step (d) is carried out in eight minutes or less. In certain embodiments, the reconstitution is carried out in six minutes or less. In certain embodiments, the reconstitution is carried out in four minutes or less. In certain embodiments, the reconstitution 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 is carried out in one minute or less.

[0054] In certain embodiments, the mixing in step (d) is carried out in 120 minutes or less. In certain embodiments, the mixing is carried out in 90 minutes or less. In certain embodiments, the mixing is carried out in 60 minutes or less. In certain embodiments, the mixing is carried out in 30 minutes or less. In certain embodiments, the mixing is carried out in 10 minutes or less.

[0055] In certain embodiments, the conjugate solution comprises 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, the conjugate solution comprises 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, the conjugate solution comprises 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.

[0056] 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.

[0057] In certain embodiments, the conjugate has a carrier protein lysine loss value greater than 5 (moles / mole).

[0058] 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).

[0059] In certain embodiments, the buffer is a histidine, succinate, MES, MOPS, HEPES or acetate buffer having a pH range of 5.0 - 7.0.

[0060] In certain embodiments, the buffer is a phosphate or citrate buffer having a pH range of 5.0 - 7.0.

[0061] 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.

[0062] In certain embodiments, the Streptococcus pneumoniae polysaccharide is activated by reaction with an oxidizing agent.

[0063] 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 .

[0064] In certain embodiments, the conjugate solution is sterile filtered.

[0065] The present invention also provides a method for preparing 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, the method comprising:

[0066] (a) providing

[0067] (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

[0068] (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;

[0069] (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;

[0070] (c) separately drying the two or more first aqueous solutions and the two or more second aqueous solutions in a sublimation drying method for producing freeze-dried spheres to produce two or more first freeze-dried sphere compositions and two or more second freeze-dried sphere compositions, the first freeze-dried sphere compositions each comprising the dried polysaccharide and the second freeze-dried sphere compositions each comprising the dried carrier protein;

[0071] (d) independently combining an amount of a first freeze-dried sphere composition with an amount of a second freeze-dried sphere composition to provide a plurality of freeze-dried sphere mixtures, each freeze-dried sphere mixture having a predetermined ratio of activated polysaccharide to carrier protein;

[0072] (e) reconstituting and mixing the plurality of freeze-dried sphere mixtures in an organic solvent to provide a plurality of reconstituted mixtures;

[0073] (f) adding a reducing agent to the plurality of reconstituted mixtures to produce a plurality of conjugate solutions; and

[0074] (g) combining two or more of the plurality of 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.

[0075] In a specific embodiment of the method, the sublimation drying method is selected from freeze-drying and radiant energy vacuum (REV) dehydration.

[0076] In certain embodiments, the first lyophilized bead composition and the second lyophilized bead composition each have a final moisture content of about 6% or less. In certain embodiments, the first lyophilized bead composition and the second lyophilized bead composition each have a final moisture content of about 5% or less. In certain embodiments, the first lyophilized bead composition and the second lyophilized bead composition each have a final moisture content of about 4% or less. In certain embodiments, the first lyophilized bead composition and the second lyophilized bead composition each have a final moisture content of about 3% or less. In certain embodiments, the first lyophilized bead composition and the second lyophilized bead composition each have a final moisture content of about 2% or less.

[0077] In certain embodiments, the first aqueous solution and the second aqueous solution contain about 0.5% or more sucrose. In certain embodiments, the first aqueous solution contains about 4% to 6% sucrose and the second aqueous solution contains about 4% to 8% sucrose.

[0078] In certain embodiments, the first aqueous solution contains the polysaccharide at a concentration of about 6 to 9 mg / mL and the second aqueous solution contains the carrier protein at a concentration of about 6 to 12 mg / mL. In certain embodiments, the first aqueous solution contains the polysaccharide at a concentration of about 6 or 9 mg / mL and the second aqueous solution contains the carrier protein at a concentration of about 6, 9, or 12 mg / mL.

[0079] 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.

[0080] In certain embodiments of the method, the reconstitution in step (e) is carried out in eight minutes or less. In certain embodiments, the reconstitution is carried out in six minutes or less. In certain embodiments, the reconstitution is carried out in four minutes or less. In certain embodiments, the reconstitution 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 is carried out in one minute or less.

[0081] In certain embodiments, the mixing in step (e) is carried out in 120 minutes or less. In certain embodiments, the mixing is carried out in 90 minutes or less. In certain embodiments, the mixing is carried out in 60 minutes or less. In certain embodiments, the mixing is carried out in 30 minutes or less. In certain embodiments, the mixing is carried out in 10 minutes or less.

[0082] In certain embodiments, each conjugate solution comprises 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 comprises 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 comprises 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.

[0083] 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.

[0084] In certain embodiments, the conjugate has a carrier protein lysine loss value greater than 5 (moles / mole).

[0085] In certain embodiments, each conjugate solution comprises 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).

[0086] In certain embodiments, the buffer is a histidine, succinate, MES, MOPS, HEPES or acetate buffer having a pH range of 5.0 - 7.0.

[0087] In certain embodiments, the buffer is a phosphate or citrate buffer having a pH range of 5.0 - 7.0.

[0088] 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.

[0089] In certain embodiments, the Streptococcus pneumoniae polysaccharide is activated by reaction with an oxidizing agent.

[0090] 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 .

[0091] In certain embodiments, the conjugate solution is sterile filtered.

[0092] 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:

[0093] (a) Provide 23 carrier protein lyophilized bead compositions and 23 activated polysaccharide lyophilized bead compositions, each of the activated polysaccharide lyophilized bead compositions comprising dried activated polysaccharides from Streptococcus pneumoniae serotypes 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 activated polysaccharide lyophilized bead compositions comprise activated polysaccharides from the same Streptococcus pneumoniae serotype;

[0094] (b) Combine each of the 23 carrier protein lyophilized bead compositions with each of the 23 activated polysaccharide lyophilized bead compositions, respectively, to provide 23 lyophilized bead mixtures, each lyophilized bead mixture having a predetermined ratio of activated polysaccharide to carrier protein, wherein none of the 23 lyophilized bead mixtures comprise activated polysaccharides from the same Streptococcus pneumoniae serotype;

[0095] (c) Reconstitute and mix each of the 23 lyophilized bead mixtures with an organic solvent, respectively, to produce a plurality of reconstituted mixtures, wherein none of the 23 reconstituted mixtures comprise activated polysaccharides from the same Streptococcus pneumoniae serotype;

[0096] (d) Add a reducing agent to each of the 23 reconstituted mixtures to produce 23 conjugate solutions, each conjugate solution comprising a carrier protein conjugated to a polysaccharide of a specific Streptococcus pneumoniae serotype, wherein none of the 23 conjugate solutions comprise activated polysaccharides from the same Streptococcus pneumoniae serotype; and

[0097] (e) Combine the 23 conjugate solutions to provide a multivalent immunogenic complex or vaccine against 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.

[0098] The present invention also includes a method for preparing a multivalent immunogenic complex or vaccine against Streptococcus pneumoniae serotype polysaccharides 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, and 33F, the method comprising:

[0099] (a) Provide 15 carrier protein lyophilized bead compositions and 15 activated polysaccharide lyophilized bead compositions, each of the activated polysaccharide lyophilized bead compositions comprising dried activated polysaccharides from Streptococcus pneumoniae serotypes selected from 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, and 33F, wherein none of the 15 activated polysaccharide lyophilized bead compositions comprises activated polysaccharides from the same Streptococcus pneumoniae serotype;

[0100] (b) Combine each of the 15 carrier protein lyophilized bead compositions with each of the 15 activated polysaccharide lyophilized bead compositions, respectively, to provide 15 lyophilized bead mixtures, each lyophilized bead mixture having a predetermined ratio of activated polysaccharide to carrier protein, wherein none of the 15 lyophilized bead mixtures comprises activated polysaccharides from the same Streptococcus pneumoniae serotype;

[0101] (c) Reconstitute each of the 15 lyophilized bead mixtures with an organic solvent and mix them to produce a plurality of reconstituted mixtures, wherein none of the 15 reconstituted mixtures comprises activated polysaccharides from the same Streptococcus pneumoniae serotype;

[0102] (d) Add a reducing agent to each of the 15 reconstituted mixtures to produce 15 conjugate solutions, each conjugate solution comprising a carrier protein conjugated to a polysaccharide of a specific Streptococcus pneumoniae serotype, wherein none of the 15 conjugate solutions comprises activated polysaccharides from the same Streptococcus pneumoniae serotype; and

[0103] (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.

[0104] The present invention also includes 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:

[0105] (a) Provide 13 carrier protein lyophilized bead compositions and 13 activated polysaccharide lyophilized bead compositions, each of the activated polysaccharide lyophilized bead compositions comprising dried activated polysaccharides 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 activated polysaccharide lyophilized bead compositions comprises activated polysaccharides from the same Streptococcus pneumoniae serotype;

[0106] (b) Combine each of the 13 carrier protein lyophilized bead compositions with each of the 13 activated polysaccharide lyophilized bead compositions to provide 13 lyophilized bead mixtures, each lyophilized bead mixture having a predetermined ratio of activated polysaccharide to carrier protein, wherein none of the 13 lyophilized bead mixtures contains activated polysaccharide from the same Streptococcus pneumoniae serotype;

[0107] (c) Reconstitute and mix each of the 13 lyophilized bead mixtures with an organic solvent to produce a plurality of reconstituted mixtures, wherein none of the 13 reconstituted mixtures contains activated polysaccharide from the same Streptococcus pneumoniae serotype;

[0108] (d) Add a reducing agent to each of the 13 reconstituted 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 contains activated polysaccharide from the same Streptococcus pneumoniae serotype; and

[0109] (e) Combine the 13 conjugate solutions to provide the multivalent pneumococcal conjugate vaccine containing polysaccharides of Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F conjugated to a carrier protein.

[0110] The present invention also provides a method for preparing a multivalent pneumococcal conjugate vaccine containing polysaccharides of Streptococcus pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, and 23F conjugated to a carrier protein, the method comprising:

[0111] (a) Provide 10 carrier protein lyophilized bead compositions and 10 activated polysaccharide lyophilized bead compositions, wherein each of the activated polysaccharide lyophilized bead compositions contains dried activated polysaccharide from a Streptococcus pneumoniae serotype selected from 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, and 23F, and wherein none of the 10 activated polysaccharide lyophilized bead compositions contains activated polysaccharide from the same Streptococcus pneumoniae serotype;

[0112] (b) Combine each of the 10 carrier protein lyophilized bead compositions with each of the 10 activated polysaccharide lyophilized bead compositions to provide 10 lyophilized bead mixtures, each lyophilized bead mixture having a predetermined ratio of activated polysaccharide to carrier protein, wherein none of the 10 lyophilized bead mixtures contains activated polysaccharide from the same Streptococcus pneumoniae serotype;

[0113] (c) Reconstitute each of the 10 lyophilized bead mixtures with an organic solvent and mix them to produce a plurality of reconstituted mixtures, wherein none of the 10 reconstituted mixtures contains an activated polysaccharide from the same Streptococcus pneumoniae serotype;

[0114] (d) Add a reducing agent to each of the 10 reconstituted 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

[0115] (e) Combine 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 a carrier protein.

[0116] In certain embodiments, the first lyophilized bead composition and the second lyophilized bead composition each have a final moisture content of about 6% or less. In certain embodiments, the first lyophilized bead composition and the second lyophilized bead composition each have a final moisture content of about 5% or less. In certain embodiments, the first lyophilized bead composition and the second lyophilized bead composition each have a final moisture content of about 4% or less. In certain embodiments, the first lyophilized bead composition and the second lyophilized bead composition each have a final moisture content of about 3% or less. In certain embodiments, the first lyophilized bead composition and the second lyophilized bead composition each have a final moisture content of about 2% or less.

[0117] In certain embodiments of the method, the sublimation drying method includes separately freezing each aqueous solution containing the carrier protein and each aqueous solution containing the polysaccharide in the form of lyophilized beadlets.

[0118] In certain embodiments, a composition of lyophilized bead carrier protein and lyophilized bead polysaccharide is prepared by sublimation drying a plurality of separate aqueous polysaccharide solutions and a plurality of aqueous carrier protein solutions, each aqueous polysaccharide solution containing an activated Streptococcus pneumoniae polysaccharide from a specific enumerated Streptococcus pneumoniae serotype as shown above, each carrier protein solution containing 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 polysaccharide and carrier protein each contain about 0.5% or more of sucrose, and wherein the sublimation drying is selected from freeze-drying and radiant energy vacuum (REV) dehydration. In certain embodiments, the aqueous polysaccharide solutions each contain about 4% to 6% of sucrose and the aqueous carrier protein solutions each contain about 4% to 8% of sucrose.

[0119] In certain embodiments, the aqueous polysaccharide solution contains the polysaccharide at a concentration of about 6 to 9 mg / mL and the aqueous carrier protein solution contains the carrier protein at a concentration of about 6 to 12 mg / mL. In certain embodiments, the aqueous polysaccharide solution contains the polysaccharide at a concentration of about 6 or 9 mg / mL and the aqueous carrier protein solution contains the carrier protein at a concentration of about 6, 9, 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 hexamethylphosphoric triamide. In certain embodiments, the organic solvent is DMSO.

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

[0122] In certain embodiments, the mixing in step (c) is carried out in 120 minutes or less. In certain embodiments, the mixing is carried out in 90 minutes or less. In certain embodiments, the mixing is carried out in 60 minutes or less. In certain embodiments, the mixing is carried out in 30 minutes or less. In certain embodiments, the mixing is carried out in 10 minutes or less.

[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 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 for 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 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 。

[0128] In certain embodiments, the Streptococcus pneumoniae polysaccharide is activated by reaction with an oxidizing agent.

[0129] In certain embodiments, the conjugate solution is sterile filtered. BRIEF DESCRIPTION OF THE DRAWINGS

[0130] Figure 1 Shows a general flow chart for preparing a carrier protein (Pr)-polysaccharide (Ps) conjugate. DETAILED DESCRIPTION

[0131] I. DEFINITIONS

[0132] As used herein, the term "polysaccharide" (Ps) is intended to include any antigenic sugar moiety (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 may be singular or plural.

[0133] As used herein, when used in connection with the immunogenic compositions of the invention, the term "comprising" means including any other components (subject to the limitations of the term "consisting of" for antigen mixtures), such as adjuvants and excipients. When used in connection with a multivalent polysaccharide-protein conjugate mixture, the term "consisting of" means a mixture having those specific Streptococcus pneumoniae polysaccharide-protein conjugates and no other Streptococcus pneumoniae polysaccharide-protein conjugates from different serotypes.

[0134] 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. Thus, after reconstitution, the solution is physically agitated to provide a homogeneous solution of the reconstituted substance.

[0135] As used herein, the term "mixing" is used to refer to the physical agitation of a solution by shaking, stirring, swirling, rotating, etc.

[0136] As used herein, the term "homogeneous solution" refers to a solution in which all components are well mixed such that there is no concentration gradient or layer between the components in the solution.

[0137] 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 the 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 force, freeze-thaw, dehydration, heavy metals, phenolic compounds, silicone oil, denaturing agents, etc.

[0138] 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 not substantially 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.

[0139] 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 antigen 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.

[0140] As used herein, the term "radiant energy vacuum (REV) dehydration" is also referred to as microwave vacuum drying (MVD).

[0141] II. Process

[0142] 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) freeze-dried using various sublimation methods, and then the discretely freeze-dried polysaccharide and carrier protein are mixed under certain conditions that result in the formation of a conjugate composition having a lower level of free polysaccharide (e.g., less than 15% free polysaccharide).

[0143] The present invention is an improvement over the 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-freezing of the carrier protein and the polysaccharide because it has been shown that co-freezing 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 freeze-dried polysaccharide and carrier protein has a composition having approximately 31% free polysaccharide.

[0144]

[0145] However, the inventors of the present invention have found that under certain conditions, separately (or discretely) freeze-dried carrier protein and activated polysaccharide can be conjugated to produce a composition comprising high molecular weight conjugates, which is different from that disclosed in U.S. Patent Application Publication No. 20110201791 and has a lower level of free polysaccharide. These specific conditions include combining a predetermined amount of carrier protein freeze-dried spheres with a predetermined amount of polysaccharide freeze-dried spheres to form a freeze-dried sphere mixture, and reconstituting and mixing the freeze-dried sphere mixture in an organic solvent to provide a conjugate solution. In a specific embodiment, the organic solvent is dimethyl sulfoxide (DMSO). Reconstituting these freeze-dried sphere mixtures rather than separately reconstituting the carrier protein and polysaccharide freeze-dried spheres substantially reduces or eliminates the concentration gradients that typically occur when two solutions are combined by adding one solution to another or dissolving a dry substance into a solution. The reduction or elimination of the concentration gradient increases the yield of a composition comprising high molecular weight conjugates and a low amount of free polysaccharide (e.g., 15% or less free polysaccharide). The present invention has various advantages compared to the co-freezing of polysaccharide and carrier protein in a single composition, 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 with the desired solution specifications.

[0146] Figure 1 Shows a general flow chart for preparing a carrier proteoglycan conjugate, in which a carrier protein and a polysaccharide are mixed in the form of dry lyophilized spheres as taught herein.

[0147] Under normal circumstances, purified pneumococcal capsular polysaccharide (Ps) powder is separately dissolved in water, 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 Ps. The size of serotype 18C is reduced by acid hydrolysis at 90 °C or higher. Since its initial size is relatively low, the size of serotype 19A is not reduced. The homogenization pressure and the number of passes through the homogenizer are controlled for serotype-specific targets to achieve serotype-specific molecular weights. The polysaccharides are separately filtered 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 filtrate 1.

[0148] Then, filtrate 1 can be adjusted to a serotype-specific temperature (between 4–22 °C) and pH (4 - 5) using sodium acetate buffer to minimize the reduction in polysaccharide size during the activation step. The polysaccharide is activated by periodate oxidation. For serotype 4, before activation, the solution is incubated at about 50 °C and pH 4 to partially deketalize the polysaccharide. Polysaccharide activation is initiated by adding a sodium metaperiodate solution. The amount of sodium metaperiodate added is serotype-specific, ranging from about 0.1 to 0.5 moles of sodium metaperiodate per mole of polysaccharide repeating unit. The serotype-specific amount (charge) of sodium metaperiodate is selected to achieve the target level of polysaccharide activation (moles of aldehyde per mole of polysaccharide repeating unit).

[0149] In ultrafiltration step 2, the activated polysaccharides can be diafiltered for all serotypes and then concentrated by tangential flow ultrafiltration using a 10 kDa ultrafiltration membrane with open channels to produce filtrate 2. At the end of diafiltration, filtrate 2 can be concentrated to a target of 15 g Ps / L. Preferably, ultrafiltration for all serotypes is carried out at 2 - 8 °C.

[0150] Then, filtrate 2 is diluted in water containing sucrose to a final concentration of about 4 to 12 mg / mL polysaccharide and 0.5% to 6% (w / v) sucrose and lyophilized during the process for producing lyophilized spheres to produce lyophilized spheres of dry polysaccharide with a final water content of 6% or less. The amount of sucrose added to the solution before lyophilization is serotype-specific. In certain embodiments, two or more polysaccharides can be dried together to produce a dry polysaccharide mixture.

[0151] Using a 5 kDa tangential flow ultrafiltration membrane, the purified carrier protein was diafiltered with 2 mM phosphate buffer (pH 7.0) and then filtered through a 0.22 micron filter. The filtered solution was diluted in water containing sucrose such that the final concentration was about 6 to 12 mg / mL carrier protein and 4 to 10% (w / v) sucrose, and lyophilized during the process of producing the lyophilized beads to produce lyophilized beads of the dried carrier protein with a final water content of 6% or less. The lyophilization method for producing the lyophilized beads may include freeze-drying or radiant energy vacuum (REV) dehydration.

[0152] In certain embodiments, the final water content of the lyophilized beads is about 5% or less. In certain embodiments, the final water content of the lyophilized beads is about 4% or less. In certain embodiments, the final water content of the lyophilized beads is about 3% or less. In certain embodiments, the final water content of the lyophilized beads is about 2% or less.

[0153] The dried polysaccharide lyophilized beads and the dried carrier protein lyophilized beads are combined to provide a lyophilized bead mixture comprising the polysaccharide lyophilized beads and the carrier protein lyophilized beads. The polysaccharide lyophilized beads and the carrier protein lyophilized beads are combined in such a way as to obtain the final concentration of the serotype-specific polysaccharide and the ratio of polysaccharide:carrier protein. Generally, the carrier protein and the polysaccharide are mixed in an amount that will provide a final conjugated polysaccharide:carrier protein ratio of from about 0.6 to 1.3 (w / w).

[0154] The lyophilized bead mixture is reconstituted or redissolved in an organic solvent (e.g., dimethyl sulfoxide (DMSO)) to provide a reconstituted mixture. In a preferred embodiment, the organic solvent is anhydrous, and in certain embodiments, the anhydrous organic solvent is anhydrous DMSO.

[0155] In certain embodiments, after reconstitution, the reconstituted 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 over a period of 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 reconstituted mixture such that the final concentration is 1.0 mM sodium phosphate.

[0156] For performing the conjugation reaction, an aqueous solution of sodium cyanoborohydride was prepared and 1.0 meq of sodium cyanoborohydride (1.0 mole of sodium cyanoborohydride per mole of polysaccharide repeating unit) was added to the reconstituted mixture to provide a conjugation solution. The molar concentration of the sodium cyanoborohydride solution was based on a target amount of about 0.5% total water content in the conjugation process. The conjugation solution was allowed to react at the serotype-specific temperature for the serotype-specific duration to produce a carrier protein:polysaccharide conjugate intermediate.

[0157] Next, an aqueous solution of sodium borohydride was prepared and 2.0 meq of sodium borohydride (relative to the polysaccharide repeating unit) was added to the conjugation solution. The molar concentration of the sodium borohydride solution was based on a target amount of about 1.0% total water content in the conjugation solution after adding sodium borohydride. The conjugation solution was allowed to react at room temperature for three hours (except for certain serotypes, such as serotype 7F, which was allowed to react for two hours) to produce a carrier protein:polysaccharide conjugate.

[0158] To quench the conjugation reaction, for conjugates containing polysaccharides from a specific serotype, the conjugation solution was diluted to 20% (v / v) or less of an organic solvent 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 a particular embodiment, the temperature was maintained at 15 °C or lower during the dilution step. After about 1 hour, 1.5 M potassium phosphate (pH 6.0) was added to the solution to give 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.

[0159] In ultrafiltration step 3, the quenched conjugate solution can be concentrated to about 2.5 g / L and diafiltered using a 30 kDa tangential flow ultrafiltration membrane with 150 mM sodium chloride or 150 mM sodium chloride containing 25 mM potassium phosphate at 2 - 8 °C using up to about 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 about 120 hours.

[0160] 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 tangential flow ultrafiltration with a 300 kDa membrane can be used to diafilter at 2 - 8 °C with 20 diafiltration volumes of 150 mM sodium chloride containing 10 mM L-histidine (pH 7.0) to produce retentate 4. For conjugates containing polysaccharides from certain serotypes (e.g., serotype 7F), a 100 kDa tangential flow ultrafiltration membrane can be used to diafilter the conjugate; for example, conjugates containing serotype 6A, 6B, and 18C polysaccharides can be concentrated to about 3.5 g / L and tangential flow ultrafiltration with a 300 kDa membrane can be used to diafilter at 2 - 8 °C with a buffer containing 150 mM sodium chloride and 0.03% (w / v) polysorbate 20 (pH 7.0) to produce retentate 4. Conjugates containing serotype 7F, 19A, 19F, and 23F polysaccharides can be concentrated to about 2.0 g / L and tangential flow ultrafiltration with a 300 kDa regenerated cellulose membrane can be used to diafilter at 2 - 8 °C with a buffer containing 150 mM sodium chloride and 0.015% (w / v) polysorbate 20 (pH 7.0) 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 particular embodiment, the buffer is 10 mM L-histidine.

[0161] 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 with 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.

[0162] 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 with additional 10 mM L-histidine in 150 mM sodium chloride, 0.03% (w / v) polysorbate 20, pH 7.0. This provides an MBC or monovalent drug substance. The MBC can be aliquoted and frozen at -60 °C to -80 °C.

[0163] 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.

[0164] III. Polysaccharide

[0165] The 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, for example, European Patent Nos. EP497524 and EP497525); and in certain embodiments, microfluidization done by using a homogenizer or chemical hydrolysis. In one embodiment, Streptococcus pneumoniae strains are grown in a soy - based medium. Then the various polysaccharides are purified by standard steps including centrifugation, precipitation, and ultrafiltration. See, for example, U.S. Patent Application Publication No. 2008 / 0286838 and U.S. Patent No. 5,847,112. The polysaccharides can be sized to reduce viscosity and / or improve the filterability of the subsequent conjugate products. 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.

[0166] IV. Carrier Protein

[0167] In a particular embodiment of the present invention, CRM 197 is used as the carrier protein. CRM 197 is a non - toxic variant (i.e., a toxoid) of diphtheria toxin. 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 .

[0168] 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. WO 01 / 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.

[0169] 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.

[0170] V. Lyophilization

[0171] For example, lyophilized spheres can be prepared in a continuous or semi-continuous manner by freeze-drying or dehydration by radiant energy vacuum (REV) in the form of a traveling wave (microwave vacuum drying), spray freeze-drying, vacuum drying, RF drying, etc. 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).

[0172] For example, lyophilized beads can be prepared by loading aliquots of an aqueous solution containing one or more polysaccharide serotypes or carrier proteins in the form of droplets (e.g., about 20, 50, 100, or 250 microliters) onto a solid surface 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, 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 through 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 onto 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.

[0173] VI. Polysaccharide-Protein Conjugation

[0174] The purified polysaccharide is chemically activated to introduce functional groups capable of reacting with the carrier protein. Once activated, each capsular polysaccharide is conjugated to the carrier protein separately to form a glycoconjugate according to the method of the invention.

[0175] In one embodiment, chemical activation of a polysaccharide can be achieved by the methods described in U.S. Patent Nos. 4,365,170; 4,673,574; and 4,902,506. Briefly, reacting a pneumococcal polysaccharide with an oxidizing agent (e.g., a periodate-based oxidizing agent such as sodium periodate, potassium periodate, or periodic acid) results in random oxidative cleavage of vicinal hydroxyl groups to generate reactive aldehyde groups. Direct amide coupling of the oxidized polysaccharide with primary amine groups (primarily lysine residues) on a protein carrier can be achieved by reductive amination. For example, conjugation can be 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 addition of a strong reducing agent such as sodium borohydride.

[0176] In one embodiment, chemical activation of a polysaccharide and subsequent conjugation to a carrier protein are achieved by the methods described in U.S. Patent Nos. 4,365,170, 4,673,574, and 4,902,506. Briefly, reacting the polysaccharide with a periodate-based oxidizing agent such as sodium periodate, potassium periodate, or periodic acid results in random oxidative cleavage of vicinal hydroxyl groups to generate reactive aldehyde groups. Then, direct amide coupling of the oxidized polysaccharide with primary amine groups (primarily lysine residues) on a protein carrier can be achieved by reductive amination. For example, conjugation 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 addition of a strong reducing agent such as sodium borohydride.

[0177] 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 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.

[0178] It is known that transition metals can form stable complexes with cyanide, and it is known that the reductive methylation of protein amines 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 surprisingly found that by complexing the residual interfering cyanide, the addition of nickel increased the consumption of protein during the conjugation process and led to the formation of larger and possibly more immunogenic conjugates.

[0179] Variations in the free cyanide levels in commercially available batches of sodium cyanoborohydride reagent can lead to inconsistent conjugation performance, and thus to variations in conjugate properties, including molecular weight and the polysaccharide-to-protein ratio. The addition of nickel to the conjugation reaction reduces the level of free cyanide, thereby improving the degree of conjugate consistency between batches.

[0180] 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.

[0181] 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 through thioether bonds obtained after reaction with maleimide-activated carrier proteins (such as using GMBS) or haloacetylated carrier proteins (such as using iodoacetamide [e.g., iodoacetamide 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.

[0182] 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.

[0183] The following examples are intended to facilitate further understanding of the present invention.

[0184] Example 1

[0185] Preparation of Streptococcus pneumoniae capsular polysaccharides 6A, 6B, 7F, 18C, 19A, 19F and 23F.

[0186] 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 Streptococcus pneumoniae subtypes are available from the American Type Culture Collection (Manassas, VA). The bacterium is identified as an encapsulated, non-motile, Gram-positive, lancet-shaped diplococcus with α-hemolysis on blood agar. Subtypes can be differentiated using specific antisera according to the Quelling reaction. See, e.g., U.S. Patent No. 5,847,112.

[0187] A cell bank representing each Streptococcus pneumoniae serotype present was obtained from frozen vials of the Merck Culture Collection (Rahway, NJ). The thawed seed culture was transferred to a seed fermentor containing pre-sterilized growth medium suitable for Streptococcus pneumoniae. The culture was grown in a seed fermentor with temperature and pH control. The entire volume of the seed fermentor was transferred to a production fermentor containing pre-sterilized growth medium. The production fermentation is the final cell growth stage of the process. Temperature, pH and agitation rate are controlled.

[0188] The fermentation process was terminated by adding an inactivator. After inactivation, the solution was transferred to an inactivation tank where it was kept under controlled temperature and agitation. Cell debris was removed using a combination of centrifugation and filtration. The solution was ultrafiltered and diafiltered. Then, the solution was fractionated based on solvents to remove impurities and recover the polysaccharide.

[0189] Example 2

[0190] The polysaccharide size reduction and activation were carried out as follows.

[0191] At room temperature, approximately 6 g of purified pneumococcal polysaccharide (Ps) powder was dissolved in water for injection (WFI) to a target concentration of approximately 4 g / L. Then, the solution was filtered through a 0.45-μm filter to reduce the bioburden. The Ps concentration of the filtered Ps solution was determined by HPSEC UV-MALS-RI.

[0192] For all serotypes except serotypes 18C and 19A, the solution was 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 except serotype 19A were homogenized to reduce the molecular weight of the polysaccharide. The size of serotype 19A was 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) were controlled for serotype-specific targets to achieve serotype-specific molecular weights. The size-reduced polysaccharide was 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 was controlled using cooling water supplied to a heat exchanger at the outlet of the homogenizer.

[0193] Acid hydrolysis was 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 was increased to approximately 96 °C and 90 °C, respectively. Then, the solution was adjusted with glacial acetic acid (17.4 M) to a final concentration of 0.2 M and kept for approximately 180 minutes and 160 minutes for batch A and batch B, respectively. 1.5 M potassium phosphate (pH 7.0) was added to a final concentration of 0.46 M to terminate the acid hydrolysis by increasing the solution pH, and then the solution was cooled to room temperature.

[0194] Serotype 19A was not filtered through a 0.45-μm filter and sized down. Due to its relatively low initial size, size reduction was not required. After dissolution, serotype 19A was filtered through a 0.22-μm filter as described in the next paragraph.

[0195] 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 produced 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.

[0196] 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).

[0197] 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).

[0198]

[0199] 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 produced 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.

[0200] Example 3

[0201] The CRM can be carried out as shown below 197 Preparation of carrier protein.

[0202] Using a 5 kDa NMWCO tangential flow ultrafiltration membrane, diafilter with 10 diafiltration volumes of 2 mM phosphate buffer (pH 7.2) the frozen, purified CRM obtained by expression in Pseudomonas fluorescens as described previously (see International Patent Application Publication No. WO 2012 / 173876) 197 and perform a 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). Table 3 below provides the representative sucrose concentrations for CRM 197 conjugated to polysaccharides of specific serotypes.

[0203] Example 4

[0204] Produce lyospheres of polysaccharide (Ps) and CRM 197 (Pr) as shown below.

[0205] Before lyophilization, dilute the CRM 197 and Ps solutions as shown below.

[0206] 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.

[0207] 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.

[0208] Use serotype-specific sucrose and phosphate concentrations (Table 3). As shown below, process the diluted CRM 197 and UF2-FR solutions into lyospheres.

[0209]

[0210] Example 5

[0211] This example shows a method for producing polysaccharide (Ps) and CRM 197Development of freeze-dryer conditions for (Pr; CRM) freeze-dried beads.

[0212] Prepare discrete solutions of CRM and activated polysaccharides from serotypes 6A and 23F as described in Examples 3 and 4 and having the Ps, CRM, and sucrose concentrations shown in the table in this example.

[0213] Use a modified Biomek FX pipetting robot (Cryomek) 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 is carried out in a freeze-dryer or by microwave vacuum drying. For freeze-dryer drying, the beads are dispensed in a single layer onto drying trays. Set the cabinet pressure, shelf temperature, and cycle time. After drying the beads, store the freeze-dried beads at 2 - 8 °C (see Example 5 for specific parameters).

[0214] The preliminary drying cycle (Lyo1) takes 18 hours as shown in Table 4.1. The residual moisture content of the freeze-dried beads determined by Karl Fisher titration is shown in Table 4.2.

[0215]

[0216]

[0217] The results show that the moisture content in the freeze-dried beads is high. In addition, the freeze-dried beads are very fragile and hygroscopic. Increase the solid content by increasing the polysaccharide, protein, and sucrose concentrations as shown in Table 5.2. Improve the drying cycle (Lyo 2) by adding a secondary drying cycle and increasing the primary drying time as shown in Table 5.1.

[0218]

[0219]

[0220] Due to the improvement of the 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).

[0221] At this time, the total drying cycle time of the freeze-dryer is 45 hours. Change some parameters such as pressure and temperature to further shorten the drying cycle time (method: Lyo 3) as shown in Tables 6.1 and 6.2.

[0222]

[0223]

[0224] Example 6

[0225] This example shows the development of radiant energy vacuum (REV) dehydration (microwave vacuum drying (MVD)) conditions for the production of polysaccharide (Ps) and CRM 197 (Pr; CRM) freeze-dried beads.

[0226] Prepare solutions of CRM and activated polysaccharides from serotypes 6A and 23F as described in Examples 3 and 4, and having the Ps, CRM, and sucrose concentrations shown in Tables 7.1 and 7.2.

[0227] 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 were 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 were dispensed in a single layer into the container. The power, pressure, and cycle time were set. After drying the beads, the freeze-dried beads were stored at 2 - 8 °C (see Example 6 for specific parameters).

[0228] 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 power applied.

[0229]

[0230]

[0231] The MVD1 cycle took 4 hours 30 minutes, but it was not sufficient to reduce the residual moisture content to at most 2%. The MVD2 cycle took longer and required higher power, and thus provided a significantly reduced residual moisture content.

[0232] Example 7

[0233] When preparing for conjugation, the polysaccharide (Ps) freeze-dried beads and CRM 197The protein (Ps) lyophilized spheres are combined in a manner to obtain a specific Ps to Pr (Ps:Pr) ratio and a final Ps concentration to provide a mixture of lyophilized spheres dissolved in the above anhydrous DMSO. Sodium cyanoborohydride is added to the Ps:Pr blend, and the solution is incubated at the desired conjugation temperature for an appropriate reaction time, both 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.

[0234] 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 repeating unit) to the solution. The molar concentration of the sodium cyanoborohydride solution (Table 8) 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 8) to produce a conjugate product intermediate (CP).

[0235] Prepare a solution of sodium borohydride in WFI and add 2.0 meq of sodium borohydride (relative to the Ps repeating unit) to the solution. The molar concentration of the sodium borohydride solution (Table 2) is based on a target value of approximately 1.0% total water content of the solution after adding the borohydride. React the solution at room temperature for 3 hours (except for V1147FPB1401, which is reacted for 2 hours) to produce a conjugate product quenching intermediate (CPQ).

[0236]

[0237] Then, dilute the conjugation solution to 20% or less (v / v) anhydrous DMSO by slowly adding the solution to 150 mM sodium chloride (for conjugates in some embodiments, 150 mM sodium chloride plus 0.025% w / v polysorbate 20). 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.

[0238] Concentrate the conjugation solution to approximately 2.5 g / L and use a 30 kDa NMWCO tangential flow ultrafiltration membrane to perform diafiltration at 2 - 8°C with 10 diafiltration volumes of 150 mM sodium chloride (for batches in WL00067190) or 150 mM sodium chloride containing 25 mM potassium phosphate (for batches in WL00067191). This produces an ultrafiltration 3 process intermediate (UF3-FR). Determine the Ps concentration of UF3-FR by HPSEC UV-MALS-RI.

[0239] For 19F in some embodiments, UF3-FR is filtered through a 0.22 micron filter and then incubated at 22 °C for approximately 120 hours.

[0240] Then, the UF3-FR solution is processed in the ultrafiltration 4 step. During the ultrafiltration 4 step in some embodiments, the solution is concentrated to a Ps concentration of approximately 2.5 g / L and diafiltered at 2-8 °C with 20 diafiltration volumes 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 used, the UF3-FR solution is concentrated to approximately 3.5 g / L and diafiltered at 2-8 °C with 10 mM L-histidine in 150 mM sodium chloride, 0.03% (w / v) PS-20, pH 7.0. In some embodiments, serotype 7F, 19A, 19F, and 23F UF3-FR solutions are concentrated to approximately 2.0 g / L and diafiltered at 2-8 °C with 10 mM L-histidine in 150 mM sodium chloride, 0.015% (w / v) PS-20, pH 7.0 using a 300 kDa NMWCO UltraCel regenerated cellulose tangential flow ultrafiltration membrane. This yields the ultrafiltration 4 process intermediate (UF4-FR). The Ps concentration of UF4-FR is determined by HPSEC UV-MALS-RI.

[0241] The UF4-FR is filtered through a PVDF filter with a 0.22 micron 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 yields the monovalent bulk conjugate intermediate (MBC). The MBC is aliquoted and frozen at -60 °C to -80 °C.

[0242] For serotypes 6A, 6B, and 18C, UF4-FR was filtered through a dual-membrane PES filter with 0.5 / 0.2 µm. 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, 0.03% w / v PS-20, pH 7.0. This yielded a monovalent bulk conjugate intermediate (MBC). The MBC was aliquoted and frozen at -60 °C to -80 °C.

[0243] For serotypes 7F, 19A, 19F, and 23F, UF4-FR was filtered through a PVDF filter with 0.22 µm. 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 yielded a monovalent bulk conjugate intermediate (MBC). The MBC was aliquoted and frozen at -60 °C to -80 °C.

[0244] Example 8

[0245] This example shows a method for conjugating Ps from 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, or 38 to CRM 197 (Pr) by reductive amination in anhydrous DMSO. Different serotype polysaccharides were conjugated to purified CRM 197 carrier protein using a common process flow.

[0246] The polysaccharide (Ps) lyophilized beads and CRM 197 protein (Ps) lyophilized beads were combined in a manner to obtain a specific Ps to Pr (Ps:Pr) ratio and final Ps concentration to provide a lyophilized bead mixture dissolved in the above anhydrous DMSO. Sodium cyanoborohydride was added to the Ps:Pr reconstituted mixture and the solution was incubated at the desired conjugation temperature for an appropriate reaction time, 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.

[0247] Reduction using sodium borohydride

[0248] 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 approximately 4 °C, the solution was diluted to 150 mM sodium chloride, 0.025% (w / v) polysorbate 20. Then phosphate buffer was added to neutralize the pH. The solution was concentrated and diafiltered at approximately 4 °C using a 10 kDa NMWCO tangential flow ultrafiltration membrane with 150 mM sodium chloride.

[0249] Final Filtration and Product Storage

[0250] Then, each solution was concentrated and diafiltered at 4 °C in 150 mM sodium chloride containing 10 mM histidine (pH 7.0) using a 300 kDa NMWCO tangential flow ultrafiltration membrane. The retentate solution was filtered through a 0.22 micron filter.

[0251] Serotype 19F was incubated for approximately 5 days, diafiltered at approximately 4 °C in 150 mM sodium chloride containing 10 mM histidine (pH 7.0) using a 300 kDa NMWCO tangential flow ultrafiltration membrane, and filtered through a 0.22 micron filter.

[0252] Serotype 18C was diafiltered at approximately 4 °C in 150 mM sodium chloride containing 10 mM histidine (pH 7.0) using a 300 kDa NMWCO tangential flow ultrafiltration membrane and filtered through a 0.22 micron filter.

[0253] The solution was diluted with additional 150 mM sodium chloride containing 10 mM L-histidine (pH 7.0), aliquoted and frozen at ≤ -60 °C.

[0254] Example 9

[0255] This example shows the formulation of a 15-valent pneumococcal conjugate vaccine with different surfactants and stabilizers.

[0256] The pneumococcal polysaccharide-protein conjugate prepared as described above was used to formulate a 15-valent pneumococcal conjugate vaccine (PCV15) with serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, and 33F.

[0257] As previously discussed, pneumococcal polysaccharide-CRM generated by reductive amination in DMSO was used 197Conjugates are used to prepare the formulation. Calculate the volume of the bulk conjugate required to obtain the target final concentration of a single serotype based on the solution volume and the bulk polysaccharide concentration. Combine 15 conjugates with excipients selected from sodium chloride, L-histidine, pH 5.8 buffer containing polysorbate (PS)-20, PS-80, or poloxamer (P) 188.

[0258] During this period, gently mix the aseptically formulated materials, and then mix them with the bulk aluminum phosphate adjuvant (APA), which may or may not contain propylene glycol (PG) and polyethylene glycol 400 (PEG400). Two concentrations of conjugates 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.

[0259] APA is an aqueous suspension of aluminum hydroxyphosphate. APA is prepared by mixing aluminum chloride and sodium phosphate in a volume ratio of 1:1 to precipitate aluminum hydroxyphosphate. After the mixing process, reduce the size of the material with a high-shear mixer to achieve a monodisperse particle size distribution. Then, use normal saline to diafilter the product and perform steam sterilization.

[0260] Although the present invention has been described herein with reference to the illustrated embodiments, it should be understood that the 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 claims appended hereto.

Claims

1. A method for preparing a multivalent pneumococcal composition, the composition comprising two or more conjugates, each conjugate comprising a pneumococcal polysaccharide from one or more serotypes covalently linked to a carrier protein, the method comprising: (a) providing (i) two or more first aqueous solutions, each first aqueous solution comprising (i) 4% to 6% sucrose and (ii) an activated polysaccharide of a specific pneumococcal 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 (ii) two or more first aqueous solutions, each first aqueous solution comprising (i) 4% to 6% sucrose and (ii) activated polysaccharides of two or more pneumococcal 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; (b) providing two or more second aqueous solutions, each comprising (i) 4% to 8% sucrose, (ii) a carrier protein, and (iii) 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; (c) separately drying the two or more first aqueous solutions and the two or more second aqueous solutions in a sublimation drying method for producing freeze-dried beads to produce two or more first freeze-dried bead compositions and two or more second freeze-dried bead compositions, the first freeze-dried bead compositions each comprising the dried polysaccharide and the second freeze-dried bead compositions each comprising the dried carrier protein; (d) independently combining an amount of a first freeze-dried bead composition with an amount of a second freeze-dried bead composition to provide a plurality of freeze-dried bead mixtures, each freeze-dried bead mixture having a predetermined ratio of activated polysaccharide to carrier protein between 1.2 and 2.0; (e) reconstituting and mixing the plurality of freeze-dried bead mixtures in an organic solvent to provide a plurality of reconstituted mixtures, wherein the reconstituted mixtures comprise an activated polysaccharide at a concentration between 1.1 g / L and 3.8 g / L; (f) adding a reducing agent to the plurality of reconstituted mixtures to produce a plurality of conjugate solutions; and (g) combining two or more of the plurality of conjugate solutions to produce a multivalent pneumococcal composition comprising two or more conjugates, each conjugate comprising a pneumococcal polysaccharide from one or more serotypes covalently linked to a carrier protein, wherein each conjugate solution comprises an amount of free polysaccharide less than 15% of the total polysaccharide in the solution.

2. The method according to claim 1, wherein the sublimation drying method is selected from freeze-drying and radiant energy vacuum (REV) dehydration.

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

4. The method according to claim 1, wherein the reconstitution is carried out in eight minutes or less and the mixing is carried out in 120 minutes or less.

5. The method according to claim 1, 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 weight for weight.

6. The method according to claim 1, wherein each conjugate solution comprises a free polysaccharide amount of less than 10% of the total polysaccharide in the solution.

7. The method according to claim 1, 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 1, 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 one or more polysaccharides are obtained from one or more 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.

10. The method according to claim 1, wherein the carrier protein is an inactivated bacterial toxoid selected from: tetanus toxoid, diphtheria toxoid, pertussis toxoid, bacteriolysin or pneumolysin.

11. The method according to claim 10, wherein the inactivated bacterial toxoid is CRM 197 .

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

13. 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) Provide 23 carrier protein lyophilized bead compositions and 23 activated polysaccharide lyophilized bead compositions, each of the activated polysaccharide lyophilized bead compositions comprising dried 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 activated polysaccharide lyophilized bead compositions comprises activated polysaccharide from the same Streptococcus pneumoniae serotype; (b) Combine each of the 23 carrier protein lyophilized bead compositions with each of the 23 activated polysaccharide lyophilized bead compositions, respectively, to provide 23 lyophilized bead mixtures, each lyophilized bead mixture having a predetermined ratio of activated polysaccharide to carrier protein between 1.2 and 2.0, wherein none of the 23 lyophilized bead mixtures comprises activated polysaccharide from the same Streptococcus pneumoniae serotype; (c) Reconstitute and mix each of the 23 lyophilized bead mixtures with an organic solvent, respectively, to produce a plurality of reconstituted mixtures, wherein none of the 23 reconstituted mixtures comprises activated polysaccharide from the same Streptococcus pneumoniae serotype, and wherein the reconstituted mixtures comprise activated polysaccharide at a concentration between 1.1 g / L and 3.8 g / L; (d) Add a reducing agent to each of the 23 reconstituted mixtures to produce 23 conjugate solutions, each conjugate solution comprising a carrier protein conjugated to a polysaccharide of a specific Streptococcus pneumoniae serotype, wherein none of the 23 conjugate solutions comprises activated polysaccharide from the same Streptococcus pneumoniae serotype; and (e) Combine 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 each conjugate solution comprises a free polysaccharide amount of less than 15% of the total polysaccharide in the solution.

14. 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) Provide 15 carrier protein lyophilized bead compositions and 15 activated polysaccharide lyophilized bead compositions, each of the activated polysaccharide lyophilized bead compositions comprising dried 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 activated polysaccharide lyophilized bead compositions comprises activated polysaccharide from the same Streptococcus pneumoniae serotype; (b) Combine each of the 15 carrier protein lyophilized bead compositions with each of the 15 activated polysaccharide lyophilized bead compositions respectively to provide 15 lyophilized bead mixtures, each lyophilized bead mixture having a predetermined ratio of activated polysaccharide to carrier protein between 1.2 and 2.0, wherein none of the 15 lyophilized bead mixtures contains activated polysaccharide from the same Streptococcus pneumoniae serotype; (c) Reconstitute and mix each of the 15 lyophilized bead mixtures with an organic solvent respectively to produce a plurality of reconstituted mixtures, wherein none of the 15 reconstituted mixtures contains activated polysaccharide from the same Streptococcus pneumoniae serotype, and wherein the reconstituted mixtures contain activated polysaccharide at a concentration between 1.1 g / L and 3.8 g / L; (d) Add a reducing agent to each of the 15 reconstituted 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 contains activated polysaccharide from the same Streptococcus pneumoniae serotype; and (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, wherein each conjugate solution contains a free polysaccharide amount of less than 15% of the total polysaccharide in the solution.

15. 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) Provide 13 carrier protein lyophilized bead compositions and 13 activated polysaccharide lyophilized bead compositions, each of the activated polysaccharide lyophilized bead compositions containing dried 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 activated polysaccharide lyophilized bead compositions contains activated polysaccharide from the same Streptococcus pneumoniae serotype; (b) Combine each of the 13 carrier protein lyophilized bead compositions with each of the 13 activated polysaccharide lyophilized bead compositions respectively to provide 13 lyophilized bead mixtures, each lyophilized bead mixture having a predetermined ratio of activated polysaccharide to carrier protein between 1.2 and 2.0, wherein none of the 13 lyophilized bead mixtures contains activated polysaccharide from the same Streptococcus pneumoniae serotype; (c) Reconstitute and mix each of the 13 lyophilized bead mixtures with an organic solvent respectively to produce a plurality of reconstituted mixtures, wherein none of the 13 reconstituted mixtures contains activated polysaccharide from the same Streptococcus pneumoniae serotype, and wherein the reconstituted mixtures contain activated polysaccharide at a concentration between 1.1 g / L and 3.8 g / L; (d) A reducing agent is added to each of the 13 reconstituted mixtures to produce 13 conjugate solutions, each conjugate solution comprising a carrier protein conjugated to a polysaccharide of a specific Streptococcus pneumoniae serotype, wherein none of the 13 conjugate solutions contains an activated polysaccharide from the same Streptococcus pneumoniae serotype; and (e) The 13 conjugate solutions are combined 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, wherein each conjugate solution contains a free polysaccharide amount of less than 15% of the total polysaccharide in the solution.

16. 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 carrier protein lyophilized bead compositions and 10 activated polysaccharide lyophilized bead compositions, wherein each of the activated polysaccharide lyophilized bead compositions contains a dried activated polysaccharide from a Streptococcus pneumoniae serotype selected from 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, and 23F, and wherein none of the 10 activated polysaccharide lyophilized bead compositions contains an activated polysaccharide from the same Streptococcus pneumoniae serotype; (b) Combining each of the 10 carrier protein lyophilized bead compositions with each of the 10 activated polysaccharide lyophilized bead compositions respectively to provide 10 lyophilized bead mixtures, each lyophilized bead mixture having a predetermined ratio of activated polysaccharide to carrier protein between 1.2 and 2.0, and wherein none of the 10 lyophilized bead mixtures contains an activated polysaccharide from the same Streptococcus pneumoniae serotype; (c) Reconstituting and mixing each of the 10 lyophilized bead mixtures with an organic solvent respectively to produce a plurality of reconstituted mixtures, wherein none of the 10 reconstituted mixtures contains an activated polysaccharide from the same Streptococcus pneumoniae serotype, and wherein the reconstituted mixtures contain an activated polysaccharide at a concentration between 1.1 g / L and 3.8 g / L; (d) Adding a reducing agent to each of the 10 reconstituted mixtures to produce 10 conjugate solutions, each conjugate solution comprising 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 a carrier protein, wherein each conjugate solution contains a free polysaccharide amount of less than 15% of the total polysaccharide in the solution.

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