Method for producing preservative-containing vaccine formulation

Abstract

The present invention relates to a method for producing a conjugate vaccine comprising a preservative. In particular, the present invention relates to a method for producing a conjugate vaccine, wherein the preservative is hydrophobic and viscous (e.g., 2-phenoxyethanol (2-PE)).

Description

Field of the Invention

[0001] The present invention relates to a method for producing a conjugate vaccine containing a preservative. The present invention particularly relates to a method for producing a conjugate vaccine, wherein the preservative is hydrophobic and viscous (eg 2-phenoxyethanol (2-PE)). Background of the Invention

[0003] Bacterial cell surface polysaccharides, especially capsular polysaccharides, have become increasingly important as therapeutic agents. In general, cell surface polysaccharides are associated with inducing immune responses in vivo.

[0004] Although polysaccharides themselves are immunogenic, conjugation of polysaccharides to protein carriers (glycoconjugates) has been used to improve immunogenicity, especially in infants and the elderly. Sugar conjugate vaccines are usually obtained by covalently linking poorly immunogenic sugar antigens to protein carriers and play an important role in preventing many deadly infectious diseases. During the preparation of conjugate vaccines, selected bacterial strains are cultured to provide the polysaccharides required for vaccine production. Cells are usually cultured in fermenters and lysis is induced at the end of fermentation. The lysate is then collected for downstream purification and recovery of the capsular polysaccharides surrounding the bacterial cells. After conjugation with the carrier protein, the polysaccharide is included in the final vaccine product and confers immunity to the vaccine target population against bacteria.

[0005] Pneumococcal disease, caused by Streptococcus pneumoniae, is one of the more important bacterial pathogens worldwide. Pneumococcal disease is a complex group of diseases and includes invasive infections such as bacteremia / sepsis, meningitis, pneumonia, and otitis media, which affect children and adults. Prevnar 13 (also referred to as "Prevenar 13" and herein as "Prev(e)nar 13") is a polysaccharide formulation from thirteen pneumococcal serotypes (1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F), which are respectively conjugated to CRM 197 (Cross-reacting substances from mutant strains of Corynebacterim diphtheriae) conjugation. See, for example, WO2006 / 110381, WO2008 / 079653, WO2008 / 079732, WO2008 / 143709 and WO 2011 / 151760.

[0006] By manufacturing and using only single-dose vaccine formulations, the need to add preservatives to vaccines can be reduced or avoided. However, the use of single-dose formulations without preservatives increases the overall cost of vaccination and jeopardizes the effectiveness of immunization programs, especially in developing countries. In addition, removing preservatives from multi-dose bottles altogether is not considered a preferred option, especially in countries with limited refrigeration and suboptimal health care standards (Drain et al., Bull World Health Organ 81(10):726–731(2003). Therefore, although multi-dose bottles appear best suited for producing cheaper vaccines, it is still desirable to formulate multi-dose vaccines with at least one preservative to protect subjects from microorganisms that are inadvertently introduced into the vaccine during multiple uses or after one or more non-sterile events.

[0007] WO 2011 / 151760 discloses multivalent immunogenic compositions comprising various capsular polysaccharides from S. pneumoniae serotypes and 2-phenoxyethanol (2-PE).

[0008] The inventors have found that the preparation of conjugate vaccines containing preservatives (e.g., 2-PE) faces significant challenges, especially in large-scale production. In the prior art, 2-PE has been added to the final stage of vaccine preparation (added to drug products, for example, see WO 00 / 62801, WO 00 / 56360 or Khandke L. et al. Vaccine 29 (2011) 7144–7153). However, the addition of 2-PE in the final stage requires aseptic filtration of relatively high concentrations of 2-PE. It has been found that aseptic filtration of such high concentrations of 2-PE poses challenges to filter wetting and the integrity of the filter before use.

[0009] Therefore, there is a need for improved methods for formulating multivalent conjugate vaccines comprising preservatives such as 2-PE. In particular, a method that is less time consuming and has fewer steps is desired. SUMMARY OF THE INVENTION

[0011] In one aspect, the present invention provides a method for producing a conjugate vaccine comprising a preservative, the method comprising the steps of:

[0012] (a) adding a preservative from a bulk solution to a solution comprising one or more conjugates, wherein the preservative is added without sterile filtration; and

[0013] (b) the mixture comprising the preservative and the conjugate is then sterile filtered.

[0014] In one aspect, the method further comprises the step (c) of subsequently adding an adjuvant.

[0015] In one aspect, the preservative is hydrophobic and viscous at bulk solution concentrations.

[0016] Preferably, the bulk solution is pure 2-PE. DETAILED DESCRIPTION OF THE INVENTION

[0018] Methods for adding preservatives to complex vaccine formulations include adding the preservatives at the final stage of the formulation process. Adding the preservatives at the final stage of the process minimizes the risk of potential effects of interactions of the preservatives with other components of the vaccine (e.g., antigens) and allows for flexibility in the process.

[0019] Preservatives are defined as compounds added to vaccines to kill or prevent the growth of microorganisms, especially bacteria and fungi. They are added to vaccine preparations to prevent the growth of microorganisms if the vaccine becomes accidentally contaminated, such as might occur when a multi-dose vial is repeatedly punctured.

[0020] The inventors have found that adding preservatives (particularly hydrophobic and viscous preservatives) as one of the last steps of the method may be challenging, especially in large-scale production. It has been found that due to the hydrophobicity and high viscosity of some preservatives (such as 2-PE), it is challenging to filter the preservatives through a sterile filter at the desired concentration, and this step is required if the preservative is added at the end of the formulation process. It has been found that ultrahigh pressure is required to completely and continuously wet the sterile filter (i.e., a filter with a nominal pore size of about 0.2 μm) with the preservative of the desired concentration, which may impair the microbial retention performance of the membrane. If such a high pressure is not used, wetting the filter with the preservative at the desired concentration may become incomplete and inconsistent, which means that the wetting step may fail and special tests need to be performed to ensure that wetting is suitable and may waste materials. Therefore, this test may be time-consuming and wasteful of resources.

[0021] Therefore, there is a need for improved methods to formulate multivalent conjugate vaccines comprising preservatives such as 2-PE. In particular, the methods should be less time consuming (hopefully with fewer preparation steps).

[0022] It has been surprisingly found that adding preservatives in the earlier stage of the method has advantages. In such a method, since the preservative is added earlier (before the final dilution), the preservative exists with a concentration higher than the concentration in the final medicine. It has been surprisingly found that adding preservatives in the earlier stage of the method, particularly before the need for aseptic filtration, is possible and allows to avoid the problems associated with aseptic filtration with high concentrations of hydrophobic and viscous preservatives, because downstream only needs to aseptically filter the diluted solution. Such a method allows the addition of preservatives without aseptic filtration. It has been found that the aseptic filtration of hydrophobic and viscous preservatives has problems (especially for 2-PE).

[0023] Therefore, the present invention relates to a method for producing a conjugate vaccine comprising a preservative, said method comprising the following steps

[0024] (a) adding a preservative from a bulk solution to a solution comprising one or more conjugates, wherein the preservative is added without sterile filtration; and

[0025] (b) the mixture comprising the preservative and the conjugate is then sterile filtered.

[0026] In one embodiment, an adjuvant is also added after the sterile filtration step (b). Therefore, in one embodiment, the present invention relates to a method for producing a conjugate vaccine comprising a preservative, the method comprising the following steps

[0027] (a) adding a preservative from a bulk solution to a solution comprising one or more conjugates, wherein the preservative is added without sterile filtration;

[0028] (b) subsequently subjecting the mixture comprising the preservative and the conjugate to sterile filtration;

[0029] (c) An adjuvant is then added to the mixture.

[0030] Preferably, the preservative used in the present invention is hydrophobic and viscous at bulk solution concentrations. In one embodiment, the viscosity of the preservative at bulk solution concentrations is at least 10 centistokes at 25°C. In another embodiment, the viscosity is at least 15 centistokes at 25°C. In a preferred embodiment, the viscosity is at least 20 centistokes at 25°C.

[0031] In one embodiment, the viscosity of the preservative used in the present invention is from about 10 to about 50 centistokes at 25° C. Preferably, the viscosity of the preservative used in the present invention is from about 15 to about 25 centistokes at 25° C.

[0032] In one embodiment, the preservative is 2-phenoxyethanol (2-PE), phenol, m-cresol, methylparaben, propylparaben or thimerosal. In a preferred embodiment, the preservative is 2-phenoxyethanol (2-PE) or thimerosal.

[0033] In one embodiment, the preservative is added in undiluted form. Thus, in one embodiment, the preservative is added in undiluted form (i.e., the bulk solution is undiluted preservative). Thus, in one embodiment, the bulk solution is undiluted pure preservative (wherein "pure" means pharmaceutical grade).

[0034] In the most preferred embodiment, the preservative used in the present invention is 2-phenoxyethanol (2-PE). 2-PE is a colorless oily liquid and can be added neat (undiluted pharmaceutical grade). Therefore, in one embodiment, 2-PE is added in undiluted form (i.e., the bulk solution is neat 2-PE). In one embodiment, the bulk solution is therefore neat 2-PE.

[0035] In one embodiment, the bulk solution is pure 2-PE, and after step (a), 2-PE is diluted in solution by about 10 times to about 200 times. In another embodiment, the bulk solution is pure 2-PE, and after step (a), 2-PE is diluted in solution by about 10 times to about 100 times. In another embodiment, the bulk solution is pure 2-PE, and after step (a), 2-PE is diluted in solution by about 50 times, about 60 times, about 70 times or about 80 times. In a preferred embodiment, the bulk solution is pure 2-PE, and after step (a), 2-PE is diluted in solution by about 73 times to 74 times.

[0036] In one embodiment, the concentration of 2-PE in the solution after step (a) is between about 1 mg / ml and about 25 mg / ml. Preferably, the concentration of 2-PE in the solution after step (a) is about 10 mg / ml to about 20 mg / ml. Even more preferably, the concentration of 2-PE in the solution after step (a) is about 15 mg / ml to about 17 mg / ml.

[0037] In a specific embodiment, the concentration of 2-PE in the solution after step (a) is about 15 mg / ml.

[0038] In a specific embodiment, the concentration of 2-PE in the solution after step (a) is about 17 mg / ml.

[0039] In one embodiment, 2-PE is added at an addition rate between about 0.5 ml / min / L of solution comprising one or more conjugates to about 5.0 ml / min / L of solution comprising one or more conjugates. In a preferred embodiment, 2-PE is added in undiluted form.

[0040] In one embodiment, 2-PE is added at an addition rate between about 1 ml / min / L of solution comprising one or more conjugates to about 4.0 ml / min / L of solution comprising one or more conjugates. In a preferred embodiment, 2-PE is added in undiluted form (pure 2-PE).

[0041] In one embodiment, pure 2-PE is added using a pump, preferably a peristaltic pump. In one embodiment, pure 2-PE is added using a peristaltic pump using thermoplastic elastomer tubing. Preferably, pure 2-PE is added using a peristaltic pump using thermoplastic elastomer tubing.

[0042] In one embodiment, the final concentration of 2-PE in the vaccine is between about 5 mg / ml and about 15 mg / ml. Preferably, the final concentration of 2-PE in the vaccine is between about 7 mg / ml and about 12 mg / ml.

[0043] In one embodiment, the final concentration of 2-PE in the vaccine is about 10 mg / ml.

[0044] In a preferred embodiment, the final concentration of 2-PE in the vaccine is about 9 mg / ml.

[0045] In one embodiment, the volume of the solution after step (a) is between about 10 L and about 2,000 L. In another embodiment, the volume of the solution after step (a) is between about 100 L and about 1,000 L. In another embodiment, the volume of the solution after step (a) is between about 100 L and about 500 L. In another embodiment, the volume of the solution after step (a) is between about 150 L and about 200 L. In a preferred embodiment, the volume of the solution after step (a) is about 180 L.

[0046] Preferably, the preservative is added with continuous mixing. In a preferred embodiment, there is no delay between adding the preservative and starting mixing.

[0047] In one embodiment, the preservative is added under continuous mixing at an angular speed of about 50 rpm to about 500 rpm. Preferably, the preservative is added under continuous mixing at an angular speed of about 100 rpm to about 400 rpm. More preferably, the preservative is added under continuous mixing at an angular speed of about 150 rpm to about 300 rpm.

[0048] Most preferably, the preservative is added with continuous mixing at an angular speed of about 150 rpm to about 200 rpm.

[0049] In one embodiment, the preservative is added with continuous mixing at an angular speed of about 150 rpm.

[0050] In one embodiment, the preservative is added with continuous mixing at an angular speed of about 200 rpm.

[0051] In one embodiment, after adding the preservative and before aseptic filtration, the solution is mixed for about 15 minutes to about 5 hours. Preferably, after adding the preservative and before aseptic filtration, the solution is mixed for about 30 minutes to about 3 hours. Even more preferably, after adding the preservative and before aseptic filtration, the solution is mixed for about 1 hour to about 3 hours.

[0052] In one embodiment, after the preservative is added, the solution is mixed for about 2 hours.

[0053] In one embodiment, after adding the preservative and before sterile filtration, the solution is mixed at an angular speed between about 50 rpm and about 500 rpm. Preferably, between about 100 rpm and about 400 rpm. More preferably, between about 150 rpm and about 300 rpm.

[0054] A solution comprising one or more conjugates of step (a)

[0055] The solution to which a preservative is added contains one or more conjugates and may contain other ingredients.

[0056] For the purposes of the present invention, the term "conjugate" or "glycoconjugate" refers to a bacterial capsular saccharide linked to a carrier protein. In one embodiment, the capsular saccharide is directly linked to the carrier protein. In a second embodiment, the capsular saccharide is linked to the protein via a spacer / linker.

[0057] In one embodiment, the solution to which the preservative is added is a monovalent composition. In such an embodiment, the solution comprises only one conjugate. In such an embodiment, the conjugate may be a conjugated Haemophilus influenzae type b capsular saccharide (Hib). In another embodiment, the conjugate is a conjugated N. meningitidis serogroup Y capsular saccharide (MenC). In yet another embodiment, the conjugate is a conjugated N. meningitidis serogroup Y capsular saccharide (MenY).

[0058] In one embodiment, the solution to which the preservative is added is a divalent, trivalent, tetravalent, pentavalent, or hexavalent composition.

[0059] In one embodiment, the trivalent composition comprises three conjugated capsular saccharides of Streptococcus agalactiae. Streptococcus agalactiae is also known as "Group B Streptococcus", or simply "GBS". In one embodiment, the three conjugates consist of conjugated capsular saccharides of GBS serotypes Ia, Ib and III. In a preferred embodiment, the capsular saccharide is conjugated to a CRM. 197 Conjugation.

[0060] In one embodiment, the hexavalent composition comprises six conjugated S. agalactiae capsular saccharides. In one embodiment, the six conjugates consist of conjugated GBS serotypes Ia, Ib, II, III, IV and V capsular saccharides. In a preferred embodiment, the capsular saccharides are conjugated to CRM 197 Conjugation.

[0061] In one embodiment, the tetravalent composition comprises four conjugated Neisseria meningitidis capsular saccharides. In one embodiment, the four conjugates consist of a conjugated Neisseria meningitidis serogroup A capsular saccharide (MenA), a conjugated Neisseria meningitidis serogroup W135 capsular saccharide (MenW135), a conjugated Neisseria meningitidis serogroup Y capsular saccharide (MenY), and a conjugated Neisseria meningitidis serogroup C capsular saccharide (MenC).

[0062] Preferably, the solution to which the preservative is added is a multivalent composition comprising seven or more conjugates. Preferably, the solution is a 7 to 25 valent composition (i.e., it contains 7 to 25 conjugates). Even more preferably, the multivalent solution is a 13 to 25 valent composition.

[0063] In one embodiment, the solution to which the preservative is added is a 15-valent composition.

[0064] In one embodiment, the solution to which the preservative is added is a 16-valent composition.

[0065] In one embodiment, the solution to which the preservative is added is a 20-valent composition.

[0066] In one embodiment, the solution to which the preservative is added is a 21-valent composition.

[0067] Preferably, the solution to which the preservative is added is a multivalent pneumococcal conjugate composition.

[0068] In one embodiment, the solution to which a preservative is added is a pneumococcal conjugate composition comprising 7 to 25 saccharide conjugates from different S. pneumoniae serotypes (7 to 25 Pneumococcal Conjugates).

[0069] In one embodiment, the solution to which a preservative is added is a pneumococcal conjugate composition comprising conjugates from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 different S. pneumoniae serotypes.

[0070] In one embodiment, the solution to which the preservative is added is a pneumococcal conjugate composition comprising conjugates from 20 different S. pneumoniae serotypes.

[0071] In one embodiment, the solution to which a preservative is added is a 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 valent pneumococcal conjugate composition.

[0072] In one embodiment, the solution to which the preservative is added is a 15-valent pneumococcal conjugate composition.

[0073] In one embodiment, the solution to which a preservative is added is a 20-valent pneumococcal conjugate composition.

[0074] In one embodiment, the solution to which the preservative is added is a 21-valent pneumococcal conjugate composition.

[0075] In one embodiment, the solution to which the preservative is added is a 22-valent pneumococcal conjugate composition.

[0076] In one embodiment, the solution to which a preservative is added is a 21, 22, 23, 24 or 25 valent pneumococcal conjugate composition.

[0077] In one embodiment, the solution to which a preservative is added comprises glycoconjugates from S. pneumoniae serotypes 4, 6B, 9V, 14, 18C, 19F and 23F.

[0078] In one embodiment, the solution to which a preservative is added comprises glycoconjugates from S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F.

[0079] In one embodiment, the solution to which a preservative is added comprises glycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F.

[0080] In one embodiment, the solution to which a preservative is added comprises glycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F.

[0081] In one embodiment, the solution to which a preservative is added comprises glycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, and 33F.

[0082] In one embodiment, the solution to which a preservative is added comprises glycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F.

[0083] In one embodiment, the solution to which a preservative is added comprises glycoconjugates from S. pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F.

[0084] In one embodiment, the solution to which a preservative is added comprises glycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F.

[0085] In one embodiment, the solution to which a preservative is added is a 10-valent pneumococcal conjugate composition, wherein the 10 saccharide conjugates are from S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F.

[0086] In one embodiment, the solution to which a preservative is added is a 13-valent pneumococcal conjugate composition, wherein the 13 saccharide conjugates are from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F.

[0087] In one embodiment, the solution to which a preservative is added is a 15-valent pneumococcal conjugate composition, wherein the 15 saccharide conjugates are from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F.

[0088] In one embodiment, the solution to which a preservative is added is a 20-valent pneumococcal conjugate composition, wherein the 20 saccharide conjugates are from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F.

[0089] In one embodiment, the solution to which a preservative is added is a 21-valent pneumococcal conjugate composition, wherein the 21 saccharide conjugates are from S. pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F.

[0090] In one embodiment, the solution to which a preservative is added is a 21-valent pneumococcal conjugate composition, wherein the 21 saccharide conjugates are from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F.

[0091] In one embodiment, the solution to which a preservative is added is a 22-valent pneumococcal conjugate composition, wherein the 22 saccharide conjugates are from S. pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F.

[0092] In one embodiment, the solution to which a preservative is added is a 21-valent pneumococcal conjugate composition, wherein the 21 saccharide conjugates are from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23F and 33F.

[0093] In one embodiment, the solution to which a preservative is added is a 21-valent pneumococcal conjugate composition, wherein the 21 saccharide conjugates are from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23A, 23F and 33F.

[0094] In one embodiment, the solution to which a preservative is added is a 21-valent pneumococcal conjugate composition, wherein the 21 saccharide conjugates are from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23B, 23F and 33F.

[0095] In one embodiment, the solution to which a preservative is added is a 21-valent pneumococcal conjugate composition, wherein the 21 saccharide conjugates are from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 24F and 33F.

[0096] In one embodiment, the solution to which a preservative is added is a 21-valent pneumococcal conjugate composition, wherein the 21 saccharide conjugates are from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 33F and 35B.

[0097] In a preferred embodiment, the solution to which a preservative is added is a 20-valent pneumococcal conjugate composition, wherein the 20 saccharide conjugates are from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F.

[0098] The component of the glycoconjugate is a carrier protein conjugated to a sugar. The terms "protein carrier" or "carrier protein" or "carrier" are used interchangeably herein. The carrier protein should be suitable for standard conjugation procedures. In one embodiment, the carrier protein of the glycoconjugate of the invention is DT (diphtheria toxoid), TT (tetanus toxoid), fragment C of TT, CRM 197 detoxifying pneumococcal hemolysin, PhtA, PhtB, PhtD, PhtE, PhtDE fusion, PhtBE fusion, OMPC, Por), PD (Haemophilus influenzae protein D) or C5a peptidase (SCP) from Streptococcus. In one embodiment, the carrier protein of the glycoconjugate of the present invention is DT (diphtheria toxoid). In another embodiment, the carrier protein of the glycoconjugate of the present invention is TT (tetanus toxoid). In another embodiment, the carrier protein of the glycoconjugate of the present invention is PD (Haemophilus influenzae protein D; see, e.g., EP0594610 B).

[0099] In a preferred embodiment, the carrier protein of the glycoconjugate of the present invention is TT, CRM 197 or C5a peptidase (SCP) from Streptococcus. In an even more preferred embodiment, the carrier protein of the glycoconjugate of the present invention is CRM197 or C5a peptidase (SCP) from Streptococcus.

[0100] In a very preferred embodiment, the carrier protein of the glycoconjugate of the invention is CRM 197 In any of the above composition embodiments, the capsular polysaccharides are all individually combined with the CRM 197 Conjugation.

[0101] In an embodiment, capsular polysaccharides of Streptococcus pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14 and / or 23F from any of the above compositions are conjugated to PD, respectively, capsular polysaccharides from Streptococcus pneumoniae serotype 18C are conjugated to TT, and capsular polysaccharides from Streptococcus pneumoniae serotype 19F are conjugated to DT.

[0102] In any of the above composition embodiments, at least one capsular polysaccharide is conjugated to TT and the other capsular polysaccharides are conjugated to CRM. 197 Conjugation.

[0103] In any of the above composition embodiments, one capsular polysaccharide is conjugated to TT and the other capsular polysaccharides are conjugated to CRM. 197 Conjugation.

[0104] In any of the above composition embodiments, at least two capsular polysaccharides are conjugated to TT, and the other capsular polysaccharides are conjugated to CRM. 197 Conjugation.

[0105] In any of the above composition embodiments, two capsular polysaccharides are conjugated to TT and the other capsular polysaccharides are conjugated to CRM. 197 Conjugation.

[0106] In any of the above composition embodiments, at least three capsular polysaccharides are conjugated to TT, and the other capsular polysaccharides are conjugated to CRM. 197 Conjugation.

[0107] In any of the above composition embodiments, three capsular polysaccharides are conjugated to TT, and the other capsular polysaccharides are conjugated to CRM. 197 Conjugation.

[0108] In any of the above composition embodiments, four capsular polysaccharides are conjugated to TT, and the other capsular polysaccharides are conjugated to CRM. 197 Conjugation.

[0109] In one embodiment, the solution to which a preservative is added is a 21-valent pneumococcal conjugate composition, wherein the 21 saccharide conjugates are from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F, wherein four capsular polysaccharides are conjugated to TT, wherein the four capsular polysaccharides conjugated to TT are serotypes 15B, 22F, and the other two serotypes are selected from the group consisting of serotypes 1, 3 and 5, and the other saccharide conjugates are conjugated to CRM. 197 Conjugation.

[0110] In one embodiment, the solution to which the preservative is added is a 21-valent pneumococcal conjugate composition, wherein the 21 saccharide conjugates are from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F, wherein four capsular polysaccharides are conjugated to TT, wherein the four capsular polysaccharides conjugated to TT are serotypes 1, 5, 15B and 22F, and the other saccharide conjugates are conjugated to CRM. 197 Conjugation.

[0111] In one embodiment, the solution to which the preservative is added is a 21-valent pneumococcal conjugate composition, wherein the 21 saccharide conjugates are from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F, wherein four capsular polysaccharides are conjugated to TT, wherein the four capsular polysaccharides conjugated to TT are serotypes 1, 3, 15B and 22F, and the other saccharide conjugates are conjugated to CRM. 197 Conjugation.

[0112] In one embodiment, the solution to which a preservative is added is a 21-valent pneumococcal conjugate composition, wherein the 21 saccharide conjugates are from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F, wherein four capsular polysaccharides are conjugated to TT, wherein the four capsular polysaccharides conjugated to TT are serotypes 3, 5, 15B and 22F, and the other saccharide conjugates are conjugated to CRM. 197 Conjugation.

[0113] In one embodiment, the solution to which the preservative is added is a 21-valent pneumococcal conjugate composition, wherein the 21 saccharide conjugates are from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F of Streptococcus pneumoniae, wherein two saccharide conjugates from serotypes 1, 3 and 5 are conjugated to TT, and the other saccharide conjugates are conjugated to CRM. 197 Conjugation.

[0114] In one embodiment, the solution to which the preservative is added is a 21-valent pneumococcal conjugate composition, wherein the 21 saccharide conjugates are from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F of Streptococcus pneumoniae, wherein the saccharide conjugates from serotypes 1 and 3 are conjugated to TT, and the other saccharide conjugates are conjugated to CRM. 197 Conjugation.

[0115] In one embodiment, the solution to which the preservative is added is a 21-valent pneumococcal conjugate composition, wherein the 21 saccharide conjugates are from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F of Streptococcus pneumoniae, wherein the saccharide conjugates from serotypes 1 and 5 are conjugated to TT, and the other saccharide conjugates are conjugated to CRM. 197 Conjugation.

[0116] In one embodiment, the solution to which the preservative is added is a 21-valent pneumococcal conjugate composition, wherein the 21 saccharide conjugates are from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F of Streptococcus pneumoniae, wherein the saccharide conjugates from serotypes 3 and 5 are conjugated to TT, and the other saccharide conjugates are conjugated to CRM. 197 Conjugation.

[0117] In one embodiment, the solution to which the preservative is added is a 15-valent pneumococcal conjugate composition, wherein the 15 saccharide conjugates are from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F, wherein all saccharide conjugates are conjugated to CRM 197 Conjugation.

[0118] In a most preferred embodiment, the solution to which the preservative is added is a 20-valent pneumococcal conjugate composition, wherein the 20 saccharide conjugates are from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F, wherein all saccharide conjugates are conjugated to CRM 197 Conjugation.

[0119] In one embodiment, the solution to which a preservative is added comprises any combination of a glycoconjugate disclosed herein and a pharmaceutically acceptable excipient, carrier, or diluent.

[0120] In addition to the conjugate, the preservative-added solution may also contain one or more of a buffer, a salt, a divalent cation, a nonionic detergent, a cryoprotectant (eg, a sugar), and an antioxidant (eg, a free radical scavenger or a chelator), or any various combinations thereof.

[0121] In one embodiment, the solution to which the preservative is added comprises a buffer. In one embodiment, the buffer has a pKa of about 3.5 to about 7.5. In some embodiments, the buffer is phosphate, succinate, histidine or citrate. In some embodiments, the buffer is succinate. In a preferred embodiment, the buffer is histidine.

[0122] In certain embodiments, the buffer is histidine at a concentration of 10 mM to 30 mM. In certain embodiments, the buffer is histidine at a concentration of 15 mM to 25 mM. In a specific embodiment, the concentration of the histidine buffer is about 22 mM.

[0123] In certain embodiments, the buffer is succinate at a concentration of 1 mM to 10 mM. In a specific embodiment, the concentration of the succinate buffer is about 5.5 mM.

[0124] In one embodiment, the solution to which the preservative is added comprises a salt. In some embodiments, the salt is magnesium chloride, potassium chloride, sodium chloride, or a combination thereof. In a specific embodiment, the salt is sodium chloride. In a specific embodiment, the solution to which the preservative is added comprises about 150 mM sodium chloride.

[0125] In one embodiment, the solution to which the preservative is added comprises a surfactant. In one embodiment, the surfactant is polysorbate 20 (TWEEN™ 20), polysorbate 40 (TWEEN™ 40), polysorbate 60 (TWEEN™ 60), polysorbate 70 (TWEEN™ 70), polysorbate 80 (TWEEN™ 80), polysorbate 90 (TWEEN™ 90), polysorbate 100 (TWEEN™ 100), polysorbate 110 (TWEEN™ 100), polysorbate 120 (TWEEN™ 100), polysorbate 130 (TWEEN™ 100), polysorbate 140 (TWEEN™ 100), polysorbate 150 (TWEEN TM 60), polysorbate 65 (TWEEN TM 65) Polysorbate 80 (TWEEN TM 80), polysorbate 85 (TWEEN TM 85)TRITON TM N-101, TRITON TM X-100, oxtoxynol 40, nonoxynol-9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate (PEG-15, Solutol H 15), polyoxyethylene-35-ricinoleate ( EL), soy lecithin or poloxamer.

[0126] In a preferred embodiment, the surfactant is polysorbate 80 or polysorbate 20.

[0127] In a particular embodiment, the surfactant is polysorbate 20. Preferably, the concentration of polysorbate 20 in the solution is 0.01% to 1% polysorbate 20 by weight (w / w). More preferably, the concentration of polysorbate 20 in the solution is 0.1% to 0.5% polysorbate 20 by weight (w / w).

[0128] In other embodiments, the concentration of polysorbate 20 in the solution is about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, or about 0.6% polysorbate 20 (w / w). In a most preferred embodiment, the concentration of polysorbate 20 in the solution is about 0.33% polysorbate 20 (w / w).

[0129] In a preferred embodiment, the surfactant is polysorbate 80. Preferably, the concentration of polysorbate 80 in the solution is 0.001% to 1% polysorbate 80 by weight (w / w). More preferably, the concentration of polysorbate 80 in the solution is 0.01% to 0.5% polysorbate 80 by weight (w / w).

[0130] In other embodiments, the concentration of polysorbate 80 in the solution is about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, or about 0.06% polysorbate 80 (w / w). In a most preferred embodiment, the concentration of polysorbate 80 in the solution is about 0.033% polysorbate 80 (w / w).

[0131] In certain embodiments, the solution to which the preservative is added has a pH between 5.5 and 7.5, more preferably between 5.6 and 7.0, even more preferably between 5.8 and 6.0. In a preferred embodiment, the solution to which the preservative is added has a pH of about 5.8.

[0132] The concentration of a particular glycoconjugate can be calculated based on the amount of polysaccharide in the conjugate. Sugar concentration can be determined by uronic acid assay.

[0133] In one embodiment, the preservative-added solution comprises about 2 μg / ml to about 20 μg / ml of a polysaccharide from a glycoconjugate of S. pneumoniae serotype 1, 2, 3, 4, 5, 6A, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, 33F and / or 35B. Preferably, between about 5 μg / ml to about 10 μg / ml.

[0134] In one embodiment, the preservative-added solution comprises about 7.5 μg / ml of a polysaccharide from a glycoconjugate of S. pneumoniae serotype 1, 2, 3, 4, 5, 6A, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, 33F and / or 35B.

[0135] In one embodiment, the solution to which the preservative is added comprises about 4 μg / ml to about 40 μg / ml of a polysaccharide from a glycoconjugate of Streptococcus pneumoniae serotype 6B. Preferably, between about 10 μg / ml to about 20 μg / ml.

[0136] In one embodiment, the preservative-added solution comprises about 15 μg / ml of a polysaccharide from a glycoconjugate of S. pneumoniae serotype 6B.

[0137] In one embodiment, the solution to which the preservative is added is a 20-valent pneumococcal conjugate composition, wherein the 20 saccharide conjugates are from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F, wherein all of the saccharide conjugates are conjugated to CRM 197 The solution comprises about 5 μg / ml to about 10 μg / ml of each capsular polysaccharide serotype 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F, and about 10 μg / ml to about 20 μg / ml of serotype 6B capsular polysaccharide, about 0.01% to about 0.05% (w / w) polysorbate 80, about 150 mM sodium chloride, and about 1 mM to about 10 mM succinate buffer pH 5.8.

[0138] In one embodiment, the solution to which the preservative is added is a 15-valent pneumococcal conjugate composition, wherein the 15 saccharide conjugates are from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F, wherein the saccharide conjugates are all conjugated to CRM 197The solution comprises about 5 μg / ml to about 10 μg / ml of each capsular polysaccharide serotype 1, 3, 4, 5, 6A, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F, and about 10 μg / ml to about 20 μg / ml of capsular polysaccharide serotype 6B, about 150 mM sodium chloride, about 0.1% to about 0.5% (w / w) polysorbate 20, and about 15 mM to about 25 mM L-histidine buffer pH 5.8.

[0139] In a preferred embodiment, the solution to which the preservative is added is a 20-valent pneumococcal conjugate composition, wherein the 20 saccharide conjugates are from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F of Streptococcus pneumoniae, wherein all of the saccharide conjugates are conjugated to CRM 197 The solution comprises about 7.5 μg / ml of each capsular polysaccharide serotype 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F and about 7.5 μg / ml of capsular polysaccharide serotype 6B, about 0.033% (w / w) polysorbate 80, about 150 mM sodium chloride and about 5.5 mM succinate buffer pH 5.8.

[0140] Sterile Filtration

[0141] After step (a), the mixture comprising the preservative and the conjugate is sterile filtered.

[0142] In one embodiment, the filter has a nominal retention range between about 0.05-0.2 μm or about 0.1-0.2 μm. Preferably about 0.15-0.2 μm.

[0143] In one embodiment, the filter has a nominal retention range of about 0.1, about 0.15, or about 0.2 μm.

[0144] In a preferred embodiment, the filter has a nominal retention range of about 0.2 μm.

[0145] In a preferred embodiment, the filter comprises a pre-filter. In some embodiments, the pre-filter has a nominal retention range between about 0.1 μm and about 1 μm. In a preferred embodiment, the pre-filter has a nominal retention range of about 0.5 μm. In another preferred embodiment, the pre-filter has a nominal retention range of about 0.5 μm, and the filter has a nominal retention range of about 0.2 μm.

[0146] In one embodiment, the filter has a filtration capacity of about 25-1000 L / m2, 50-1000 L / m2, 75-1000 L / m2, 100-1000 L / m2, 150-1000 L / m2, 200-1000 L / m2, 250-1000 L / m2, 300-1000 L / m2, 350-1000 L / m2, 400-1000 L / m2, 500-1000 L / m2, or 750-1000 L / m2.

[0147] In one embodiment, the filter has a filtration capacity of 200-1000 L / m2, 250-1000 L / m2, or 300-1000 L / m2.

[0148] In a preferred embodiment, the filter has a filtration capacity of about 300-1000 L / m2.

[0149] In one embodiment, the filter comprises polysulfone. In one embodiment, the filter comprises polyphenylene sulfone. In a preferred embodiment, the filter comprises polyethersulfone (PES). In one embodiment, the filter is a disc filter. In a preferred embodiment, the filter is a capsule filter. In a further preferred embodiment, the filter is a capsule filter comprising PES.

[0150] Conjugate vaccine of the present invention

[0151] The conjugate vaccine obtained by the method of the present invention comprises one or more conjugates and a preservative. Preferably, the preservative is 2-PE.

[0152] For the purposes of the present invention, the term "conjugate" refers to a capsular saccharide linked to a carrier protein. In one embodiment, the capsular saccharide is directly linked to the carrier protein. In a second embodiment, the capsular saccharide is linked to the protein via a spacer / linker.

[0153] In one embodiment, the conjugate vaccine of the present invention is a monovalent conjugate vaccine. In such an embodiment, the conjugate vaccine comprises only one conjugate. In such an embodiment, the conjugate may be a conjugated capsular saccharide of Haemophilus influenzae type b (Hib). In another embodiment, the conjugate is a conjugated capsular saccharide of Neisseria meningitidis serogroup Y (MenC). In yet another embodiment, the conjugate is a conjugated capsular saccharide of Neisseria meningitidis serogroup Y (MenY).

[0154] In another embodiment, the conjugate vaccine of the invention is a bivalent, trivalent, tetravalent, pentavalent or hexavalent conjugate vaccine.

[0155] In one embodiment, the trivalent conjugate vaccine comprises three conjugated capsular saccharides of Streptococcus agalactiae. Streptococcus agalactiae is also known as "Group B Streptococcus", or simply "GBS". In one embodiment, the three conjugates consist of conjugated capsular saccharides of GBS serotypes Ia, Ib and III. In a preferred embodiment, the capsular saccharides are conjugated to CRM 197 Conjugation.

[0156] In one embodiment, the hexavalent conjugate vaccine comprises six conjugated capsular saccharides of Streptococcus agalactiae. In one embodiment, the six conjugates consist of conjugated capsular saccharides of GBS serotypes Ia, Ib, II, III, IV and V. In a preferred embodiment, the capsular saccharides are conjugated to CRM 197 Conjugation.

[0157] In one embodiment, the tetravalent conjugate vaccine comprises four conjugated Neisseria meningitidis capsular saccharides. In one embodiment, the four conjugates consist of conjugated Neisseria meningitidis serogroup A capsular saccharide (MenA), conjugated Neisseria meningitidis serogroup W135 capsular saccharide (MenW135), conjugated Neisseria meningitidis serogroup Y capsular saccharide (MenY) and conjugated Neisseria meningitidis serogroup C capsular saccharide (MenC).

[0158] In a preferred embodiment, the conjugate vaccine of the present invention is a multivalent conjugate vaccine comprising seven or more conjugates. Preferably, the multivalent conjugate vaccine of the present invention is a 7 to 25 valent conjugate vaccine (i.e., contains 7 to 25 conjugates). Even more preferably, the multivalent conjugate vaccine of the present invention is a 13 to 25 valent conjugate vaccine.

[0159] In one embodiment, the multivalent conjugate vaccine of the invention is a 15-valent conjugate vaccine.

[0160] In one embodiment, the multivalent conjugate vaccine of the invention is a 16-valent conjugate vaccine.

[0161] In one embodiment, the multivalent conjugate vaccine of the invention is a 20-valent conjugate vaccine.

[0162] In one embodiment, the multivalent conjugate vaccine of the present invention is a 21-valent conjugate vaccine.

[0163] Preferably, the multivalent conjugate vaccine of the present invention is a pneumococcal conjugate vaccine.

[0164] In one embodiment, the multivalent conjugate vaccine of the invention is a pneumococcal conjugate vaccine comprising 7 to 25 saccharide conjugates from different serotypes of S. pneumoniae (7 to 25 pneumococcal conjugates).

[0165] In one embodiment, the multivalent conjugate vaccine of the invention is a pneumococcal conjugate vaccine comprising saccharide conjugates from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 different serotypes of S. pneumoniae.

[0166] In one embodiment, the multivalent conjugate vaccine of the invention is a pneumococcal conjugate vaccine comprising saccharide conjugates from 20 different serotypes of S. pneumoniae.

[0167] In one embodiment, the pneumococcal conjugate vaccine of the invention is a 7-, 8-, 9-, 10-, 11-, 12-, 13-, 14-, 15-, 16-, 17-, 18-, 19- or 20-valent pneumococcal conjugate vaccine.

[0168] In one embodiment, the pneumococcal conjugate vaccine of the present invention is a 15-valent pneumococcal conjugate vaccine.

[0169] In a preferred embodiment, the pneumococcal conjugate vaccine of the present invention is a 20-valent pneumococcal conjugate vaccine.

[0170] In one embodiment, the pneumococcal conjugate vaccine of the invention is a 21-, 22-, 23-, 24- or 25-valent pneumococcal conjugate vaccine.

[0171] In one embodiment, the pneumococcal conjugate vaccine of the present invention is a 21-valent pneumococcal conjugate vaccine.

[0172] In one embodiment, the pneumococcal conjugate vaccine of the present invention is a 22-valent pneumococcal conjugate vaccine.

[0173] In one embodiment, the pneumococcal conjugate vaccine of the invention comprises saccharide conjugates from S. pneumoniae serotypes 4, 6B, 9V, 14, 18C, 19F and 23F.

[0174] In one embodiment, the pneumococcal conjugate vaccine of the invention comprises saccharide conjugates from S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F.

[0175] In one embodiment, the pneumococcal conjugate vaccine of the invention comprises saccharide conjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F.

[0176] In one embodiment, the pneumococcal conjugate vaccine of the invention comprises saccharide conjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F.

[0177] In one embodiment, the pneumococcal conjugate vaccine of the invention comprises saccharide conjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F.

[0178] In one embodiment, the pneumococcal conjugate vaccine of the invention comprises saccharide conjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F.

[0179] In one embodiment, the pneumococcal conjugate vaccine of the invention comprises saccharide conjugates from S. pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F.

[0180] In one embodiment, the pneumococcal conjugate vaccine of the invention comprises saccharide conjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F.

[0181] In one embodiment, the pneumococcal conjugate vaccine of the invention is a 10-valent pneumococcal conjugate, wherein the 10 saccharide conjugates are from S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F.

[0182] In one embodiment, the pneumococcal conjugate vaccine of the invention is a 13-valent pneumococcal conjugate, wherein the 13 saccharide conjugates are from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F.

[0183] In one embodiment, the pneumococcal conjugate vaccine of the invention is a 15-valent pneumococcal conjugate, wherein the 15 saccharide conjugates are from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F.

[0184] In one embodiment, the pneumococcal conjugate vaccine of the invention is a 20-valent pneumococcal conjugate, wherein the 20 saccharide conjugates are from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F.

[0185] In one embodiment, the pneumococcal conjugate vaccine of the invention is a 21-valent pneumococcal conjugate, wherein the 21 saccharide conjugates are from S. pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F.

[0186] In one embodiment, the pneumococcal conjugate vaccine of the invention is a 21-valent pneumococcal conjugate, wherein the 21 saccharide conjugates are from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F.

[0187] In one embodiment, the pneumococcal conjugate vaccine of the invention is a 22-valent pneumococcal conjugate, wherein the 22 saccharide conjugates are from S. pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F.

[0188] In one embodiment, the pneumococcal conjugate vaccine of the invention is a 21-valent pneumococcal conjugate, wherein the 21 saccharide conjugates are from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23F and 33F.

[0189] In one embodiment, the pneumococcal conjugate vaccine of the invention is a 21-valent pneumococcal conjugate, wherein the 21 saccharide conjugates are from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23A, 23F and 33F.

[0190] In one embodiment, the pneumococcal conjugate vaccine of the invention is a 21-valent pneumococcal conjugate, wherein the 21 saccharide conjugates are from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23B, 23F and 33F.

[0191] In one embodiment, the pneumococcal conjugate vaccine of the invention is a 21-valent pneumococcal conjugate, wherein the 21 saccharide conjugates are from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 24F and 33F.

[0192] In one embodiment, the pneumococcal conjugate vaccine of the invention is a 21-valent pneumococcal conjugate, wherein the 21 saccharide conjugates are from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 33F and 35B.

[0193] The component of the glycoconjugate is a carrier protein conjugated to a sugar. The terms "protein carrier" or "carrier protein" or "carrier" are used interchangeably herein. The carrier protein should be suitable for standard conjugation procedures.

[0194] In one embodiment, the carrier protein of the glycoconjugate of the present invention is selected from the group consisting of: DT (diphtheria toxoid), TT (tetanus toxoid), fragment C of TT, CRM 197(a non-toxic but antigenically identical variant of diphtheria toxoid), other DT mutants (e.g., CRM176, CRM228, CRM45 (Uchida et al. (1973) J. Biol. Chem. 218:3838-3844), CRM9, CRM102, CRM103 or CRM107, pneumolysin (ply) (Kuo et al. (1995) Infect lmmun 63:2706-2713), including ply detoxified in some manner, such as dPLY-GMBS (WO 2004 / 081515, WO 2006 / 032499) or dPLY-formol, PhtX, including PhtA, PhtB, PhtD, PhtE (the sequences of PhtA, PhtB, PhtD or PhtE are disclosed in WO 00 / 37105 and WO 2006 / 032499). 00 / 39299) and a fusion of a Pht protein, such as a PhtDE fusion, a PhtBE fusion, a Pht AE (WO 01 / 98334, WO 03 / 054007, WO 2009 / 000826), OMPC (meningococcal outer membrane protein), which is usually extracted from Neisseria meningitidis serogroup B (EP0372501), PorB (from Neisseria meningitidis), PD (Haemophilus influenzae protein D; see, e.g., EP0594610 B), or an immunologically functional equivalent thereof, a synthetic peptide (EP0378881, EP0427347), a heat shock protein (WO 93 / 17712, WO 94 / 03208), a pertussis protein (WO 98 / 58668, EP0471177), a cytokine, a lymphokine, a growth factor or a hormone (WO 91 / 01146), artificial proteins containing multiple human CD4+ T cell epitopes from various pathogen-derived antigens (Falugi et al. (2001) Eur J Immunol 31:3816-3824) such as N19 protein (Baraldoi et al., (2004) Infect lmmun 72:4884-4887), pneumococcal surface protein PspA (WO 02 / 091998), iron uptake proteins (WO 01 / 72337), Clostridium difficile toxin A or B (WO 00 / 61761), transferrin binding proteins, pneumococcal adhesion proteins (PsaA), recombinant Pseudomonas aeruginosa (Pseudomonas aeruginosa) exotoxin A (particularly non-toxic mutants thereof, such as exotoxin A with a substitution at glutamic acid 553 (Douglas et al. (1987) J. Bacteriol. 169(11):4967-4971)).Other proteins, such as ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or purified protein derivative of tuberculin (PPD) can also be used as carrier proteins. Other suitable carrier proteins include inactivated bacterial toxins, such as cholera toxoid (e.g., as described in WO 2004 / 083251), E. coli LT, E. coli ST, and exotoxin A from Pseudomonas aeruginosa. Another suitable carrier protein is C5a peptidase (SCP) from Streptococcus.

[0195] In one embodiment, the carrier protein of the glycoconjugate of the present invention is DT (diphtheria toxoid). In another embodiment, the carrier protein of the glycoconjugate of the present invention is TT (tetanus toxoid).

[0196] In another embodiment, the carrier protein of the glycoconjugate of the invention is PD (Haemophilus influenzae protein D; see, eg, EP0594610 B).

[0197] In a preferred embodiment, the carrier protein of the glycoconjugate of the present invention is TT, CRM 197 or C5a peptidase (SCP) from Streptococcus.

[0198] In a preferred embodiment, the carrier protein of the glycoconjugate of the present invention is CRM197 or C5a peptidase (SCP) from Streptococcus.

[0199] In a very preferred embodiment, the carrier protein of the glycoconjugate of the invention is CRM 197 . The CRM197 protein is a non-toxic form of diphtheria toxin, but it cannot be distinguished from diphtheria toxin immunologically. CRM197 is produced by Corynebacterium diphtheriae infected with the non-toxigenic phage β197tox, which is produced by nitrosoguanidine mutagenesis of toxigenic Corynebacterium phage β (Uchida et al. (1971) Nature New Biology 233: 8-11). The CRM197 protein has the same molecular weight as the diphtheria toxin, but differs from it by a base change in the structural gene (guanine becomes adenine). This single base change leads to an amino acid substitution in the mature protein (glutamic acid replaces glycine) and eliminates the toxicity of the diphtheria toxin. The CRM197 protein is a safe and effective T cell-dependent sugar carrier. More details about CRM197 and its production can be found in, for example, U.S. Patent No. 5,614,382.

[0200] In one embodiment, the carrier protein of the glycoconjugate of the present invention is the A chain of CRM197 (see CN103495161). In one embodiment, the carrier protein of the glycoconjugate of the present invention is the A chain of CRM197 obtained by genetic recombination expression in Escherichia coli (see CN103495161).

[0201] In one embodiment of any of the above vaccines, the capsular polysaccharide is all alone with the CRM 197 Conjugation.

[0202] In one embodiment, the capsular polysaccharide of S. pneumoniae serotype 1, 4, 5, 6B, 7F, 9V, 14 and / or 23F of any one of the above vaccines is conjugated to PD, respectively.

[0203] In one embodiment, the capsular polysaccharide of S. pneumoniae serotype 18C of any of the above vaccines is conjugated to TT.

[0204] In one embodiment, the capsular polysaccharide of S. pneumoniae serotype 19F of any of the above vaccines is conjugated to DT.

[0205] In one embodiment, the capsular polysaccharide of Streptococcus pneumoniae serotype 1, 4, 5, 6B, 7F, 9V, 14 and / or 23F of any of the above vaccines is conjugated to PD, respectively, the capsular polysaccharide of Streptococcus pneumoniae serotype 18C is conjugated to TT, and the capsular polysaccharide of Streptococcus pneumoniae serotype 19F is conjugated to DT.

[0206] In one embodiment of any of the above vaccines, at least one capsular polysaccharide is conjugated to TT and the other capsular polysaccharides are conjugated to CRM. 197 Conjugation.

[0207] In one embodiment of any of the above vaccines, one capsular polysaccharide is conjugated to TT and the other capsular polysaccharides are conjugated to CRM. 197 Conjugation.

[0208] In one embodiment of any of the above vaccines, at least two capsular polysaccharides are conjugated to TT, and the other capsular polysaccharides are conjugated to CRM. 197 Conjugation.

[0209] In one embodiment of any of the above vaccines, two capsular polysaccharides are conjugated to TT and the other capsular polysaccharides are conjugated to CRM. 197 Conjugation.

[0210] In one embodiment of any of the above vaccines, at least three capsular polysaccharides are conjugated to TT, and the other capsular polysaccharides are conjugated to CRM. 197 Conjugation.

[0211] In one embodiment of any of the above vaccines, three capsular polysaccharides are conjugated to TT and the other capsular polysaccharides are conjugated to CRM. 197 Conjugation.

[0212] In one embodiment of any of the above vaccines, four capsular polysaccharides are conjugated to TT and the other capsular polysaccharides are conjugated to CRM. 197 Conjugation.

[0213] In one embodiment, the pneumococcal conjugate vaccine of the invention is a 21-valent pneumococcal conjugate, wherein the 21 saccharide conjugates are from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F, wherein four capsular polysaccharides are conjugated to TT, wherein the four capsular polysaccharides conjugated to TT are serotypes 15B, 22F, and the other two serotypes are selected from the group consisting of serotypes 1, 3 and 5, and the other saccharide conjugates are conjugated to CRM. 197 Conjugation.

[0214] In one embodiment, the pneumococcal conjugate vaccine of the invention is a 21-valent pneumococcal conjugate, wherein the 21 saccharide conjugates are from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F, wherein four capsular polysaccharides are conjugated to TT, wherein the four capsular polysaccharides conjugated to TT are serotypes 1, 5, 15B and 22F, and the other saccharide conjugates are conjugated to CRM. 197 Conjugation.

[0215] In one embodiment, the pneumococcal conjugate vaccine of the invention is a 21-valent pneumococcal conjugate, wherein the 21 saccharide conjugates are from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F, wherein four capsular polysaccharides are conjugated to TT, wherein the four capsular polysaccharides conjugated to TT are serotypes 1, 3, 15B and 22F, and the other saccharide conjugates are conjugated to CRM. 197 Conjugation.

[0216] In one embodiment, the pneumococcal conjugate vaccine of the invention is a 21-valent pneumococcal conjugate, wherein the 21 saccharide conjugates are from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F, wherein four capsular polysaccharides are conjugated to TT, wherein the four capsular polysaccharides conjugated to TT are serotypes 3, 5, 15B and 22F, and the other saccharide conjugates are conjugated to CRM. 197 Conjugation.

[0217] In one embodiment, the pneumococcal conjugate vaccine of the present invention is a 21-valent pneumococcal conjugate, wherein the 21 saccharide conjugates are from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F of Streptococcus pneumoniae, wherein two saccharide conjugates selected from serotypes 1, 3 and 5 are conjugated to TT, and the other saccharide conjugates are conjugated to CRM. 197 Conjugation.

[0218] In one embodiment, the pneumococcal conjugate vaccine of the present invention is a 21-valent pneumococcal conjugate, wherein the 21 sugar conjugates are from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F of Streptococcus pneumoniae, wherein the sugar conjugates selected from serotypes 1 and 3 are conjugated to TT, and the other sugar conjugates are conjugated to CRM. 197 Conjugation.

[0219] In one embodiment, the pneumococcal conjugate vaccine of the present invention is a 21-valent pneumococcal conjugate, wherein the 21 saccharide conjugates are from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F of Streptococcus pneumoniae, wherein the saccharide conjugates selected from serotypes 1 and 5 are conjugated to TT, and the other saccharide conjugates are conjugated to CRM. 197 Conjugation.

[0220] In one embodiment, the pneumococcal conjugate vaccine of the present invention is a 21-valent pneumococcal conjugate, wherein the 21 saccharide conjugates are from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F of Streptococcus pneumoniae, wherein the saccharide conjugates selected from serotypes 3 and 5 are conjugated to TT, and the other saccharide conjugates are conjugated to CRM. 197 Conjugation.

[0221] In one embodiment, the pneumococcal conjugate vaccine of the invention is a 15-valent pneumococcal conjugate, wherein the 15 saccharide conjugates are from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F of Streptococcus pneumoniae, wherein all saccharide conjugates are conjugated to CRM 197 Conjugation.

[0222] In a preferred embodiment, the pneumococcal conjugate vaccine of the invention is a 20-valent pneumococcal conjugate, wherein the 20 saccharide conjugates are from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F, wherein all saccharide conjugates are conjugated to CRM 197 Conjugation.

[0223] The vaccine of the present invention may include a small amount of free carrier. When a given carrier protein is present in the vaccine of the present invention in free form and in conjugated form, the non-conjugated form preferably does not exceed 5% of the total amount of carrier protein in the whole composition, and more preferably less than 2% by weight.

[0224] In some embodiments, the vaccines of the present invention may include at least one adjuvant. In some embodiments, the vaccines of the present invention may include an adjuvant. The term "adjuvant" refers to a compound or mixture that enhances the immune response to an antigen. An antigen may primarily serve as a delivery system, primarily serve as an immunomodulator, or have the powerful characteristics of both.

[0225] Suitable adjuvants include those suitable for mammals, including humans. Examples of known suitable delivery system types of adjuvants that can be used in humans include, but are not limited to, alum (e.g., aluminum phosphate, aluminum sulfate, or aluminum hydroxide), calcium phosphate, liposomes, oil-in-water emulsions, such as MF59 (4.3% w / v squalene, 0.5% w / v polysorbate 80 (Tween 80), 0.5% w / v sorbitan trioleate (Span 85)), water-in-oil emulsions (e.g., Montanide), and poly (D, L-lactide-co-glycolide) (PLG) microparticles or nanoparticles.

[0226] In one embodiment, the vaccine of the invention comprises an aluminum salt (alum) as an adjuvant (e.g., aluminum phosphate, aluminum sulfate, or aluminum hydroxide). In a preferred embodiment, the vaccine of the invention comprises aluminum phosphate or aluminum hydroxide as an adjuvant. In an even more preferred embodiment, the vaccine of the invention comprises aluminum phosphate as an adjuvant.

[0227] In some embodiments, the vaccines of the present disclosure comprise aluminum phosphate as an adjuvant and have a final aluminum phosphate concentration between about 0.1 mg / mL to about 1 mg / ml. In some embodiments, the vaccines of the present disclosure comprise aluminum phosphate as an adjuvant and have a final aluminum phosphate concentration between about 0.1 mg / mL to about 0.5 mg / ml. In a preferred embodiment, the vaccines of the present disclosure comprise aluminum phosphate as an adjuvant and have a final aluminum phosphate concentration of about 0.25 mg / mL.

[0228] In one embodiment, the adjuvant is added to the vaccine after the preservative is added. In a preferred embodiment, the adjuvant is added to the vaccine after the 2-PE preservative is added. In another preferred embodiment, the aluminum phosphate adjuvant is added to the vaccine after the 2-PE preservative is added.

[0229] Other exemplary adjuvants for enhancing the effectiveness of the vaccines of the present invention include, but are not limited to: (1) oil-in-water emulsion formulations (with or without other specific immunostimulants, such as muramyl peptides (see below) or bacterial cell wall components), such as (a) SAF containing 10% squalene, 0.4% Tween 80, 5% pluronic block polymer L121 and thr-MDP, which is microfluidized into a submicron emulsion or vortexed to produce an emulsion with larger particle size, and (b) RIBI TM Adjuvant system (RAS), (RibiImmunochem, Hamilton, MT), which contains 2% squalene, 0.2% Tween 80 and one or more bacterial cell wall components, such as monophosphoryl lipid A (MPL), trehalose dimycolate (TDM) and cell wall skeleton (CWS), preferably MPL + CWS (DETOX TM ); (2) Saponin adjuvants such as QS21, STIMULON TM (Cambridge Bioscience, Worcester, MA), (Isconova, Sweden) or (Commonwealth Serum Laboratories, Australia), or particles produced therefrom, such as ISCOMs (immunostimulatory complexes), which may not contain additional detergents (e.g. WO 00 / 07621); (3) complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA); (4) cytokines, such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12 (e.g. WO 99 / 44636)), interferons (e.g. gamma interferon), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), etc.; (5) monophosphoryl lipid A (MPL) or 3-O-deacetyl MPL (3dMPL) (see, e.g. GB-2220221, EP0689454), when used with pneumococcal saccharides, optionally in the substantial absence of alum (see, e.g. WO 00 / 56358); (6) 3dMPL in combination with, for example, QS21 and / or an oil-in-water emulsion (see, for example, EP0835318, EP0735898, EP0761231); (7) polyoxyethylene ethers or polyoxyethylene esters (see, for example, WO 99 / 52549); (8) a combination of a polyoxyethylene sorbitan ester surfactant and octoxynol (e.g., WO 01 / 21207) or a combination of a polyoxyethylene alkyl ether or ester surfactant and at least one additional nonionic surfactant, such as octoxynol (e.g., WO 01 / 21152); (9) saponin and an immunostimulatory oligonucleotide (e.g., CpG oligonucleotide) (e.g., WO 00 / 62800); (10) an immunostimulatory agent and metal salt particles (see, for example, WO 00 / 23105); (11) a saponin and an oil-in-water emulsion (e.g., WO 99 / 11241); (12) saponin (e.g., QS21) + 3dMPL + IM2 (optionally + sterol) (e.g., WO 98 / 57659); (13) other substances that act as immunostimulants to enhance the efficacy of the composition. Muramyl peptides include N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-25 acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutamyl-L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE), etc.

[0230] In one embodiment of the invention, the vaccine of the present invention comprises CpG oligonucleotide as adjuvant.CpG oligonucleotide used herein refers to immunostimulatory CpG oligodeoxynucleotide (CpG ODN), therefore, unless otherwise indicated, these terms are used interchangeably.Immunostimulatory CpG oligodeoxynucleotide contains one or more immunostimulatory CpG motifs, which are unmethylated cytosine-guanine dinucleotides, optionally located within certain preferred base ranges.The methylation state of CpG immunostimulatory motifs generally refers to the cytosine residues in the dinucleotides.An immunostimulatory oligonucleotide containing at least one unmethylated CpG dinucleotide is an oligonucleotide containing a 5' unmethylated cytosine connected to 3' guanine by a phosphate bond, and it activates the immune system by binding to Toll-like receptor 9 (TLR-9).In another embodiment, the immunostimulatory oligonucleotide may contain one or more methylated CpG dinucleotides, which will activate the immune system by TLR9, but the intensity is not as strong as when the CpG motif is unmethylated. CpG immunostimulatory oligonucleotides may comprise one or more palindromes, which in turn may comprise CpG dinucleotides. CpG oligonucleotides have been described in a number of issued patents, published patent applications, and other publications, including U.S. Pat. Nos. 6,194,388, 6,207,646, 6,214,806, 6,218,371, 6,239,116, and 6,339,068.

[0231] In one embodiment of the present invention, the vaccine of the present invention comprises any one of the CpG oligonucleotides described on page 3, line 22 to page 12, line 36 of WO 2010 / 125480.

[0232] Different classes of CpG immunostimulatory oligonucleotides have been identified. These are referred to as classes A, B, C and P and are described in more detail in WO 2010 / 125480, page 3, line 22 to page 12, line 36. The methods of the present invention include the use of these different classes of CpG immunostimulatory oligonucleotides.

[0233] In one embodiment, the vaccine of the invention comprises any combination of the saccharide conjugate disclosed herein and a pharmaceutically acceptable excipient, carrier or diluent.

[0234] In addition to the conjugate and preservative, the vaccines of the present disclosure may also contain one or more of a buffer, a salt, a divalent cation, a non-ionic detergent, a cryoprotectant (e.g., a sugar), and an antioxidant (e.g., a free radical scavenger or a chelator), or any various combinations thereof.

[0235] In one embodiment, the vaccine of the present disclosure comprises a buffer. In one embodiment, the buffer has a pKa of about 3.5 to about 7.5. In some embodiments, the buffer is phosphate, succinate, histidine or citrate. In some embodiments, the buffer is succinate. In some embodiments, the buffer is histidine. In certain embodiments, the buffer is succinate with a final concentration of 1mM to 10mM. In a specific embodiment, the final concentration of the succinate buffer is about 5mM.

[0236] In one embodiment, the vaccine of the present disclosure comprises a salt. In some embodiments, the salt is selected from the group consisting of magnesium chloride, potassium chloride, sodium chloride, and combinations thereof. In a specific embodiment, the salt is sodium chloride. In a specific embodiment, the immunogenic composition of the present invention comprises 150 mM sodium chloride.

[0237] In one embodiment, the vaccine of the present disclosure comprises a surfactant. In one embodiment, the surfactant is selected from polysorbate 20 (TWEEN 20), polysorbate 40 (TWEEN 40), polysorbate 60 (TWEEN 60), polysorbate 70 (TWEEN 70), polysorbate 80 (TWEEN 70), polysorbate 90 (TWEEN 70), polysorbate 100 (TWEEN 70), polysorbate 110 (TWEEN 70), polysorbate 120 (TWEEN 70), polysorbate 130 (TWEEN 70), polysorbate 140 (TWEEN 70), polysorbate 150 (TWEEN 70), polysorbate 160 (TWEEN 70), polysorbate 170 (TWEEN TM 60), polysorbate 65 (TWEEN TM 65) Polysorbate 80 (TWEEN TM 80), polysorbate 85 (TWEEN TM 85)TRITON TM N-101, TRITON TM X-100, oxtoxynol 40, nonoxynol-9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate (PEG-15, Solutol H 15), polyoxyethylene-35-ricinoleate ( EL), soybean lecithin and poloxamer.

[0238] In a specific embodiment, the surfactant is polysorbate 80. In some of the embodiments, the final concentration of polysorbate 80 in the formulation is at least 0.0001% to 10% polysorbate 80 by weight (w / w). In some of the embodiments, the final concentration of polysorbate 80 in the formulation is at least 0.001% to 1% polysorbate 80 by weight (w / w). In some of the embodiments, the final concentration of polysorbate 80 in the formulation is at least 0.01% to 1% polysorbate 80 by weight (w / w). In other embodiments, the final concentration of polysorbate 80 in the formulation is 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09% or 0.1% polysorbate 80 (w / w). In a preferred embodiment, the final concentration of polysorbate 80 in the formulation is 0.01% to 0.03%. In another embodiment, the final concentration of polysorbate 80 in the formulation is 0.02% polysorbate 80 (w / w). In another embodiment, the final concentration of polysorbate 80 in the formulation is 0.01% polysorbate 80 (w / w). In another embodiment, the final concentration of polysorbate 80 in the formulation is 0.03% polysorbate 80 (w / w). In another embodiment, the final concentration of polysorbate 80 in the formulation is 0.04% polysorbate 80 (w / w). In another embodiment, the final concentration of polysorbate 80 in the formulation is 0.05% polysorbate 80 (w / w). In another embodiment, the final concentration of polysorbate 80 in the formulation is 1% polysorbate 80 (w / w).

[0239] In a specific embodiment, the surfactant is polysorbate 20. In some of the embodiments, the final concentration of polysorbate 20 in the formulation is at least 0.01% to 10% polysorbate 20 by weight (w / w). In some of the embodiments, the final concentration of polysorbate 20 in the formulation is at least 0.1% to 1% polysorbate 20 by weight (w / w). In some of the embodiments, the final concentration of polysorbate 20 in the formulation is at least 0.1% to 0.5% polysorbate 20 by weight (w / w). In other embodiments, the final concentration of polysorbate 20 in the formulation is 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% or 1% polysorbate 20 (w / w). In another embodiment, the final concentration of polysorbate 20 in the formulation is 0.2% polysorbate 20 (w / w). In another embodiment, the final concentration of polysorbate 20 in the formulation is 0.1% polysorbate 20 (w / w). In another embodiment, the final concentration of polysorbate 20 in the formulation is 0.3% polysorbate 20 (w / w). In another embodiment, the final concentration of polysorbate 20 in the formulation is 0.4% polysorbate 80 (w / w). In another embodiment, the final concentration of polysorbate 20 in the formulation is 0.5% polysorbate 20 (w / w). In a preferred embodiment, the final concentration of polysorbate 20 in the formulation is 0.2% polysorbate 20 (w / w).

[0240] In a specific embodiment, the surfactant is polysorbate 40. In some of the embodiments, the final concentration of polysorbate 40 in the formulation is at least 0.0001% to 10% polysorbate 40 by weight (w / w). In some of the embodiments, the final concentration of polysorbate 40 in the formulation is at least 0.001% to 1% polysorbate 40 by weight (w / w). In some of the embodiments, the final concentration of polysorbate 40 in the formulation is at least 0.01% to 1% polysorbate 40 by weight (w / w). In other embodiments, the final concentration of polysorbate 40 in the formulation is 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09% or 0.1% polysorbate 40 (w / w). In another embodiment, the final concentration of polysorbate 40 in the formulation is 1% polysorbate 40 (w / w).

[0241] In a specific embodiment, the surfactant is polysorbate 60. In some of the embodiments, the final concentration of polysorbate 60 in the formulation is at least 0.0001% to 10% polysorbate 60 by weight (w / w). In some of the embodiments, the final concentration of polysorbate 60 in the formulation is at least 0.001% to 1% polysorbate 60 by weight (w / w). In some of the embodiments, the final concentration of polysorbate 60 in the formulation is at least 0.01% to 1% polysorbate 60 by weight (w / w). In other embodiments, the final concentration of polysorbate 60 in the formulation is 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09% or 0.1% polysorbate 60 (w / w). In another embodiment, the final concentration of polysorbate 60 in the formulation is 1% polysorbate 60 (w / w).

[0242] In a specific embodiment, the surfactant is polysorbate 65. In some of the embodiments, the final concentration of polysorbate 65 in the formulation is at least 0.0001% to 10% polysorbate 65 weight ratio (w / w). In some of the embodiments, the final concentration of polysorbate 65 in the formulation is at least 0.001% to 1% polysorbate 65 weight ratio (w / w). In some of the embodiments, the final concentration of polysorbate 65 in the formulation is at least 0.01% to 1% polysorbate 65 weight ratio (w / w). In other embodiments, the final concentration of polysorbate 65 in the formulation is 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09% or 0.1% polysorbate 65 (w / w). In another embodiment, the final concentration of polysorbate 65 is 1% polysorbate 65 (w / w).

[0243] In a specific embodiment, the surfactant is polysorbate 85. In some of the embodiments, the final concentration of polysorbate 85 in the formulation is at least 0.0001% to 10% polysorbate 85 weight ratio (w / w). In some of the embodiments, the final concentration of polysorbate 85 in the formulation is at least 0.001% to 1% polysorbate 85 weight ratio (w / w). In some of the embodiments, the final concentration of polysorbate 85 in the formulation is at least 0.01% to 1% polysorbate 85 weight ratio (w / w). In other embodiments, the final concentration of polysorbate 85 in the formulation is 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09% or 0.1% polysorbate 85 (w / w). In another embodiment, the final concentration of polysorbate 85 in the formulation is 1% polysorbate 85 (w / w).

[0244] In certain embodiments, the vaccine of the present disclosure has a final pH of 5.5 to 7.5, more preferably a final pH of 5.6 to 7.0, and even more preferably a final pH of 5.3 to 6.3. In a preferred embodiment, the vaccine of the present disclosure has a final pH of 5.8 to 6.0. In another preferred embodiment, the vaccine of the present disclosure has a final pH of 5.8.

[0245] In some embodiments, the vaccine of the present disclosure has a final protein concentration between about 50 μg / ml and about 150 μg / ml. In a preferred embodiment, the vaccine of the present disclosure has a final protein concentration between about 60 μg / ml and about 130 μg / ml. In another preferred embodiment, the vaccine of the present disclosure has a final protein concentration between about 90 μg / ml and about 99 μg / ml. In another preferred embodiment, the vaccine of the present disclosure has a final protein concentration of about 99 μg / ml.

[0246] In some embodiments, the vaccine of the present disclosure has a final D10 particle size distribution between about 3,000 and about 6,000. In another embodiment, the vaccine of the present disclosure has a final D10 particle size distribution between about 4,000 and about 5,000. In a preferred embodiment, the vaccine of the present disclosure has a final D10 particle size distribution between about 4,300 and about 4,700. In some embodiments, the vaccine of the present disclosure has a final D50 particle size distribution between about 6,000 and about 8,000. In another embodiment, the vaccine of the present disclosure has a final D50 particle size distribution between about 7,000 and about 8,000. In a preferred embodiment, the vaccine of the present disclosure has a final D50 particle size distribution between about 7,500 and about 7,900. In some embodiments, the vaccine of the present disclosure has a final D90 particle size distribution between about 10,000 and about 16,000. In another embodiment, the vaccine of the present disclosure has a final D90 particle size distribution between about 12,000 and about 15,000. In a preferred embodiment, the vaccines of the present disclosure have a final D90 particle size distribution of between about 12,300 and about 14,900.

[0247] The typical dose of the vaccine of the present invention for injection has a volume of 0.1 mL to 2 mL. In one embodiment, the vaccine for injection of the present invention has a volume of 0.2 mL to 1 mL, even more preferably a volume of about 0.5 mL. Most preferably, the vaccine for injection of the present invention has a volume of 0.5 mL.

[0248] The amount of glycoconjugate in each dose is selected to be an amount that induces an immunoprotective response without significant adverse side effects in a typical vaccine recipient. This amount will vary depending on which specific immunogen is used and how it is presented.

[0249] The amount of a particular glycoconjugate in a vaccine can be calculated based on the total polysaccharide of the conjugate (conjugated and unconjugated). For example, a glycoconjugate containing 20% ​​free polysaccharide will contain about 80 μg of conjugated polysaccharide and about 20 μg of unconjugated polysaccharide in a 100 μg polysaccharide dose. The amount of glycoconjugate will vary depending on the pneumococcal serotype. The sugar concentration can be determined by uronic acid assay.

[0250] The "immunogenic amount" of different polysaccharide components in the vaccine may vary. In general, each dose will contain 0.1 μg to 100 μg of polysaccharide for a given serotype. In one embodiment, each dose will contain 0.1 μg to 100 μg of polysaccharide for a given serotype. In a preferred embodiment, each dose will contain 0.5 μg to 20 μg. In a preferred embodiment, each dose will contain 1.0 μg to 10 μg. In an even preferred embodiment, each dose will contain 2.0 μg to 5.0 μg of polysaccharide for a given serotype. Any whole number integer within any of the above ranges is considered an embodiment of the present disclosure.

[0251] In one embodiment, each dose will contain about 1.1 μg, about 1.2 μg, about 1.3 μg, about 1.4 μg, about 1.5 μg, about 1.6 μg, about 1.7 μg, about 1.8 μg, about 1.9 μg, about 2.0 μg, about 2.1 μg, about 2.2 μg, about 2.3 μg, about 2.4 μg, about 2.5 μg, about 2.6 μg, about 2.7 μg, about 2.8 μg, about 2.9 μg, or about 3.0 μg of a polysaccharide from a glycoconjugate of S. pneumoniae serotype 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and / or 33F.

[0252] In one embodiment, each dose will contain about 2.0 μg of a polysaccharide from a glycoconjugate of S. pneumoniae serotype 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and / or 33F.

[0253] In one embodiment, each dose will contain about 2.2 μg of a polysaccharide from a glycoconjugate of S. pneumoniae serotype 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and / or 33F.

[0254] In one embodiment, each dose will contain about 4.0 μg of a polysaccharide from a glycoconjugate of S. pneumoniae serotype 6B.

[0255] In one embodiment, each dose will contain about 4.4 μg of a polysaccharide from a glycoconjugate of S. pneumoniae serotype 6B.

[0256] In one embodiment, each dose will contain from about 1.5 μg to about 3.0 μg of polysaccharide from each of the glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F, and from about 3.0 μg to about 6.0 μg of polysaccharide from the glycoconjugate of S. pneumoniae serotype 6B.

[0257] In one embodiment, each dose will contain from about 2.0 μg to about 2.5 μg of polysaccharide from each glycoconjugate of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F, and from about 4.0 μg to about 4.8 μg of polysaccharide from a glycoconjugate of S. pneumoniae serotype 6B.

[0258] In one embodiment, each dose will contain about 2.2 μg of polysaccharide from glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F, and about 4.4 μg of polysaccharide from glycoconjugates of S. pneumoniae serotype 6B.

[0259] In one embodiment, each dose will contain from about 1.5 μg to about 3.0 μg of polysaccharide from each of the glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, and 33F, and from about 3 μg to about 6 μg of polysaccharide from the glycoconjugate of S. pneumoniae serotype 6B.

[0260] In one embodiment, each dose will contain from about 2.0 μg to about 2.5 μg of polysaccharide from each of the glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, and 33F, and from about 4.0 μg to about 4.8 μg of polysaccharide from the glycoconjugate of S. pneumoniae serotype 6B.

[0261] In one embodiment, each dose will contain about 2.0 μg of polysaccharide from each glycoconjugate of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F, and about 4.0 μg of polysaccharide from a glycoconjugate of S. pneumoniae serotype 6B.

[0262] In one embodiment, each dose will contain from about 1.5 μg to about 3.0 μg of polysaccharide from each of the glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 7F, 9N, 9V, 14, 18C, 19A, 19F, 22F, 23F, and 33F, and from about 3 μg to about 6 μg of polysaccharide from the glycoconjugate of S. pneumoniae serotype 6B.

[0263] In one embodiment, each dose will contain from about 2.0 μg to about 2.5 μg of polysaccharide from each of the glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 7F, 9N, 9V, 14, 18C, 19A, 19F, 22F, 23F, and 33F, and from about 4.0 μg to about 4.8 μg of polysaccharide from the glycoconjugate of S. pneumoniae serotype 6B.

[0264] In one embodiment, each dose will contain about 2.0 μg of polysaccharide from each glycoconjugate of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 7F, 9N, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F, and about 4.0 μg of polysaccharide from a glycoconjugate of S. pneumoniae serotype 6B.

[0265] Generally, each dose will contain a total amount of 10 μg to 150 μg of carrier protein. In one embodiment, each dose will contain a total amount of 10 μg to 150 μg of carrier protein. In one embodiment, each dose will contain a total amount of 25 μg to 75 μg of carrier protein. In a preferred embodiment, each dose will contain a total amount of 40 μg to 60 μg of carrier protein. In one embodiment, the carrier protein is CRM 197 .

[0266] In one embodiment, each dose will contain about 30 μg of carrier protein. In one embodiment, each dose will contain about 31 μg of carrier protein. In one embodiment, each dose will contain about 32 μg of carrier protein. In one embodiment, each dose will contain about 33 μg of carrier protein. In a preferred embodiment, each dose will contain about 34 μg of carrier protein.

[0267] In a preferred embodiment, the vaccine is a 13-valent pneumococcal conjugate vaccine and each dose contains about 34 μg of carrier protein. In one embodiment, the carrier protein is CRM 197 .

[0268] In another preferred embodiment, the vaccine is a 15-valent pneumococcal conjugate vaccine and each dose contains about 30 μg of carrier protein. In one embodiment, the carrier protein is CRM 197 .

[0269] In one embodiment, each dose will contain about 40 μg of carrier protein. In one embodiment, each dose will contain about 41 μg of carrier protein. In one embodiment, each dose will contain about 42 μg of carrier protein. In one embodiment, each dose will contain about 43 μg of carrier protein. In one embodiment, each dose will contain about 44 μg of carrier protein. In one embodiment, each dose will contain about 45 μg of carrier protein.

[0270] In a preferred embodiment, the vaccine is a 16-valent pneumococcal conjugate vaccine and each dose contains about 42 μg of carrier protein. In one embodiment, the carrier protein is CRM 197 .

[0271] In one embodiment, each dose will contain about 48 μg of carrier protein. In one embodiment, each dose will contain about 49 μg of carrier protein. In one embodiment, each dose will contain about 50 μg of carrier protein. In one embodiment, each dose will contain about 51 μg of carrier protein. In one embodiment, each dose will contain about 52 μg of carrier protein. In one embodiment, each dose will contain about 53 μg of carrier protein.

[0272] In a preferred embodiment, the vaccine is a 20-valent pneumococcal conjugate vaccine and each dose contains about 50 μg of carrier protein. In one embodiment, the carrier protein is CRM 197 .

[0273] In a preferred embodiment, the pneumococcal conjugate vaccine of the present invention is a 20-valent pneumococcal conjugate vaccine, wherein the 20 saccharide conjugates are from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F, wherein all of the saccharide conjugates are conjugated to CRMs. 197 conjugated, and each dose contains about 2.2 μg of each capsular polysaccharide serotype 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F and about 4.4 μg of capsular polysaccharide serotype 6B, about 50 μg of CRM 197 , 2-PE, 0.125 mg elemental aluminum (as aluminum phosphate adjuvant), 100 μg polysorbate 80, sodium chloride and succinate buffer.

[0274] In one embodiment, the pneumococcal conjugate vaccine of the present invention is a 15-valent pneumococcal conjugate vaccine, wherein the 15 saccharide conjugates are from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F of Streptococcus pneumoniae, wherein all of the saccharide conjugates are conjugated to CRM 197 conjugated, and each dose contains about 2.0 μg of each capsular polysaccharide serotypes 1, 3, 4, 5, 6A, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F and about 4.0 μg of serotype 6B capsular polysaccharide, about 30 μg of CRM 197 , 2-PE, 0.125 mg elemental aluminum (as aluminum phosphate adjuvant), sodium chloride and L-histidine buffer.

[0275] In one embodiment, the pneumococcal conjugate vaccine of the present invention is a 15-valent pneumococcal conjugate vaccine, wherein the 15 saccharide conjugates are from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F of Streptococcus pneumoniae, wherein all of the saccharide conjugates are conjugated to CRM 197 conjugated, and each dose contains about 2.0 μg of each capsular polysaccharide serotypes 1, 3, 4, 5, 6A, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F and about 4.0 μg of serotype 6B capsular polysaccharide, about 30 μg of CRM 197 , 2-PE, 0.125 mg elemental aluminum (as aluminum phosphate adjuvant), polysorbate 20, sodium chloride and L-histidine buffer.

[0276] In some embodiments, the vaccine is a 21-valent pneumococcal conjugate vaccine and each dose contains about 1 μg to about 30 μg TT and about 20 μg to about 85 μg CRM 197 .

[0277] In some embodiments, the vaccine is a 21-valent pneumococcal conjugate vaccine and each dose contains about 2 μg to about 25 μg TT and about 40 μg to about 75 μg CRM 197 .

[0278] In some embodiments, the vaccine is a 21-valent pneumococcal conjugate vaccine and each dose comprises about 2 μg to about 2.5 μg of each capsular polysaccharide serotype 1, 5, 6A, 7F, 8, 9N, 10A, 11A, 12F, 14, 15B, 18C, 22F, 23F and 33F, and about 4 μg to about 5 μg of capsular polysaccharides of serotypes 3, 4, 6B, 9V, 19A and 19F.

[0279] In some embodiments, the vaccine is a 21-valent pneumococcal conjugate vaccine and each dose comprises about 2 μg to about 2.5 μg of each capsular polysaccharide serotype 1, 4, 5, 6A, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F and about 4 to about 5 μg of capsular polysaccharides of serotypes 3 and 6B.

[0280] In one embodiment, the pneumococcal conjugate vaccine of the present invention is a 21-valent pneumococcal conjugate vaccine, wherein the 21 sugar conjugates are from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F of Streptococcus pneumoniae, wherein the sugar conjugates from serotypes 1 and 5 are conjugated to TT, and the other sugar conjugates are conjugated to CRM. 197 conjugated, and each dose contains about 2 μg to about 2.5 μg of each capsular polysaccharide serotype 1, 3, 4, 5, 6A, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F and about 4 to about 5 μg of the capsular polysaccharide of serotype 6B.

[0281] In one embodiment, the pneumococcal conjugate vaccine of the present invention is a 21-valent pneumococcal conjugate vaccine, wherein the 21 sugar conjugates are from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F of Streptococcus pneumoniae, wherein the sugar conjugates from serotypes 1 and 5 are conjugated to TT, and the other sugar conjugates are conjugated to CRM. 197 conjugated, and each dose contains about 2.2 μg of each capsular polysaccharide serotype 1, 3, 4, 5, 6A, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F and about 4.4 μg of capsular polysaccharide of serotype 6B.

[0282] In one embodiment, the pneumococcal conjugate vaccine of the present invention is a 21-valent pneumococcal conjugate vaccine, wherein the 21 sugar conjugates are from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F of Streptococcus pneumoniae, wherein the sugar conjugates from serotypes 1 and 5 are conjugated to TT, and the other sugar conjugates are conjugated to CRM. 197conjugated, and each dose contains about 2 μg to about 2.5 μg of each capsular polysaccharide serotype 1, 5, 6A, 7F, 8, 9N, 10A, 11A, 12F, 14, 15B, 18C, 22F, 23F and 33F, and about 4 to about 5 μg of each capsular polysaccharide serotype 3, 4, 6B, 9V, 19A and 19F.

[0283] In one embodiment, the pneumococcal conjugate vaccine of the present invention is a 21-valent pneumococcal conjugate vaccine, wherein the 21 sugar conjugates are from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F of Streptococcus pneumoniae, wherein the sugar conjugates from serotypes 1 and 5 are conjugated to TT, and the other sugar conjugates are conjugated to CRM. 197 conjugated, and each dose contains approximately 2.2 μg of each capsular polysaccharide serotype 1, 5, 6A, 7F, 8, 9N, 10A, 11A, 12F, 14, 15B, 18C, 22F, 23F and 33F, and approximately 4.4 μg of each capsular polysaccharide serotype 3, 4, 6B, 9V, 19A and 19F.

[0284] In one embodiment, the pneumococcal conjugate vaccine of the present invention is a 21-valent pneumococcal conjugate vaccine, wherein the 21 sugar conjugates are from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F of Streptococcus pneumoniae, wherein the sugar conjugates from serotypes 1 and 5 are conjugated to TT, and the other sugar conjugates are conjugated to CRM. 197 conjugated, and each dose contains about 2 μg to about 2.5 μg of each capsular polysaccharide serotype 1, 3, 4, 5, 6A, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F, and about 4 to about 5 μg of capsular polysaccharide serotype 6B, about 2 μg to about 25 μg of TT and about 40 μg to about 75 μg of CRM 197 .

[0285] In one embodiment, the pneumococcal conjugate vaccine of the present invention is a 21-valent pneumococcal conjugate vaccine, wherein the 21 sugar conjugates are from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F of Streptococcus pneumoniae, wherein the sugar conjugates from serotypes 1 and 5 are conjugated to TT, and the other sugar conjugates are conjugated to CRM. 197conjugated, and each dose contains about 2 μg to about 2.5 μg of each capsular polysaccharide serotype 1, 3, 4, 5, 6A, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F, and about 4 to about 5 μg of capsular polysaccharide serotype 6B, about 2 μg to about 25 μg of TT, about 40 μg to about 75 μg of CRM 197 , 2-PE, 0.125 to 0.250 mg elemental aluminum adjuvant, sodium chloride, and succinate buffer.

[0286] In one embodiment, the pneumococcal conjugate vaccine of the present invention is a 21-valent pneumococcal conjugate vaccine, wherein the 21 sugar conjugates are from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F of Streptococcus pneumoniae, wherein the sugar conjugates from serotypes 1 and 5 are conjugated to TT, and the other sugar conjugates are conjugated to CRM. 197 conjugated, and each dose contains about 2.2 μg of each capsular polysaccharide serotype 1, 3, 4, 5, 6A, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F, and about 4.4 μg of capsular polysaccharide serotype 6B, about 2 μg to about 25 μg of TT, about 40 μg to about 75 μg of CRM 197 , 2-PE, 0.125 to 0.250 mg elemental aluminum adjuvant, sodium chloride, and succinate buffer.

[0287] In one embodiment, the pneumococcal conjugate vaccine of the present invention is a 21-valent pneumococcal conjugate vaccine, wherein the 21 sugar conjugates are from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F of Streptococcus pneumoniae, wherein the sugar conjugates from serotypes 1 and 5 are conjugated to TT, and the other sugar conjugates are conjugated to CRM. 197 conjugated, and each dose contains about 2.2 μg of each capsular polysaccharide serotype 1, 3, 4, 5, 6A, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F, and about 4.4 μg of capsular polysaccharide serotype 6B, about 2 μg to about 25 μg of TT, about 40 μg to about 75 μg of CRM 197 , 2-PE, 0.125 to 0.250 mg elemental aluminum adjuvant, polysorbate 80, sodium chloride, and succinate buffer.

[0288] In a preferred embodiment, the dose of the vaccine of the present invention is a 0.5 mL dose.

[0289] As used herein, the term "about" refers to a statistically significant range of values, such as a specified amount of 2-PE, polysaccharides, carrier proteins, buffer concentrations, volumes or pH. Such a range can be within an order of magnitude, typically within 20% of a given value or range, more typically within 10%, even more typically within 5% or within 1%. Sometimes, such a range will be within the typical experimental error of a standard method for measuring and / or determining a given value or range. The permissible variations encompassed by the term "about" will depend on the particular system being studied, and can be easily understood by those skilled in the art. Whenever a range is mentioned in this application, each numeral within the range is also considered an embodiment of the present disclosure.

[0290] The inventors intend that the terms "comprising", "comprise" and "comprises" herein be optionally replaced with the terms "consisting essentially of", "consist essentially of", "consists essentially of", "consisting of" and "consists of", in each instance respectively.

[0291] An "immunogenic amount," "immunologically effective amount," "therapeutically effective amount," "prophylactically effective amount," or "dose," each used interchangeably herein, generally refers to an amount of an antigen or immunogenic composition sufficient to elicit an immune response, either a cellular (T cell) or humoral (B cell or antibody) response, or both, as measured by standard assays known to those of skill in the art.

[0292] Any whole number integer within any range in this document is considered an embodiment of the present disclosure.

[0293] All references or patent applications cited in this patent specification are incorporated herein by reference.

[0294] The present invention is illustrated by the accompanying examples. The following examples were performed using standard techniques, which are well known and routine to those skilled in the art unless otherwise described in detail. These examples are illustrative, but not limiting, of the present invention. Example

[0295] Example 1 Solubility of 2-phenoxyethanol (2-PE)

[0296] The purpose of this study was to evaluate the dissolution time and solubility of 2-phenoxyethanol (2-PE). The dissolution and concentration of 2-PE were evaluated in succinate buffer solution (SBS) (5 mM succinic acid, 150 mM NaCl, pH 5.8) at the following concentrations: 11 g / L, 16.5 g / L, and 20 g / L.

[0297] Weigh an appropriate amount of 2-PE into a 400 mL beaker and add an appropriate amount of succinic acid buffered saline. Stir at 300 rpm and the solution temperature is about 20°C. Record the complete dissolution time based on visual observation. Measure the 2-PE concentration by RP-HPLC.

[0298] The dissolution time results are shown in Table 1, and the 2-PE concentration results are shown in Table 2. The overall dissolution time (15 minutes) of 20 mg / mL 2-PE in succinic acid buffered saline is longer than that of 11 mg / mL (7 minutes) and 16.5 mg / mL (9 minutes) samples. A few small particles were noted after completing the first two dissolution tests (11 mg / mL and 16.5 mg / mL), but complete dissolution was not tracked under the initial mixing speed (300 rpm). Based on these observations, the complete dissolution time of the 20 mg / mL sample was monitored. For the sample of 20 mg / mL, the complete dissolution time (of all small particles) was 35 minutes.

[0299] Table 1. Dissolution time

[0300]

[0301] *Except for up to about 5 small particles, all 2-PE was dissolved

[0302] Table 2.2-PE concentration

[0303]

[0304] *Calculation: % = (measured value - target value) / target value x 100

[0305] The 2-PE concentration data indicated that 2-PE was soluble at 19.1 mg / mL or higher in succinate buffered saline (pH 5.8) at 20°C.

[0306] The measured 2-PE concentration results were all approximately 5% below the target concentration. Since the percentage difference was consistent at all concentrations, the 20 mg / mL sample's measurement result of 5% below the target value would not be expected to indicate reaching the solubility limit of 2-PE in succinate buffered saline (20°C).

[0307] Example 2 Effect on filter integrity of polyethersulfone (PES) membrane (0.5 μm / 0.2 μm) filters when exposed to a multivalent conjugate pool containing 2-phenoxyethanol

[0308] The aim of this study was to evaluate the impact on filter integrity of PES membrane (0.5 μm / 0.2 μm) filters when exposed to a 20-valent conjugate pool formulation for at least 48 hours. Testing has been performed on both the filter membrane and the filter device. Compatibility testing has also been performed for other filter sizes in the same filter family.

[0309] A 20-valent conjugate pool (S. pneumoniae polysaccharide serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F were each mixed with CRM at a concentration of approximately 7.5 μg / ml) was prepared with 17 mg / mL 2-phenoxyethanol (final concentration). 197 The conjugate was 6B at a concentration of 15 μg / ml with CRM 197 Conjugate of 5.5 mM succinate buffer pH 5.8, 0.033% (w / w) PS80, about 150 mM NaCl, see WO2015110941). The concentration of the conjugate depends on the sugar content (anthrone sugar concentration).

[0310] 17 mg / mL of 2-PE in the conjugate pool is equivalent to 10 mg / mL of 2-PE in the final drug product after final dilution with buffer and aluminum phosphate (AlPO4) addition.

[0311] Each filter of the two capsule filters was thoroughly wetted by filtering with 1L of deionized (DI) water. Then, the two filters were subjected to a pre-use filter integrity test (FIT) at ambient temperature (22±4°C). After the pre-FIT of both filters, the conjugate pool material containing 17mg / mL 2-PE was filled into each filtration device. The conjugate pool material was kept static at 22±4°C in each filtration device for at least 48 hours. After 48 hours, the conjugate pool material was discharged from the filtration device and then rinsed with 2L DI water. Then both filtration devices were subjected to post-FIT at 22±4°C.

[0312] The acceptance criterion for passing the FIT is the rated bubble point of the capsule filter, which is provided by the manufacturer [≥4000 mbar (≥58 psi) for air and water].

[0313] The results are provided in Table 3.

[0314] Table 3. Filter integrity test results of capsule filters

[0315]

[0316] a. Bubble point at 23℃

[0317] b. Post-use testing occurred 48 hours after exposure to the PCV20 MDV conjugate pool

[0318] The results of this study showed that there was no effect on the filtration integrity of polyethersulfone (PES) membrane (0.5 μm / 0.2 μm) filters when exposed to the conjugate pool formulation for at least 48 hours. Both filters passed the integrity tests before and after use.

[0319] Compatibility testing is also available for other filter sizes within the same filter family.

[0320] Example 3 Addition and mixing rates of 2-phenoxyethanol in a multivalent conjugate pool

[0321] The purpose of this study was to determine the addition rate and mixing speed required to dissolve 2-PE in a conjugate pool without significantly increasing the turbidity of the intermediate solution.

[0322] The results show that the turbidity of the formulations containing 17 mg / mL and 20 mg / mL 2-PE 20-valent conjugate pool (see Example 2) has a significant increase. For these two formulations, 2-PE was added to the conjugate pool without controlling the rate of 2-PE addition or mixing the conjugate pool during and after addition. It is assumed that these two factors play a role in the observed increase in turbidity.

[0323] Studies were performed to understand whether the addition of 2-PE to the conjugate pool needed to be controlled.

[0324] This study evaluated the potential effects of 2-PE addition rate and mixing speed on 2-PE solubilization in the conjugate pool.

[0325] 1.97 L of the 20-valent conjugate pool (see Example 2) was prepared without the addition of 2-PE, with a final volume target of 2 L and a final 2-PE concentration target of 15 mg / mL. The entire conjugate pool was divided into 197 mL aliquots, and 2-PE was added to each aliquot at different addition and mixing rates.

[0326] Table 4 lists the aliquots evaluated:

[0327] Table 4. Conjugate pool aliquots

[0328]

[0329] For each aliquot, 197 mL of the conjugate pool was added to a 250 mL glass beaker. Each beaker was placed on a mixing plate and magnetic stirring was added. An aliquot of 2.71 mL of 2-PE (3 g of 2-PE was added, and the density of 2-PE was 1.107 g / ml) was manually added to the conjugate pool immediately (i.e., once) using a 5 mL disposable syringe equipped with a 21G 1" stainless steel needle. For a controlled rate of adding aliquots, 2-PE (same amount) was added to the mixed conjugate pool using the same syringe and needle, but an automatic syringe pump was used to control the rate of dispensing 2-PE. The syringe pump was programmed to add 2-PE from the syringe at the rate specified above. For all aliquots, the mixing plate was set to 200 RPM for 2 hours. Immediate addition and delayed mixing allowed mixing to be delayed for 30 minutes after the addition of 2-PE was complete.

[0330] A qualitative approach was utilized by assessing the visual appearance of the aliquoted conjugate pool after 2-PE was dissolved. A video was taken to capture the addition of 2-PE to the aliquoted conjugate pool. All aliquots were photographed after adding 2-PE and mixing for 2 hours, and the appearance was compared to each other in an appearance box.

[0331] Although the 1 mL / min addition with immediate mixing and 0.1 mL / min addition with immediate mixing aliquots appeared to eliminate the observed turbidity, a slight increase in turbidity was observed in these aliquots compared to the control conjugate pool (without 2-PE) when placed near a light source.

[0332] The results of this study indicate that the degree of turbidity of the conjugate pool after the addition of 2-PE depends on the addition rate and mixing time. The visual appearance of the immediate addition with delayed mixing aliquot was significantly more turbid than the immediate addition with immediate mixing aliquot, suggesting that mixing while adding 2-PE to the conjugate pool has a direct effect on turbidity development.

[0333] The turbidity of the aliquot added at a controlled rate was significantly lower than that of the aliquot added immediately. In addition, the lower rate of 2-PE addition correlated with reduced turbidity in the conjugate pool after 2-PE dissolution was complete.

[0334] Example 4 Evaluation of the addition of 2-phenoxyethanol (2-PE) to formulate a multivalent conjugate vaccine

[0335] The aim of this study was to evaluate the addition of 2-PE during the formulation of a 20-valent conjugate vaccine adjuvanted with alum.

[0336] 2-PE has been added at two different rates: 1- bulk vaccine before final formulation or 2- and 3- to the conjugate pool before final formulation.

[0337] The main process steps evaluated were 2-PE addition, filtration and adjuvant (AlPO4) addition.

[0338] 1: Add 2-PE to the final formulated vaccine batch

[0339] The 20-valent conjugate pool (see Example 2) was mixed at 200 rpm for at least 5 minutes.

[0340] Obtain a pre-filtration sample (135 ml). Filter the conjugate pool sample (0.5 / 0.2 μm polyethersulfone (PES) filter) using a product volume:filter surface area ratio (L / m2) of approximately 76.3. Start filtration using a peristaltic pump. After approximately half of the total volume has been filtered off, pause the filtration operation by stopping the peristaltic pump. Leave the product in the filter for at least 30 minutes before restarting the filtration operation to evaluate filtration pauses during manufacturing.

[0341] Obtain the filtered sample.

[0342] Succinate buffered saline and AlPO4 (final concentration 5 mM succinate buffer (pH 5.8), 150 mM sodium chloride, and 0.25 mg / mL aluminum (as aluminum phosphate (AlPO4)) were added and the final drug product was mixed for at least 120 minutes.

[0343] 2-PE was added using a syringe pump and the total time of 2-PE addition was recorded – see Table 5 for 2-PE addition parameters.

[0344] After mixing for 120 minutes after the addition of 2-PE, mixing was stopped.

[0345] 2: Before final preparation, add 2-PE to the conjugate pool at a rate of 5.0 mL / min / L (add 5.5 g / min / L of 2-PE)

[0346] In a 5 L glass vessel 4 L of 20-valent conjugate pool (see Example 2) has been added. Mixing has been started at 200 rpm and maintained for at least 5 minutes.

[0347] 2-PE has been added at 20 mL / min using a syringe pump - see Table 5 for 2-PE addition parameters.

[0348] After the 2-PE addition was complete, samples were taken from the top / bottom of the vessel.

[0349] The 2-PE dissolution time determined by visual observation was recorded - see Table 7.

[0350] After mixing for 120 minutes, mixing was stopped.

[0351] A pre-filtration sample (135 ml) was obtained and photographed.

[0352] The conjugate pool sample was filtered (0.5 / 0.2 μm polyethersulfone (PES) filter) using a product volume: filter surface area ratio (L / m2) of approximately 76.3. Filtration was started using a peristaltic pump. After approximately half of the total volume had been filtered, the filtration operation was paused by stopping the peristaltic pump. The product was retained in the filter for at least 30 minutes and the filter retention time was determined before restarting the filtration operation.

[0353] Obtain the filtered sample.

[0354] Succinate buffered saline and AlPO4 (final concentration 5 mM succinate buffer (pH 5.8), 150 mM sodium chloride, and 0.25 mg / mL aluminum (as aluminum phosphate (AlPO4)) were added and the final drug product was mixed for at least 120 minutes.

[0355] 3: Rapidly add 2-PE to the conjugate pool before final formulation

[0356] 400 mL of 20-valent conjugate pool (see Example 2) has been added to a 500 mL glass container. Mixing has been started at 200 rpm and maintained for at least 5 minutes.

[0357] 2-PE has been added using a syringe (added as fast as possible) and the total time and amount of 2-PE addition recorded - see Table 5 for 2-PE addition parameters.

[0358] After mixing for 120 minutes, mixing was stopped.

[0359] A pre-filtration sample (135 ml) was obtained and photographed.

[0360] The conjugate pool sample was filtered (0.5 / 0.2 μm polyethersulfone (PES) filter) using a product volume: filter surface area ratio (L / m2) of approximately 76.3. Filtration was started using a peristaltic pump. After approximately half of the total volume had been filtered, the filtration operation was paused by stopping the peristaltic pump. The product was retained in the filter for at least 30 minutes and the filter retention time was determined before restarting the filtration operation.

[0361] Obtain the filtered sample.

[0362] Succinate buffered saline and AlPO4 (final concentration 5 mM succinate buffer (pH 5.8), 150 mM sodium chloride, and 0.25 mg / mL aluminum (as aluminum phosphate (AlPO4)) were added and the final drug product (DP) was mixed for at least 120 minutes.

[0363] Table 5.2-PE added parameters

[0364]

[0365] result:

[0366] This study evaluated the options of 2-PE addition by formulating batches, including adding 2-PE to the conjugate pool, quickly adding 2-PE to the conjugate pool, and adding 2-PE to the final drug product. For adding 2-PE to the conjugate pool, the data showed that 2-PE was uniformly diffused into the batch material. No significant turbidity was observed in any of the three pre-filtered samples. Multiple quality attributes of the drug product were evaluated, and the drug product met all acceptance criteria. These results demonstrate the feasibility of adding 2-PE to the conjugate pool (before adding AlPO4 and before sterile filtration).

[0367] Aspects of the Invention

[0368] The following clauses describe additional embodiments of the invention:

[0369] C1. A method for producing a conjugate vaccine comprising a preservative, the method comprising the following steps:

[0370] (a) adding a preservative from a bulk solution to a solution comprising one or more conjugates, wherein the preservative is added without sterile filtration; and

[0371] (b) the mixture comprising the preservative and the conjugate is then sterile filtered.

[0372] C2. The method of C1, further comprising the step (c) of subsequently adding an adjuvant.

[0373] C3. The method of C2, wherein the adjuvant is aluminum phosphate.

[0374] C4. The method of C3, wherein the final concentration of aluminum phosphate in the vaccine is between about 0.1 mg / mL and about 0.5 mg / ml.

[0375] C5. The method of C4, wherein the final concentration of aluminum phosphate in the vaccine is about 0.25 mg / ml.

[0376] C6. The method of any of C1-C5, wherein the preservative is hydrophobic and viscous at the concentration in the bulk solution.

[0377] C7. The method of any one of C1-C6, wherein the viscosity of the preservative at the concentration in bulk solution is at least 10 centistokes at 25°C.

[0378] C8. The method of any one of C1-C6, wherein the viscosity of the preservative at the concentration in bulk solution is at least 15 centistokes at 25°C.

[0379] C9. The method of any one of C1-C6, wherein the viscosity of the preservative at the concentration in bulk solution is at least 20 centistokes at 25°C.

[0380] C10. The method of any one of C1-C6, wherein the viscosity of the preservative at a concentration in bulk solution is between about 10 and about 50 centistokes at 25°C.

[0381] C11. The method of any one of C1-C6, wherein the viscosity of the preservative at a concentration in bulk solution is between about 15 and about 25 centistokes at 25°C.

[0382] C12. The method of any one of C1-C11, wherein the preservative is 2-phenoxyethanol (2-PE), phenol, m-cresol, methylparaben, propylparaben or thimerosal.

[0383] C13. The method of any one of C1-C11, wherein the preservative is 2-phenoxyethanol (2-PE) or thimerosal.

[0384] C14. The method of any one of C1-C13, wherein the preservative is added in undiluted form.

[0385] C15. The method of any one of C1-C14, wherein the bulk solution of preservative is undiluted pure preservative.

[0386] C16. The method of any one of C1-C15, wherein the preservative is 2-phenoxyethanol (2-PE).

[0387] C17. The method of any one of C1-C16, wherein the bulk solution is pure 2-phenoxyethanol (2-PE).

[0388] C18. The method of C17, wherein the 2-PE is diluted about 10 to 200 times in the solution after step (a).

[0389] C19. The method of C17, wherein after step (a), the 2-PE is diluted in the solution by about 50 times, about 60 times, about 70 times, or about 80 times.

[0390] C20. The method of C17, wherein the 2-PE is diluted about 73 to 74 times in the solution after step (a).

[0391] C21. The method of any one of C16-C20, wherein the concentration of 2-PE in the solution after step (a) is between about 1 mg / ml and about 25 mg / ml.

[0392] C22. The method of any one of C16-C20, wherein the concentration of 2-PE in the solution after step (a) is between about 10 mg / ml and about 20 mg / ml.

[0393] C23. The method of any one of C16-C20, wherein the concentration of 2-PE in the solution after step (a) is between about 15 mg / ml and about 17 mg / ml.

[0394] C24. The method of any one of C16-C20, wherein the concentration of 2-PE in the solution after step (a) is about 15 mg / ml.

[0395] C25. The method of any one of C16-C20, wherein the concentration of 2-PE in the solution after step (a) is about 17 mg / ml.

[0396] C26. The method of any one of C16-C25, wherein 2-PE is added at an addition rate between about 0.5 ml / min / L of the solution comprising one or more conjugates to about 5.0 ml / min / L of the solution comprising one or more conjugates.

[0397] C27. The method of any one of C16-C25, wherein 2-PE is added at an addition rate between about 1 ml / min / L of the solution comprising one or more conjugates to about 4.0 ml / min / L of the solution comprising one or more conjugates.

[0398] C28. The method of any one of C17-C27, wherein pure 2-PE is added using a pump.

[0399] C29. The method of any one of C17-C27, wherein pure 2-PE is added using a peristaltic pump.

[0400] C30. The method of any of C17-C27, wherein neat 2-PE is added using a peristaltic pump using silicone or polyurethane tubing.

[0401] C31. The method of any of C17-C27, wherein neat 2-PE is added using a peristaltic pump employing thermoplastic elastomer tubing.

[0402] C32. The method of any one of C16-C31, wherein the final concentration of 2-PE in the vaccine is between about 5 mg / ml and about 15 mg / ml.

[0403] C33. The method of any one of C16-C31, wherein the final concentration of 2-PE in the vaccine is between about 7 mg / ml and about 12 mg / ml.

[0404] C34. The method of any one of C16-C31, wherein the final concentration of 2-PE in the vaccine is about 10 mg / ml.

[0405] C35. The method of any one of C16-C31, wherein the final concentration of 2-PE in the vaccine is about 9 mg / ml.

[0406] C36. The method of any one of C1-C35, wherein the volume of the solution after step (a) is between about 100 L and about 500 L.

[0407] C37. The method of any one of C1-C35, wherein the volume of the solution after step (a) is about 180L.

[0408] C38. The method of any of C1-C37, wherein the preservative is added under continuous mixing.

[0409] C39. The method of C38, wherein there is no delay between adding the preservative and starting mixing.

[0410] C40. The method of any of C1-C39, wherein the preservative is added under continuous mixing at an angular speed of about 50 rpm to about 500 rpm.

[0411] C41. The method of any of C1-C39, wherein the preservative is added under continuous mixing at an angular speed of about 100 rpm to about 400 rpm.

[0412] C42. The method of any of C1-C39, wherein the preservative is added under continuous mixing at an angular speed of about 150 rpm to about 300 rpm.

[0413] C43. The method of any of C1-C39, wherein the preservative is added under continuous mixing at an angular speed of about 150 rpm to about 200 rpm.

[0414] C44. The method of any of C1-C39, wherein the preservative is added under continuous mixing at an angular speed of about 150 rpm.

[0415] C45. The method of any of C1-C39, wherein the preservative is added under continuous mixing at an angular speed of about 200 rpm.

[0416] C46. The method of any of C1-C45, wherein the solution is mixed for about 15 minutes to about 5 hours after adding the preservative and before sterile filtration.

[0417] C47. The method of any of C1-C45, wherein the solution is mixed for about 30 minutes to about 3 hours after adding the preservative and before sterile filtration.

[0418] C48. The method of any one of C1-C45, wherein the solution is mixed for about 1 hour to about 3 hours after adding the preservative and before sterile filtration.

[0419] C49. The method of any of C1-C45, wherein the solution is mixed for about 2 hours after adding the preservative and before sterile filtration.

[0420] C50. The method of any one of C1-C49, wherein the solution is mixed at an angular speed between about 50 rpm to about 500 rpm after adding the preservative and before sterile filtration.

[0421] C51. The method of any of C1-C49, wherein the solution is mixed at an angular speed between about 100 rpm and about 400 rpm after adding the preservative and before sterile filtration.

[0422] C52. The method of any of C1-C49, wherein the solution is mixed at an angular speed between about 150 rpm and about 300 rpm after adding the preservative and before sterile filtration.

[0423] C53. The method of any one of C1-C52, wherein the solution to which the preservative is added is a monovalent composition.

[0424] C54. The method of any one of C1-C52, wherein the solution to which the preservative is added is a divalent, trivalent, tetravalent, pentavalent or hexavalent composition.

[0425] C55. The method of any one of C1-C52, wherein the solution to which a preservative is added is a multivalent composition comprising seven or more conjugates.

[0426] C56. The method of any one of C1-C52, wherein the solution to which the preservative is added is a 7 to 25 valent composition.

[0427] C57. The method of any one of C1-C52, wherein the solution to which the preservative is added is a 13 to 25 valent composition.

[0428] C58. The method of any one of C1-C52, wherein the solution to which the preservative is added is a 15-valent composition.

[0429] C59. The method of any one of C1-C52, wherein the solution to which the preservative is added is a 16-valent composition.

[0430] C60. The method of any one of C1-C52, wherein the solution to which the preservative is added is a 20-valent composition.

[0431] C61. The method of any one of C1-C52, wherein the solution to which the preservative is added is a 21-valent composition.

[0432] C62. The method of any one of C1-C52, wherein the solution to which a preservative is added is a multivalent pneumococcal conjugate composition.

[0433] C63. The method of any one of C1-C52, wherein the solution to which a preservative is added is a pneumococcal conjugate composition comprising 7 to 25 saccharide conjugates from different S. pneumoniae serotypes.

[0434] C64. The method of any one of C1-C52, wherein the solution to which a preservative is added is a pneumococcal conjugate composition comprising conjugates from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 different S. pneumoniae serotypes.

[0435] C65. The method of any one of C1-C52, wherein the solution to which a preservative is added is a pneumococcal conjugate composition comprising conjugates from 20 different S. pneumoniae serotypes.

[0436] C66. The method of any one of C1-C52, wherein the solution to which a preservative is added is a 7-, 8-, 9-, 10-, 11-, 12-, 13-, 14-, 15-, 16-, 17-, 18-, 19-, or 20-valent pneumococcal conjugate composition.

[0437] C67. The method of any one of C1-C52, wherein the solution to which a preservative is added is a 15-valent pneumococcal conjugate composition.

[0438] C68. The method of any one of C1-C52, wherein the solution to which a preservative is added is a 20-valent pneumococcal conjugate composition.

[0439] C69. The method of any one of C1-C52, wherein the solution to which a preservative is added is a 21-valent pneumococcal conjugate composition.

[0440] C70. The method of any one of C1-C52, wherein the solution to which a preservative is added is a 22-valent pneumococcal conjugate composition.

[0441] C71. The method of any one of C1-C52, wherein the solution to which a preservative is added is a 21-, 22-, 23-, 24-, or 25-valent pneumococcal conjugate composition.

[0442] C72. The method of any one of C1-C52, wherein the solution to which a preservative is added comprises glycoconjugates from S. pneumoniae serotypes 4, 6B, 9V, 14, 18C, 19F and 23F.

[0443] C73. The method of any one of C1-C52, wherein the solution to which a preservative is added comprises glycoconjugates from S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F.

[0444] C74. The method of any one of C1-C52, wherein the solution to which a preservative is added comprises glycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F.

[0445] C75. The method of any one of C1-C52, wherein the solution to which a preservative is added comprises glycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F.

[0446] C76. The method of any one of C1-C52, wherein the solution to which a preservative is added comprises glycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, and 33F.

[0447] C77. The method of any one of C1-C52, wherein the solution to which a preservative is added comprises glycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F.

[0448] C78. The method of any one of C1-C52, wherein the solution to which a preservative is added comprises glycoconjugates from S. pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F.

[0449] C79. The method of any one of C1-C52, wherein the solution to which a preservative is added comprises glycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F.

[0450] C80. The method of any one of C1-C52, wherein the solution to which a preservative is added is a 10-valent pneumococcal saccharide conjugate composition, wherein the 10 saccharide conjugates are from Streptococcus pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F.

[0451] C81. The method of any one of C1-C52, wherein the solution to which a preservative is added is a 13-valent pneumococcal saccharide conjugate composition, wherein the 13 saccharide conjugates are from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F.

[0452] C82. The method of any one of C1-C52, wherein the solution to which a preservative is added is a 15-valent pneumococcal saccharide conjugate composition, wherein the 15 saccharide conjugates are from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F.

[0453] C83. The method of any one of C1-C52, wherein the solution to which a preservative is added is a 20-valent pneumococcal saccharide conjugate composition, wherein the 20 saccharide conjugates are from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F.

[0454] C84. The method of any one of C1-C52, wherein the solution to which a preservative is added is a 21-valent pneumococcal saccharide conjugate composition, wherein the 21 saccharide conjugates are from Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F.

[0455] C85. The method of any one of C1-C52, wherein the solution to which a preservative is added is a 21-valent pneumococcal saccharide conjugate composition, wherein the 21 saccharide conjugates are from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F.

[0456] C86. The method of any one of C1-C52, wherein the solution to which a preservative is added is a 22-valent pneumococcal saccharide conjugate composition, wherein the 22 saccharide conjugates are from Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F.

[0457] C87. The method of any one of C1-C52, wherein the solution to which a preservative is added is a 21-valent pneumococcal saccharide conjugate composition, wherein the 21 saccharide conjugates are from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23F and 33F.

[0458] C88. The method of any one of C1-C52, wherein the solution to which a preservative is added is a 21-valent pneumococcal saccharide conjugate composition, wherein the 21 saccharide conjugates are from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23A, 23F and 33F.

[0459] C89. The method of any one of C1-C52, wherein the solution to which a preservative is added is a 21-valent pneumococcal saccharide conjugate composition, wherein the 21 saccharide conjugates are from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23B, 23F and 33F.

[0460] C90. The method of any one of C1-C52, wherein the solution to which a preservative is added is a 21-valent pneumococcal saccharide conjugate composition, wherein the 21 saccharide conjugates are from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 24F and 33F.

[0461] C91. The method of any one of C1-C52, wherein the solution to which a preservative is added is a 21-valent pneumococcal saccharide conjugate composition, wherein the 21 saccharide conjugates are from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 33F and 35B.

[0462] C92. The method of any one of C1-C52, wherein the solution to which a preservative is added is a 20-valent pneumococcal saccharide conjugate composition, wherein the 20 saccharide conjugates are from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F.

[0463] C93. The method of any one of C63-C92, wherein the carrier protein of the glycoconjugate is TT, CRM197, or C5a peptidase (SCP) from Streptococcus.

[0464] C94. The method of any one of C63-C92, wherein the carrier protein of the glycoconjugate is CRM 197 .

[0465] The method of any one of C95.C63-C92, wherein at least one capsular polysaccharide is conjugated to TT and the other capsular polysaccharides are conjugated to CRM 197 Conjugation.

[0466] C96. The method of any one of C63-C92, wherein one capsular polysaccharide is conjugated to TT and the other capsular polysaccharides are conjugated to CRM 197 Conjugation.

[0467] The method of any one of C97.C63-C92, wherein at least two capsular polysaccharides are conjugated to TT and the other capsular polysaccharides are conjugated to CRM 197 Conjugation.

[0468] C98. The method of any one of C63-C92, wherein two of the capsular polysaccharides are conjugated to TT and the other capsular polysaccharides are conjugated to CRM. 197 Conjugation.

[0469] The method of any one of C99.C63-C92, wherein at least three capsular polysaccharides are conjugated to TT and the other capsular polysaccharides are conjugated to CRM 197 Conjugation.

[0470] The method of any one of C100.C63-C92, wherein three capsular polysaccharides are conjugated to TT and the other capsular polysaccharides are conjugated to CRM 197 Conjugation.

[0471] C101. The method of any one of C63-C92, wherein four capsular polysaccharides are conjugated to TT and the other capsular polysaccharides are conjugated to CRM 197 Conjugation.

[0472] C102. The method of any one of C1-C52, wherein the solution to which a preservative is added is a 21-valent pneumococcal conjugate composition, wherein the 21 saccharide conjugates are from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F, wherein four capsular polysaccharides are conjugated to TT, wherein the four capsular polysaccharides conjugated to TT are serotypes 15B and 22F, and the other two serotypes are selected from the group consisting of serotypes 1, 3 and 5, and the other saccharide conjugates are conjugated to CRM.197 Conjugation.

[0473] C103. The method of any one of C1-C52, wherein the solution to which a preservative is added is a 21-valent pneumococcal conjugate composition, wherein the 21 saccharide conjugates are from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F, wherein four capsular polysaccharides are conjugated to TT, and wherein the four capsular polysaccharides conjugated to TT are serotypes 1, 5, 15B and 22F, and the other saccharide conjugates are conjugated to CRM. 197 Conjugation.

[0474] C104. The method of any one of C1-C52, wherein the solution to which a preservative is added is a 21-valent pneumococcal conjugate composition, wherein the 21 saccharide conjugates are from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F, wherein four capsular polysaccharides are conjugated to TT, and wherein the four capsular polysaccharides conjugated to TT are serotypes 1, 3, 15B and 22F, and the other saccharide conjugates are conjugated to CRM. 197 Conjugation.

[0475] C105. The method of any one of C1-C52, wherein the solution to which a preservative is added is a 21-valent pneumococcal conjugate composition, wherein the 21 saccharide conjugates are from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F, wherein four capsular polysaccharides are conjugated to TT, and wherein the four capsular polysaccharides conjugated to TT are serotypes 3, 5, 15B and 22F, and the other saccharide conjugates are conjugated to CRM. 197 Conjugation.

[0476] C106. The method of any one of C1-C52, wherein the solution to which a preservative is added is a 21-valent pneumococcal conjugate composition, wherein the 21 saccharide conjugates are from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F, wherein two saccharide conjugates selected from serotypes 1, 3 and 5 are conjugated to TT, and the other saccharide conjugates are conjugated to CRM. 197 Conjugation.

[0477] C107. The method of any one of C1-C52, wherein the solution to which a preservative is added is a 21-valent pneumococcal conjugate composition, wherein the 21 saccharide conjugates are from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F, wherein the saccharide conjugates from serotypes 1 and 3 are conjugated to TT, and the other saccharide conjugates are conjugated to CRM. 197 Conjugation.

[0478] C108. The method of any one of C1-C52, wherein the solution to which a preservative is added is a 21-valent pneumococcal conjugate composition, wherein the 21 saccharide conjugates are from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F, wherein the saccharide conjugates from serotypes 1 and 5 are conjugated to TT, and the other saccharide conjugates are conjugated to CRM. 197 Conjugation.

[0479] C109. The method of any one of C1-C52, wherein the solution to which a preservative is added is a 21-valent pneumococcal conjugate composition, wherein the 21 saccharide conjugates are from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F, wherein the saccharide conjugates from serotypes 3 and 5 are conjugated to TT, and the other saccharide conjugates are conjugated to CRM. 197 Conjugation.

[0480] C110. The method of any one of C1-C52, wherein the solution to which the preservative is added is a 15-valent pneumococcal conjugate composition, wherein the 15 saccharide conjugates are from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F, wherein all saccharide conjugates are in combination with CRM 197 Conjugation.

[0481] C111. The method of any one of C1-C52, wherein the solution to which the preservative is added is a 20-valent pneumococcal conjugate composition, wherein the 20 saccharide conjugates are from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F, wherein all saccharide conjugates are in combination with CRM 197 Conjugation.

[0482] C112. The method of any one of C1-C111, wherein the solution to which the preservative is added comprises a pharmaceutically acceptable excipient, carrier or diluent.

[0483] C113. The method of any one of C1-C111, wherein the solution to which the preservative is added comprises one or more of a buffer, a salt, a divalent cation, a non-ionic detergent, a cryoprotectant (e.g., a sugar), and an antioxidant (e.g., a free radical scavenger or a chelating agent), or any multiple combinations thereof.

[0484] C114. The method of any one of C1-C111, wherein the solution to which the preservative is added comprises a buffer.

[0485] C115. The method of C114, wherein the buffer has a pKa of about 3.5 to about 7.5.

[0486] C116. The method of C114, wherein the buffer is phosphate, succinate, histidine or citrate.

[0487] C117. The method of C114, wherein the buffer is succinate.

[0488] C118. The method of C114, wherein the buffer is histidine.

[0489] C119. The method of C114, wherein the buffer is histidine at a concentration of 10 mM to 30 mM.

[0490] C120. The method of C114, wherein the buffer is histidine at a concentration of 15 mM to 25 mM.

[0491] C121. The method of C114, wherein the buffer is histidine at a concentration of about 22 mM.

[0492] C122. The method of C114, wherein the buffer is succinate at a concentration of 1 mM to 10 mM.

[0493] C123.The method of C114, wherein the buffer is succinate at a concentration of about 5.5 mM.

[0494] C124. The method of any one of C1-C123, wherein the solution to which the preservative is added comprises a salt.

[0495] C125. The method of C124, wherein the salt is magnesium chloride, potassium chloride, sodium chloride, or a combination thereof.

[0496] C126. The method of C124, wherein the salt is sodium chloride.

[0497] C127. The method of any one of C1-C123, wherein the solution to which the preservative is added comprises about 150 mM sodium chloride.

[0498] C128. The method of any one of C1-C127, wherein the solution to which the preservative is added comprises a surfactant.

[0499] The method of C129.C128, wherein the surfactant is polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, TRITON TM N-101, TRITON TM X-100, oxtoxynol 40, nonoxynol-9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate (PEG-15, Solutol H15), polyoxyethylene-35-ricinoleate ( EL), soy lecithin or poloxamer.

[0500] C130. The method of C129, wherein the surfactant is polysorbate 80 or polysorbate 20.

[0501] C131. The method of C129, wherein the surfactant is polysorbate 20.

[0502] C132. The method of C131, wherein the concentration of polysorbate 20 in the solution is 0.01% to 1% polysorbate 20 by weight (w / w).

[0503] C133. The method of C131, wherein the concentration of polysorbate 20 in the solution is 0.1% to 0.5% polysorbate 20 by weight (w / w).

[0504] C134. The method of C131, wherein the concentration of polysorbate 20 in the solution is about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5% or about 0.6% polysorbate 20 (w / w).

[0505] C135. The method of C131, wherein the concentration of polysorbate 20 in the solution is about 0.33% polysorbate 20 (w / w).

[0506] C136. The method of C129, wherein the surfactant is polysorbate 80.

[0507] C137. The method of C136, wherein the concentration of polysorbate 80 in the solution is 0.001% to 1% polysorbate 80 by weight (w / w).

[0508] C138. The method of C136, wherein the concentration of polysorbate 80 in the solution is 0.01% to 0.5% polysorbate 80 by weight (w / w).

[0509] C139. The method of C136, wherein the concentration of polysorbate 80 in the solution is about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05% or about 0.06% polysorbate 80 (w / w).

[0510] C140. The method of C136, wherein the concentration of polysorbate 80 in the solution is about 0.033% polysorbate 80 (w / w).

[0511] The method of C141.C136, wherein the final concentration of polysorbate 80 in the vaccine is between about 0.01% and about 0.03%.

[0512] C142. The method of C136, wherein the final concentration of polysorbate 80 in the vaccine is about 0.02%.

[0513] C143. The method of any one of claims C1-C142, wherein the solution to which the preservative is added has a pH between 5.5 and 7.5.

[0514] C144. The method of any of C1-C142, wherein the solution to which the preservative is added has a pH between 5.6 and 7.0.

[0515] C145. The method of any of C1-C142, wherein the solution to which the preservative is added has a pH between 5.8 and 6.0.

[0516] C146. The method of any of C1-C142, wherein the solution to which the preservative is added has a pH of about 5.8.

[0517] C147. The method of any of C1-C142, wherein the final pH in the vaccine is between about 5.3 and about 6.3.

[0518] C148. The method of any one of C1-C142, wherein the final pH in the vaccine is about 5.8.

[0519] C149. The method of any one of C1-C52, wherein the solution to which the preservative is added is a 20-valent pneumococcal conjugate composition, wherein the 20 saccharide conjugates are from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F, wherein all of the saccharide conjugates are conjugated to CRM197 The solution comprises about 5 μg / ml to about 10 μg / ml of each capsular polysaccharide serotype 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F, and about 10 μg / ml to about 20 μg / ml of capsular polysaccharide of serotype 6B, about 0.01% to about 0.05% (w / w) polysorbate 80, about 150 mM sodium chloride, and about 1 mM to about 10 mM succinate buffer pH 5.8.

[0520] C150. The method of any one of C1-C52, wherein the solution to which the preservative is added is a 15-valent pneumococcal conjugate composition, wherein the 15 saccharide conjugates are from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F, wherein the saccharide conjugates are all conjugated to CRM 197 Conjugation, the solution comprises about 5 μg / ml to about 10 μg / ml of each capsular polysaccharide serotypes 1, 3, 4, 5, 6A, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F and about 10 μg / ml to about 20 μg / ml of capsular polysaccharide of serotype 6B, about 150 mM sodium chloride, about 0.1% to about 0.5% (w / w) polysorbate 20, and about 15 mM to about 25 mM L-histidine buffer pH 5.8.

[0521] C151. The method of any one of C1-C52, wherein the solution to which the preservative is added is a 20-valent pneumococcal conjugate composition, wherein the 20 saccharide conjugates are from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F, wherein all of the saccharide conjugates are conjugated to CRM 197 The solution comprises about 7.5 μg / ml of each capsular polysaccharide serotype 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F and about 7.5 μg / ml of capsular polysaccharide serotype 6B, about 0.033% (w / w) polysorbate 80, about 150 mM sodium chloride and about 5.5 mM succinate buffer pH 5.8.

[0522] C152. The method of any of C1-C151, wherein the final protein concentration in the vaccine is between about 60 μg / ml and about 130 μg / ml.

[0523] C153. The method of any of C1-C151, wherein the final protein concentration in the vaccine is between about 90 μg / ml and about 99 μg / ml.

[0524] C154. The method of any of C1-C153, wherein the final D10 particle size distribution of the vaccine is between about 4,000 and about 5,000.

[0525] C155. The method of any of C1-C153, wherein the final D10 particle size distribution of the vaccine is between about 4,300 and about 4,700.

[0526] C156. The method of any of C1-C153, wherein the final D50 particle size distribution of the vaccine is between about 7,000 and about 8,000.

[0527] C157. The method of any of C1-C153, wherein the final D50 particle size distribution of the vaccine is between about 7,500 and about 7,900.

[0528] C158. The method of any of C1-C153, wherein the final D90 particle size distribution of the vaccine is between about 12,000 and about 15,000.

[0529] C159. The method of any of C1-C153, wherein the final D90 particle size distribution of the vaccine is between about 12,300 and about 14,900.

[0530] C160. The method of any one of C1-C159, wherein after step (a), the mixture comprising the preservative and the conjugate is sterile filtered.

[0531] C161. The method of C160, wherein the filter has a nominal retention range of about 0.05-0.2 μm.

[0532] C162. The method of C160, wherein the filter has a nominal retention range of about 0.1-0.2 μm.

[0533] C163. The method of C160, wherein the filter has a nominal retention range of about 0.15-0.2 μm.

[0534] C164. The method of C160, wherein the filter has a nominal retention range of about 0.1, about 0.15, or about 0.2 μm.

[0535] C165. The method of C160, wherein the filter has a nominal retention range of approximately 0.2 μm.

[0536] C166. The method of any one of C160-C165, wherein the filter comprises a pre-filter.

[0537] C167. The method of C166, wherein the pre-filter has a nominal retention range between about 0.1 μm and 1 μm.

[0538] C168. The method of C166, wherein the pre-filter has a nominal retention range of about 0.5 μm.

[0539] C169. The method of C166, wherein the pre-filter has a nominal retention range of about 0.5 μm and the filter has a nominal retention range of about 0.2 μm.

[0540] C170. The method of any one of C160-C169, wherein the filter has a filtration capacity of about 25-1000 L / m2, 50-1000 L / m2, 75-1000 L / m2, 100-1000 L / m2, 150-1000 L / m2, 200-1000 L / m2, 250-1000 L / m2, 300-1000 L / m2, 350-1000 L / m2, 400-1000 L / m2, 500-1000 L / m2 or 750-1000 L / m2.

[0541] C171. The method of any one of C160-C169, wherein the filter has a filtration capacity of 200-1000 L / m2, 250-1000 L / m2 or 300-1000 L / m2.

[0542] C172. The method of any one of C160-C169, wherein the filter has a filtration capacity of about 300-1000 L / m2.

[0543] C173. The method of any one of C160-C172, wherein the filter comprises polysulfone.

[0544] C174. The method of any one of C160-C172, wherein the filter comprises polyethersulfone (PES).

[0545] C175. The method of any one of C160-C174, wherein the filter is a capsule filter.

[0546] C176. The method of any one of C1-C175, wherein the combined vaccine obtained by the method comprises one or more conjugates and a preservative.

Claims

1. A method for producing a conjugate vaccine comprising a preservative, the method comprising the following steps: (a) adding a preservative from a bulk solution to a solution comprising one or more conjugates, a buffer, a salt and a surfactant; and (b) subsequently sterile filtering the mixture comprising the preservative, conjugate, buffer, salt and surfactant, wherein the preservative is not sterile filtered before being added to the solution, and wherein the preservative is hydrophobic and viscous at the bulk solution concentration.

2. The method of claim 1, further comprising the step (c) of subsequently adding an adjuvant.

3. The method of claim 2, wherein the adjuvant is aluminum phosphate.

4. The method of claim 3, wherein the final concentration of aluminum phosphate in the vaccine is about 0.25 mg / ml.

5. The method of any one of claims 1-4, wherein the viscosity of the preservative at the bulk solution concentration is from about 15 to about 25 centistokes at 25°C.

6. The method of any one of claims 1 to 5, wherein the preservative is 2-phenoxyethanol (2-PE), phenol, m-cresol, methylparaben, propylparaben or thimerosal.

7. The method of any one of claims 1 to 6, wherein the bulk solution is pure 2-phenoxyethanol (2-PE).

8. The method of any one of claims 1 to 7, wherein the preservative is 2-PE and the concentration of 2-PE in the solution after step (a) is from about 15 mg / ml to about 17 mg / ml.

9. The method of any one of claims 1-8, wherein the final concentration of 2-PE in the vaccine is about 9 mg / ml or about 10 mg / ml.

10. The method of any one of claims 1-9, wherein the buffer comprises succinate, the salt comprises sodium chloride, and the surfactant comprises polysorbate 80.

11. The method of claim 10, wherein: (a) the concentration of succinate is between about 1 mM and about 10 mM; (b) a sodium chloride concentration of between about 100 mM and about 200 mM; and (c) Polysorbate 80 is present in a concentration between about 0.01% and about 0.05%.

12. The method of any one of claims 1-11, wherein the solution to which the preservative is added has a pH of about 5.8 to about 6.

0.

13. The method of any one of claims 1-12, wherein the preservative is added at an addition rate of about 0.5 ml / min / L to about 5.0 ml / min / L of the solution comprising one or more conjugates, buffer, salt, and surfactant.

14. The method of any one of claims 1 to 13, wherein the preservative is added using a peristaltic pump.

15. The method of any one of claims 1 to 14, wherein the preservative is added under continuous mixing and there is no delay between adding the preservative and starting mixing, and wherein: (a) mixing at an angular speed of about 150 rpm to about 200 rpm; or (b) After adding the preservative and prior to sterile filtration, the solution is mixed for about 30 minutes to about 3 hours.

16. The method of any one of claims 1-15, wherein the filtering of step (b) comprises using a filter having a nominal retention range of about 0.1 μm, about 0.15 μm, or about 0.2 μm.

17. The method of claim 16, wherein the filter comprises a pre-filter having a nominal retention range of about 0.5 μm.

18. The method of any one of claims 1 to 17, wherein the filtration of step (b) comprises using a filtration apparatus having a flow rate of 200 to 1000 L / m 2 、250-1000L / m 2 or 300-1000L / m 2 The filter has a filtering capacity of 100%.

19. The method of any one of claims 1 to 18, wherein the filtering of step (b) comprises using a filter comprising polyethersulfone (PES).

20. The method of any one of claims 1 to 19, wherein the solution to which a preservative is added is a pneumococcal conjugate composition comprising 7 to 25 saccharide conjugates from different S. pneumoniae serotypes.

21. The method of any one of claims 1 to 19, wherein the solution to which a preservative is added is a 20-valent pneumococcal saccharide conjugate composition, wherein the 20 saccharide conjugates are from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F.

22. The method of claim 20 or 21, wherein all glycoconjugates are conjugated to CRM 197 Conjugation.

23. The method of any one of claims 1 to 19, wherein the solution to which a preservative is added is a 20-valent pneumococcal conjugate composition, wherein the 20 saccharide conjugates are from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F, wherein all of the saccharide conjugates are conjugated to CRMs. 197 The solution comprises about 7.5 μg / ml of each capsular polysaccharide serotype 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F and about 7.5 μg / ml of capsular polysaccharide serotype 6B, about 0.033% (w / w) polysorbate 80, about 150 mM sodium chloride and about 5.5 mM succinate buffer pH 5.8.

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