Process for producing a vaccine formulation using a preservative

Abstract

The present invention relates to a process for producing a conjugate vaccine comprising a preservative. The present invention relates in particular to a process for producing a conjugate vaccine in which the preservative is hydrophobic and viscous (e.g. 2-phenoxyethanol (2-PE)).

Description

Technical Field

[0001] The present invention relates to a process for preparing a conjugate vaccine comprising a preservative. The present invention relates in particular to a process for preparing a conjugate vaccine in which the preservative is hydrophobic and viscous (e.g., 2-phenoxyethanol (2-PE)).

Background Art

[0002] Bacterial cell surface polysaccharides, particularly capsular polysaccharides, are becoming increasingly important as therapeutic agents. In general, cell surface polysaccharides are associated with the induction of an immune response in vivo.

[0003] Polysaccharides are immunogenic per se, but glycoconjugation (glycoconjugate) of polysaccharides with protein carriers has been used to improve immunogenicity, particularly for infants and the elderly. Glycoconjugate vaccines are generally obtained by covalently linking poorly immunogenic sugar antigens to protein carriers and play an important role in the prevention of many life-threatening infectious diseases. In the preparation of conjugate vaccines, a selected bacterial strain is grown until it supplies the polysaccharides required for vaccine production. Cell growth is often carried out in a fermenter and lysis is induced at the end of fermentation. The lysate is collected for downstream purification and recovery of the capsular polysaccharides surrounding the bacterial cells. The polysaccharides are included in the final vaccine formulation after conjugation with the carrier protein, conferring immunity to the bacteria on the target population of the vaccine.

[0004] Diseases caused by Streptococcus pneumoniae, also known as pneumococcal diseases, are one of the more important ones worldwide caused by bacterial pathogens. Pneumococcal diseases are a complex group of diseases, including invasive infections such as bacteremia / sepsis, meningitis, pneumonia, and otitis media that affect both children and adults. Prevnar 13 (also known as "Prevenar 13" and referred to as "Prev(e)nar 13" herein) is a preparation in which polysaccharides derived from 13 pneumococcal serotypes (1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F)) are individually conjugated with CRM 197 (Cross Reactive Material derived from a mutant strain of Corynebacterium diphtheriae). See, for example, WO2006 / 110381; WO2008 / 079653; WO2008 / 079732; WO2008 / 143709 and WO2011 / 151760.

[0005] The need to add preservatives to vaccines can be reduced or eliminated by manufacturing and using only single-dose vaccine formulations. However, using single-dose preservative-free formulations increases the overall cost of vaccination and, particularly in developing countries, jeopardizes the effectiveness of immunization programs. Furthermore, removing preservatives from multi-dose vials is generally not considered a preferred option, especially in countries where cold storage is limited and health care standards are not optimal (Drain et al., Bull World Health Organ 81(10):726 - 731 (2003)). Therefore, although multi-dose vials are considered most appropriate for low-cost vaccine production, it is desirable to formulate multi-dose vaccines with at least one preservative to protect subjects from microorganisms inadvertently introduced into the vaccine during multiple uses or after one or more non-sterile events.

[0006] WO2011 / 151760 discloses a multivalent immunogenic composition comprising multiple capsular polysaccharides derived from Streptococcus pneumoniae serotypes and 2-phenoxyethanol (2-PE).

[0007] The inventors have found that formulating conjugate vaccines using preservatives such as 2-PE presents significant problems, especially on a large scale. In the prior art, 2-PE is added at the final stage of vaccine formulation (for pharmaceutical products, see, for example, WO00 / 62801, WO00 / 56360 or Khandke L. et al., Vaccine 29 (2011) 7144-7153). However, when adding 2-PE at the final stage, it is necessary to filter-sterilize 2-PE at a relatively high concentration. It has been found that filtering and sterilizing 2-PE at such a high concentration raises problems regarding wetting of the filter and filter integrity prior to use.

Summary of the Invention

Problems to be Solved by the Invention

[0008] Therefore, there is a need for an improved process for formulating multivalent conjugate vaccines containing preservatives such as 2-PE. In particular, a process with a shorter time and fewer steps is desirable.

Means for Solving the Problems

[0009] In one aspect, the present invention provides a process for preparing a conjugate vaccine containing a preservative, comprising: (a) adding the preservative from a bulk solution to a solution containing one or more conjugates without filter-sterilizing the preservative; and (b) subsequently subjecting the mixture containing the preservative and the conjugate to sterile filtration. The process is provided.

[0010] In one aspect, the process further includes the step of (c) subsequently adding an adjuvant.

[0011] In one aspect, the preservative is hydrophobic and viscous at the concentration of the bulk solution.

[0012] The bulk solution is preferably neat 2-PE. **DETAILED DESCRIPTION OF THE INVENTION**

[0013] The technique used to add a preservative to the composite vaccine formulation consists of adding the preservative at the final stage of the formulation process. By adding the preservative as the final stage of the process, the risk of potential effects of the interaction between the preservative and other components (such as antigens) of the vaccine is minimized, and the flexibility of the process is enabled.

[0014] A preservative is defined as a compound added to a vaccine to kill microorganisms, particularly bacteria and fungi, or to prevent their growth. The preservative is added to the vaccine formulation to prevent the growth of microorganisms in the event of accidental contamination of the vaccine that may occur by repeatedly piercing the needle into a multi-dose vial.

[0015] The inventors have found that adding a preservative, particularly a hydrophobic and viscous preservative, as one of the final steps of the process can be particularly difficult on a large scale. Due to the hydrophobicity and high viscosity of some preservatives (such as 2-PE), it has been found that filtration by sterile filtration of the preservative at the required concentration, which is a necessary step when adding the preservative at the end of the formulation process, is difficult. To completely and consistently wet a sterile filtration filter (i.e., a filter with a nominal pore size of about 0.2 μm) with the preservative at the required concentration, extremely high pressure is required, and it has been found that thereby the microbial retention properties of the membrane are impaired. If such high pressure is not used, wetting of the filter with the preservative at the required concentration can be incomplete and inconsistent, which means that the wetting step may be unsuccessful and special tests and potential waste of materials are required to ensure proper wetting. Thus, such tests are time-consuming and may cause waste.

[0016] Therefore, there is a need for an improved process for formulating multivalent conjugate vaccines containing preservatives such as 2-PE. In particular, the process should be less time-consuming (a preparation with a reduced number of steps is desirable).

[0017] Surprisingly, it has been found that there are advantages to adding the preservative at an early stage of the process. In such a process, since the preservative is added at an early stage (before final dilution), the preservative is present at a higher concentration than the concentration present in the final pharmaceutical product. Surprisingly, adding the preservative at an early stage of the process, particularly before sterile filtration, is necessary, possible, and also enables avoidance of the problems associated with sterile filtration of a high-concentration hydrophobic and viscous preservative since only the diluted solution needs to be sterile filtered downstream. Such a process makes it possible to add the preservative without sterile filtration. It has been found that there are problems with sterile filtration of hydrophobic and viscous preservatives (particularly with regard to 2-PE).

[0018] Accordingly, the present invention relates to a process for producing a conjugate vaccine comprising a preservative, (a) adding the preservative from a bulk solution to a solution comprising one or more conjugates, the step of adding the preservative without filter sterilization, (b) subsequently, sterile filtering the mixture comprising the preservative and the conjugate relates to a process comprising.

[0019] In certain embodiments, an adjuvant is further added after the sterile filtration step (b). Accordingly, in certain embodiments, the present invention relates to a process for producing a conjugate vaccine comprising a preservative, (a) adding the preservative from a bulk solution to a solution comprising one or more conjugates, the step of adding the preservative without filter sterilization, (b) subsequently, sterile filtering the mixture comprising the preservative and the conjugate, (c) subsequently, adding an adjuvant to the mixture relates to a process comprising.

[0020] The preservative used in the present invention is preferably hydrophobic and viscous at the concentration of the bulk solution. In certain embodiments, the viscosity of the preservative at the concentration of the bulk solution 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.

[0021] In certain embodiments, the viscosity of the preservative used in the present invention is between about 10 centistokes and about 50 centistokes at 25°C. The viscosity of the preservative used in the present invention is preferably between about 15 centistokes and about 25 centistokes at 25°C.

[0022] In one embodiment, the preservative is 2-phenoxyethanol (2-PE), phenol, metacresol, methylparaben, propylparaben, or thimerosal. In a preferred embodiment, the preservative is 2-phenoxyethanol (2-PE) or thimerosal.

[0023] In one embodiment, the preservative is added without dilution. Thus, in one embodiment, the preservative is added without dilution (i.e., the bulk solution is the undiluted preservative). In one embodiment, thus the bulk solution is a pure undiluted preservative (where "pure" refers to pharmaceutical grade).

[0024] 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 pure (pharmaceutical grade without dilution). Thus, in one embodiment, 2-PE is added without dilution (i.e., the bulk solution is undiluted 2-PE). In one embodiment, thus the bulk solution is undiluted 2-PE.

[0025] In one embodiment, the bulk solution is undiluted 2-P, and 2-PE is diluted between about 10-fold and about 200-fold in the solution after step (a). In another embodiment, the bulk solution is undiluted 2-PE, and 2-PE is diluted between about 10-fold and about 100-fold in the solution after step (a). In another embodiment, the bulk solution is undiluted 2-PE, and 2-PE is diluted about 50-fold, about 60-fold, about 70-fold, or about 80-fold in the solution after step (a). In a preferred embodiment, the bulk solution is undiluted 2-PE, and 2-PE is diluted between about 73-fold and 74-fold in the solution after step (a).

[0026] In certain embodiments, the concentration of 2-PE in the solution after step (a) is between about 1 mg / ml and about 25 mg / ml. The concentration of 2-PE in the solution after step (a) is between about 10 mg / ml and about 20 mg / ml. Even more preferably, the concentration of 2-PE in the solution after step (a) is between about 15 mg / ml and about 17 mg / ml.

[0027] In certain embodiments, the concentration of 2-PE in the solution after step (a) is about 15 mg / ml.

[0028] In certain embodiments, the concentration of 2-PE in the solution after step (a) is about 17 mg / ml.

[0029] In certain embodiments, 2-PE is added at a rate between about 0.5 ml / min per liter of solution containing one or more conjugates and about 5.0 ml / min per liter of solution containing one or more conjugates. In preferred embodiments, 2-PE is added without dilution.

[0030] In certain embodiments, 2-PE is added at a rate between about 1 ml / min per liter of solution containing one or more conjugates and about 4.0 ml / min per liter of solution containing one or more conjugates. In preferred embodiments, 2-PE is added without dilution (undiluted 2-PE).

[0031] In certain embodiments, undiluted 2-PE is added using a pump, preferably a peristaltic pump. In certain embodiments, undiluted 2-PE is added using a peristaltic pump using a thermoplastic elastomer tube. It is preferred that undiluted 2-PE is added using a peristaltic pump using a thermoplastic elastomer tube.

[0032] In certain embodiments, 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.

[0033] In one embodiment, the final concentration of 2-PE in the vaccine is about 10 mg / ml.

[0034] In a preferred embodiment, the final concentration of 2-PE in the vaccine is about 9 mg / ml.

[0035] 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.

[0036] The preservative is preferably added under continuous mixing. In a preferred embodiment, there is no delay between the addition of the preservative and the start of mixing.

[0037] In one embodiment, the preservative is added under continuous mixing and the angular velocity is between about 50 rpm and about 500 rpm. The preservative is preferably added under continuous mixing and the angular velocity is between about 100 rpm and about 400 rpm. More preferably, the preservative is added under continuous mixing and the angular velocity is between about 150 rpm and about 300 rpm.

[0038] Most preferably, the preservative is added under continuous mixing and the angular velocity is between about 150 rpm and about 200 rpm.

[0039] In one embodiment, the preservative is added under continuous mixing and the angular velocity is about 150 rpm.

[0040] In one embodiment, the preservative is added under continuous mixing and the angular velocity is about 200 rpm.

[0041] In certain embodiments, the solution is mixed for about 15 minutes to about 5 hours after the addition of the preservative and before filter sterilization. It is preferred that the solution be mixed for about 30 minutes to about 3 hours after the addition of the preservative and before filter sterilization. Even more preferably, the solution is mixed for about 1 hour to about 3 hours after the addition of the preservative and before filter sterilization.

[0042] In certain embodiments, the solution is mixed for about 2 hours after the addition of the preservative.

[0043] In certain embodiments, the solution is mixed at an angular velocity between about 50 rpm and about 500 rpm after the addition of the preservative and before filter sterilization. Between about 100 rpm and about 400 rpm is preferred. Between about 150 rpm and about 300 rpm is even more preferred.

[0044] A solution containing one or more conjugates of step (a) The solution to which the preservative is added contains one or more conjugates and may contain other components.

[0045] For the purposes of the present invention, the term "conjugate" or "saccharide conjugate" refers to a bacterial capsular polysaccharide linked to a carrier protein. In one embodiment, the capsular polysaccharide is linked directly to the carrier protein. In a second embodiment, the capsular polysaccharide is linked to the protein through a spacer / linker.

[0046] In certain embodiments, the solution to which the preservative is added is a monovalent composition. In such embodiments, the solution contains only one conjugate. In such embodiments, the conjugate may be a conjugated Haemophilus influenzae type b (Hib) capsular polysaccharide. In another embodiment, the conjugate is a conjugated Neisseria meningitidis serogroup C capsular polysaccharide (MenC). In yet another embodiment, the conjugate is a conjugated Neisseria meningitidis serogroup Y capsular polysaccharide (MenY).

[0047] In one embodiment, the solution to which the preservative is added is a divalent, trivalent, tetravalent, pentavalent or hexavalent composition.

[0048] In one embodiment, the trivalent composition comprises three conjugated Streptococcus agalactiae capsular polysaccharides. Streptococcus agalactiae is also known as "Group B streptococcus" or simply "GBS". In one embodiment, the three conjugates consist of conjugated GBS serotypes Ia, Ib and III capsular polysaccharides. In a preferred embodiment, the capsular polysaccharide is CRM 197 conjugated.

[0049] In one embodiment, the hexavalent composition comprises six conjugated Streptococcus agalactiae capsular polysaccharides. In one embodiment, the six conjugates consist of conjugated GBS serotypes Ia, Ib, II, III, IV and V capsular polysaccharides. In a preferred embodiment, the capsular polysaccharide is CRM 197 conjugated.

[0050] In one embodiment, the tetravalent composition comprises four conjugated Neisseria meningitidis capsular polysaccharides. In one embodiment, the four conjugates consist of conjugated Neisseria meningitidis serogroup A capsular polysaccharide (MenA), conjugated Neisseria meningitidis serogroup W135 capsular polysaccharide (MenW135), conjugated Neisseria meningitidis serogroup Y capsular polysaccharide (MenY), and conjugated Neisseria meningitidis serogroup C capsular polysaccharide (MenC).

[0051] The solution to which the preservative is added is preferably a multivalent composition containing seven or more conjugates. The solution is preferably a 7- to 25-valent composition (i.e., containing 7 to 25 conjugates). The multivalent solution is even more preferably a 13- to 25-valent composition.

[0052] In one embodiment, the solution to which the preservative is added is a 15-valent composition.

[0053] In one embodiment, the solution to which the preservative is added is a 16-valent composition.

[0054] In one embodiment, the solution to which the preservative is added is a 20-valent composition.

[0055] In one embodiment, the solution to which the preservative is added is a 21-valent composition.

[0056] The solution to which the preservative is added is preferably a multivalent pneumococcal conjugate composition.

[0057] In one embodiment, the solution to which the preservative is added is a pneumococcal conjugate composition containing 7 to 25 sugar conjugates (7 to 25 pneumococcal conjugates) derived from different serotypes of Streptococcus pneumoniae (S. pneumoniae).

[0058] In one embodiment, the solution to which the preservative is added is a pneumococcal conjugate composition containing conjugates derived from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 different serotypes of Streptococcus pneumoniae (S. pneumoniae).

[0059] In one embodiment, the solution to which the preservative is added is a pneumococcal conjugate composition containing conjugates derived from 20 different serotypes of Streptococcus pneumoniae (S. pneumoniae).

[0060] In one embodiment, the solution to which the preservative is added is a heptavalent, octavalent, nonavalent, decavalent, undecavalent, dodecavalent, tridecavalent, tetradecavalent, pentadecavalent, hexadecavalent, heptadecavalent, octadecavalent, nonadecavalent or icosavalent pneumococcal conjugate composition.

[0061] In one embodiment, the solution to which the preservative is added is a 15-valent pneumococcal conjugate composition.

[0062] In one embodiment, the solution to which the preservative is added is a 20-valent pneumococcal conjugate composition.

[0063] In one embodiment, the solution to which the preservative is added is a 21-valent pneumococcal conjugate composition.

[0064] In one embodiment, the solution to which the preservative is added is a 22-valent pneumococcal conjugate composition.

[0065] In one embodiment, the solution to which the preservative is added is a 21-valent, 22-valent, 23-valent, 24-valent or 25-valent pneumococcal conjugate composition.

[0066] In one embodiment, the solution to which the preservative is added contains saccharide conjugates derived from Streptococcus pneumoniae (S. pneumoniae) serotypes 4, 6B, 9V, 14, 18C, 19F and 23F.

[0067] In one embodiment, the solution to which the preservative is added contains saccharide conjugates derived from Streptococcus pneumoniae (S. pneumoniae) serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F.

[0068] In one embodiment, the solution to which the preservative is added contains saccharide conjugates derived from Streptococcus pneumoniae (S. pneumoniae) serotypes 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F.

[0069] In certain embodiments, the solution to which the preservative is added comprises sugar conjugates derived from Streptococcus pneumoniae (S. pneumoniae) serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F.

[0070] In certain embodiments, the solution to which the preservative is added comprises sugar conjugates derived from Streptococcus pneumoniae (S. pneumoniae) serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, and 33F.

[0071] In certain embodiments, the solution to which the preservative is added comprises sugar conjugates derived from Streptococcus pneumoniae (S. pneumoniae) serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F.

[0072] In certain embodiments, the solution to which the preservative is added comprises sugar conjugates derived from Streptococcus pneumoniae (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.

[0073] In certain embodiments, the solution to which the preservative is added comprises sugar conjugates derived from Streptococcus pneumoniae (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.

[0074] In certain embodiments, the solution to which the preservative is added is a 10-valent pneumococcal conjugate composition, and the above 10 sugar conjugates are derived from Streptococcus pneumoniae (S. pneumoniae) serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, and 23F.

[0075] In one embodiment, the solution to which the preservative is added is a 13-valent pneumococcal conjugate composition, and the above 13 sugar conjugates are derived from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F.

[0076] In one embodiment, the solution to which the preservative is added is a 15-valent pneumococcal conjugate composition, and the above 15 sugar conjugates are derived from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, and 33F.

[0077] In one embodiment, the solution to which the preservative is added is a 20-valent pneumococcal conjugate composition, and the above 20 sugar conjugates are derived 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.

[0078] In one embodiment, the solution to which the preservative is added is a 21-valent pneumococcal conjugate composition, and the above 21 sugar conjugates are derived 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.

[0079] In one embodiment, the solution to which the preservative is added is a 21-valent pneumococcal conjugate composition, and the above 21 sugar conjugates are derived 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.

[0080] In certain embodiments, the solution to which the preservative is added is a 22-valent pneumococcal conjugate composition, and the above 22 sugar conjugates are derived 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.

[0081] In certain embodiments, the solution to which the preservative is added is a 21-valent pneumococcal conjugate composition, and the above 21 sugar conjugates are derived 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.

[0082] In certain embodiments, the solution to which the preservative is added is a 21-valent pneumococcal conjugate composition, and the above 21 sugar conjugates are derived 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.

[0083] In certain embodiments, the solution to which the preservative is added is a 21-valent pneumococcal conjugate composition, and the above 21 sugar conjugates are derived 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.

[0084] In certain embodiments, the solution to which the preservative is added is a 21-valent pneumococcal conjugate composition, and the above 21 sugar conjugates are derived 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.

[0085] In one embodiment, the solution to which the preservative is added is a 21-valent pneumococcal conjugate composition, and the above 21 sugar conjugates are derived 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.

[0086] In a preferred embodiment, the solution to which the preservative is added is a 20-valent pneumococcal conjugate composition, and the above 20 sugar conjugates are derived 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.

[0087] One component of the sugar conjugate is a carrier protein conjugated to the sugar. The terms "protein carrier" or "carrier protein" or "carrier" may be used interchangeably herein. The carrier protein should be one to which standard conjugation procedures are applicable. In one embodiment, the carrier protein of the sugar conjugate of the present invention is DT (diphtheria toxoid), TT (tetanus toxoid), fragment C of TT, CRM 197 Detoxified neuraminidase, PhtA, PhtB, PhtD, PhtE, PhtDE fusion, PhtBE fusion, OMPC, Por), PD (Haemophilus influenzae protein D) or C5a peptidase (SCP) derived from Streptococcus. In one embodiment, the carrier protein of the sugar conjugate of the present invention is DT (diphtheria toxoid). In another embodiment, the carrier protein of the sugar conjugate of the present invention is TT (tetanus toxoid). In another embodiment, the carrier protein of the sugar conjugate of the present invention is PD (Haemophilus influenzae (H. influenzae) protein D; see, for example, EP0594610B).

[0088] In a preferred embodiment, the carrier protein of the glycoconjugate of the present invention is TT, CRM 197 or C5a peptidase (SCP) derived from Streptococcus. In a more preferred embodiment, the carrier protein of the glycoconjugate of the present invention is CRM 197 or C5a peptidase (SCP) derived from Streptococcus.

[0089] In a highly preferred embodiment, the carrier protein of the glycoconjugate of the present invention is CRM 197 In any embodiment of the above compositions, all of the capsular polysaccharides are individually conjugated to CRM 197

[0090] In one embodiment, the capsular polysaccharides derived from any of Streptococcus pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14 and / or 23F of the above compositions are individually conjugated to PD, the capsular polysaccharide derived from Streptococcus pneumoniae serotype 18C is conjugated to TT, and the capsular polysaccharide derived from Streptococcus pneumoniae serotype 19F is conjugated to DT.

[0091] In any embodiment of the above compositions, at least one capsular polysaccharide is conjugated to TT and all other capsular polysaccharides are conjugated to CRM 197

[0092] In any embodiment of the above compositions, one capsular polysaccharide is conjugated to TT and all other capsular polysaccharides are conjugated to CRM 197

[0093] In any embodiment of the above compositions, at least two capsular polysaccharides are conjugated to TT and all other capsular polysaccharides are conjugated to CRM 197

[0094] ​​​​ In some embodiments of any of the above compositions, two capsular polysaccharides are conjugated to TT, and all other capsular polysaccharides are CRM 197 conjugated thereto.

[0095] In some embodiments of any of the above compositions, at least three capsular polysaccharides are conjugated to TT, and all other capsular polysaccharides are CRM 197 conjugated thereto.

[0096] In some embodiments of any of the above compositions, three capsular polysaccharides are conjugated to TT, and all other capsular polysaccharides are CRM 197 conjugated thereto.

[0097] In some embodiments of any of the above compositions, four capsular polysaccharides are conjugated to TT, and all other capsular polysaccharides are CRM 197 conjugated thereto.

[0098] In one embodiment, the solution to which the preservative is added is a 21-valent pneumococcal conjugate composition, and the above 21 sugar conjugates are derived 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. Four capsular polysaccharides are conjugated to TT, and the four capsular polysaccharides conjugated to TT are serotype 15B, 22F and two other serotypes selected from the group consisting of serotypes 1, 3 and 5. All other sugar conjugates are conjugated to CRM 197 thereto.

[0099] In one embodiment, the solution to which the preservative is added is a 21-valent pneumococcal conjugate composition. The above 21 sugar conjugates are derived 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. Four capsular polysaccharides are conjugated with TT, and the four capsular polysaccharides conjugated with TT are serotypes 1, 5, 15B, and 22F. All other sugar conjugates are conjugated with CRM 197 and conjugated ones.

[0100] In one embodiment, the solution to which the preservative is added is a 21-valent pneumococcal conjugate composition. The above 21 sugar conjugates are derived 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. Four capsular polysaccharides are conjugated with TT, and the four capsular polysaccharides conjugated with TT are serotypes 1, 3, 15B, and 22F. All other sugar conjugates are conjugated with CRM 197 and conjugated ones.

[0101] In one embodiment, the solution to which the preservative is added is a 21-valent pneumococcal conjugate composition. The above 21 sugar conjugates are derived 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. Four capsular polysaccharides are conjugated with TT, and the four capsular polysaccharides conjugated with TT are serotypes 3, 5, 15B, and 22F. All other sugar conjugates are conjugated with CRM 197 and conjugated ones.

[0102] In one embodiment, the solution to which the preservative is added is a 21-valent pneumococcal conjugate composition, and the above 21 sugar conjugates are derived 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. Two sugar conjugates selected from serotypes 1, 3 and 5 are conjugated with TT, and all other sugar conjugates are conjugated with CRM 197 and conjugated.

[0103] In one embodiment, the solution to which the preservative is added is a 21-valent pneumococcal conjugate composition, and the above 21 sugar conjugates are derived 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. The sugar conjugates derived from serotypes 1 and 3 are conjugated with TT, and all other sugar conjugates are conjugated with CRM 197 and conjugated.

[0104] In one embodiment, the solution to which the preservative is added is a 21-valent pneumococcal conjugate composition, and the above 21 sugar conjugates are derived 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. The sugar conjugates derived from serotypes 1 and 5 are conjugated with TT, and all other sugar conjugates are conjugated with CRM 197 and conjugated.

[0105] In one embodiment, the solution to which the preservative is added is a 21-valent pneumococcal conjugate composition, and the above 21 sugar conjugates are derived 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. The sugar conjugates derived from serotypes 3 and 5 are conjugated with TT, and all other sugar conjugates are conjugated with CRM 197 thereof.

[0106] In one embodiment, the solution to which the preservative is added is a 15-valent pneumococcal conjugate composition, and the above 15 sugar conjugates are derived from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, and 33F. All of the sugar conjugates are conjugated with CRM 197 thereof.

[0107] In the most preferred embodiment, the solution to which the preservative is added is a 20-valent pneumococcal conjugate composition, and the above 20 sugar conjugates are derived 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. All of the sugar conjugates are conjugated with CRM 197 thereof.

[0108] In one embodiment, the solution to which the preservative is added comprises any combination of the sugar conjugates disclosed herein, and a pharmaceutically acceptable excipient, carrier, or diluent.

[0109] The solution to which the preservative is added may contain, in addition to the conjugate(s), a buffer, a salt, a divalent cation, a nonionic surfactant, a cryoprotective substance such as a sugar, and an antioxidant such as a free radical scavenger or a chelating agent, or one or more of any plurality of combinations thereof.

[0110] In certain embodiments, the solution to which the preservative is added contains a buffer. In certain embodiments, the pKa of the buffer is from about 3.5 to about 7.5. In some embodiments, the buffer is a phosphate buffer, a succinate buffer, a histidine buffer, or a citrate buffer. In some embodiments, the buffer is a succinate buffer. In a preferred embodiment, the buffer is a histidine buffer.

[0111] In certain embodiments, the buffer is a histidine buffer at a concentration of 10 mM to 30 mM. In certain embodiments, the buffer is a histidine buffer at a concentration of 15 mM to 25 mM. In one particular embodiment, the concentration of the histidine buffer is about 22 mM.

[0112] In certain embodiments, the buffer is a succinate buffer at a concentration of 1 mM to 10 mM. In one particular embodiment, the concentration of the succinate buffer is about 5.5 mM.

[0113] In certain embodiments, the solution to which the preservative is added contains a salt. In some embodiments, the salt is magnesium chloride, potassium chloride, sodium chloride, or a combination thereof. In one particular embodiment, the salt is sodium chloride. In one particular embodiment, the solution to which the preservative is added contains about 150 mM of sodium chloride.

[0114] In certain embodiments, the solution to which the preservative is added contains a surfactant. In certain embodiments, the surfactant is polysorbate 20 (TWEEN™ 20), polysorbate 40 (TWEEN™ 40), polysorbate 60 (TWEEN™ 60), polysorbate 65 (TWEEN™ 65), polysorbate 80 (TWEEN™ 80), polysorbate 85 (TWEEN™ 85), TRITON™ N-101, TRITON™ X-100, octoxynol 40, nonoxynol-9, triethanolamine, triethanolamine oleate polypeptide, polyoxyethylene-660 hydroxystearate (PEG-15, Solutol H15), polyoxyethylene-35-ricinoleic acid (CREMOPHOR® EL), soy lecithin or poloxamer.

[0115] In a preferred embodiment, the surfactant is polysorbate 80 or polysorbate 20.

[0116] In a particular embodiment, the surfactant is polysorbate 20. The concentration of polysorbate 20 in the solution is preferably between 0.01% and 1% polysorbate 20 weight by weight (w / w). The concentration of polysorbate 20 in the solution is more preferably between 0.1% and 0.5% polysorbate 20 weight by weight (w / w).

[0117] 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 the most preferred embodiment, the concentration of polysorbate 20 in the solution is about 0.33% polysorbate 20 (w / w).

[0118] In a preferred embodiment, the surfactant is polysorbate 80. The concentration of polysorbate 80 in the solution is preferably between 0.001% and 1% polysorbate 80 weight by weight (w / w). More preferably, the concentration of polysorbate 80 in the solution is between 0.01% and 0.5% polysorbate 80 weight by weight (w / w).

[0119] 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 the most preferred embodiment, the concentration of polysorbate 80 in the solution is about 0.033% polysorbate 80 (w / w).

[0120] In certain embodiments, the pH of the solution to which the preservative is added is between 5.5 and 7.5, more preferably between pH 5.6 and 7.0, even more preferably between pH 5.8 and 6.0. In a preferred embodiment, the pH of the solution to which the preservative is added is about 5.8.

[0121] The concentration of a particular sugar conjugate can be calculated based on the amount of polysaccharide for that conjugate. The sugar concentration can be determined by an uronic acid assay.

[0122] In one embodiment, the solution to which the preservative is added contains between about 2 μg / ml and about 20 μg / ml of polysaccharide for sugar conjugates derived from Streptococcus pneumoniae (S. pneumoniae) serotypes 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. Between about 5 μg / ml and about 10 μg / ml is preferred.

[0123] In certain embodiments, the solution to which the preservative is added contains about 7.5 μg / ml of polysaccharide for sugar conjugates derived from Streptococcus pneumoniae (S. pneumoniae) serotypes 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.

[0124] In certain embodiments, the solution to which the preservative is added contains from about 4 μg / ml to about 40 μg / ml of polysaccharide for sugar conjugates derived from Streptococcus pneumoniae (S. pneumoniae) serotype 6B. Between about 10 μg / ml and about 20 μg / ml is preferred.

[0125] In certain embodiments, the solution to which the preservative is added contains about 15 μg / ml of polysaccharide for sugar conjugates derived from Streptococcus pneumoniae (S. pneumoniae) serotype 6B.

[0126] In certain embodiments, the solution to which the preservative is added is a 20-valent pneumococcal conjugate composition, and the above 20 sugar conjugates are derived from Streptococcus pneumoniae (S. pneumoniae) serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F. The sugar conjugates are all conjugated with CRM 197 and the solution to which the preservative is added contains, respectively, capsular polysaccharides of serotypes 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F between about 5 μg / ml and about 10 μg / ml and capsular polysaccharide of serotype 6B between about 10 μg / ml and about 20 μg / ml, about 0.01% to about 0.05% (w / w) of polysorbate 80, about 150 mM of sodium chloride, and about 1 mM to about 10 mM of succinic acid buffer, pH 5.8.

[0127] In one embodiment, the solution to which the preservative is added is a 15-valent pneumococcal conjugate composition, and the above 15 sugar conjugates are derived from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, and 33F, and all the sugar conjugates are conjugated with CRM 197 The solution to which the preservative is added contains capsular polysaccharides of serotypes 1, 3, 4, 5, 6A, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, and 33F each at about 5 μg / ml to about 10 μg / ml, capsular polysaccharide of serotype 6B at about 10 μg / ml to about 20 μg / ml, about 150 mM sodium chloride, polysorbate 20 at about 0.1% to about 0.5% (w / w), and L-histidine buffer at about 15 mM to about 25 mM, pH 5.8.

[0128] In a preferred embodiment, the solution to which the preservative is added is a 20-valent pneumococcal conjugate composition, and the above 20 sugar conjugates are derived 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, and all the sugar conjugates are conjugated with CRM 197 The solution to which the preservative is added contains capsular polysaccharides of serotypes 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F each at about 7.5 μg / ml and capsular polysaccharide of serotype 6B at about 7.5 μg / ml, about 0.033% (w / w) polysorbate 80, about 150 mM sodium chloride, and succinic acid buffer at about 5.5 mM, pH 5.8.

[0129] Filter sterilization After step (a), the mixture containing the preservative and the conjugate is sterile filtered.

[0130] In one embodiment, the nominal retention range of the filter is from about 0.05 to 0.2 μm or from about 0.1 to 0.2 μm. About 0.15 to 0.2 μm is preferred.

[0131] In one embodiment, the nominal retention range of the filter is about 0.1 μm, about 0.15 μm or about 0.2 μm.

[0132] In a preferred embodiment, the nominal retention range of the filter is about 0.2 μm.

[0133] In a preferred embodiment, the filter includes a prefilter. In some embodiments, the nominal retention range of the prefilter is between about 0.1 μm and about 1 μm. In a preferred embodiment, the nominal retention range of the prefilter is about 0.5 μm. In another preferred embodiment, the nominal retention range of the prefilter is about 0.5 μm and the nominal retention range of the filter is about 0.2 μm.

[0134] In one embodiment, the filtration capacity of the filter is about 25 - 1000 L / m 2 , 50 - 1000 L / m 2 , 75 - 1000 L / m 2 , 100 - 1000 L / m 2 , 150 - 1000 L / m 2 , 200 - 1000 L / m 2 , 250 - 1000 L / m 2 , 300 - 1000 L / m 2 , 350 - 1000 L / m 2 , 400 - 1000 L / m 2 , 500 - 1000 L / m 2 or 750 - 1000 L / m 2 is.

[0135] In one embodiment, the filtration capacity of the filter is 200 - 1000 L / m 2 , 250 - 1000 L / m 2 or 300 - 1000 L / m 2 is.

[0136] In a preferred embodiment, the filtration capacity of the filter is about 300 to 1000 L / m 2 is.

[0137] In some embodiments, the filter comprises polysulfone. In one embodiment, the filter comprises polyphenylene sulfone. In a preferred embodiment, the filter comprises polyethersulfone (PES). In some embodiments, the filter is a disk filter. In a preferred embodiment, the filter is a capsule filter. In a more preferred embodiment, the filter is a capsule filter comprising PES.

[0138] The conjugate vaccine of the present invention The conjugate vaccine obtained by the process of the present invention comprises one or more conjugates and a preservative. The preservative is preferably 2-PE.

[0139] For the purposes of the present invention, the term "conjugate" refers to a capsular polysaccharide linked to a carrier protein. In one embodiment, the capsular polysaccharide is directly linked to the carrier protein. In a second embodiment, the capsular polysaccharide is linked to the protein through a spacer / linker.

[0140] In some embodiments, the conjugate vaccine of the present invention is a monovalent conjugate vaccine. In such embodiments, the conjugate vaccine comprises only one type of conjugate. In such embodiments, the conjugate may be a conjugated Haemophilus influenzae type b (Hib) capsular polysaccharide. In another embodiment, the conjugate is a conjugated Neisseria meningitidis serogroup Y capsular polysaccharide (MenC). In yet another embodiment, the conjugate is a conjugated Neisseria meningitidis serogroup Y capsular polysaccharide (MenY).

[0141] In another embodiment, the conjugate vaccine of the present invention is a bivalent, trivalent, tetravalent, pentavalent or hexavalent conjugate vaccine.

[0142] In one embodiment, the trivalent conjugate vaccine contains three conjugated Streptococcus agalactiae capsular polysaccharides. Streptococcus agalactiae is also known as "Group B Streptococcus" or simply "GBS". In one embodiment, the three conjugates consist of conjugated GBS serotypes Ia, Ib and III capsular polysaccharides. In a preferred embodiment, the capsular polysaccharides are conjugated to CRM 197 In one embodiment, the hexavalent conjugate vaccine contains six conjugated Streptococcus agalactiae capsular polysaccharides. In one embodiment, the six conjugates consist of conjugated GBS serotypes Ia, Ib, II, III, IV and V capsular polysaccharides. In a preferred embodiment, the capsular polysaccharides are conjugated to CRM 197 In one embodiment, the tetravalent conjugate vaccine contains four conjugated Neisseria meningitidis capsular polysaccharides. In one embodiment, the four conjugates consist of conjugated Neisseria meningitidis serogroup A capsular polysaccharide (MenA), conjugated Neisseria meningitidis serogroup W135 capsular polysaccharide (MenW135), conjugated Neisseria meningitidis serogroup Y capsular polysaccharide (MenY), and conjugated Neisseria meningitidis serogroup C capsular polysaccharide (MenC).

[0143]

[0144] In a preferred embodiment, the conjugate vaccine of the present invention is a multivalent conjugate vaccine containing 7 or more conjugates. The multivalent conjugate vaccine of the present invention is preferably a 7- to 25-valent conjugate vaccine (i.e., containing 7 to 25 conjugates). More preferably, the multivalent conjugate vaccine of the present invention is a 13- to 25-valent conjugate vaccine.

[0145] In one embodiment, the multivalent conjugate vaccine of the present invention is a 15-valent conjugate vaccine.

[0146] In one embodiment, the multivalent conjugate vaccine of the present invention is a 16-valent conjugate vaccine.

[0147] In one embodiment, the multivalent conjugate vaccine of the present invention is a 20-valent conjugate vaccine.

[0148] In one embodiment, the multivalent conjugate vaccine of the present invention is a 21-valent conjugate vaccine.

[0149] The multivalent conjugate vaccine of the present invention is preferably a pneumococcal conjugate vaccine.

[0150] In one embodiment, the multivalent conjugate vaccine of the present invention is a pneumococcal conjugate vaccine containing 7 to 25 sugar conjugates (7 to 25 pneumococcal conjugates) derived from different serotypes of Streptococcus pneumoniae (S. pneumoniae). In one embodiment, the multivalent conjugate vaccine of the present invention is a pneumococcal conjugate vaccine containing sugar conjugates derived from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 different serotypes of Streptococcus pneumoniae (S. pneumoniae). In one embodiment, the multivalent conjugate vaccine of the present invention is a pneumococcal conjugate vaccine containing sugar conjugates derived from 20 different serotypes of Streptococcus pneumoniae (S. pneumoniae).

[0151] In certain embodiments, the pneumococcal conjugate vaccine of the present invention is a 7-valent, 8-valent, 9-valent, 10-valent, 11-valent, 12-valent, 13-valent, 14-valent, 15-valent, 16-valent, 17-valent, 18-valent, 19-valent or 20-valent pneumococcal conjugate vaccine.

[0152] In certain embodiments, the pneumococcal conjugate vaccine of the present invention is a 15-valent pneumococcal conjugate vaccine.

[0153] In a preferred embodiment, the pneumococcal conjugate vaccine of the present invention is a 20-valent pneumococcal conjugate vaccine.

[0154] In certain embodiments, the pneumococcal conjugate vaccine of the present invention is a 21-valent, 22-valent, 23-valent, 24-valent or 25-valent pneumococcal conjugate vaccine.

[0155] In certain embodiments, the pneumococcal conjugate vaccine of the present invention is a 21-valent pneumococcal conjugate vaccine.

[0156] In certain embodiments, the pneumococcal conjugate vaccine of the present invention is a 22-valent pneumococcal conjugate vaccine.

[0157] In certain embodiments, the pneumococcal conjugate vaccine of the present invention comprises saccharide conjugates derived from Streptococcus pneumoniae (S. pneumoniae) serotypes 4, 6B, 9V, 14, 18C, 19F and 23F.

[0158] In certain embodiments, the pneumococcal conjugate vaccine of the present invention comprises saccharide conjugates derived from Streptococcus pneumoniae (S. pneumoniae) serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F.

[0159] In certain embodiments, the pneumococcal conjugate vaccine of the present invention comprises saccharide conjugates derived from Streptococcus pneumoniae (S. pneumoniae) serotypes 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, and 23F.

[0160] In certain embodiments, the pneumococcal conjugate vaccine of the present invention comprises saccharide conjugates derived from Streptococcus pneumoniae (S. pneumoniae) serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F.

[0161] In certain embodiments, the pneumococcal conjugate vaccine of the present invention comprises saccharide conjugates derived from Streptococcus pneumoniae (S. pneumoniae) serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, and 33F.

[0162] In certain embodiments, the pneumococcal conjugate vaccine of the present invention comprises saccharide conjugates derived from Streptococcus pneumoniae (S. pneumoniae) serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F.

[0163] In certain embodiments, the pneumococcal conjugate vaccine of the present invention comprises saccharide conjugates derived from Streptococcus pneumoniae (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.

[0164] In certain embodiments, the pneumococcal conjugate vaccine of the present invention comprises saccharide conjugates derived from Streptococcus pneumoniae (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.

[0165] In certain embodiments, the pneumococcal conjugate vaccine of the present invention is a 10-valent pneumococcal conjugate, and the above 10 sugar conjugates are derived from Streptococcus pneumoniae (S. pneumoniae) serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, and 23F.

[0166] In certain embodiments, the pneumococcal conjugate vaccine of the present invention is a 13-valent pneumococcal conjugate, and the above 13 sugar conjugates are derived from Streptococcus pneumoniae (S. pneumoniae) serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F.

[0167] In certain embodiments, the pneumococcal conjugate vaccine of the present invention is a 15-valent pneumococcal conjugate, and the above 15 sugar conjugates are derived from Streptococcus pneumoniae (S. pneumoniae) serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, and 33F.

[0168] In certain embodiments, the pneumococcal conjugate vaccine of the present invention is a 20-valent pneumococcal conjugate, and the above 20 sugar conjugates are derived from Streptococcus pneumoniae (S. pneumoniae) serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F.

[0169] In certain embodiments, the pneumococcal conjugate vaccine of the present invention is a 21-valent pneumococcal conjugate, and the above 21 sugar conjugates are derived from Streptococcus pneumoniae (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.

[0170] In certain embodiments, the pneumococcal conjugate vaccine of the present invention is a 21-valent pneumococcal conjugate, and the above 21 sugar conjugates are derived from Streptococcus pneumoniae (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.

[0171] In certain embodiments, the pneumococcal conjugate vaccine of the present invention is a 22-valent pneumococcal conjugate, and the above 22 sugar conjugates are derived from Streptococcus pneumoniae (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.

[0172] In certain embodiments, the pneumococcal conjugate vaccine of the present invention is a 21-valent pneumococcal conjugate, and the above 21 sugar conjugates are derived from Streptococcus pneumoniae (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.

[0173] In certain embodiments, the pneumococcal conjugate vaccine of the present invention is a 21-valent pneumococcal conjugate, and the above 21 sugar conjugates are derived from Streptococcus pneumoniae (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.

[0174] In certain embodiments, the pneumococcal conjugate vaccine of the present invention is a 21-valent pneumococcal conjugate, and the above 21 sugar conjugates are derived from Streptococcus pneumoniae (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.

[0175] In certain embodiments, the pneumococcal conjugate vaccine of the present invention is a 21-valent pneumococcal conjugate, and the above 21 sugar conjugates are derived from Streptococcus pneumoniae (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.

[0176] In certain embodiments, the pneumococcal conjugate vaccine of the present invention is a 21-valent pneumococcal conjugate, and the above 21 sugar conjugates are derived from Streptococcus pneumoniae (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.

[0177] One component of the sugar conjugate is a carrier protein conjugated to the sugar. The terms "protein carrier" or "carrier protein" or "carrier" may be used interchangeably herein. The carrier protein should be one to which standard conjugation procedures are applicable.

[0178] In certain embodiments, the carrier protein of the sugar conjugate of the present invention is: DT (diphtheria toxoid), TT (tetanus toxoid), fragment C of TT, CRM 197(A non-toxic but antigenically identical variant of diphtheria toxin), other DT variants (e.g., CRM176, CRM228, CRM45 (Uchida et al. (1973) J. Biol. Chem. 218: 3838-3844), CRM9, CRM102, CRM103 or CRM107), ply detoxified in some way, e.g., dPLY-GMBS (WO2004 / 081515, WO2006 / 032499) or pneumococcal pneumolysin (ply) including dPLY-formol (Kuo et al. (1995) Infect Immun 63: 2706-2713), PhtA, PhtB, PhtD, PhtE (the sequences of PhtA, PhtB, PhtD or PhtE are disclosed in WO00 / 37105 and WO00 / 39299) and fusions of Pht proteins, e.g., PhtDE fusion, PhtBE fusion, Pht A-E (WO01 / 98334, WO03 / 054007, WO2009 / 000826) including PhtX, OMPC (meningococcal outer membrane protein) usually extracted from Neisseria meningitidis serogroup B (EP0372501), PorB (Neisseria meningitidis (N.derived from meningitidis), PD (Haemophilus influenzae protein D; see, for example, EP0594610B), or immunologically functional equivalents thereof, synthetic peptides (EP0378881, EP0427347), heat shock proteins (WO93 / 17712, WO94 / 03208), pertussis proteins (WO98 / 58668, EP0471177), cytokines, lymphokines, growth factors or hormones (WO91 / 01146), artificial proteins containing multiple human CD4+ T cell epitopes derived from antigens from various pathogens (Falugi et al. (2001) Eur J Immunol 31:3816-3824), for example, N19 protein (Baraldoi et al. (2004) Infect Immun 72:4884-4887), pneumococcal surface protein PspA (WO02 / 091998), iron uptake proteins (WO01 / 72337), toxins A or B of Clostridium difficile (WO00 / 61761), transferrin-binding proteins, pneumococcal adhesion protein (PsaA), recombinant Pseudomonas aeruginosa exotoxin A (especially its non-toxic variants (e.g., exotoxin A with a substitution at glutamic acid 553 (Douglas et al. (1987) J. Bacteriol. 169(11):4967-4971)) are selected from the group consisting of. Other proteins, such as ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or purified tuberculin protein (PPD) can also be used as carrier proteins. Other suitable carrier proteins include inactivated bacterial toxins, such as cholera toxoid (as described, for example, in WO2004 / 083251), Escherichia coli LT, Escherichia coli ST, and exotoxin A from Pseudomonas aeruginosa. Another suitable carrier protein is C5a peptidase (SCP) from the genus Streptococcus.

[0179] 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).

[0180] In another embodiment, the carrier protein of the glycoconjugate of the present invention is PD (Haemophilus influenzae protein D; see, for example, EP0594610B).

[0181] In a preferred embodiment, the carrier protein of the glycoconjugate of the present invention is TT, CRM 197 or C5a peptidase (SCP) derived from Streptococcus.

[0182] In a preferred embodiment, the carrier protein of the glycoconjugate of the present invention is CRM 197 or C5a peptidase (SCP) derived from Streptococcus.

[0183] In a highly preferred embodiment, the carrier protein of the glycoconjugate of the present invention is CRM 197 . The CRM 197 protein is a non-toxic form of diphtheria toxin but is immunologically indistinguishable from diphtheria toxin. CRM 197 is produced by Corynebacterium diphtheriae infected with the non-toxic phage β197tox created by nitrosoguanidine mutagenesis of the toxin-producing corynephage beta (Uchida et al. (1971) Nature New Biology 233:8-11). The CRM 197 protein has the same molecular weight as diphtheria toxin but differs by a single base change (from guanine to adenine) in the structural gene. This single base change causes an amino acid substitution in the mature protein (from glycine to glutamic acid), eliminating the toxicity of diphtheria toxin. CRM 197The protein is a safe and effective T cell-dependent carrier for sugars. CRM 197 Further details regarding it and its preparation can be found, for example, in U.S. Patent No. 5,614,382.

[0184] In certain embodiments, the carrier protein of the sugar conjugate of the present invention is the A chain of CRM 197 (see CN103495161). In certain embodiments, the carrier protein of the sugar conjugate of the present invention is the A chain of CRM obtained by expression in recombinant E. coli 197 (see CN103495161).

[0185] In any of the above vaccine embodiments, all of the capsular polysaccharides are individually conjugated to CRM 197 .

[0186] In certain embodiments, the capsular polysaccharides derived from any of the above vaccines of Streptococcus pneumoniae (S. pneumoniae) serotypes 1, 4, 5, 6B, 7F, 9V, 14 and / or 23F are individually conjugated to PD.

[0187] In certain embodiments, the capsular polysaccharide derived from Streptococcus pneumoniae (S. pneumoniae) serotype 18C of any of the above vaccines is conjugated to TT.

[0188] In certain embodiments, the capsular polysaccharide derived from Streptococcus pneumoniae (S. pneumoniae) serotype 19F of any of the above vaccines is conjugated to DT.

[0189] In one embodiment, the capsular polysaccharides derived from any of the above-mentioned pneumococcal (S. pneumoniae) serotypes 1, 4, 5, 6B, 7F, 9V, 14 and / or 23F of the vaccine are individually conjugated to PD, and the capsular polysaccharide derived from pneumococcal (S. pneumoniae) serotype 18C is conjugated to TT, and the capsular polysaccharide derived from pneumococcal (S. pneumoniae) serotype 19F is conjugated to DT.

[0190] In any embodiment of the above vaccine, at least one capsular polysaccharide is conjugated to TT and all other capsular polysaccharides are conjugated to CRM 197 to.

[0191] In any embodiment of the above vaccine, one capsular polysaccharide is conjugated to TT and all other capsular polysaccharides are conjugated to CRM 197 to.

[0192] In any embodiment of the above vaccine, at least two capsular polysaccharides are conjugated to TT and all other capsular polysaccharides are conjugated to CRM 197 to.

[0193] In any embodiment of the above vaccine, two capsular polysaccharides are conjugated to TT and all other capsular polysaccharides are conjugated to CRM 197 to.

[0194] In any embodiment of the above vaccine, at least three capsular polysaccharides are conjugated to TT and all other capsular polysaccharides are conjugated to CRM 197 to.

[0195] In any embodiment of the above vaccine, three capsular polysaccharides are conjugated to TT and all other capsular polysaccharides are conjugated to CRM 197 to.

[0196] In some embodiments of the above vaccines, four capsular polysaccharides are conjugated to TT, and all other capsular polysaccharides are conjugated to CRM 197 and conjugated.

[0197] In some embodiments, the pneumococcal conjugate vaccine of the present invention is a 21-valent pneumococcal conjugate, and the above 21 sugar conjugates are derived 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. Four capsular polysaccharides are conjugated to TT, and the four capsular polysaccharides conjugated to TT are serotype 15B, 22F and two other serotypes selected from the group consisting of serotypes 1, 3 and 5. All other sugar conjugates are conjugated to CRM 197 and conjugated.

[0198] In some embodiments, the pneumococcal conjugate vaccine of the present invention is a 21-valent pneumococcal conjugate, and the above 21 sugar conjugates are derived 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. Four capsular polysaccharides are conjugated to TT, and the four capsular polysaccharides conjugated to TT are serotypes 1, 5, 15B and 22F. All other sugar conjugates are conjugated to CRM 197 and conjugated.

[0199] In one embodiment, the pneumococcal conjugate vaccine of the present invention is a 21-valent pneumococcal conjugate, and the above 21 sugar conjugates are derived from Streptococcus pneumoniae (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. Four capsular polysaccharides are conjugated with TT, and the four capsular polysaccharides conjugated with TT are serotypes 1, 3, 15B and 22F. All other sugar conjugates are CRM 197 conjugated thereto.

[0200] In one embodiment, the pneumococcal conjugate vaccine of the present invention is a 21-valent pneumococcal conjugate, and the above 21 sugar conjugates are derived from Streptococcus pneumoniae (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. Four capsular polysaccharides are conjugated with TT, and the four capsular polysaccharides conjugated with TT are serotypes 3, 5, 15B and 22F. All other sugar conjugates are CRM 197 conjugated thereto.

[0201] In one embodiment, the pneumococcal conjugate vaccine of the present invention is a 21-valent pneumococcal conjugate, and the above 21 sugar conjugates are derived from Streptococcus pneumoniae (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. Two sugar conjugates selected from serotypes 1, 3 and 5 are conjugated with TT, and all other sugar conjugates are CRM 197 conjugated thereto.

[0202] In certain embodiments, the pneumococcal conjugate vaccine of the present invention is a 21-valent pneumococcal conjugate, and the above 21 sugar conjugates are derived from Streptococcus pneumoniae (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. The sugar conjugates derived from serotypes 1 and 3 are conjugated with TT, and all other sugar conjugates are conjugated with CRM 197 and conjugated.

[0203] In certain embodiments, the pneumococcal conjugate vaccine of the present invention is a 21-valent pneumococcal conjugate, and the above 21 sugar conjugates are derived from Streptococcus pneumoniae (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. The sugar conjugates derived from serotypes 1 and 5 are conjugated with TT, and all other sugar conjugates are conjugated with CRM 197 and conjugated.

[0204] In certain embodiments, the pneumococcal conjugate vaccine of the present invention is a 21-valent pneumococcal conjugate, and the above 21 sugar conjugates are derived from Streptococcus pneumoniae (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. The sugar conjugates derived from serotypes 3 and 5 are conjugated with TT, and all other sugar conjugates are conjugated with CRM 197 and conjugated.

[0205] In one embodiment, the pneumococcal conjugate vaccine of the present invention is a 15-valent pneumococcal conjugate, and the above 15 sugar conjugates are derived from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F, and all of the sugar conjugates are CRM 197 conjugated thereto.

[0206] In a preferred embodiment, the pneumococcal conjugate vaccine of the present invention is a 20-valent pneumococcal conjugate, and the above 20 sugar conjugates are derived 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, and all of the sugar conjugates are CRM 197 conjugated thereto.

[0207] The vaccine of the present invention may contain a small amount of free carrier. When a given carrier protein is present in both free and conjugated forms in the vaccine of the present invention, the unconjugated form is preferably 5% or less of the total amount of the carrier protein in the whole composition, and more preferably present at less than 2% by weight.

[0208] In some embodiments, the vaccine of the present invention may contain at least one adjuvant. In some embodiments, the vaccine of the present invention may contain one adjuvant. The term "adjuvant" refers to a compound or mixture that enhances the immune response to an antigen. Adjuvants can act primarily as delivery systems, primarily as immune modulators, or can have both strong characteristics.

[0209] Suitable adjuvants include those suitable for use in mammals, including humans. Examples of known suitable delivery system-type 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, water-in-oil 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)), oil-in-water emulsions such as Montanide, and poly(D,L-lactide-co-glycolide) (PLG) microparticles or nanoparticles.

[0210] In certain embodiments, the vaccine of the invention comprises an aluminum salt (alum) (e.g., aluminum phosphate, aluminum sulfate, or aluminum hydroxide) as an adjuvant. In preferred embodiments, the vaccine of the invention comprises aluminum phosphate or aluminum hydroxide as an adjuvant. In more preferred embodiments, the vaccine of the invention comprises aluminum phosphate as an adjuvant.

[0211] In some embodiments, the vaccine of the present disclosure comprises aluminum phosphate as an adjuvant, and the final concentration of aluminum phosphate is between about 0.1 mg / mL and about 1 mg / ml. In some embodiments, the vaccine of the present disclosure comprises aluminum phosphate as an adjuvant, and the final concentration of aluminum phosphate is between about 0.1 mg / mL and about 0.5 mg / ml. In a preferred embodiment, the vaccine of the present disclosure comprises aluminum phosphate as an adjuvant, and the final concentration of aluminum phosphate is about 0.25 mg / ml.

[0212] In certain embodiments, the addition of the adjuvant to the vaccine is performed after the addition of the preservative. In preferred embodiments, the addition of the adjuvant to the vaccine is performed after the addition of the 2-PE preservative. In another preferred embodiment, the addition of the aluminum phosphate adjuvant to the vaccine is performed after the addition of the 2-PE preservative.

[0213] Additional exemplary adjuvants for enhancing the effect of the vaccines of the present invention include, but are not limited to: (1) oil-in-water emulsion formulations (with or without other specific immunostimulatory agents such as muramyl peptides (see below) or bacterial cell wall components), for example, (a) SAF containing 10% squalene, 0.4% Tween™ 80, 5% Pluronic® block polymer L121, and thr-MDP, microfluidized or vortexed into a submicron emulsion to produce an emulsion with a larger particle size, and (b) RIBI™ Adjuvant System (RAS) (Ribi Immunochem, Hamilton, MT) containing 2% squalene, 0.2% Tween™ 80, and one or more bacterial cell wall components, for example, monophosphoryl lipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL + CWS (DETOX™); (2) saponin adjuvants, for example, QS21, STIMULON™ (Cambridge Bioscience, Worcester, MA), ABISCO® (Isconova, Sweden), or ISCOMATRIX® (Commonwealth Serum Laboratories, Australia) can be used or particles generated therefrom, for example, ISCOM (immunostimulatory complex), ISCOMs may be lacking additional surfactants (e.g., WO00 / 07621); (3) complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA); (4) cytokines, for example, interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12 (e.g., WO99 / 44636)), interferons (e.g., gamma interferon), macrophage colony-stimulating factor (M-CSF), tumor necrosis factor (TNF), etc.(5) When used with monophosphoryl lipid A (MPL) or 3-O-deacylated MPL (3dMPL) (see, for example, GB-2220221, EP0689454), alum may not be substantially present (see, for example, WO00 / 56358); (6) A combination of 3dMPL with, for example, QS21 and / or an oil-in-water emulsion (see, for example, EP0835318, EP0735898, EP0761231); (7) Polyoxyethylene ether or polyoxyethylene ester (see, for example, WO99 / 52549); (8) A combination of a polyoxyethylene sorbitan ester surfactant and octoxynol (see, for example, WO01 / 21207) or a combination of a polyoxyethylene alkyl ether or ester surfactant and at least one additional nonionic surfactant, such as octoxynol (see, for example, WO01 / 21152); (9) Saponin and an immunostimulatory oligonucleotide (e.g., a CpG oligonucleotide) (see, for example, WO00 / 62800); (10) Immunostimulants and particles of metal salts (see, for example, WO00 / 23105); (11) Saponin and an oil-in-water emulsion (see, for example, WO99 / 11241); (12) Saponin (e.g., QS21) + 3dMPL + IM2 (sterol may be added) (see, for example, WO98 / 57659); (13) Other substances that act as immunostimulants and enhance the effectiveness of the composition. Examples of 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-isoglutaminyl-L-alanine-2-(1’-2’-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine MTP-PE), etc.;

[0214] In certain embodiments of the present invention, the vaccine of the present invention comprises a CpG oligonucleotide as an adjuvant. As used herein, CpG oligonucleotide refers to an immunostimulatory CpG oligodeoxynucleotide (CpG ODN), and thus these terms are used interchangeably unless otherwise specified. Immunostimulatory CpG oligodeoxynucleotides contain one or more immunostimulatory CpG motifs that are unmethylated cytosine-guanine dinucleotides, optionally within a certain preferred range of base contexts. The methylation state of the CpG immunostimulatory motif generally refers to the cytosine residue of the dinucleotide. An immunostimulatory oligonucleotide containing at least one unmethylated CpG dinucleotide is an oligonucleotide containing a 5'-unmethylated cytosine linked by a phosphate bond to a 3'-guanine, and 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 and activates the immune system through TLR9, but is not as potent as when the CpG motif(s) are not methylated. CpG immunostimulatory oligonucleotides may contain one or more palindromes and thus may include CpG dinucleotides. CpG oligonucleotides are described in numerous registered patents, published patent applications, and other publications, including U.S. Patent Nos. 6,194,388; 6,207,646; 6,214,806; 6,218,371; 6,239,116; and 6,339,068.

[0215] In certain embodiments of the present invention, the vaccine of the present invention comprises any of the CpG oligonucleotides described in lines 22 to 36 on page 12 of WO2010 / 125480.

[0216] Different classes of CpG immunostimulatory oligonucleotides have been identified. They are referred to as classes A, B, C, and P and are described in more detail on pages 3, line 22 to page 12, line 36 of WO2010 / 125480. The processes of the present invention include the use of these different classes of CpG immunostimulatory oligonucleotides.

[0217] In certain embodiments, the vaccines of the present invention comprise any of the combinations of sugar conjugates disclosed herein and a pharmaceutically acceptable excipient, carrier, or diluent.

[0218] The vaccines of the present disclosure may include, in addition to the conjugate(s) and preservative, a buffer, salts, divalent cations, nonionic surfactants, cryoprotective substances such as sugars, and antioxidants such as free radical scavengers or chelating agents, or one or more of any combination thereof.

[0219] In certain embodiments, the vaccines of the present disclosure include a buffer. In certain embodiments, the pKa of the buffer is from about 3.5 to about 7.5. In some embodiments, the buffer is a phosphate buffer, a succinate buffer, a histidine buffer, or a citrate buffer. In some embodiments, the buffer is a succinate buffer. In some embodiments, the buffer is a histidine buffer. In certain specific embodiments, the buffer is a succinate buffer at a final concentration of 1 mM to 10 mM. In a specific embodiment, the final concentration of the succinate buffer is about 5 mM.

[0220] In certain embodiments, the vaccines of the present disclosure include salts. In some embodiments, the salts are 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 includes 150 mM sodium chloride.

[0221] In certain embodiments, the vaccines of the present disclosure include a surfactant. In certain embodiments, the surfactant is selected from the group consisting of polysorbate 20 (TWEEN™ 20), polysorbate 40 (TWEEN™ 40), polysorbate 60 (TWEEN™ 60), polysorbate 65 (TWEEN™ 65), polysorbate 80 (TWEEN™ 80), polysorbate 85 (TWEEN™ 85), TRITON™ N-101, TRITON™ X-100, octoxynol 40, nonoxynol-9, triethanolamine, triethanolamine oleate polypeptide, polyoxyethylene-660 hydroxystearate (PEG-15, Solutol H15), polyoxyethylene-35-ricinoleate (CREMOPHOR® EL), soy lecithin, and poloxamer.

[0222] In a particular embodiment, the surfactant is polysorbate 80. In some of the above embodiments, the final concentration of polysorbate 80 in the formulation is at least 0.0001% to 10% polysorbate 80 weight by weight (w / w). In some of the above embodiments, the final concentration of polysorbate 80 in the formulation is at least 0.001% to 1% polysorbate 80 weight by weight (w / w). In some of the above embodiments, the final concentration of polysorbate 80 in the formulation is at least 0.01% to 1% polysorbate 80 weight 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 between 0.01% and 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).

[0223] In a particular embodiment, the surfactant is polysorbate 20. In some of the above embodiments, the final concentration of polysorbate 20 in the formulation is at least 0.01% to 10% polysorbate 20 weight by weight (w / w). In some of the above embodiments, the final concentration of polysorbate 20 in the formulation is at least 0.1% to 1% polysorbate 20 weight by weight (w / w). In some of the above embodiments, the final concentration of polysorbate 20 in the formulation is at least 0.1% to 0.5% polysorbate 20 weight 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).

[0224] In a particular embodiment, the surfactant is polysorbate 40. In some of the above embodiments, the final concentration of polysorbate 40 in the formulation is at least 0.0001% to 10% polysorbate 40 weight by weight (w / w). In some of the above embodiments, the final concentration of polysorbate 40 in the formulation is at least 0.001% to 1% polysorbate 40 weight by weight (w / w). In some of the above embodiments, the final concentration of polysorbate 40 in the formulation is at least 0.01% to 1% polysorbate 40 weight 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).

[0225] In a particular embodiment, the surfactant is polysorbate 60. In some of the above embodiments, the final concentration of polysorbate 60 in the formulation is at least 0.0001% to 10% polysorbate 60 weight by weight (w / w). In some of the above embodiments, the final concentration of polysorbate 60 in the formulation is at least 0.001% to 1% polysorbate 60 weight by weight (w / w). In some of the above embodiments, the final concentration of polysorbate 60 in the formulation is at least 0.01% to 1% polysorbate 60 weight 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).

[0226] In a particular embodiment, the surfactant is polysorbate 65. In some of the above embodiments, the final concentration of polysorbate 65 in the formulation is at least 0.0001% to 10% polysorbate 65 weight by weight (w / w). In some of the above embodiments, the final concentration of polysorbate 65 in the formulation is at least 0.001% to 1% polysorbate 65 weight by weight (w / w). In some of the above embodiments, the final concentration of polysorbate 65 in the formulation is at least 0.01% to 1% polysorbate 65 weight by weight (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 in the formulation is 1% polysorbate 65 (w / w).

[0227] In a particular embodiment, the surfactant is polysorbate 85. In some of the above embodiments, the final concentration of polysorbate 85 in the formulation is at least 0.0001% to 10% polysorbate 85 weight by weight (w / w). In some of the above embodiments, the final concentration of polysorbate 85 in the formulation is at least 0.001% to 1% polysorbate 85 weight by weight (w / w). In some of the above embodiments, the final concentration of polysorbate 85 in the formulation is at least 0.01% to 1% polysorbate 85 weight by weight (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).

[0228] In certain embodiments, the final pH of the vaccines of the present disclosure is from 5.5 to 7.5, more preferably the final pH is from 5.6 to 7.0, and even more preferably the final pH is from 5.3 to 6.3. In preferred embodiments, the final pH of the vaccines of the present disclosure is from 5.8 to 6.0. In another preferred embodiment, the final pH of the vaccines of the present disclosure is 5.8.

[0229] In some embodiments, the final protein concentration of the vaccines of the present disclosure is between about 50 μg / ml and about 150 μg / ml. In preferred embodiments, the final protein concentration of the vaccines of the present disclosure is between about 60 μg / ml and about 130 μg / ml. In another preferred embodiment, the final protein concentration of the vaccines of the present disclosure is between about 90 μg / ml and about 99 μg / ml. In yet another preferred embodiment, the final protein concentration of the vaccines of the present disclosure is about 99 μg / ml.

[0230] In some embodiments, the final D10 particle size distribution of the vaccines of the present disclosure is between about 3,000 and about 6,000. In another embodiment, the final D10 particle size distribution of the vaccines of the present disclosure is between about 4,000 and about 5,000. In preferred embodiments, the final D10 particle size distribution of the vaccines of the present disclosure is between about 4,300 and about 4,700. In some embodiments, the final D50 particle size distribution of the vaccines of the present disclosure is between about 6,000 and about 8,000. In another embodiment, the final D50 particle size distribution of the vaccines of the present disclosure is between about 7,000 and about 8,000. In preferred embodiments, the final D50 particle size distribution of the vaccines of the present disclosure is between about 7,500 and about 7,900. In some embodiments, the final D90 particle size distribution of the vaccines of the present disclosure is between about 10,000 and about 16,000. In another embodiment, the final D90 particle size distribution of the vaccines of the present disclosure is between about 12,000 and about 15,000. In preferred embodiments, the final D90 particle size distribution of the vaccines of the present disclosure is between about 12,300 and about 14,900.

[0231] The typical volume of the vaccine of the present invention for injection is 0.1 mL to 2 mL. In certain embodiments, the volume of the vaccine of the present invention for injection is 0.2 mL to 1 mL, and more preferably the volume is about 0.5 mL. Most preferably, the volume of the vaccine of the present invention for injection is 0.5 mL.

[0232] The amount of the sugar conjugate(s) in each dose is selected as the amount that induces an immunoprotective response without significant adverse side effects in a typical vaccine recipient. Such amount varies depending on which specific immunogen is used and how the immunogen is provided.

[0233] The amount of a specific sugar conjugate in the vaccine can be calculated based on the total polysaccharide (conjugated and unconjugated) to that conjugate. For example, a sugar conjugate having 20% free polysaccharide would have approximately 80 μg of conjugated polysaccharide and approximately 20 μg of unconjugated polysaccharide in a 100 μg polysaccharide dose. The amount of the sugar conjugate can vary depending on the pneumococcal serotype. The sugar concentration can be determined by an uronic acid assay.

[0234] The “immunogenic amount” of different polysaccharide components in the vaccine can be different. Generally, each dose includes 0.1 μg to 100 μg of polysaccharide for a given serotype. In certain embodiments, each dose includes 0.1 μg to 100 μg of polysaccharide for a given serotype. In preferred embodiments, each dose includes 0.5 μg to 20 μg of polysaccharide. In preferred embodiments, each dose includes 1.0 μg to 10 μg of polysaccharide. In more preferred embodiments, each dose includes 2.0 μg to 5.0 μg of polysaccharide for a given serotype. Any integer falling within the above ranges is intended as an embodiment of the present disclosure.

[0235] In certain embodiments, each dose includes 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 polysaccharide from a saccharide conjugate derived from Streptococcus pneumoniae (S. pneumoniae) serotypes 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and / or 33F.

[0236] In certain embodiments, each dose includes about 2.0 μg of polysaccharide from a saccharide conjugate derived from Streptococcus pneumoniae (S. pneumoniae) serotypes 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and / or 33F.

[0237] In certain embodiments, each dose includes about 2.2 μg of polysaccharide from a saccharide conjugate derived from Streptococcus pneumoniae (S. pneumoniae) serotypes 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and / or 33F.

[0238] In certain embodiments, each dose includes about 4.0 μg of polysaccharide from a saccharide conjugate derived from Streptococcus pneumoniae (S. pneumoniae) serotype 6B.

[0239] In certain embodiments, each dose includes about 4.4 μg of polysaccharide from a saccharide conjugate derived from Streptococcus pneumoniae (S. pneumoniae) serotype 6B.

[0240] In certain embodiments, each dose includes about 1.5 μg to about 3.0 μg of polysaccharide for each sugar conjugate derived from Streptococcus pneumoniae (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 3.0 μg to about 6.0 μg of polysaccharide for the sugar conjugate derived from Streptococcus pneumoniae (S. pneumoniae) serotype 6B.

[0241] In certain embodiments, each dose includes about 2.0 μg to about 2.5 μg of polysaccharide for each sugar conjugate derived from Streptococcus pneumoniae (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 4.0 μg to about 4.8 μg of polysaccharide for the sugar conjugate derived from Streptococcus pneumoniae (S. pneumoniae) serotype 6B.

[0242] In certain embodiments, each dose includes about 2.2 μg of polysaccharide for each sugar conjugate derived from Streptococcus pneumoniae (S. pneumoniae) serotypes 1, 3, 4, 5, 6A, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, and 33F, and about 4.4 μg of polysaccharide for the sugar conjugate derived from Streptococcus pneumoniae (S. pneumoniae) serotype 6B.

[0243] In certain embodiments, each dose includes about 1.5 μg to about 3.0 μg of polysaccharide for each sugar conjugate derived from Streptococcus pneumoniae (S. pneumoniae) serotypes 1, 3, 4, 5, 6A, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, and 33F, and about 3 μg to about 6 μg of polysaccharide for the sugar conjugate derived from Streptococcus pneumoniae (S. pneumoniae) serotype 6B.

[0244] In certain embodiments, each dose includes about 2.0 μg to about 2.5 μg of polysaccharide for each sugar conjugate derived from Streptococcus pneumoniae (S. pneumoniae) serotypes 1, 3, 4, 5, 6A, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, and 33F, and about 4.0 μg to about 4.8 μg of polysaccharide for the sugar conjugate derived from Streptococcus pneumoniae (S. pneumoniae) serotype 6B.

[0245] In certain embodiments, each dose includes about 2.0 μg of polysaccharide for each sugar conjugate derived from Streptococcus pneumoniae (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 for the sugar conjugate derived from Streptococcus pneumoniae (S. pneumoniae) serotype 6B.

[0246] In certain embodiments, each dose includes about 1.5 μg to about 3.0 μg of polysaccharide for each sugar conjugate derived from Streptococcus pneumoniae (S. pneumoniae) serotypes 1, 3, 4, 5, 6A, 7F, 9N, 9V, 14, 18C, 19A, 19F, 22F, 23F, and 33F, and about 3 μg to about 6 μg of polysaccharide for the sugar conjugate derived from Streptococcus pneumoniae (S. pneumoniae) serotype 6B.

[0247] In certain embodiments, each dose includes about 2.0 μg to about 2.5 μg of polysaccharide for each sugar conjugate derived from Streptococcus pneumoniae (S. pneumoniae) serotypes 1, 3, 4, 5, 6A, 7F, 9N, 9V, 14, 18C, 19A, 19F, 22F, 23F, and 33F, and about 4.0 μg to about 4.8 μg of polysaccharide for the sugar conjugate derived from Streptococcus pneumoniae (S. pneumoniae) serotype 6B.

[0248] In certain embodiments, each dose includes about 2.0 μg of polysaccharide for each sugar conjugate derived from Streptococcus pneumoniae (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 for the sugar conjugate derived from Streptococcus pneumoniae (S. pneumoniae) serotype 6B.

[0249] Generally, each dose includes a carrier protein in a total amount of 10 μg to 150 μg. In certain embodiments, each dose includes a carrier protein in a total amount of 10 μg to 150 μg. In certain embodiments, each dose includes a carrier protein in a total amount of 25 μg to 75 μg. In a preferred embodiment, each dose includes a carrier protein in a total amount of 40 μg to 60 μg. In certain embodiments, the carrier protein is CRM 197 is.

[0250] In certain embodiments, each dose includes about 30 μg of carrier protein. In certain embodiments, each dose includes about 31 μg of carrier protein. In certain embodiments, each dose includes about 32 μg of carrier protein. In certain embodiments, each dose includes about 33 μg of carrier protein. In a preferred embodiment, each dose includes about 34 μg of carrier protein.

[0251] In a preferred embodiment, the vaccine is a 13-valent pneumococcal conjugate vaccine and each dose contains about 34 μg of carrier protein. In certain embodiments, the carrier protein is CRM 197 is.

[0252] In another preferred embodiment, the vaccine is a 15-valent pneumococcal conjugate vaccine and each dose contains about 30 μg of carrier protein. In certain embodiments, the carrier protein is CRM 197 is.

[0253] In certain embodiments, each dose includes about 40 μg of carrier protein. In certain embodiments, each dose includes about 41 μg of carrier protein. In certain embodiments, each dose includes about 42 μg of carrier protein. In certain embodiments, each dose includes about 43 μg of carrier protein. In certain embodiments, each dose includes about 44 μg of carrier protein. In certain embodiments, each dose includes about 45 μg of carrier protein.

[0254] In a preferred embodiment, the vaccine is a 16-valent pneumococcal conjugate vaccine and each dose contains about 42 μg of carrier protein. In certain embodiments, the carrier protein is CRM 197 and.

[0255] In certain embodiments, each dose includes about 48 μg of carrier protein. In certain embodiments, each dose includes about 49 μg of carrier protein. In certain embodiments, each dose includes about 50 μg of carrier protein. In certain embodiments, each dose includes about 51 μg of carrier protein. In certain embodiments, each dose includes about 52 μg of carrier protein. In certain embodiments, each dose includes about 53 μg of carrier protein.

[0256] In a preferred embodiment, the vaccine is a 20-valent pneumococcal conjugate vaccine and each dose contains about 50 μg of carrier protein. In certain embodiments, the carrier protein is CRM 197 and.

[0257] In a preferred embodiment, the pneumococcal conjugate vaccine of the present invention is a 20-valent pneumococcal conjugate vaccine, and the above 20 sugar conjugates are derived 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, and all the sugar conjugates are CRM 197It is conjugated, and each dose is approximately 2.2 μg of capsular polysaccharides of serotypes 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F and approximately 4.4 μg of capsular polysaccharide of serotype 6B, approximately 50 μg of CRM 197 , 2-PE, 0.125 mg of elemental aluminum as aluminum phosphate adjuvant, 100 μg of polysorbate 80, sodium chloride, and succinic acid buffer.

[0258] In certain embodiments, the pneumococcal conjugate vaccine of the present invention is a 15-valent pneumococcal conjugate vaccine, and the above 15 sugar conjugates are derived from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, and 33F, and all the sugar conjugates are CRM 197 It is conjugated, and each dose is approximately 2.0 μg of capsular polysaccharides of serotypes 1, 3, 4, 5, 6A, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, and 33F, and approximately 4.0 μg of capsular polysaccharide of serotype 6B, approximately 30 μg of CRM 197 , 2-PE, 0.125 mg of elemental aluminum as aluminum phosphate adjuvant, sodium chloride, and L-histidine buffer.

[0259] In certain embodiments, the pneumococcal conjugate vaccine of the present invention is a 15-valent pneumococcal conjugate vaccine, and the above 15 sugar conjugates are derived from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, and 33F, and all the sugar conjugates are CRM 197 It is conjugated, and each dose is approximately 2.0 μg of capsular polysaccharides of serotypes 1, 3, 4, 5, 6A, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, and 33F, and approximately 4.0 μg of capsular polysaccharide of serotype 6B, approximately 30 μg of CRM 197, 2-PE, 0.125 mg of elemental aluminum as aluminum phosphate adjuvant, containing polysorbate 20, sodium chloride, and L-histidine buffer.

[0260] In some embodiments, the vaccine is a 21-valent pneumococcal conjugate vaccine, and each dose contains about 1 μg to about 30 μg of TT and about 20 μg to about 85 μg of CRM 197 and contains.

[0261] In some embodiments, the vaccine is a 21-valent pneumococcal conjugate vaccine, and each dose contains about 2 μg to about 25 μg of TT and about 40 μg to about 75 μg of CRM 197 and contains.

[0262] In some embodiments, the vaccine is a 21-valent pneumococcal conjugate vaccine, and each dose contains about 2 μg to about 2.5 μg of capsular polysaccharide serotypes 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.

[0263] In some embodiments, the vaccine is a 21-valent pneumococcal conjugate vaccine, and each dose contains about 2 μg to about 2.5 μg of capsular polysaccharide serotypes 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.

[0264] In one embodiment, the pneumococcal conjugate vaccine of the present invention is a 21-valent pneumococcal conjugate vaccine. The above 21 sugar conjugates are derived from Streptococcus pneumoniae (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. The sugar conjugates derived from serotypes 1 and 5 are conjugated with TT, and all other sugar conjugates are conjugated with CRM 197 and conjugated. Each dose contains approximately 2 μg to approximately 2.5 μg of capsular polysaccharide serotypes 1, 3, 4, 5, 6A, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F, and approximately 4 to approximately 5 μg of capsular polysaccharide of serotype 6B.

[0265] In one embodiment, the pneumococcal conjugate vaccine of the present invention is a 21-valent pneumococcal conjugate vaccine. The above 21 sugar conjugates are derived from Streptococcus pneumoniae (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. The sugar conjugates derived from serotypes 1 and 5 are conjugated with TT, and all other sugar conjugates are conjugated with CRM 197 and conjugated. Each dose contains approximately 2.2 μg of capsular polysaccharide serotypes 1, 3, 4, 5, 6A, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F, and approximately 4.4 μg of capsular polysaccharide of serotype 6B.

[0266] In certain embodiments, the pneumococcal conjugate vaccine of the present invention is a 21-valent pneumococcal conjugate vaccine, and the above 21 sugar conjugates are derived 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. The sugar conjugates derived from serotypes 1 and 5 are conjugated with TT, and all other sugar conjugates are conjugated with CRM 197 and conjugated. Each dose contains approximately 2 μg to approximately 2.5 μg of capsular polysaccharide serotypes 1, 5, 6A, 7F, 8, 9N, 10A, 11A, 12F, 14, 15B, 18C, 22F, 23F, and 33F, and approximately 4 to approximately 5 μg of capsular polysaccharide serotypes 3, 4, 6B, 9V, 19A and 19F, respectively.

[0267] In certain embodiments, the pneumococcal conjugate vaccine of the present invention is a 21-valent pneumococcal conjugate vaccine, and the above 21 sugar conjugates are derived 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. The sugar conjugates derived from serotypes 1 and 5 are conjugated with TT, and all other sugar conjugates are conjugated with CRM 197 and conjugated. Each dose contains approximately 2.2 μg of capsular polysaccharide serotypes 1, 5, 6A, 7F, 8, 9N, 10A, 11A, 12F, 14, 15B, 18C, 22F, 23F, and 33F, and approximately 4.4 μg of capsular polysaccharide serotypes 3, 4, 6B, 9V, 19A and 19F, respectively.

[0268] In one embodiment, the pneumococcal conjugate vaccine of the present invention is a 21-valent pneumococcal conjugate vaccine, and the above 21 sugar conjugates are derived from Streptococcus pneumoniae (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. The sugar conjugates derived from serotypes 1 and 5 are conjugated with TT, and all other sugar conjugates are conjugated with CRM 197 and each dose contains approximately 2 μg to approximately 2.5 μg of capsular polysaccharide serotypes 1, 3, 4, 5, 6A, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F, and approximately 4 to approximately 5 μg of capsular polysaccharide of serotype 6B, approximately 2 μg to approximately 25 μg of TT, and approximately 40 μg to approximately 75 μg of CRM 197 .

[0269] In one embodiment, the pneumococcal conjugate vaccine of the present invention is a 21-valent pneumococcal conjugate vaccine, and the above 21 sugar conjugates are derived from Streptococcus pneumoniae (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. The sugar conjugates derived from serotypes 1 and 5 are conjugated with TT, and all other sugar conjugates are conjugated with CRM 197 and each dose contains approximately 2 μg to approximately 2.5 μg of capsular polysaccharide serotypes 1, 3, 4, 5, 6A, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F, and approximately 4 to approximately 5 μg of capsular polysaccharide of serotype 6B, approximately 2 μg to approximately 25 μg of TT, approximately 40 μg to approximately 75 μg of CRM 197 , 2-PE, 0.125 - 0.250 mg of elemental aluminum adjuvant, sodium chloride, and succinic acid buffer.

[0270] In one embodiment, the pneumococcal conjugate vaccine of the present invention is a 21-valent pneumococcal conjugate vaccine, and the above 21 sugar conjugates are derived from Streptococcus pneumoniae (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. The sugar conjugates derived from serotypes 1 and 5 are conjugated with TT, and all other sugar conjugates are conjugated with CRM 197 and each dose contains approximately 2.2 μg of capsular polysaccharide of serotypes 1, 3, 4, 5, 6A, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F, and approximately 4.4 μg of capsular polysaccharide of serotype 6B, approximately 2 μg to approximately 25 μg of TT, approximately 40 μg to approximately 75 μg of CRM 197 , 2-PE, 0.125 - 0.250 mg of elemental aluminum adjuvant, sodium chloride, and succinic acid buffer.

[0271] In one embodiment, the pneumococcal conjugate vaccine of the present invention is a 21-valent pneumococcal conjugate vaccine, and the above 21 sugar conjugates are derived from Streptococcus pneumoniae (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. The sugar conjugates derived from serotypes 1 and 5 are conjugated with TT, and all other sugar conjugates are conjugated with CRM 197 and each dose contains approximately 2.2 μg of capsular polysaccharide of serotypes 1, 3, 4, 5, 6A, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F, and approximately 4.4 μg of capsular polysaccharide of serotype 6B, approximately 2 μg to approximately 25 μg of TT, approximately 40 μg to approximately 75 μg of CRM 197 , 2-PE, 0.125 - 0.250 mg of elemental aluminum adjuvant, polysorbate 80, sodium chloride, and succinic acid buffer.

[0272] In a preferred embodiment, the dosage of the vaccine of the present invention is a dosage of 0.5 mL.

[0273] As used herein, the term "about" means within a statistically meaningful range of a value, e.g., an amount of a disclosed 2-PE, polysaccharide, carrier protein, concentration, volume or pH of a buffer. Such ranges may be within one digit of a given value or range, generally within 20%, more generally within 10%, even more generally within 5% or 1% of a given value or range. Sometimes such ranges may be within the range of experimental error typical of the standard methods used to measure and / or determine a given value or range. The allowable variation encompassed by the term "about" depends on the particular system under test and can be readily understood by one of ordinary skill in the art. Whenever a range is described in this application, every number within that range is also intended as an embodiment of the present disclosure.

[0274] The terms "comprising", "comprise" and "comprises" are, in this specification, in each case, intended by the inventors to be interchangeable with the terms "consisting essentially of", "consist essentially of", "consists essentially of", "consisting of", "consist of" and "consists of" as the case may be.

[0275] The terms "immunogenic amount", "immunologically effective amount", "therapeutically effective amount", "prophylactically effective amount", or "dosage" are each used interchangeably herein and generally refer 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 ordinary skill in the art.

[0276] Any integer falling within any of the ranges in this specification is intended as an embodiment of the present disclosure.

[0277] All references or patent applications cited within this patent specification are hereby incorporated by reference into this specification.

[0278] The present invention is illustrated by the accompanying examples. The following examples are carried out using standard techniques well known and conventional to those skilled in the art, unless otherwise described in detail. The examples are illustrative and do not limit the present invention.

Examples

[0279] (Example 1) Solubility of 2-Phenoxyethanol (2-PE) The purpose of this test was to evaluate the dissolution time and solubility of 2-phenoxyethanol (2-PE). The 2-PE dissolution and concentration were evaluated in succinic acid buffer solution (SBS) (5 mM succinic acid buffer, 150 mM NaCl, pH 5.8) at the following concentrations: 11 g / L, 16.5 g / L, and 20 g / L.

[0280] An appropriate amount of 2-PE was weighed and placed in a 400 mL beaker, and an appropriate amount of succinic acid buffer solution was added. The stirring speed was set at 300 rpm, and the solution temperature was set at approximately 20°C. The time required for complete dissolution was recorded based on visual observation. The 2-PE concentration was measured by RP-HPLC.

[0281] The results of the dissolution time are shown in Table 1, and the results of the 2-PE concentration are shown in Table 2. The dissolution time of most of the 20 mg / mL 2-PE in succinic acid buffer solution (15 minutes) was longer than that of the 11 mg / mL sample (7 minutes) and the 16.5 mg / mL sample (9 minutes). After the completion of the first two dissolution tests (11 mg / mL and 16.5 mg / mL), a small amount of small particles were observed, but the complete dissolution at the initial mixing speed (300 rpm) was not followed. Based on these findings, the complete dissolution time of the 20 mg / mL sample was monitored. The complete dissolution (dissolution of all small particles) time for the 20 mg / mL sample was 35 minutes.

[0282] [Table 1]

[0283] [Table 2]

[0284] From the 2-PE concentration data, it is demonstrated that 2-PE is soluble at 19.1 mg / mL or higher in succinic acid buffer solution (pH 5.8) at 20 °C.

[0285] All the measured 2-PE concentration results were approximately 5% below the target concentration. Since the percent differences were consistent at all concentrations, it was not expected that the measurement result of being 5% below the target for the 20 mg / mL sample would indicate that the dissolution limit of 2-PE in succinic acid buffer solution (at 20 °C) had been reached.

[0286] (Example 2) Effect on filter integrity when a polyethersulfone (PES) membrane (0.5 μm / 0.2 μm) filter is exposed to a polyvalent conjugate pool containing 2-phenoxyethanol The objective of this test was to evaluate the effect on filter integrity when a PES membrane (0.5 μm / 0.2 μm) filter was exposed to a 20-valent conjugate pool formulation for at least 48 hours. The test was performed on both the filtration membrane and the filtration device. This compatibility test is applicable to other filter sizes of the same filter family.

[0287] 20-valent conjugate pool (Streptococcus pneumoniae (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 conjugated to CRM, each at a concentration of approximately 7.5 μg / ml except for 6B which was approximately 15 μg / ml, 5.5 mM succinic acid buffer pH 5.8, 0.033% (w / w) PS80, approximately 150 mM NaCl, see WO2015110941) was formulated with 17 mg / mL of 2-phenoxyethanol (final concentration). The concentration of the conjugate is based on the sugar content (sugar concentration by anthrone). 197 Conjugate pool (Streptococcus pneumoniae (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 conjugated to CRM, each at a concentration of approximately 7.5 μg / ml except for 6B which was approximately 15 μg / ml, 5.5 mM succinic acid buffer pH 5.8, 0.033% (w / w) PS80, approximately 150 mM NaCl, see WO2015110941) was formulated with 17 mg / mL of 2-phenoxyethanol (final concentration). The concentration of the conjugate is based on the sugar content (sugar concentration by anthrone).

[0288] 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 by buffer and addition of aluminum phosphate (AlPO 4 )).

[0289] Two capsule filters were sufficiently wetted by passing 1 L of deionized (DI) water through each filter. Then, both filters were subjected to a pre-use filter integrity test (FIT) at ambient temperature (22 ± 4°C). If both filters passed the pre-use FIT, the conjugate pool material containing 17 mg / mL of 2-PE was filled into each filtration device. The conjugate pool material was left standing in each filtration device at 22 ± 4°C for a minimum of 48 hours. After 48 hours, the conjugate pool material was drained from the filtration device and then rinsed with 2 L of DI water. Then, both filtration devices were subjected to a post-use FIT at 22 ± 4°C.

[0290] The criterion for FIT qualification is the rated bubble point provided by the manufacturer for the capsule filter [air containing water with ≧ 4000 mbar (≧ 58 psi)].

[0291] The results are presented in Table 3.

[0292]

Table 3

[0293] From the results of this test, it is demonstrated that when a polyethersulfone (PES) membrane (0.5 μm / 0.2 μm) filter is exposed to the conjugate pool formulation for at least 48 hours, there is no impact on filter integrity. Both filters passed the pre-use integrity test and the post-use integrity test.

[0294] This compatibility test is applicable to other filter sizes of the same filter family.

[0295] (Example 3) Rate of addition and mixing of 2-phenoxyethanol to the multivalent conjugate pool The objective of this test was to determine the addition rate and mixing speed required to dissolve 2-PE in the conjugate pool without significantly increasing the turbidity of the intermediate solution.

[0296] Formulations of the 20-valent conjugate pool (see Example 2) with 17 mg / mL and 20 mg / mL of 2-PE were shown to have a significant increase in turbidity. For both formulations, the addition of 2-PE to the conjugate pool was carried out without controlling the rate of addition of 2-PE or the mixing of the conjugate pool during and after addition. These two factors were hypothesized to play a role in the observed increase in turbidity.

[0297] Tests were conducted to understand whether it is necessary to control the addition of 2-PE to the conjugate pool.

[0298] In this test, the potential roles of the 2-PE addition rate and mixing speed in the dissolution of 2-PE into the conjugate pool were evaluated.

[0299] 1.97 L of the 20-valent conjugate pool (see Example 2) was formulated without adding 2-PE, targeting a final volume of 2 L and a final 2-PE concentration of 15 mg / mL. The bulk conjugate pool was separated into 197-mL aliquots, and 2-PE was added to the individual aliquots at various addition and mixing rates.

[0300] The evaluated aliquots are listed in Table 4.

[0301] [Table 4]

[0302] 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 a magnetic stir bar was added. As the immediate addition aliquot, 2.71 mL of 2-PE (3 g of 2-PE added, 2-PE density 1.107 g / ml) was manually added immediately (i.e., all at once) to the conjugate pool using a 5-mL disposable syringe with a 21 G 1” stainless steel needle. For the addition rate control aliquots, 2-PE (the same amount) was added to the mixed conjugate pool using the same syringe and needle, but a syringe pump was used to control the dispersion rate of 2-PE for addition. The syringe pump was programmed so that 2-PE was added from the syringe at the specified rate above. For all aliquots, the mixing plate was set at 200 RPM for 2 hours. For immediate addition and delayed mixing, mixing was delayed 30 minutes after 2-PE addition was complete.

[0303] A qualitative method was utilized by evaluating the appearance after dissolution of 2-PE in the conjugate pools divided into aliquots. Videos were taken to capture the addition of 2-PE to the conjugate pools divided into aliquots. For all aliquots, after mixing for 2 hours following the addition of 2-PE, images were acquired and the appearances were compared with each other within the appearance box.

[0304] In the aliquots with addition and immediate mixing at 1 mL / min and addition and immediate mixing at 0.1 mL / min, the observed turbidity seemed to be removed. However, when these aliquots were held near the light source, a slight increase in turbidity was observed compared to the control conjugate pool (without 2-PE).

[0305] From the results of this test, it is demonstrated that the degree of turbidity of the conjugate pool after the addition of 2-PE depends on the addition rate and mixing time. The appearances of the immediate addition and delayed mixing aliquots were significantly more turbid than those of the immediate addition and immediate mixing aliquots, suggesting that mixing during the addition of 2-PE to the conjugate pool directly affects turbidity formation. The addition rate control aliquots had significantly less turbidity than the immediate addition aliquots. Furthermore, a slower addition rate of 2-PE was correlated with a decrease in the turbidity of the conjugate pool after the completion of 2-PE dissolution.

[0306] (Example 4) Evaluation of the Addition of 2-Phenoxyethanol (2-PE) for Formulating a Multivalent Conjugate Vaccine The objective of this test was to evaluate the addition of 2-PE in the process of formulating a 20-valent conjugate vaccine with alum as an adjuvant. 2-PE was added at two different rates either to the 1-final formulated bulk vaccine or to the 2- and 3-conjugate pools before the final formulation.

[0307] The main process steps evaluated were 2-PE addition, filtration, and the addition of the adjuvant (AlPO 4 )).

[0308] 1: Addition of 2-PE to the final formulated bulk vaccine The 20-valent conjugate pool (see Example 2) was mixed at 200 rpm and maintained for at least 5 minutes.

[0309] A pre-filtered sample was obtained (135 ml). The conjugate pool sample was filtered using a product volume: filter surface area ratio (L / m 2 ) of approximately 76.3 (0.5 / 0.2 μm polyethersulfone (PES) filter). Filtration was started using a peristaltic pump. After filtering approximately half of the total volume, the filtration operation was paused by stopping the peristaltic pump. The product was held in the filter for at least 30 minutes and then the filtration operation was restarted to evaluate the pause in filtration during production.

[0310] A post-filtration sample was obtained.

[0311] Succinic acid buffer solution and AlPO 4 were added (succinic acid buffer (pH 5.8) at a final concentration of 5 mM, sodium chloride at 150 mM, and aluminum phosphate (AlPO 4 ) as 0.25 mg / mL of aluminum), and the final drug product was mixed for at least 120 minutes.

[0312] 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.

[0313] After mixing for 120 minutes after 2-PE addition, mixing was stopped.

[0314] 2: 2-PE was added to the conjugate pool at a rate of 5.0 mL / min / L before the final formulation (5.5 g / min / L of 2-PE was added) 4 L of the 20-valent conjugate pool (see Example 2) was added to a 5 L glass container. Mixing at 200 rpm was started and maintained for at least 5 minutes. 2-PE was added using a syringe pump at 20 mL / min. See Table 5 for 2-PE addition parameters.

[0315] After the addition of 2-PE was completed, samples were taken from the top / bottom of the container.

[0316] The 2-PE dissolution time determined by visual observation was recorded. See Table 7.

[0317] After mixing for 120 minutes, the mixing was stopped.

[0318] Preliminary filtered samples were taken (135 ml) and photographs were taken.

[0319] The conjugate pool sample was filtered using a product volume: filter surface area ratio (L / m 2 ) of approximately 76.3 (0.5 / 0.2 μm polyethersulfone (PES) filter). Filtration was started using a peristaltic pump. After filtering approximately half of the total volume, the filtration operation was paused by stopping the peristaltic pump. After holding the product in the filter for at least 30 minutes, the filtration operation was restarted and the filter hold time was determined.

[0320] Post-filter samples were taken.

[0321] Succinic acid buffer solution and AlPO 4 were added (succinic acid buffer (pH 5.8) at a final concentration of 5 mM, sodium chloride at 150 mM, and aluminum phosphate (AlPO 4 ) as 0.25 mg / mL aluminum), and the final drug product was mixed for at least 120 minutes.

[0322] 3: High-speed 2-PE addition to the conjugate pool before final formulation 400 mL of the 20-valent conjugate pool (see Example 2) was added to a 500 mL glass container. Mixing was started at 200 rpm and maintained for at least 5 minutes.

[0323] 2-PE was added using a syringe (added as quickly as possible), and the total time and amount of 2-PE addition were recorded. See Table 5 for 2-PE addition parameters.

[0324] After mixing for 120 minutes, the mixing was stopped.

[0325] A pre-filtered sample was taken (135 ml) and a photo was taken. The conjugate pool sample was filtered using a product volume: filter surface area ratio (L / m 2 ) of approximately 76.3 (0.5 / 0.2 μm polyethersulfone (PES) filter). Filtration was started using a peristaltic pump. After filtering approximately half of the total volume, the filtration operation was paused by stopping the peristaltic pump. The product was held in the filter for at least 30 minutes, then the filtration operation was restarted and the filter hold time was determined.

[0326] A post-filtered sample was taken.

[0327] Succinic acid buffer solution and AlPO 4 were added (succinic acid buffer (pH 5.8) at a final concentration of 5 mM, sodium chloride at 150 mM, and aluminum as aluminum phosphate (AlPO 4 ) at 0.25 mg / mL), and the final drug product (DP) was mixed for at least 120 minutes.

[0328]

Table 5

[0329] Results: In this study, options for adding 2-PE by batch formulation, including adding 2-PE to the conjugate pool, adding 2-PE rapidly to the conjugate pool, and adding 2-PE to the final drug product, were evaluated. Regarding the addition of 2-PE to the conjugate pool, it was demonstrated from the data that 2-PE was uniformly diffused in the bulk material. No significant turbidity was observed in any of the three prefiltered samples. Various quality characteristics of the drug product were evaluated, and the drug product met all the criteria. From these results, the feasibility of adding 2-PE to the conjugate pool (before addition of AlPO 4 and before filtration sterilization) was demonstrated.

[0330] Aspects of the invention The following clauses describe additional embodiments of the invention. C1. A process for preparing a conjugate vaccine comprising a preservative, comprising: (a) adding the preservative from a bulk solution to a solution comprising one or more conjugates without filter sterilizing the preservative; (b) subsequently, sterile filtering the mixture comprising the preservative and the conjugate; and

[0331] C2. The process according to C1, further comprising (c) subsequently adding an adjuvant.

[0332] C3. The process according to C2, wherein the adjuvant is aluminum phosphate.

[0333] C4. The process according to C3, wherein the final concentration of aluminum phosphate in the vaccine is between about 0.1 mg / mL and about 0.5 mg / mL.

[0334] C5. The process according to C4, wherein the final concentration of aluminum phosphate in the vaccine is about 0.25 mg / mL.

[0335] Process C6. Any one of the processes C1 to C5, wherein the preservative is hydrophobic and viscous at the concentration of the bulk solution.

[0336] Process C7. Any one of the processes C1 to C6, wherein the viscosity of the preservative at the concentration of the bulk solution is at least 10 centistokes at 25°C.

[0337] Process C8. Any one of the processes C1 to C6, wherein the viscosity of the preservative at the concentration of the bulk solution is at least 15 centistokes at 25°C.

[0338] Process C9. Any one of the processes C1 to C6, wherein the viscosity of the preservative at the concentration of the bulk solution is at least 20 centistokes at 25°C.

[0339] Process C10. Any one of the processes C1 to C6, wherein the viscosity of the preservative at the concentration of the bulk solution is between about 10 centistokes and about 50 centistokes at 25°C.

[0340] Process C11. Any one of the processes C1 to C6, wherein the viscosity of the preservative at the concentration of the bulk solution is between about 15 centistokes and about 25 centistokes at 25°C.

[0341] Process C12. Any one of the processes C1 to C11, wherein the preservative is 2-phenoxyethanol (2-PE), phenol, metacresol, methylparaben, propylparaben or thimerosal.

[0342] Process C13. Any one of the processes C1 to C11, wherein the preservative is 2-phenoxyethanol (2-PE) or thimerosal.

[0343] Process C14. Any one of the processes C1 to C13, wherein the preservative is added without dilution.

[0344] Process C15. Any one of the processes C1 to C14, wherein the bulk solution of the preservative is pure undiluted preservative.

[0345] Process of any one of C1 to C15 where the preservative is 2-phenoxyethanol (2-PE).

[0346] Process of any one of C1 to C16 where the bulk solution is undiluted 2-phenoxyethanol (2-PE).

[0347] Process according to C17 where 2-PE is diluted in the solution after step (a) between about 10-fold and 200-fold.

[0348] Process according to C17 where 2-PE is diluted in the solution after step (a) to about 50-fold, about 60-fold, about 70-fold, or about 80-fold.

[0349] Process according to C17 where 2-PE is diluted in the solution after step (a) between about 73-fold and 74-fold.

[0350] Process of any one of C16 to C20 where the concentration of 2-PE in the solution after step (a) is between about 1 mg / ml and about 25 mg / ml.

[0351] Process of any one of C16 to C20 where the concentration of 2-PE in the solution after step (a) is between about 10 mg / ml and about 20 mg / ml.

[0352] Process of any one of C16 to C20 where the concentration of 2-PE in the solution after step (a) is between about 15 mg / ml and about 17 mg / ml.

[0353] Process of any one of C16 to C20 where the concentration of 2-PE in the solution after step (a) is about 15 mg / ml.

[0354] Process of any one of C16 to C20 where the concentration of 2-PE in the solution after step (a) is about 17 mg / ml.

[0355] A process from C16 to C25, wherein C26.2-PE is added at an addition rate between about 0.5 ml / min per liter of the solution containing one or more conjugates and about 5.0 ml / min per liter of the solution containing one or more conjugates.

[0356] A process from C16 to C25, wherein C27.2-PE is added at an addition rate between about 1 ml / min per liter of the solution containing one or more conjugates and about 4.0 ml / min per liter of the solution containing one or more conjugates.

[0357] A process from C17 to C27, wherein undiluted 2-PE is added using a pump.

[0358] A process from C17 to C27, wherein undiluted 2-PE is added using a peristaltic pump.

[0359] A process from C17 to C27, wherein undiluted 2-PE is added using a peristaltic pump with a silicone or polyurethane tube.

[0360] A process from C17 to C27, wherein undiluted 2-PE is added using a peristaltic pump with a thermoplastic elastomer tube.

[0361] A process from C16 to C31, wherein the final concentration of 2-PE in the vaccine is between about 5 mg / ml and about 15 mg / ml.

[0362] A process from C16 to C31, wherein the final concentration of 2-PE in the vaccine is between about 7 mg / ml and about 12 mg / ml.

[0363] A process from C16 to C31, wherein the final concentration of 2-PE in the vaccine is about 10 mg / ml.

[0364] Process of any one of C16 to C31, wherein the final concentration of 2-PE in the vaccine is about 9 mg / ml.

[0365] Process of any one of C1 to C35, wherein the volume of the solution after step (a) is between about 100 L and about 500 L.

[0366] Process of any one of C1 to C35, wherein the volume of the solution after step (a) is about 180 L.

[0367] Process of any one of C1 to C37, wherein the preservative is added under continuous mixing.

[0368] Process according to C38, wherein there is no delay between the addition of the preservative and the start of mixing.

[0369] Process of any one of C1 to C39, wherein the preservative is added under continuous mixing and the angular velocity is between about 50 rpm and about 500 rpm.

[0370] Process of any one of C1 to C39, wherein the preservative is added under continuous mixing and the angular velocity is between about 100 rpm and about 400 rpm.

[0371] Process of any one of C1 to C39, wherein the preservative is added under continuous mixing and the angular velocity is between about 150 rpm and about 300 rpm.

[0372] Process of any one of C1 to C39, wherein the preservative is added under continuous mixing and the angular velocity is between about 150 rpm and about 200 rpm.

[0373] Process of any one of C1 to C39, wherein the preservative is added under continuous mixing and the angular velocity is about 150 rpm.

[0374] Process C1 to C39, where the preservative is added under continuous mixing and the angular velocity is about 200 rpm.

[0375] Process C1 to C45, where the solution is mixed for about 15 minutes to about 5 hours after the addition of the preservative and before filtration sterilization.

[0376] Process C1 to C45, where the solution is mixed for about 30 minutes to about 3 hours after the addition of the preservative and before filtration sterilization.

[0377] Process C1 to C45, where the solution is mixed for about 1 hour to about 3 hours after the addition of the preservative and before filtration sterilization.

[0378] Process C1 to C45, where the solution is mixed for about 2 hours after the addition of the preservative and before filtration sterilization.

[0379] Process C1 to C49, where the solution is mixed at an angular velocity between about 50 rpm and about 500 rpm after the addition of the preservative and before filtration sterilization.

[0380] Process C1 to C49, where the solution is mixed at an angular velocity between about 100 rpm and about 400 rpm after the addition of the preservative and before filtration sterilization.

[0381] Process C1 to C49, where the solution is mixed at an angular velocity between about 150 rpm and about 300 rpm after the addition of the preservative and before filtration sterilization.

[0382] Process C1 to C52, where the solution to which the preservative is added is a monovalent composition.

[0383] Process C1 to C52, where the solution to which the preservative is added is a divalent, trivalent, tetravalent, pentavalent or hexavalent composition.

[0384] Process of any one of C1 to C52, wherein the solution to which the preservative is added is a multivalent composition containing seven or more conjugates.

[0385] Process of any one of C1 to C52, wherein the solution to which the preservative is added is a 7- to 25-valent composition.

[0386] Process of any one of C1 to C52, wherein the solution to which the preservative is added is a 13- to 25-valent composition.

[0387] Process of any one of C1 to C52, wherein the solution to which the preservative is added is a 15-valent composition.

[0388] Process of any one of C1 to C52, wherein the solution to which the preservative is added is a 16-valent composition.

[0389] Process of any one of C1 to C52, wherein the solution to which the preservative is added is a 20-valent composition.

[0390] Process of any one of C1 to C52, wherein the solution to which the preservative is added is a 21-valent composition.

[0391] Process of any one of C1 to C52, wherein the solution to which the preservative is added is a multivalent pneumococcal conjugate composition.

[0392] Process of any one of C1 to C52, wherein the solution to which the preservative is added is a pneumococcal conjugate composition containing 7 to 25 sugar conjugates derived from different serotypes of Streptococcus pneumoniae (S. pneumoniae).

[0393] Process according to any one of C1 to C52, wherein the solution to which the preservative is added is a pneumococcal conjugate composition comprising conjugates derived from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 different serotypes of Streptococcus pneumoniae (S. pneumoniae).

[0394] Process according to any one of C1 to C52, wherein the solution to which the preservative is added is a pneumococcal conjugate composition comprising conjugates derived from 20 different serotypes of Streptococcus pneumoniae (S. pneumoniae).

[0395] Process according to any one of C1 to C52, wherein the solution to which the preservative is added is a 7-valent, 8-valent, 9-valent, 10-valent, 11-valent, 12-valent, 13-valent, 14-valent, 15-valent, 16-valent, 17-valent, 18-valent, 19-valent or 20-valent pneumococcal conjugate composition.

[0396] Process according to any one of C1 to C52, wherein the solution to which the preservative is added is a 15-valent pneumococcal conjugate composition.

[0397] Process according to any one of C1 to C52, wherein the solution to which the preservative is added is a 20-valent pneumococcal conjugate composition.

[0398] Process according to any one of C1 to C52, wherein the solution to which the preservative is added is a 21-valent pneumococcal conjugate composition.

[0399] Process according to any one of C1 to C52, wherein the solution to which the preservative is added is a 22-valent pneumococcal conjugate composition.

[0400] Process according to any one of C1 to C52, wherein the solution to which the preservative is added is a 21-valent, 22-valent, 23-valent, 24-valent or 25-valent pneumococcal conjugate composition.

[0401] A process according to any one of C1 to C52, wherein the solution to which the preservative is added contains sugar conjugates derived from Streptococcus pneumoniae (S. pneumoniae) serotypes 4, 6B, 9V, 14, 18C, 19F and 23F.

[0402] A process according to any one of C1 to C52, wherein the solution to which the preservative is added contains sugar conjugates derived from Streptococcus pneumoniae (S. pneumoniae) serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F.

[0403] A process according to any one of C1 to C52, wherein the solution to which the preservative is added contains sugar conjugates derived from Streptococcus pneumoniae (S. pneumoniae) serotypes 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F.

[0404] A process according to any one of C1 to C52, wherein the solution to which the preservative is added contains sugar conjugates derived from Streptococcus pneumoniae (S. pneumoniae) serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F.

[0405] A process according to any one of C1 to C52, wherein the solution to which the preservative is added contains sugar conjugates derived from Streptococcus pneumoniae (S. pneumoniae) serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F.

[0406] A process according to any one of C1 to C52, wherein the solution to which the preservative is added contains sugar conjugates derived from Streptococcus pneumoniae (S. pneumoniae) serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F.

[0407] Process C1 to C52, wherein the solution to which the preservative is added contains sugar conjugates derived from Streptococcus pneumoniae (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.

[0408] Process C1 to C52, wherein the solution to which the preservative is added contains sugar conjugates derived from Streptococcus pneumoniae (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.

[0409] Process C1 to C52, wherein the solution to which the preservative is added is a 10-valent pneumococcal sugar conjugate composition, and the above 10 sugar conjugates are derived from Streptococcus pneumoniae (S. pneumoniae) serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F.

[0410] Process C1 to C52, wherein the solution to which the preservative is added is a 13-valent pneumococcal sugar conjugate composition, and the above 13 sugar conjugates are derived from Streptococcus pneumoniae (S. pneumoniae) serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F.

[0411] Process C1 to C52, wherein the solution to which the preservative is added is a 15-valent pneumococcal sugar conjugate composition, and the above 15 sugar conjugates are derived from Streptococcus pneumoniae (S. pneumoniae) serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F.

[0412] The solution to which the preservative is added is a 20-valent pneumococcal conjugate composition, and the above 20 sugar conjugates are derived 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, and the process is any one of C1 to C52.

[0413] The solution to which the preservative is added is a 21-valent pneumococcal conjugate composition, and the above 21 sugar conjugates are derived 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, and the process is any one of C1 to C52.

[0414] The solution to which the preservative is added is a 21-valent pneumococcal conjugate composition, and the above 21 sugar conjugates are derived 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, and the process is any one of C1 to C52.

[0415] The solution to which the preservative is added is a 22-valent pneumococcal conjugate composition, and the above 22 sugar conjugates are derived 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, and the process is any one of C1 to C52.

[0416] The solution to which the preservative is added is a 21-valent pneumococcal polysaccharide conjugate composition, and the above 21 polysaccharide conjugates are derived 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, and it is any one process from C1 to C52.

[0417] C88. The solution to which the preservative is added is a 21-valent pneumococcal polysaccharide conjugate composition, and the above 21 polysaccharide conjugates are derived 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, and it is any one process from C1 to C52.

[0418] C89. The solution to which the preservative is added is a 21-valent pneumococcal polysaccharide conjugate composition, and the above 21 polysaccharide conjugates are derived 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, and it is any one process from C1 to C52.

[0419] C90. The solution to which the preservative is added is a 21-valent pneumococcal polysaccharide conjugate composition, and the above 21 polysaccharide conjugates are derived 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, and it is any one process from C1 to C52.

[0420] The solution to which the preservative is added is a 21-valent pneumococcal conjugate composition, and the 21 sugar conjugates are derived 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, and any one of the processes C1 to C52.

[0421] The solution to which the preservative is added is a 20-valent pneumococcal conjugate composition, and the 20 sugar conjugates are derived 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, and any one of the processes C1 to C52.

[0422] C93. The carrier protein of the sugar conjugate is TT, CRM 197 or C5a peptidase (SCP) derived from the genus Streptococcus, and any one of the processes C63 to C92.

[0423] C94. The carrier protein of the sugar conjugate is CRM 197 and any one of the processes C63 to C92.

[0424] C95. At least one capsular polysaccharide is conjugated to TT, and all other capsular polysaccharides are conjugated to CRM 197 and any one of the processes C63 to C92.

[0425] C96. One capsular polysaccharide is conjugated to TT, and all other capsular polysaccharides are conjugated to CRM 197 and any one of the processes C63 to C92.

[0426] C97. At least two capsular polysaccharides are conjugated to TT, and all other capsular polysaccharides are conjugated to CRM 197A process of conjugating with any one of C63 to C92.

[0427] C98. Two types of capsular polysaccharides are conjugated with TT, and all other capsular polysaccharides are CRM 197 A process of conjugating with any one of C63 to C92.

[0428] C99. At least three types of capsular polysaccharides are conjugated with TT, and all other capsular polysaccharides are CRM 197 A process of conjugating with any one of C63 to C92.

[0429] C100. Three types of capsular polysaccharides are conjugated with TT, and all other capsular polysaccharides are CRM 197 A process of conjugating with any one of C63 to C92.

[0430] C101. Four types of capsular polysaccharides are conjugated with TT, and all other capsular polysaccharides are CRM 197 A process of conjugating with any one of C63 to C92.

[0431] C102. The solution to which a preservative is added is a 21-valent pneumococcal conjugate composition. The above 21 sugar conjugates are derived 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. Four types of capsular polysaccharides are conjugated with TT, and the four types of capsular polysaccharides conjugated with TT are two other serotypes selected from the group consisting of serotypes 15B, 22F, and serotypes 1, 3, and 5. All other sugar conjugates are conjugated with CRM 197 A process of any one of C1 to C52 that is conjugated with.

[0432] The solution to which the preservative is added is a 21-valent pneumococcal conjugate composition. The above 21 sugar conjugates are derived 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. Four capsular polysaccharides are conjugated with TT, and the four capsular polysaccharides conjugated with TT are serotypes 1, 5, 15B, and 22F. All other sugar conjugates are CRM 197 One of the processes from C1 to C52, which is a conjugate with CRM.

[0433] The solution to which the preservative is added is a 21-valent pneumococcal conjugate composition. The above 21 sugar conjugates are derived 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. Four capsular polysaccharides are conjugated with TT, and the four capsular polysaccharides conjugated with TT are serotypes 1, 3, 15B, and 22F. All other sugar conjugates are CRM 197 One of the processes from C1 to C52, which is a conjugate with CRM.

[0434] The solution to which the preservative is added is a 21-valent pneumococcal conjugate composition. The above 21 sugar conjugates are derived 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. Four capsular polysaccharides are conjugated with TT, and the four capsular polysaccharides conjugated with TT are serotypes 3, 5, 15B, and 22F. All other sugar conjugates are CRM 197 One of the processes from C1 to C52, which is a conjugate with CRM.

[0435] The solution to which the preservative is added is a 21-valent pneumococcal conjugate composition, and the above 21 sugar conjugates are derived 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. Two sugar conjugates selected from serotypes 1, 3 and 5 are conjugated with TT, and all other sugar conjugates are conjugated with CRM 197 One of the processes C1 to C52, wherein the solution to which the preservative is added is a 21-valent pneumococcal conjugate composition, and the above 21 sugar conjugates are derived 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. Two sugar conjugates selected from serotypes 1, 3 and 5 are conjugated with TT, and all other sugar conjugates are conjugated with CRM

[0436] The solution to which the preservative is added is a 21-valent pneumococcal conjugate composition, and the above 21 sugar conjugates are derived 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. The sugar conjugates derived from serotypes 1 and 3 are conjugated with TT, and all other sugar conjugates are conjugated with CRM 197 One of the processes C1 to C52, wherein the solution to which the preservative is added is a 21-valent pneumococcal conjugate composition, and the above 21 sugar conjugates are derived 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. The sugar conjugates derived from serotypes 1 and 3 are conjugated with TT, and all other sugar conjugates are conjugated with CRM

[0437] The solution to which the preservative is added is a 21-valent pneumococcal conjugate composition, and the above 21 sugar conjugates are derived 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. The sugar conjugates derived from serotypes 1 and 5 are conjugated with TT, and all other sugar conjugates are conjugated with CRM 197 One of the processes C1 to C52, wherein the solution to which the preservative is added is a 21-valent pneumococcal conjugate composition, and the above 21 sugar conjugates are derived 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. The sugar conjugates derived from serotypes 1 and 5 are conjugated with TT, and all other sugar conjugates are conjugated with CRM

[0438] The solution to which the preservative is added is a 21-valent pneumococcal conjugate composition, and the above 21 sugar conjugates are derived 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. The sugar conjugates derived from serotypes 3 and 5 are conjugated with TT, and all other sugar conjugates are conjugated with CRM 197 One of the processes from C1 to C52, wherein the sugar conjugate is conjugated with CRM

[0439] The solution to which the preservative is added is a 15-valent pneumococcal conjugate composition, and the above 15 sugar conjugates are derived from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, and 33F. All of the sugar conjugates are conjugated with CRM 197 One of the processes from C1 to C52, wherein the sugar conjugate is conjugated with CRM

[0440] The solution to which the preservative is added is a 20-valent pneumococcal conjugate composition, and the above 20 sugar conjugates are derived 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. All of the sugar conjugates are conjugated with CRM 197 One of the processes from C1 to C52, wherein the sugar conjugate is conjugated with CRM

[0441] One of the processes from C1 to C111, wherein the solution to which the preservative is added contains a pharmaceutically acceptable excipient, carrier, or diluent

[0442] Process C1 to C111, wherein the solution to which the preservative is added contains one or more of a buffer, a salt, a divalent cation, a nonionic surfactant, a cryoprotective substance such as a sugar, and an antioxidant such as a free radical scavenger or a chelating agent, or any combination of these.

[0443] Process C1 to C111, wherein the solution to which the preservative is added contains a buffer.

[0444] The process according to C114, wherein the pKa of the buffer is from about 3.5 to about 7.5.

[0445] The process according to C114, wherein the buffer is a phosphate buffer, a succinate buffer, a histidine buffer or a citrate buffer.

[0446] The process according to C114, wherein the buffer is a succinate buffer.

[0447] The process according to C114, wherein the buffer is a histidine buffer.

[0448] The process according to C114, wherein the buffer is a histidine buffer having a concentration of 10 mM to 30 mM.

[0449] The process according to C114, wherein the buffer is a histidine buffer having a concentration of 15 mM to 25 mM.

[0450] The process according to C114, wherein the buffer is a histidine buffer having a concentration of about 22 mM.

[0451] The process according to C114, wherein the buffer is a succinate buffer having a concentration of 1 mM to 10 mM.

[0452] The process according to C114, wherein the buffer is a succinate buffer having a concentration of about 5.5 mM.

[0453] Process C1 to C123 in which the solution to which the preservative is added contains a salt.

[0454] The process according to C124, wherein the salt is magnesium chloride, potassium chloride, sodium chloride, or a combination thereof.

[0455] The process according to C124, wherein the salt is sodium chloride.

[0456] Process C1 to C123 in which the solution to which the preservative is added contains about 150 mM of sodium chloride.

[0457] Process C1 to C127 in which the solution to which the preservative is added contains a surfactant.

[0458] The process according to C128, wherein the surfactant is polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, TRITON™ N-101, TRITON™ X-100, octoxynol 40, nonoxynol-9, triethanolamine, triethanolamine oleate polypeptide, polyoxyethylene-660 hydroxystearate (PEG-15, Solutol H15), polyoxyethylene-35-ricinoleate (CREMOPHOR® EL), soy lecithin, or poloxamer.

[0459] The process according to C129, wherein the surfactant is polysorbate 80 or polysorbate 20.

[0460] The process according to C129, wherein the surfactant is polysorbate 20.

[0461] The process according to C131, wherein the concentration of polysorbate 20 in the solution is between 0.01% and 1% by weight to weight (w / w) of polysorbate 20.

[0462] The process according to C131, wherein the concentration of polysorbate 20 in the solution is between 0.1% and 0.5% by weight to weight (w / w) of polysorbate 20.

[0463] The process according to 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% of polysorbate 20 (w / w).

[0464] The process according to C131, wherein the concentration of polysorbate 20 in the solution is about 0.33% of polysorbate 20 (w / w).

[0465] The process according to C129, wherein the surfactant is polysorbate 80.

[0466] The process according to C136, wherein the concentration of polysorbate 80 in the solution is between 0.001% and 1% by weight to weight (w / w) of polysorbate 80.

[0467] The process according to C136, wherein the concentration of polysorbate 80 in the solution is between 0.01% and 0.5% by weight to weight (w / w) of polysorbate 80.

[0468] The process according to 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% of polysorbate 80 (w / w).

[0469] The process according to C136, wherein the concentration of polysorbate 80 in the solution is about 0.033% of polysorbate 80 (w / w).

[0470] The process according to C136, wherein the final concentration of polysorbate 80 in the vaccine is between about 0.01% and about 0.03%.

[0471] The process according to C136, wherein the final concentration of polysorbate 80 in the vaccine is about 0.02%.

[0472] The process according to any one of C1 to C142, wherein the pH of the solution to which the preservative is added is between 5.5 and 7.5.

[0473] The process according to any one of C1 to C142, wherein the pH of the solution to which the preservative is added is between 5.6 and 7.0.

[0474] The process according to any one of C1 to C142, wherein the pH of the solution to which the preservative is added is between 5.8 and 6.0.

[0475] The process according to any one of C1 to C142, wherein the pH of the solution to which the preservative is added is about 5.8.

[0476] The process according to any one of C1 to C142, wherein the final pH of the vaccine is between about 5.3 and about 6.3.

[0477] The process according to any one of C1 to C142, wherein the final pH of the vaccine is about 5.8.

[0478] The solution to which the preservative is added is a 20-valent pneumococcal conjugate composition, and the above 20 sugar conjugates are derived 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, and all the sugar conjugates are CRM 197It is conjugated, and the solution to which the preservative is added contains capsular polysaccharides of serotypes 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F, each between about 5 μg / ml and about 10 μg / ml, capsular polysaccharide of serotype 6B between about 10 μg / ml and about 20 μg / ml, polysorbate 80 between about 0.01% and about 0.05% (w / w), about 150 mM sodium chloride, and succinic acid buffer between about 1 mM and about 10 mM, pH 5.8, and is any one of the processes C1 to C52.

[0479] C150. The solution to which the preservative is added is a 15-valent pneumococcal conjugate composition, and the above 15 sugar conjugates are derived from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, and 33F, and all the sugar conjugates are CRM 197 It is conjugated, and the solution to which the preservative is added contains capsular polysaccharides of serotypes 1, 3, 4, 5, 6A, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, and 33F, each between about 5 μg / ml and about 10 μg / ml, capsular polysaccharide of serotype 6B between about 10 μg / ml and about 20 μg / ml, about 150 mM sodium chloride, polysorbate 20 between about 0.1% and about 0.5% (w / w), and L-histidine buffer between about 15 mM and about 25 mM, pH 5.8, and is any one of the processes C1 to C52.

[0480] C151. The solution to which the preservative is added is a 20-valent pneumococcal conjugate composition, and the above 20 sugar conjugates are derived 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, and all the sugar conjugates are CRM 197It is conjugated, and the solution to which the preservative is added contains approximately 7.5 μg / ml of capsular polysaccharides of serotypes 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F, approximately 7.5 μg / ml of capsular polysaccharide of serotype 6B, approximately 0.033% (w / w) of polysorbate 80, approximately 150 mM of sodium chloride, and approximately 5.5 mM of succinic acid buffer, pH 5.8, and is any one process from C1 to C52.

[0481] C152. Any one process from C1 to C151 where the final protein concentration in the vaccine is between approximately 60 μg / ml and approximately 130 μg / ml.

[0482] C153. Any one process from C1 to C151 where the final protein concentration in the vaccine is between approximately 90 μg / ml and approximately 99 μg / ml.

[0483] C154. Any one process from C1 to C153 where the final D10 particle size distribution of the vaccine is between approximately 4,000 and approximately 5,000.

[0484] C155. Any one process from C1 to C153 where the final D10 particle size distribution of the vaccine is between approximately 4,300 and approximately 4,700.

[0485] C156. Any one process from C1 to C153 where the final D50 particle size distribution of the vaccine is between approximately 7,000 and approximately 8,000.

[0486] C157. Any one process from C1 to C153 where the final D50 particle size distribution of the vaccine is between approximately 7,500 and approximately 7,900.

[0487] C158. Any one process from C1 to C153 where the final D90 particle size distribution of the vaccine is between approximately 12,000 and approximately 15,000.

[0488] Process of any one of C1 to C153, wherein the final D90 particle size distribution of the vaccine is between about 12,300 and about 14,900.

[0489] C160. Process of any one of C1 to C159, wherein after step (a), the mixture containing the preservative and the conjugate is sterile filtered.

[0490] C161. Process according to C160, wherein the nominal retention range of the filter is about 0.05 - 0.2 μm.

[0491] C162. Process according to C160, wherein the nominal retention range of the filter is about 0.1 - 0.2 μm.

[0492] C163. Process according to C160, wherein the nominal retention range of the filter is about 0.15 - 0.2 μm.

[0493] C164. Process according to C160, wherein the nominal retention range of the filter is about 0.1 μm, about 0.15 μm or about 0.2 μm.

[0494] C165. Process according to C160, wherein the nominal retention range of the filter is about 0.2 μm.

[0495] C166. Process of any one of C160 to C165, wherein the filter includes a prefilter.

[0496] C167. Process according to C166, wherein the nominal retention range of the prefilter is between about 0.1 μm and 1 μm.

[0497] C168. Process according to C166, wherein the nominal retention range of the prefilter is about 0.5 μm.

[0498] C169. Process according to C166, wherein the nominal retention range of the prefilter is about 0.5 μm and the nominal retention range of the filter is about 0.2 μm.

[0499] C170. The filtration capacity of the filter is about 25 - 1000 L / m 2 , 50 - 1000 L / m 2 , 75 - 1000 L / m 2 , 100 - 1000 L / m 2 , 150 - 1000 L / m 2 , 200 - 1000 L / m 2 , 250 - 1000 L / m 2 , 300 - 1000 L / m 2 , 350 - 1000 L / m 2 , 400 - 1000 L / m 2 , 500 - 1000 L / m 2 or 750 - 1000 L / m 2 and is one of the processes from C160 to C169.

[0500] C171. The filtration capacity of the filter is 200 - 1000 L / m 2 , 250 - 1000 L / m 2 or 300 - 1000 L / m 2 and is one of the processes from C160 to C169.

[0501] C172. The filtration capacity of the filter is about 300 - 1000 L / m 2 and is one of the processes from C160 to C169.

[0502] C173. The filter contains polysulfone and is one of the processes from C160 to C172.

[0503] C174. The filter contains polyethersulfone (PES) and is one of the processes from C160 to C172.

[0504] C175. The filter is a capsule filter and is one of the processes from C160 to C174.

[0505] Process C176, wherein the conjugate vaccine obtained by the above process comprises one or more conjugates and a preservative, is any one of the processes C1 to C175.

Claims

1. A process for preparing a conjugate vaccine comprising a preservative, comprising: (a) adding the 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 a mixture comprising the preservative, the conjugate(s), the buffer, the salt, and the surfactant; wherein the preservative is not sterile filtered prior to addition to the solution, and the preservative is hydrophobic and viscous at the concentration of the bulk solution.

2. The process according to claim 1, further comprising (c) subsequently adding an adjuvant.

3. The process according to claim 2, wherein the adjuvant is aluminum phosphate.

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

5. The process according to any one of claims 1 to 4, wherein the viscosity of the preservative at the concentration of the bulk solution is between about 15 centistokes and about 25 centistokes at 25°C.

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

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

8. The process according to 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 between about 15 mg / ml and about 17 mg / ml.

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

10. The process according to any one of claims 1 to 9, wherein the buffer comprises a succinate buffer, the salt comprises sodium chloride, and the surfactant comprises polysorbate 80.

11. wherein (a) the concentration of the succinate buffer is between about 1 mM and about 10 mM, (b) the concentration of sodium chloride is between about 100 mM and about 200 mM, and (c) the concentration of polysorbate 80 is between about 0.01% and about 0.05%; The process according to claim 10.

12. The process according to any one of claims 1 to 11, wherein the pH of the solution to which the preservative is added is between about 5.8 and about 6.

0.

13. The process according to any one of claims 1 to 12, wherein the preservative is added at a rate of between about 0.5 ml / min per liter of solution and about 5.0 ml / min per liter of solution, the solution containing one or more conjugates, buffers, salts, and surfactants.

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

15. The preservative is added under continuous mixing, with no delay between the start of addition of the preservative and the start of mixing, (a) the angular velocity of mixing is between about 150 rpm and about 200 rpm, or (b) the solution is mixed for about 30 minutes to about 3 hours after addition of the preservative and before filter sterilization, The process according to any one of claims 1 to 14.

16. The process according to any one of claims 1 to 15, wherein the filtration in step (b) involves the use of a filter with a nominal retention range of about 0.1 μm, about 0.15 μm, or about 0.2 μm.

17. The process according to claim 16, wherein the filter includes a prefilter with a nominal retention range of about 0.5 μm.

18. The filtration in step (b) is carried out using a filter with a filtration capacity of 200 to 1000 L / m ２ , 250 to 1000 L / m ２ , or 300 to 1000 L / m ２ The process according to any one of claims 1 to 17, including the use of a filter which is such.

19. The process according to any one of claims 1 to 18, wherein the filtration in step (b) involves the use of a filter containing polyethersulfone (PES).

20. The process according to any one of claims 1 to 19, wherein the solution to which the preservative is added is a pneumococcal conjugate composition containing 7 to 25 sugar conjugates derived from different serotypes of Streptococcus pneumoniae (S. pneumoniae).

21. The process according to any one of claims 1 to 19, wherein the solution to which the preservative is added is a 20-valent pneumococcal sugar conjugate composition, and the 20 sugar conjugates are derived from Streptococcus pneumoniae (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. All of the sugar conjugates are conjugated with CRM １９７ The process according to claim 20 or 21, wherein the sugar conjugate is conjugated with CRM

23. The solution to which the preservative is added is a 20-valent pneumococcal conjugate composition, and the 20 sugar conjugates are derived 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, and all the sugar conjugates are conjugated with CRM １９７ The process according to any one of claims 1 to 19, wherein the solution to which the preservative is added contains approximately 7.5 μg / ml of capsular polysaccharides of serotypes 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F, approximately 7.5 μg / ml of capsular polysaccharide of serotype 6B, approximately 0.033% (w / w) of polysorbate 80, approximately 150 mM of sodium chloride, and approximately 5.5 mM of succinic acid buffer, pH 5.8.

Images