Field of Invention
The present disclosure relates to a novel improved multivalent pneumococcal vaccine compositions comprising capsular pneumococcal polysaccharide of one or more Streptococcus pneumoniae serotypes conjugated to two or more carrier proteins.
The present invention relates to a novel conjugated pneumococcal vaccine composition comprising up to 30 capsular polysaccharides. Specifically capsular pneumococcal polysaccharide of different S. pneumoniae serotypes conjugated to 2 or more different carrier proteins where the composition comprises capsular polysaccharides conjugated to first carrier protein and remaining capsular polysaccharides conjugated to carrier proteins other than first carrier protein. At least three of the capsular polysaccharides selected preferably from the group comprising of serotypes 3, 6A, 6B, 15A, 15B, 18C, 19A, 19F, 23F, 33F, 35F are conjugated to first carrier protein selected from the group of Diphtheria Toxoid, Tetanus toxoid, Cross-reacting material CRM197, Protein D of non-typeable H. influenzae, Exotoxin A of Pseudomonas Aeruginosa (EPA), nontoxic peptide from C. difficile toxin A (CDTA) and Pneumococcal surface protein A (PspA). And polysaccharides from remaining serotypes are conjugated to Cross-reacting material CRM197 herein called as second carrier protein. The process of manufacturing such multivalent vaccine composition using multiple carriers is also disclosed.
Typically incorporating multiple antigens in the vaccine composition also increases the amount of conjugation protein carriers. Therefore one of the aspects of the present invention is to develop a vaccine composition with broader coverage of pneumococcal conjugate composition using reduced or controlled amount of carrier proteins, especially CRM197. The percentage of serotypes conjugated to CRM197 may range from 30% to 90 % of total serotypes, preferably from 30% to 80 %, more preferably from 40% to 80 % and more preferably from
Af\Q/. +rt *7A 0/

The invention also provides a kit comprising all the antigenic components in a single vial/ prefilled syringe or a kit comprising two multi-valent pneumococcal vaccines in two different containers, vials, prefilled syringes or dual chamber syringe.
The present invention overcomes the deficits of prior pneumococcal conjugate vaccines in Asia with lower coverage of serotypes; Instant invention can cover more than 90% of pneumococcal conjugate composition and preparation method and application in all regions of the world.
A composition according to the invention may be administered by any conventional route which is used in the field of vaccines, in particular by the systemic, i.e. parenteral route, e.g. by the subcutaneous, intramuscular, intradermal route for sequential or simultaneous administration of the first and second immunogenic compositions.
Another aspect of the present invention is provide complimentary immunization to a person who is already vaccinated with 7, 10, 11, 13 or 15 valent vaccine and require additional protection against serotypes not present in 7, 10, 11, 13 or 15 valent vaccine.
An objective of the novel immunogenic compositions of the present invention is to provide for appropriate protection against S. pneumoniae serotypes not found in the marketed vaccines.
Background of Invention
Streptococcus pneumoniae is a Gram-positive bacterium and accountable for considerable morbidity and mortality (particularly in the young and aged) worldwide. Streptococcus pneumoniae is leading reason for causing invasive diseases such as pneumonia, bacteremia and meningitis, and diseases associated with colonization, such as acute Otitis media.

Pneumococcus can be divided into 90 types of serotypes. Distribution of these serotypes is determined by various factors such as age, disease syndrome, and its geographic distribution along with the time. The protective efficacy of the pneumococcal polysaccharide vaccine is known to be related to the concentration of antibody generated against a capsular polysaccharide. Pneumococcus cells are encapsulated with a polysaccharide giving rise to more than 90 different pneumococcus serotypes. The capsule is the principal virulence determinant for pneumococci.
The multivalent pneumococcal polysaccharide vaccine compositions have been licensed for many years and have valued in preventing pneumococcal disease in elderly adults and high-risk patients. However, infants and young children respond poorly to most pneumococcal polysaccharides. Children less than 2 years of age do not mount an immune response to maximum polysaccharide vaccines, so it has been necessary to render the polysaccharides immunogenic by chemical conjugation to a protein carrier.
Early step in that direction was Prevnar®-7 which demonstrated to be highly immunogenic and effective against invasive disease and otitis media in infants and young children. Prevnar®-7 is a pneumococcal polysaccharide-protein conjugate vaccine and includes the seven most frequently isolated polysaccharide serotypes (e.g., 4, 6B, 9V, 14, 18C, 19F, and 23F conjugated to CRM197). In spite of availability of 7 valent vaccine composition, there was a need for additional multivalent pneumococcal vaccines comprising alternative polysaccharide serotypes for a broader coverage of serotypes. In this context, GlaxoSmithKline introduced 10 valent conjugated pneumococcal vaccine - Synflorix®. Synflorix® is a pneumococcal vaccine that included ten polysaccharide serotypes - serotypes 1, 4, 5, 6B, 7, 9, 14, 23F - conjugated to protein D (PD), serotype 18C conjugated to tetanus toxoid (TT) and. serotype 19F conjugated to diphtheria toxoid (DT). Total amount of carrier proteins used in Prevnar®-7 is 16 fig/dose.

So, it is noted that enhancement in the valency of the conjugated pneumococcal vaccine from 7 to 10 also increases the number of conjugation proteins from 1 to 3. Basically developing a safe multivalent pneumococcal conjugated vaccine is an extremely challenging undertaking even today, one of the reasons behind this is 'immune interference'. When multiple antigens are administered concomitantly, interactions occur among immune responses by the individual antigens which may strengthen, weaken or sometimes not affect responses. Immune interference refers to such interactions that may weaken the immune response. Thus when new antigens are added to a vaccine, one cannot predict whether the immunogenicity would be achieved at the desired level for all the antigens until confirmed by the actual experimental results. Moreover, pneumococcal conjugate vaccines are usually prepared in higher valency than other infant vaccines. In this context, antigens of different serotype, which are complex polysaccharides having different chemical structures from each other, are not only distinguished immunologically from each other, but also have immunogenic characteristics distinct from those of carrier proteins. When multiple components having such distinct characteristics are combined, it becomes even harder to predict how those multiple components would interact and how they would impact on the overall immune response. Further components in a pneumococcal conjugate vaccine cannot be simply combined. Rather, they are chemically conjugated to form a new conformational structure. Thus, the level of immunogenicity may vary depending on the process and conditions under which the conjugates are prepared. The various characteristic of each polysaccharide antigen (e.g. size, structure, number and type of functional groups), activation method, presence of linkers, and conjugation chemistry may affect the conformation, and in turn the immunogenicity of the conjugates, which contributes to the particularly high unpredictability when conjugates are combined to form highly multivalent vaccines. Since immunogenicity of final pneumococcal conjugated vaccine formulation may depend on such various determinants, pneumococcal conjugated vaccines are not developed by simply choosing certain vaccine serotypes and carrier proteins. Developing such complex multivalent conjugate vaccines is quite

challenging not in terms of preparation, but more importantly in terms of immunological compatibility.
Most important parameter to consider in increasing the valency of the vaccine is to insure that any additional serotypes would not negatively impact the response to the original serotypes. It was generally understood that immune suppression was a significant issue when expanding the serotypes in conjugate vaccine. Fattom et al. clearly showed that combining conjugate vaccines on the same carrier results in interferences and suggested need for multiple carriers. Thus mixed carrier approach is designed to circumvent immune suppression.
One of such concern is that use of a single carrier protein for all serotype could lead to a decrease in carrier-Specific T helper cell support. Adding more conjugated serotypes to existing vaccines which are conjugated to same carrier protein, increases the carrier protein antigenic load in the human body. This approach hinder the generation of a balanced immune response to all serotype which often lead to a suboptimal immune response to some or majority of the serotypes. Theoretically two major mechanisms of this immunologic interference have been described: (1) antigen competition and (2) Carrier Induced Epitope Suppression. Antigen competition among combination components probably arises at the level of antigen processing or transport. Carrier Induced Epitope Suppression is a phenomenon whereby the polysaccharide antigen epitopes presented on a protein carrier are inhibited by prior or concurrent immunization with the specific protein carrier in the conjugate, e.g. when PRP conjugate vaccines were combined with DTaP vaccines and given simultaneously with IPV, it was shown that PRP antibody responses were lower; antibody levels to tetanus toxin was also reduced. Thus instead of overload of a single carrier protein it is desirable to have multicarrier vaccines in which selected serotypes are conjugated to different carrier proteins so that a safe and balanced immune response can be obtained. To overcome above mentioned challenges, a vaccine composed of a multi carrier conjugate is exemplified.

A 13-valent conjugate vaccine Prevenar-13®, containing thirteen serotypes 1 , 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, I9A, I9F and 23F conjugated to CRM!97, was developed and approved due to the limitations in serotype coverage with Prevnar®-7 in certain regions of the world.
13-valent pneumococcal vaccine covers only 74 percent burden of pneumococcal disease in Asia, coverage is significantly lower than in Europe and America, taking into account variations in the epidemiological distribution serotypes in infants and young children, development of the present invention comprising 30 monovalent pneumococcal conjugate vaccine, can provide coverage greater than 90% in infants, young children and adults for the prevention of pneumococcal disease in all parts of the world.
In light of the present epidemiology of pneumococcal disease, PCV10 or PCV13 immunization in infants has the potential for significantly reducing the global pneumococcal disease burden that exists today. However, invasive disease serotypes not covered by currently available Pneumococcal Conjugate Vaccines are already evident and might become prominent causes of reported disease. In the long run, as the geographic distribution of predominant serotypes changes, effective vaccine coverage provided by PCVs may not be optimal worldwide. However, a significant amount of disease burden, due to serotypes not contained in existing Pneumococcal Conjugate Vaccines, still exists globally.
Although the higher valent formulations of pneumococcal conjugate polysaccharides are anticipated in literature however no relevant proof of their immunogenicity is established or approved by any regulatory agencies worldwide. These are still in the development phase.
Moreover, Current pediatric immunization schedules include the administration of several vaccines simultaneously, therefore increasing the potential for immune interference. The administration of a conjugate vaccine results in antibody production against both the hapten and the protein carrier epitopes. The co-

administration of multiple vaccines based on the same protein carrier (common antigenic epitopes) requires consideration due to potential interactions and consequent impact on immune responses to the vaccine antigens.
The co-administration and/or combinations of vaccines containing a given conjugate protein can induce interference that extends to unrelated antigens that are part of the combinations in use. This has been termed 'Bystander interference' and may influence responses to non-conjugated antigens administered simultaneously, or even sequentially.
Vaccines differ in their vulnerability to bystander interference, and Hib responses appear to be particularly vulnerable to these effects. Conjugate vaccines utilizing CRM-197 when co-administered with DTaP/Hib vaccines consistently demonstrate reduced anti-Hib IgG responses compared to schedules where CRMt97 is not co-administered. The addition of further CRM197 conjugate vaccines may reduce the Hib responses further.
Lower Meningococcal C serum bacterial antibody and geometric mean titre of antibody was observed following simultaneous administration of meningococcal C-CRM197 conjugate vaccine and other CRMi97-based conjugate vaccines.
This trend was observed when total CRM197 carrier protein amount was above 47 Hg/dose. (Lee LH, Blake MS. Effect of increased CRM197 carrier protein dose on meningococcal C bactericidal antibody response. Clin Vaccine Immunol. 2012;19(4):551-556.) Carrier dose-related effects were evident when infants received a conjugate vaccine with higher CRM197 content and concomitant DTaP combination vaccines. Reduced MenC antibody responses continued to be observed after the toddler dose, which constitutes the dose that contributes to longer disease protection. Reduced meningococcal C antibody responses related to an increased total CRM197 carrier protein dose clearly impact future immunization strategies.

Prevenar7 (PCV 7) contains 20 |ig of CRM|97 per dose and Prevenar 13 contains 34 [ig of CRM197 per dose. This indicates that addition of more 6 pneumococcal capsular saccharides also increases the amount of CRM i.e. Conjugation protein. As already seen, there is always a need for additional multivalent pneumococcal vaccines. This will also increases the amount of carrier protein per dose of the vaccine. So, simultaneous, concomitant or in combination administration of another antigen conjugated to CRM 197 with PCV may lead to reduced antibody responses to an increased total CRM 197 carrier protein dose. Therefore one of the aspects of the present invention is to develop a highly multivalent pneumococcal conjugated vaccine composition with reduced amount/quantity of CRM 197 as a conjugated protein.
Therefore the present invention is about a vaccine composition with broader coverage of serotypes and careful selection of multiple carriers which can circumvent the issues like immune interference, epitopic load, Antigen competition, and carrier induced epitope suppression while delivering highly multivalent immunogenic composition
A composition according to the invention may be administered by any conventional route which is used in the field of vaccines, in particular by the systemic, i.e. parenteral route, e.g. by the subcutaneous, intramuscular, intradermal or intravenous route for simultaneous or sequential administration of the first and second immunogenic compositions.
The composition of the present invention can be formulated in a form of a unit dose vial, multiple dose vial, dual chamber syringe, or pre-filled syringe.
In one aspect of the invention is provided in a vaccine kit; comprising a vial containing an immunogenic composition (all the antigens in single vial) of the invention in aqueous form, i.e. solutions or suspensions; optionally, in a lyophilised form, and further comprising a vial containing an adjuvant as

described herein. It is intended that in this aspect of the invention, the adjuvant will be used to reconstitute the lyophilised immunogenic composition.
The vaccine may be supplied in various containers (e.g. 2 or 3). In one embodiment of this aspect of the invention there is provided a kit comprising two multi-valent vaccines for conferring protection in a host by simultaneous, or sequential administration of the first and second immunogenic compositions
Granted patent rN293947 discloses a vaccine composition comprising 12 or more pneumococcal capsular saccharides conjugated to 2 or more different carrier proteins. In this vaccine composition, 19F capsular polysaccharide is conjugated to DT or CRM and remaining 2-8 serotypes conjugated to Protein D. Only 11 valent vaccine compositions are exemplified in this patent application.
I1M293958 also describes 12 or more pneumococcal capsular saccharides conjugated to 2 or more different carrier proteins in which capsular saccharides 19A and 19F are conjugated to 2 different carrier proteins and remaining 2-8 serotypes conjugated to Protein D.
Indian patent application 3140/CHE/2015 discloses a multivalent pneumococcal conjugate vaccine (PCV) composition comprising: a) at least 12 capsular polysaccharides selected from serotypes 1, 3, 4, 5, 6B, 7F, 9N, 9V, 15B, 14,18C, 19A, 19F, 22F, 23F and 33F of S, pneumoniae conjugated to carrier protein selected from CRM 197, pneumococcal surface protein A (PspA), pneumococcal adhesin protein (PsaA) or combination thereof. Exemplified vaccine compositions are 13, 14 and 15 valent; all polysaccharides are conjugated with CRM197 yet combination of carrier proteins suggested in claims.
PCT application WO2018064444 describes a pneumococcal vaccine composition, the composition comprising two or more capsular pneumococcal polysaccharide serotypes each individually conjugated to a carrier protein pneumococcal surface adhesion protein A (PsaA) or combination of PsaA and CRM 197 as carrier

proteins. Exemplified vaccine composition in this patent application is 15 valent vaccine comprised of serotypes I, 4, 5, 7F, 9V, 14, I8C, I9A, I9F, 22F, 23F & 33F conjugated to CRM197 protein and polysaccharide for Serotypes 3, 6A and 6B conjugated to PsaA protein.
AU2012216698 discloses a Streptococcus pneumoniae immunogenic composition comprising 9 or more, 10 or more, 11 or more, 13 or more, or 14 or more capsular saccharides from different S. pneumoniae serotypes conjugated to 2 or more different carrier proteins, wherein the composition comprises serotype 19F capsular saccharide conjugated to diphtheria toxoid (DT) or CRM 197, optionally wherein 19F is the only saccharide in the composition conjugated to diphtheria toxoid (DT) or CRM19.Exemplified formulations are 11- valent, I3-valent and 14-valent serotypes conjugated to 3 different carrier proteins.
WO2018027123 claimed 16 valent pneumococcal conjugate compositions in which 2 of the serotypes conjugated to TT and remaining conjugated to CRM 197 two capsular polysaccharides that are conjugated to tetanus toxoid are selected from the group consisting of serotypes 1, 3, and 5.
WO2018027126 discloses a 20 valent pneumococcal conjugated vaccine in which any 2 of the serotypes from the serotypes 1, 3 and 5 are conjugated to TT and remaining are conjugated to CRM. About 50 (xg to about 60 \xg of CRM was used as a carrier protein.
IN266150 claimed a Streptococcus pneumoniae immunogenic composition comprising 10-23 capsular saccharides from different S. pneumoniae serotypes which are conjugated to a carrier proteins and comprising 3 different carrier proteins, wherein serotype 19F conjugated to Diphtheria toxoid, 18C conjugated to tetanus toxoid and serotypes 1, 4, 5, 6B, 7F, 9V, 14 and 23F capsular saccharides are conjugated to protein D from Haemophilus influenza.
IN271080 describes an immunogenic composition comprising capsular saccharides conjugates from serotypes 19A and 19F wherein 19A is conjugated to first bacterial toxoid selected from pneumolysin or Diphtheria toxoid or CRMi97

and 19F is conjugated to second bacterial toxoid selected from Diphtheria toxoid or CRM197 and comprising conjugates of S. pneumoniae capsular saccharides 3 1,4,5,6B, 7F,9V 14, 18C 22F and 23F. In this patent 10 valent, 11 valent, 12 valent, 13 valent and 14 valent compositions are exemplified.
All these cited prior arts indicate only up to 20 valent vaccine compositions using mixed carrier approach (2 or 3 carrier proteins). None of the cited prior art discloses a highly multivalent (more than 20- valent) immunogenic vaccine 5 composition. Also none of the prior art attempts highly multivalent (more than 20-valent) vaccine composition with reduced/controlled amount (less than 40 ^ig) of carrier proteins per dose which may suppress an immune response for another conjugated antigen administered simultaneously, concurrently, concomitantly or sequentially.
) Therefore an object of the present invention to develop an improved vaccine formulation of multiple Streptococcus pneumoniae serotype polysaccharides conjugated with 2 or more carrier proteins without compromising an immunoresponse for each serotype. One more aspect of the present invention is to develop a vaccine composition with broader coverage of pneumococcal conjugate
i composition using less than 40|ig/dose of carrier proteins, especially CRM197.
The percentage of serotypes conjugated to CRM197 may range from 30% to 90 % of total serotypes, preferably from 30% to 80 %, more preferably from 40% to 80 % and more preferably from 40% to 70 %.
Objective of the Invention
1 The main objective of the present invention is to design and develop a novel conjugated pneumococcal vaccine composition comprising up to 30 capsular polysaccharides
Typically incorporating multiple antigens in the vaccine composition also increases the amount of conjugation protein carriers. Therefore another objective of the present invention is to develop a vaccine composition with broader

coverage' of pneumococcal conjugate composition using reduced or controlled amount of carrier proteins.
Summary of Invention
The present invention relates to a novel conjugated pneumococcal vaccine ) composition comprising up to 30 capsular polysaccharides. Capsular pneumococcal polysaccharide of different S. pneumoniae serotypes conjugated to 2 or more different carrier proteins where the composition comprises capsular polysaccharides conjugated to first carrier protein and remaining capsular polysaccharides conjugated to carrier proteins other than first carrier protein. At > least three of the capsular polysaccharides selected preferably from the group comprising of serotypes 3, 6A, 6B, 15A, 15B, 18C, 19A, 19F, 23F, 33F, 35F are conjugated to first carrier protein. And polysaccharides from remaining serotypes are conjugated to second carrier protein. The process of manufacturing such multivalent vaccine composition using multiple carriers is also disclosed.
I Present invention is also relates to 30 valent pneumococcal conjugate vaccine composition using reduced or controlled amount of carrier proteins, especially
CRM]97.
Detailed Description:
The first aspect of the present invention is to provide a multivalent pneumococcal polysaccharide - protein conjugate composition; the composition comprises 30 pneumococcal capsular polysaccharides conjugated to two or more carrier proteins.
One feature of the invention is that conjugated pneumococcal polysaccharides of the present invention provides a protective effect on pneumococcal serotypes disease, in the region of the world with more than 90% of protection coverage.
The present invention provides new and improved mixed carrier, multivalent pneumococcal conjugate compositions and vaccines comprising the same. In one

aspect, this application provides a mixed carrier, multivalent pneumococcal conjugate composition, comprising up to 30 different pneumococcal capsular polysaccharide-protein conjugates, wherein each pneumococcal capsular polysaccharide-protein conjugate comprises a protein carrier conjugated to a capsular polysaccharide from a different serotype of Streptococcus pneumoniae, wherein the Streptococcus pneumoniae serotypes are selected from 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 7A, 7B, 7C, 7F, 8, 9A, 9L, 9N, 9V, 10A, 10B, IOC, 10F, 11A, 11B, 11C, 1 ID, 11F, 12A, 12B, 12F,13, 14, 15A, 15B, 15C, 15F, 16A, 16F, 17A, 17F, 18A, 18B, I8C, 18FJ9A, 19B, I9C, 19F,20 21, 22A, 22F, 23A, 23B, 23F, 24A, 24B, 24F, 25A, 25F, 27, 28A, 28F,29, 31, 32A, 32F, 33A, 33B, 33C, 33D, 33F, 34, 35A, 35B, 35C, 35F, 36, 37, 38, 39, 40, 41A, 41F, 42, 43, 44, 45, 46, 47F, 47A, 48; wherein the carrier proteins are selected from group of Diphtheria Toxoid, Tetanus toxoid, Cross-reacting material CRM197, Protein D of non-typeable H. influenzae, Exotoxin A of Pseudomonas Aeruginosa (EPA), nontoxic peptide from C. difficile toxin A (CDTA) and Pneumococcal surface protein A (PspA) and pneumococcal adhesion protein.
Amongst the polysaccharides selected from 30 serotype of Streptococcus pneumoniae, at least three of the capsular polysaccharides are conjugated to first carrier protein and remaining capsular polysaccharides are conjugated to carrier proteins other than first carrier protein.
Amongst the polysaccharides selected from 30 serotype of Streptococcus pneumoniae, at least three of the capsular polysaccharides are conjugated to first carrier protein and remaining capsular polysaccharides are conjugated to CRM197 carrier protein.
In Streptococcus pneumoniae vaccine of the present invention, at least three of the capsular polysaccharides selected preferably from the group comprising of serotypes 3, 6A, 6B, 15A, 15B, 18C, 19A, 19F, 23F, 33F, 35F are conjugated to first carrier protein and remaining capsular polysaccharides are conjugated to carrier protein CKJVI197.

Typically the Streptococcus pneumoniae vaccine of the present invention will comprise capsular saccharide antigens (preferably conjugated), wherein the saccharides are derived from at least 5 serotypes of S. pneumoniae. The number of S. pneumoniae capsular saccharides can range from 5 different serotypes to 30 different serotypes (30V). In one embodiment, there are vaccine compositions comprising 5-30 different serotypes. In another embodiment of the invention, the vaccine may comprise conjugated S. pneumoniae saccharides or unconjugated S. pneumoniae saccharides. Preferably, the total number of saccharide serotypes is less than or equal to 30.
In one embodiment, the present disclosure provides a pneumococcal vaccine composition that is a 20 valent, 21 valent, 22 valent, 23 valent, 24 valent, 25 valent, 26 valent, 27 valent, 28 valent, 29 valent, or 30 valent pneumococcal vaccine compositions.
In one embodiment, the 30 pneumococcal vaccine of the invention will be selected from the following serotypes 1, 2, 3, 4, 5, 6B, 6C, 6D, 7A, 7B, 7C, 7F, 8, 9A, 9L, 9N, 9V, 10A, 10B, 10C, 10F, HA" I IB, 11C, 11D, 1 IF, 12A, 12B, 12F,13, 14, 15A, 15B, 15C, 15F, 16A, 16F, 17A, 17F, 18A, 18B, 18C, 18F,19A, 19B, 19C, 19F,20 21, 22A, 22F, 23A, 23B, 23F, 24A, 24B, 24F, 25A, 25F, 27, 28A, 28F,29, 31, 32A, 32F, 33A, 33B, 33C, 33D, 33F, 34, 35A, 35B, 35C, 35F, 36, 37, 38, 39, 40, 41 A, 4IF, 42, 43, 44, 45, 46, 47F, 47A, 48; although it is appreciated that one or two other serotypes could be substituted depending on the age of the recipient receiving the vaccine and the geographical location where the vaccine will be administered, e.g. serotype 6A may be included on the list.
The 30 valent composition in one embodiment includes Purified Polysaccharide of S. Pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9B, 91M, 9V, 10A, 11A, 12F, 14, 15A, 15B, 16F, 18C, 19A, 19F, 20, 22F, 21A, 23F, 24B, 33F, 34, 35F, 45 conjugates.

The 29 valent composition in one embodiment includes Purified Polysaccharide of S. Pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9B, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 16F, 18C, 19A, 19F, 20, 22F, 23A, 23F, 33F, 34, 35F, 45 conjugates
The 28 valent composition in one embodiment includes Purified Polysaccharide of S. Pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9B, 9N, 9V, 10A, 11 A, 12F, 14, 15B, 16F, 18C, 19A, 19F, 20, 22F, 23A, 23F, 33F, 34, 35F, 45 conjugates
The 26 valent composition in one embodiment includes Purified Polysaccharide of S. Pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9B, 9N, 9V, 10A, 11A, 12F, 14, 15B, 16F, 18C, 19A, 19F, 20, 22F, 23A, 23F, 33F, 34, 35F conjugates
The 25 valent composition in one embodiment includes Purified Polysaccharide of S. Pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9B, 9V, 10A, 11A, 12F, 14, 15B, 16F, 18C, 19A, 19F, 20, 22F, 23A, 23F, 33F, 34, 35F conjugates.
The 23 valent composition in one embodiment includes Purified Polysaccharide of S. Pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, I9F, 20, 22F, 23A, 23F, 33F, 34, 35F conjugates.
The 22 valent composition in one embodiment includes Purified Polysaccharide of S. Pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9N, 9V, 10A, 12F, 14, 15A, 18C, 19A, 19F, 22F, 23F, 33F, 34, 35F, 45 conjugates.
The 20 valent composition in one embodiment includes Purified Polysaccharide of S. Pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9B, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 33F conjugates.
The 30 valent composition in an embodiment includes Purified Polysaccharide of S. Pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9B, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 16F, 18C, 19A, 19F, 20, 22F, 23A, 23F, 24B, 33F, 34, 35F, 45

conjugates wherein Purified Polysaccharide of S. Pneumoniae serotypes 3, 19F, 33F are conjugated to first carrier protein and Purified Polysaccharide of S. Pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 8, 9B, 9N, 9V, 10A, 11 A, I2F, 14, 15A, 15B, 16F, 18C, 19A, 20, 22F, 23A, 23F, 24B, 34, 35F, 45 are conjugated to second carrier protein.
The 29 valent composition in one embodiment includes Purified Polysaccharide of S. Pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9B, 9N, 9V, 10A, 11 A, 12F, 14, 15A, 15B, 16F, 18C, I9A, 19F, 20, 22F, 23A, 23F, 33F, 34, 35F, 45 conjugates wherein Purified Polysaccharide of S. Pneumoniae serotypes 6A, 18C, 33F are conjugated to first carrier protein and Purified Polysaccharide of S. Pneumoniae serotypes I, 3, 4, 5, 6B, 7F, 8, 9B, 9N, 9V, 10A, 11 A, 12F, 14, 15 A, 15B, 16F, 19A, 19F, 20, 22F, 23A, 23F, 34, 35F, 45 are conjugated to second carrier protein.
The 28 valent composition in one embodiment includes Purified Polysaccharide of S. Pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9B, 9N, 9V, 10A, 11 A, 12F, 14, 15B, 16F, 18C, 19A, 19F, 20, 22F, 23A, 23F, 33F, 34, 35F, 45 Conjugate wherein Purified Polysaccharide of S. Pneumoniae serotypes 6B, 19A, 23F are conjugated to first carrier protein and Purified Polysaccharide of S. Pneumoniae serotypes 1, 3, 4, 5, 6A, 7F, 8, 9B, 9N, 9V, 10A, 11A, 12F, 14, 15B, 16F, 18C, 19F, 20, 22F, 23A, 33F, 34, 35F, 45 are conjugated to second carrier protein.
The 26 valent composition in one embodiment includes Purified Polysaccharide of S. Pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9B, 9N, 9V, 10A, 11A, 12F, 14, 15B, 16F, 18C, 19A, 19F, 20, 22F, 23A, 23F, 33F, 34, 35F conjugates wherein Purified Polysaccharide of S. Pneumoniae serotypes 3, 6A, 19F are conjugated to first carrier protein and Purified Polysaccharide of S. Pneumoniae serotypes 1, 4, 5, 6B, 7F, 9B, 9N, 9V, 10A, 11 A, 12F, 14, 15B, 16F, 18C, 19A, 20, 22F, 23A, 23F, 33F, 34, 35F are conjugated to second carrier protein.

The 25 valent composition in one embodiment includes Purified Polysaccharide of S. Pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9B, 9V, 10A, 11A, 12F, 14, 15B, 16F, 18C, 19A, I9F, 20, 22F, 23A, 23F, 33F, 34, 35F conjugates wherein Purified Polysaccharide of S. Pneumoniae serotypes 19A, 23F, 33F are conjugated to first carrier protein and Purified Polysaccharide of S. Pneumoniae serotypes I, 3, 4, 5, 6A, 6B, 7F, 9B, 9V, 10A, 11 A, 12F, 14, 15B, 16F, 18C, I9F, 20, 22F, 23A, 34, 35F are conjugated to second carrier protein.
The 23 valent composition in one embodiment includes Purified Polysaccharide of S. Pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 11 A, 12F, 14, 15B, 18C, 19A, 19F, 20, 22F, 23A, 23F, 33F, 34, 35F conjugates wherein Purified Polysaccharide of S. Pneumoniae serotypes 6B, 18C, 23F are conjugated to first carrier protein and Purified Polysaccharide of S. Pneumoniae serotypes 1, 3, 4, 5, 6A, 7F, 9V, 10A, 11 A, 12F, 14, 15B, 19A, 19F, 20, 22F, 23A, 33F, 34, 35F are conjugated to second carrier protein.
The 22 valent composition in one embodiment includes Purified Polysaccharide of S, Pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9N, 9V, 10A, 12F, 14, 15A, 18C, 19A, 19F, 22F, 23F, 33F, 34, 35F, 45 conjugates wherein Purified Polysaccharide of S. Pneumoniae serotypes 6A, 19A, 33F are conjugated to first carrier protein and Purified Polysaccharide of S. Pneumoniae serotypes I, 3, 4, 5, 6B, 7F, 9N, 9V, 10A, 12F, 14, 15A, 18C, 19F, 22F, 23F, 34, 35F, 45 are conjugated to second carrier protein.
The 20 valent composition in one embodiment includes Purified Polysaccharide of S. Pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9B, 10A, 11 A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 33F conjugates wherein Purified Polysaccharide of S. Pneumoniae serotypes 6B, 19F, 23F are conjugated to first carrier protein and Purified Polysaccharide of S. Pneumoniae serotypes 1, 3, 4, 5, 6A, 7F, 8, 9B, 10A, 11A, 12F, 14, 15B, 18C, 19A, 22F, 33F are conjugated to second carrier protein

The vaccine of the present invention comprises two or more different types of carrier protein. Carrier proteins are preferably proteins that are non-toxic and nonreactogenic and obtainable in sufficient amount and purity. Carrier proteins should be amenable to standard conjugation procedures. Each Streptococcus pneumoniae capsular saccharide' may be conjugated to a carrier protein independently selected from the group consisting of Diphtheria Toxoid, Tetanus toxoid, Cross-reacting material CRM|97, Protein D of non-typeable H influenzae, Exotoxin A of Pseudomonas Aeruginosa (EPA), nontoxic peptide from C. difficile toxin A (CDTA) and Pneumococcal surface protein A (PspA).
The vaccine of the present invention comprises a protein D (PD) from Haemophilus influenzae. Haemophilus influenzae is a key causative organism of otitis media, and the present inventors have shown that including this protein in a Streptococcus pneumoniae vaccine will provide a level of protection against Haemophilus influenzae related otitis media. In one embodiment, the vaccine composition comprises protein D. In one aspect, protein D is present as a carrier protein for one or more of the saccharides. In the present invention, at least three of the capsular polysaccharides selected preferably from the group comprising of serotypes 3, 6A, 6B, 15A, 15B, 18C, 19A, 19F, 23F, 33F, 35F are conjugated to Protein D or Diphtheria Toxoid or Tetanus toxoid Exotoxin A of Pseudomonas Aeruginosa (EPA) or nontoxic peptide from C. difficile toxin A (CDTA) or Pneumococcal surface protein A (PspA).
In a particular embodiment of the present invention, CRM197 is used as the carrier protein for conjugation of remaining proteins which are not conjugated with Protein D. CRM197 is a non-toxic variant (i.e., toxoid) of diphtheria toxin isolated from cultures of Corynebacterium diphtheria strain C7 (PI97) grown in amino acids and yeast extract-based medium. CRM197 is purified through ultrafiltration, ammonium sulfate precipitation, and ion-exchange chromatography. Alternatively, CRM197 may be prepared recombinantly in accordance with U.S.

Patent No. 5,614,382, which is hereby incorporated by reference. Other diphtheria toxoids are also suitable for use as carrier proteins.
In one of the embodiment of the present invention, CRJV1]97 is used as the second carrier protein. Amount of the CRM197 included in the vaccine composition varies from 30(ig to 40 |ig/dose and more preferably from 32|ig to 38 |ig/dose. Most preferably amount of the CRM197 included in the vaccine composition is less than 35|ig/dose.
The percentage of serotypes conjugated to CRM197 may range from 30% to 90 % of total serotypes, preferably from 30% to 80 %, more preferably from 40% to 80 % and more preferably from 40% to 70 %.
In another embodiment, the present disclosure provides a pneumococcal vaccine composition comprising a pharmaceutical^ acceptable diluent, buffer, preservative, stabilizer, adjuvant, and/or a lyophilization excipient.
As defined herein, an "adjuvant" is a substance that serves to enhance the immunogenicity of an immunogenic composition of the invention. An immune adjuvant may enhance an immune response to an antigen that is weakly immunogenic when administered alone, e.g., inducing no or weak antibody titers or cell-mediated immune response, increase antibody titers to the antigen, and/or lowers the dose of the antigen effective to achieve an immune response in the individual. Thus, adjuvants are often given to boost the immune response and are well known to the skilled artisan. Suitable adjuvants to enhance effectiveness of the composition include, but are not limited to aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc.
In one embodiment, the present invention provides an immunogenic composition administered as a single 0.5 mL dose formulated to contain: > I to < 5 ng of each polysaccharide; < 60(ig, preferably < 40|ag carrier protein; NMT 1.5 mg of aluminum phosphate adjuvant and saline solution as excipients.

The percentage of serotypes conjugated to CRM]97 may range from 30% to 90 % of total serotypes, preferably from 30% to 80 %, more preferably from 40% to 80 % and more preferably from 40% to 70 %.
Another aspect of the present invention is provide complimentary immunization to a person who is already vaccinated with 7, 10, 11, 13 or 15 valent vaccine and require additional protection against serotypes not present in 7, 10, 11, 13 or 15 valent vaccine.
To cater the remaining unmet medical need felt due to immunization of marketed vaccines having limited number of serotypes the immunization of population with serotypes not found in marketed vaccines is required.
The specific serotypes causing disease beyond the 13 valent vaccine vary by region, population, and may change over time due to acquisition of antibiotic resistance, pneumococcal vaccine introduction and secular trends of unknown origin.
There is a need for immunogenic compositions that can be used to induce an immune response against additional Streptococcus pneumoniae serotypes in humans. An object of the new immunogenic compositions of the present invention is to provide for appropriate protection against & pneumoniae serotypes not found in marketed vaccines.
The following example gives the composition and process of manufacturing of pneumococcal polysaccharide conjugate vaccine.
Examples for Pneumococcal Polysaccharide Conjugate Vaccine (Multicarrier approach)
Composition: The composition of Pneumococcal Polysaccharide Conjugate Vaccine is presented in table below
Each dose of 0.5 ml contains:-

Description Qty per 0.5ml Formulation I (30) Formulation II (29)
Serotype Set 1 Conjugated to First Carrier protein >1 to<5 lag each Purified Polysaccharide of S. Pneumoniae serotypes 3, 19F, 33F conjugates Purified Polysaccharide of S. Pneumoniae serotypes 6A, I8C, 33 F conjugates
Serotype Set 2
Conjugated to Second Carrier protein >1 to<5 lag each Purified Polysaccharide of S. Pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 8, 9B, 9N, 9V, 10A, 11 A, 12F, 14, 15A, 15B, 16F, 18C, 19A, 20, 22F, 23A, 23F, 24B, 34, 35F, 45 conjugates Purified Polysaccharide of S. Pneumoniae serotypes 1, 3, 4, 5, 6B, 7F, 8, 9B, 9N, 9V, 10A, 11 A, 12F, 14, 15A, 15B, 16F, I9A, 19F, 20, 22F, 23A, 23F, 34, 35F, 45 conjugates
Adjuvant NMT 1.25 mg Aluminum content (As Aluminum phosphate)
Other ingredient q.s Saline solution
Each dose of 0.5 ml contains:-

Description Qty per 0.5ml Formulation III (28) Formulation IV (26)
Serotype Set 1
Conjugated to First Carrier protein > 1 to < 5 (ig each Purified Polysaccharide of S. Pneumoniae serotypes 6B, 19A, 23F conjugates Purified Polysaccharide of S. Pneumoniae serotypes 3, 6A, 19F conjugates
Serotype Set 2 Conjugated to > 1 to < 5 Hg each Purified Polysaccharide of S. Pneumoniae Purified Polysaccharide of S. Pneumoniae

Second Carrier protein serotypes 1, 3, 4, 5, 6A, 7F, 8, 9B, 9N, 9 V, 10A, 11 A, I2F, 14, 15B, 16F, I8C, 19F, 20, 22F, 23A, 33F, 34, 35F, 45 conjugates serotypes 1, 4, 5, 6B, 7F, 9B, 9N, 9V, 10A, 11 A, 12F, 14, 15B, 16F, 18C, 19A, 20, 22F, 23A, 23F, 33F, 34, 35F conjugates
Adjuvant NMT 1.25 mg Aluminum content (As Aluminum phosphate)
Other ingredient q.s Saline solution
Each dose of 0.5 ml contains:-

Description Qty per 0.5ml Formulation V (25) Formulation VI (23)
Serotype Set 1
Conjugated to First Carrier protein >1 to<5 |ig each Purified Polysaccharide of S. Pneumoniae serotypes 19A, 23F, 33F conjugates Purified Polysaccharide of S. Pneumoniae serotypes 6B, 18C, 23F conjugates
Serotype Set 2
Conjugated to Second Carrier protein >1 to<5 jig each Purified Polysaccharide of S. Pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9B, 9V, 10A, 11A, 12F, 14, 15B, 16F, 18C, 19F, 20, 22F, 23A, 34, 35F conjugates Purified Polysaccharide of S. Pneumoniae serotypes 1, 3, 4, 5, 6A, 7F, 9V, 10A, 11 A, 12F, 14, 15B, 19A, 19F, 20, 22F, 23A, 33F, 34, 35F conjugates
Adjuvant NMT 1.25 mg Aluminum content (As Aluminum phosphate)
Other
ingredient q.s Saline solution

Each dose of 0.5 ml contains:-

Description Qty per 0.5ml Formulation VII (22) Formulation VIII (20)
Serotype Set 1
Conjugated to First Carrier protein >1 to<5 |ig each Purified Polysaccharide of 5. Pneumoniae serotypes 6A, 19A, 33F conjugates Purified Polysaccharide of S. Pneumoniae serotypes 6B, 19F, 23F conjugates
Serotype Set 2
Conjugated to Second Carrier protein >1 to<5 |ig each Purified Polysaccharide of S. Pneumoniae serotypes 1, 3, 4, 5, 6B, 7F, 9N, 9V, 10A, 12F, 14, 15A, 18C, 19F, 22F, 23F, 34, 35F, 45 conjugates Purified Polysaccharide of S. Pneumoniae serotypes 1, 3, 4, 5, 6A, 7F, 8, 9B, 10A, 11 A, 12F, 14, 15B, 18C, 19A, 22F, 33F conjugates
Adjuvant NMT 1.25 mg Aluminum content (As Aluminum phosphate)
Other ingredient q.s Saline solution
Formulation of bulk vaccine
The required quantity of individual drug substance and other excipients was calculated based on the batch size and the label claim.
Calculated quantity of buffer solution was transferred to formulation vessel under constant stirring. The monovalent bulk of Purified Polysaccharide conjugates of S. Pneumoniae serotypes were added one after another to the above blend. After complete addition of all serotypes, the pH of blend was adjusted and calculated quantity of adjuvant was transferred to the above blend. The volume of resultant blend was made up to the required, batch size with buffer solution and it was

incubated with stirring. Post incubation the final bulk vaccine was stored at 2-8°C for pending filling.
Stored temperature
Store at 2-8°C.
Examples for Pneumococcal Polysaccharide Conjugate Vaccine (Two container approach)
Composition: The composition of Pneumococcal Polysaccharide Conjugate Vaccine is presented in table below
Each dose of 0.5 ml contains:-

Description Qty
per 0.5ml Formulation I (15+5) Formulation II (14+6)
Container 1 >1 to
<5ug Purified Polysaccharide of S. Pneumoniae serotypes 1, 3,4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, 33F conjugates Purified Polysaccharide of S. Pneumoniae serotypes 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, 33F conjugates
Container 2 >1 to
<5ug Five Purified Polysaccharide conjugates of S. Pneumoniae serotypes selected from group of 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 7A, 7B, 7C, 7F, 8, 9A, 9L, 9N, 9V, 10A, 10B, IOC, 10F, 11A, 1 IB, 11C, 1 ID, 1 IF, 12A, 12B, 12FJ3, 14, 15A, 15B, 15C, 15F, 16A, 16F, 17A, 17F, 18A, 18B, Five Purified Polysaccharide conjugates of S. Pneumoniae serotypes selected from group of 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 7A, 7B, 7C, 7F, 8, 9A, 9L, 9N, 9V, 10A, 10B, IOC, 10F, 11A, I IB, 11C, 1 ID, 1 IF, 12A, 12B, 12F,13, 14, 15A, 15B, ISC, 15F, 16A, 16F, 17A, 17F, 18A, 18B,

18C, 18F.19A, 19B, 19C, 19F,20 21, 22A, 22F, 23A, 23B, 23F, 24A, 24B, 24F, 25A, 25F, 27, 28A, 28F,29, 31, 32A, 32F, 33A, 33B, 33C, 33D, 33F, 34, 35A, 35B, 35C, 35F, 36, 37, 38, 39, 40, 41 A, 4IF, 42, 43, 44,45,46, 47F, 47A, 48 18C, 18F.19A, 19B, 19C, 19F,20 21, 22A, 22F, 23A, 23B, 23F, 24A, 24B, 24F, 25A, 25F, 27, 28A, 28F,29, 31, 32A, 32F, 33A, 33B, 33C, 33D, 33F, 34, 35A, 35B, 35C, 35F, 36, 37, 38, 39, 40, 41 A, 4IF, 42, 43, 44,45,46, 47F, 47A, 48
Adjuvant NMT
1.25
mg Aluminum content (As Aluminum phosphate)
Other ingredient q.s Saline solution
Each dose of 0.5 ml contains:-

Description Qty
per 0.5ml Formulation III (13+7) Formulation IV (11+9)
Container I >1 to
<5ng Purified Polysaccharide of S. Pneumoniae serotypes 1, 3,4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F
conjugates Purified Polysaccharide of S. Pneumoniae serotypes 1, 5, 0A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F conjugates
Container 2 >1 to
<5^g Seven Purified Polysaccharide conjugates of S. Pneumoniae serotypes selected from group of 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, Nine Purified Polysaccharide conjugates of S. Pneumoniae serotypes selected from group of 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D,

7A, 7B, 7C, 7F, 8, 9A, 9L, 9N, 9V, 10A, 10B, IOC, lOF, 11 A, IIB, 11C, IlD, IIF, 12A, 12B, 12FJ3, 14, 15A, 15B, 15C, 15F, 16A, 16F, 17A, 17F, 18A, 18B, 18C, 18FJ9A, 19B, 19C, 19F,20 21, 22A, 22F, 23A, 23B, 23F, 24A, 24B, 24F, 25A, 25F, 27, 28A, 28F,29, 31, 32A, 32F, 33A, 33B, 33C, 33D, 33F, 34, 35A, 35B, 35C, 35F, 36, 37, 38, 39, 40, 41 A, 4IF, 42, 43, 44,45,46, 47F, 47A, 48 7A, 7B, 7C, 7F, 8, 9A, 9L, 9N, 9V, 10A, 10B, IOC, 10F, 11A, IIB, 11C, IlD, IIF, 12A, 12B, 12F,13, 14, 15A, 15B, 15C, 15F, I6A, 16F, 17A, 17F, 18A, 18B, 18C, 18FJ9A, 19B, 19C, 19F,20 21, 22A, 22F, 23A, 23B, 23F, 24A, 24B, 24F, 25A, 25F, 27, 28A, 28F,29, 31, 32A, 32F, 33A, 33B, 33C, 33D, 33F, 34, 35A, 35B, 35C, 35F, 36, 37, 38, 39, 40, 41 A, 41F, 42, 43, 44, 45, 46, 47F, 47A, 48
Adjuvant NMT
1.25
mg Aluminum content (As Aluminum phosphate)
Other ingredient q.s Saline solution
Each dose of 0.5 ml contains:-

Description Qty
per 0.5ml Formulation V (10+10) Formulation VI (7+13)
Container 1 >1 to
<5^g Purified Polysaccharide of S. Pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, 23F conjugates Purified Polysaccharide of 5*. Pneumoniae serotypes 4, 6B, 9V, 14, 18C, 19F, 23F conjugates

Ten Purified Polysaccharide Thirteen Purified
conjugates of S. Polysaccharide conjugates
Pneumoniae serotypes of S. Pneumoniae serotypes
selected from group of 1, 2, selected from group of 1, 2,
3, 4, 5, 6A, 6B, 6C, 6D, 3, 4, 5, 6A, 6B, 6C, 6D,
7A, 7B, 7C, 7F, 8, 9A, 9L, 7A, 7B, 7C, 7F, 8, 9A, 9L,
9N, 9V, 10A, 10B, IOC, 9N, 9V, 10A, 10B, IOC,
10F, 11A, 1 IB, 11C, 1 ID, 10F, 11A, 1 IB, 11C, 1 ID,
11F, 12A, 12B, 12F,13, 14, 1 IF, 12A, 12B, 12F,13, 14,
Container 2 >1 to
<5ug 15A, 15B, 15C, 15F, 16A, 16F, 17A, 17F, 18A, 18B, 15A, 15B, 15C, 15F, 16A, 16F, 17A, 17F, 18A, 18B,
18C, 18F,19A, 19B, 19C, 18C, 18F,19A, 19B, 19C,
19F,20 21, 22A, 22F, 23A, 19F,20 21, 22A, 22F, 23A,
23B, 23F, 24A, 24B, 24F, 23B, 23F, 24A, 24B, 24F,
25A, 25F, 27, 28A, 28F,29, 25A, 25F, 27, 28A, 28F,29,
31, 32A, 32F, 33A, 33B, 31, 32A, 32F, 33A, 33B,
33C, 33D, 33F, 34, 35A, 33C, 33D, 33F, 34, 35A,
35B, 35C, 35F, 36, 37, 38, 35B, 35C, 35F, 36, 37, 38,
39, 40, 41 A, 4IF, 42, 43, 39, 40, 41 A, 4IF, 42, 43,
44, 45, 46, 47F, 47A, 48 44,45,46, 47F, 47A, 48
NMT
Adjuvant 1.25 mg Aluminum content (As Aluminum phosphate)
Other ingredient q.s Saline solution
Manufacturing process: As per examples 1 Store at 2-8°C.

We Claim:
1. A Streptococcus pneumonia immunogenic composition comprising up to
30 capsular saccharides from different S. pneumonia serotypes conjugated
to 2 or more different carrier proteins, wherein
a) At least three serotypes are conjugated to first carrier protein selected from Diphtheria Toxoid, Tetanus toxoid, Cross-reacting material CRM197, Protein D of non-typeable H. influenzae, Exotoxin A of Pseudomonas Aeruginosa (EPA), nontoxic peptide from C. difficile toxin A (CDTA) and Pneumococcal surface protein A (PspA)
b) Polysaccharides from remaining serotypes are conjugated to second carrier protein which is Cross-reacting material CRM197.

2. A Streptococcus pneumonia immunogenic composition of claim 1, wherein at least three serotypes conjugated to first carrier protein are selected from the group consisting of serotypes 3, 6A, 6B, 15A, 15B, 18C, 19A, 19F, 23F, 33F , 35F.
3. A Streptococcus pneumonia immunogenic composition of claim 1, comprises up to 30 capsular saccharides from S. pneumonia serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9B, 9N, 9V, 10A, 11A, 12F, 14, 15A, I5B, 16F, I8C, 19A, 19F, 20, 22F, 23A, 23F, 24B, 33F, 34, 35F, 45.
4. A Streptococcus pneumonia immunogenic composition of claim I, comprising up to 30 capsular saccharides from different S. pneumonia serotypes wherein serotypes 3, 19F, 33F are conjugated to first carrier protein and Purified Polysaccharide of S. Pneumoniae serotypes 1, 4, 5, 6A,6B, 7F, 8, 9B, 9N, 9V, 10A, 11 A, 12F, 14, 15A, 15B, 16F, 18C, 19A, 20, 22F, 23A, 23F, 24B, 34, 35F, 45 are conjugated to second carrier protein.

5. A Streptococcus pneumonia immunogenic composition of claim 1, comprising 29 capsular saccharides from different S. pneumonia serotypes wherein serotypes 6A, 18C, 33F are conjugated to first carrier protein and Purified Polysaccharide of S. Pneumoniae serotypes 1, 3, 4, 5, 6B, 7F, 8, 9B, 9N, 9V, 10A, 11 A, 12F, 14, 15 A, I5B, 16F, 19A, I9F, 20, 22F, 23 A, 23F, 34, 35F, 45 are conjugated to second carrier protein.
6. A Streptococcus pneumonia immunogenic composition of claim 1, comprising 28 capsular saccharides from different S. pneumonia serotypes wherein serotypes 6B, 19A, 23F are conjugated to first carrier protein and Purified Polysaccharide of S. Pneumoniae serotypes 1, 3, 4, 5, 6A, 7F, 8, 9B, 9N, 9V, 10A, 11 A, 12F, 14, I5B, 16F, 18C, 19F, 20, 22F, 23A, 33F, 34, 35F, 45 are conjugated to second carrier protein.
7. A Streptococcus pneumonia immunogenic composition of claim 1, comprising 26 capsular saccharides from different S. pneumonia serotypes wherein serotypes 3, 6A, 19F are conjugated to first carrier protein and Purified Polysaccharide of S. Pneumoniae serotypes 1, 4, 5, 6B, 7F, 9B, 9N, 9V, 10A, 1IA, 12F, 14, 15B, 16F, 18C, 19A, 20, 22F, 23A, 23F, 33F, 34, 35F are conjugated to second carrier protein.
8. A Streptococcus pneumonia immunogenic composition of claim 1, comprising 25 capsular saccharides from different S. pneumonia serotypes wherein serotypes 19A, 23F, 33F are conjugated to first carrier protein and Purified Polysaccharide of S. Pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9B, 9V, 10A, 1 IA, 12F, 14, 15B, 16F, 18C, 19F, 20, 22F, 23A, 34, 35F are conjugated to second carrier protein.
9. A Streptococcus pneumonia immunogenic composition of claim 1, comprising 23 capsular saccharides from different S. pneumonia serotypes wherein serotypes 6B, 18C, 23F are conjugated to first carrier protein and

Purified Polysaccharide of S. Pneumoniae serotypes 1, 3, 4, 5, 6A, 7F, 9V, 10A, 1IA, 12F, 14, 15B, 19A, 19F, 20, 22F, 23A, 33F, 34, 35F are conjugated to second carrier protein.
10. A Streptococcus pneumonia immunogenic composition of claim 1, comprising 22 capsular saccharides from different S. pneumonia serotypes wherein serotypes 6A, 19A, 33F are conjugated to first carrier protein and Purified Polysaccharide of S. Pneumoniae serotypes 1, 3, 4, 5, 6B, 7F, 9N, 9V, 10A, I2F, 14, 15A, 18C, 19F, 22F, 23F, 34, 35F, 45 are conjugated to second carrier protein.
11. A Streptococcus pneumonia immunogenic composition of claim 1, comprising 20 capsular saccharides from different S. pneumonia serotypes wherein serotypes 6B, 19F, 23F are conjugated to first carrier protein and Purified Polysaccharide of S. Pneumoniae serotypes 1, 3, 4, 5, 6A, 7F, 8, 9B, 10A, 11 A, 12F, 14, 15B, 18C, 19A, 22F, 33F are conjugated to second carrier protein.
12. A Streptococcus pneumonia immunogenic composition of claim 1, wherein the percentage of serotypes conjugated to CRM197 may range from 30% to 90% of total serotypes.
13. A Streptococcus pneumonia immunogenic composition of claim 1, wherein the amount of conjugation protein is limited to less than 40 |ig.
14. A Streptococcus pneumonia immunogenic composition of claim 1, presented in a form of a kit comprising all the antigenic components in a single vial/ prefilled syringe or a kit comprising two multi-valent pneumococcal vaccines in two different containers, vials, prefilled syringes or dual chamber syringe.