Claims:
WE CLAIM:

1. An immunogenic composition comprising at least one glycoconjugate from Streptococcus pneumoniae serotype 15A.

2. The immunogenic composition as claimed in claim 1, wherein the carrier protein of said serotype 15 A glycoconjugate is selected from the group consisting of: DT (Diph-theria toxin), TT (tetanus toxoid), CRM197, other DT mutants, or immunologically func-tional equivalents thereof.

3. The immunogenic composition as claimed in claim 2, wherein the carrier protein of serotype 15 A glycoconjugate is CRM197.

4. The immunogenic composition as claimed in claim 2, wherein the carrier protein of serotype 15 A glycoconjugate is TT (tetanus toxoid).

5.The immunogenic composition as claimed in claim 1, further comprise at least one glycoconjugate from Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 15C, 17F, 18C, 19A, 19F, 20, 22F, 23F, 33F and 39.

6. The immunogenic composition as claimed in claim 4, wherein each capsular polysac-charide is separately conjugated to a carrier protein CRM197

7. The immunogenic composition as claimed in claim 4, wherein each capsular polysac-charide is separately conjugated to a carrier protein TT (tetanus toxoid).

8. The immunogenic composition according to any of claims 1 to 7, wherein immuno-genic composition is separately activated by cyanylation and then individually conju-gated to the carrier protein.

9. The immunogenic composition according to claim 8, wherein the cyanylation agent is
CDAP.

10. The immunogenic composition according to claims 8 and 9, wherein the cyanylated polysaccharide is attached to the carrier protein by carbodiimide chemistry, preferably using EDAC.

11.The immunogenic composition of any one of claims 1-10, wherein the mixing ratio between polysaccharide to carrier protein are about 1:1, 1:2, 1:3 or 1:4.

12. The immunogenic composition according to claim 11, wherein the mixing ratio be-tween polysaccharide to carrier protein in the conjugate is between 1:1 and 1:2.

13. The immunogenic composition as claimed in claim 1, wherein said immunogenic composition further comprises at least one adjuvant.

14.The immunogenic composition as claimed in claim no 13, wherein adjuvant is alu-minium.

15. A method for producing a multivalent immunogenic composition comprising poly-saccharide-protein conjugates, wherein each of the conjugates comprises a capsular pol-ysaccharide from a different serotype of Streptococcus pneumoniae conjugated to a car-rier protein, and the capsular polysaccharides are prepared from serotype 15 A as one glycoconjugate from Streptococcus pneumoniae. the method comprising:
a) dissolution of pneumococcal polysaccharide (PnPs) of Streptococcus pneumonia Serotype 15A ,
b) activation of 15A serotype by addition of 50mg/ml of CDAP solution (75 % of the weight of total Ps weight) and the desired pH adjusted with 0.2 M TEA to get activated PnPs,
c) activation of CRM197 was carried out by mixing equal amount of 10 mg/ml EDAC solution to the concentrated and diafiltered CRM197,
d) Conjugation of pneumococcal polysaccharide (PnPs) of Streptococcus pneumonia Serotype 15A with carrier protein,
e) adding the immunogenic conjugate comprising Streptococcus pneumoni-ae serotype 15A polysaccharide and at least one glycoconjugate from Streptococ-cus pneumoniae serotype producing a multivalent immunogenic composition.

16. The method of claim 15, wherein the capsular polysaccharides further comprise at least one glycoconjugate from Streptococcus pneumoniae 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 15C, 17F, 18C, 19A, 19F, 20, 22F, 23F, 33F and 39.

17. The multivalent immunogenic composition as claimed in claim 15, which is a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26-valent pneumococcal conjugate composition.

18. The multivalent immunogenic composition as claimed in claim 17, wherein compo-sition is a 16- valent pneumococcal conjugate.

19. The method of claim 15, further comprising purifying the serotypes of the multiva-lent immunogenic composition prior to compounding with the carrier protein.
20. The method of claim 16, wherein each serotypes of the multivalent immunogenic composition are separately conjugated to a carrier protein.
21. The method of claim 17, wherein the carrier protein is CRM197 to TT (tetanus tox-oid).
22. The method of claim 15, further comprising purifying the 15A serotype, the method comprising:
a) Concentration and Diafiltration of PnPs- CRM197 conjugate,
b) followed by filtration of the concentrated-diafiltered PnPs- CRM197 conjugate,
c) the mixture is incubated, filtered and followed by concentrated and subjected to diafil
tration,
d) filtered conjugate is purified using chromatography method ,
e) followed by addition of non-ionic surfactant and emulsifier.
f) non-ionic surfactant and emulsifier treated purified PnPs- CRM197 conjugated mate-rials then passed through cartridge filter, pore size 0.22-?m to get activated 15A - CRM197 conjugated protein.
g) activated 15A - CRM197 conjugated protein is further added with at least one gly-coconjugate from Streptococcus pneumoniae to produce the multivalent immunogenic composition.

23. The multivalent immunogenic composition as claimed in claim 15, wherein the mul-tivalent pneumococcal conjugate composition as a pneumococcal conjugate vaccine to the subject.

24. The immunogenic composition as claimed in claim 23, wherein the subject is a hu-man who is at least 6 weeks old and the disease is pneumonia, invasive pneumococcal disease (IPD), or acute otitis media (AOM).

25. The immunogenic composition as claimed in claim 23, wherein the human subject is 6 weeks to 5 years of age.

26 The immunogenic composition as claimed in claim 23, wherein the human subject 6 to 17 years of age.

27. The immunogenic composition as claimed in claim 22, wherein the multivalent pneumococcal conjugate composition or vaccine administered by intramuscular injec-tion.

28. The immunogenic composition as claimed in claim 22, the multivalent pneumococ-cal conjugate composition or vaccine is administered as part of an immunization series.
, Description:FIELD OF THE INVENTION
The present invention is to provide immunogenic compositions for protection against Streptococcus polysaccharide in particular against Streptococcus pneumoniae serogroup 15A. The immunogenic composition, formulated as a vaccine, increases coverage against pneumococcal disease in infants and young children globally, and provides cov-erage for serotype 15 A is not dependent on the limitations of serogroup cross-protection.
Again, the object of the present invention is to provide a process for the production and purification of glycoconjugate from Streptococcus pneumoniae 15 A. Another object of the present invention is to provide a method for conjugation of capsular polysaccharide with carrier protein CRM197 and / or tetanus toxoid.
Another aspect the present invention relates to the use of an immunogenic composition comprising at least one glycoconjugate from Streptococcus pneumoniae serotype 15 A, wherein the immunogenic compositions is a multi-valent pneumococcal conjugate com-position.

BACKGROUND OF THE INVENTION
Background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifi-cally or implicitly referenced in this application is prior art. Disclosures of these publi-cations in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains.

According to WHO (World Health organization), diseases caused by Streptococcus pneumoniae (the pneumococcus) is a major public health problem worldwide. In the de-veloping world young children and the elderly are most affected; it is estimated that about one million children die of pneumococcal disease every year. Streptococcus pneu-moniae is an encapsulated bacteria with a polysaccharide capsule an essential factor in virulence. About 90 distinct pneumococcal serotypes have been identified throughout the world, with a small number of these serotypes accounting for most disease in infants. Pneumococci are transmitted by direct contact with respiratory secretions from patients and healthy carriers. Serious pneumococcal infections include pneumonia, meningitis and febrile bacteremia; otitis media, sinusitis and bronchitis are more common but less serious manifestations. Pneumococcal resistance to antimicrobials is a serious and rapid-ly increasing problem worldwide.
Streptococcus pneumoniae is currently the leading cause of invasive bacterial disease in children and the elderly. Streptococcus pneumoniae is known in medical microbiology as the pneumococcus, referring to its morphology and its consistent involvement in pneu-mococcal pneumonia. (Musher DM. Infections caused by Streptococcus pneumoniae: clinical spectrum, pathogenesis, immunity, and treatment. Clinical Infectious Diseases, 1992, 14:801–807)
S. pneumoniae is an important cause of otitis media, meningitis, bacteremia and pneu-monia, and a leading cause of fatal infections in the elderly and persons with underlying medical conditions, such as pulmonary disease, liver disease, alcoholism, sickle cell, cerebrospinal fluid leaks, acquired immune deficiency syndrome (AIDS), and patients undergoing immunosuppressive therapy. It is also a leading cause of morbidity in young children. The most severe pneumococcal infections involve invasive meningitis and bacteremia infections.
Antibodies to capsular polysaccharide antigens of S.pneumoniae provide serotype-specific protection against pneumococcal infections. Pneumococcal vaccines are de-signed to cover the serotypes most frequently associated with severe pneumococcal dis-ease.

Till date following Pneumococcal vaccines are in the market or about to launch.
Pfizer Prevnar 13® 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F
20vPnC 1, 3, 4, 5, 6A,6B 7F, 9V, 14, 18C, 19A, 19F, 23F, 8, 10A, 11A, 12F, 15BC, 22F, and 33F
GSK Synflorix 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F
Tergene & Au-robindo PCV15 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 18C, 19A, 19F , 23F

Merck PCV-15 (V114) 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F
PNEU-MOVAX® 23 unconjugated, polysaccharides: 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19F, 19A, 20, 22F, 23F, and 33F,
Biological E Limited Pneumococ-cal conjugate vaccine 14-valent (ad-sorbed) 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, 19A, 22F, 23F, 33F

Sinovac PPV-23 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19F, 19A, 20, 22F, 23F, and 33F
SK bioscience PCV13-14 1, 3, 4, 5, 6A 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F, and serotype 2 or 9N
Serum PNEU-MOSIL-10valent 1, 5, 6A, 6B, 7F, 9V, 14, 19A, 19F, and 23F
Eubiologics PCV15 1, 3, 4, 5, 6A, 6B, 7F, 9V, 11A, 14, 18C, 19A, 19F, 22F, and 23F
Conjugate vaccines have reduced pneumococcal disease in vaccinated children and un-vaccinated adults, but non-vaccine serotypes are of concern, particularly if antibiotic re-sistant. Infants and young children respond poorly to most pneumococcal polysaccha-rides. The 7-valent pneumococcal conjugate vaccine (7vPnC, Prevnar®) was the first of its kind demonstrated to be highly immunogenic and effective against invasive disease and otitis media in infants and young children. This vaccine is now approved in many countries around the world. Prevnar contains the capsular polysaccharides from sero-types 4, 6B, 9V, 14, 18C, 19F and 23F, each conjugated to a carrier protein designated CRM197. Prevnar covers approximately 80-90%, 60-80%, and 40-80% of invasive pneumococcal disease (IPD) in the US, Europe, and other regions of the world, respec-tively. Surveillance data gathered in the years following Prevnar's introduction has clear-ly demonstrated a reduction of invasive pneumococcal disease in infants as expected.
Globally, several PCV serotypes covered by PPV23 were identified; 15A appears to be very prominent among both paediatric and adult non-IPD cases.
Reports are there on the spread of non-PCV13 serotype 15A with penicillin-non-susceptible S. pneumoniae (PNSP; 37.8 %) in France (Janoir C et al, Open Forum Infect Dis. 2016;3(1):ofw020) .In Japan, studies on patients with IPD after the introduction of PCV7 in 2010–2013 indicated an increase in non PCV13 serotypes such as 15A with IPD in children and adults (Chiba N et al, Emerg Infect Dis. 2014;20(7):1132-1139.)
Again from recent studies in Russia (Vorobieva S. Jensen et al. BMC Infectious Diseases (2020) 20:279 and Sidorenko S, Diagn Microbiol Infect Dis. 2020;96(1)).
It is clear that there is prevalence of 15 A serotype in Russia which is not included in previous Pneumococcal Conjugate Vaccine (PCV).
The recent development of the widespread resistance of microorganisms to major antibi-otics and the increase in the number of people with impaired immunity underline the need for pneumococcal vaccines with an even wider spectrum of protection.
India has the highest mortality and morbidity due to IPD among adults aged >50 years. With the increase in the aging population and the rise in the incidence of comorbid con-ditions in the country, vaccination can play a crucial role in the prevention of pneumo-coccal diseases and in improving the quality of life of the at-risk population. Both PPV23 and PCV13 are available in the country. At present, PCV13 is approved for use only in adults aged >50 years. Although the vaccines are recommended and advocated globally for the at-risk elderly population, their usage is suboptimal in the region and is majorly dependent on clinicians' awareness of guidelines.(Koul PA et al, Lung India. 2019;36(3):216-225.)
Again, in a systematic review from India reported that 15A is one of the top 11 pre-dominant non-vaccine serotypes, which accounted for 12.1% of invasive pneumococcal disease cases in India, among 22% of the NVS isolates in under-five Indian children. (Dr Balaji Veeraraghavan , Indian Pediatr 2018;55:874-876)
Given the relative burden and importance of invasive pneumococcal disease due to sero-type 15 A and adding the serotype to the other serotypes formulation would increase coverage for invasive disease as high as 70%-80% in Asia and Latin America. Proposed vaccine would significantly expand coverage beyond that of the available vaccine and provide coverage for 15A that is not dependent on the limitations of serogroup cross-protection. Serotype 15A showed the highest proportion of multidrug resistance. (Ya-hiaoui et al, BMC Infect Dis 18, 440 (2018)
Pneumococcal serogroup 15 comprises four structurally related serotypes: 15A, 15B, 15C, and 15F. Reports are there on the cross-functional OPA response between Serotype 15B and Serotype 15C as disclosed in US20190070283A1. However there are no reports on the cross-functional OPA response between Serotype 15B and Serotype 15A.
Again, the incidence of invasive pneumococcal diseases caused by non-vaccine sero-types such as Streptococcus pneumoniae serotypes 15A, has recently increased (see for example Beall B. et al, Journal of Clinical Microbiology. 44(3):999-1017, 2006, or Ja-cobs et Al, Clin Infect Dis. (2008) 47 (1 1): 1388-1395). Reports are also there on the multidrug-resistant non-vaccine serotype 15A Streptococcus pneumoniae. Sheppard C et al, Euro Surveill. 2016;21(50):30423)

None of the currently marketed pneumococcal vaccine provides an appropriate protec-tion against serotype 15A Streptococcus pneumoniae in human and in particular in chil-dren less than 2 years old. Therefore, there is a need for immunogenic compositions that can be used to induce an immune response against serotype 15A Streptococcus pneu-moniae.

Again, pneumococcal vaccination is associated with the emergence of new serotypes and related prevalence of antimicrobial resistance might justify at the short-term, a continu-ous evaluation and adjustment of available vaccines, in order to include newly emerged serotypes. At the long-term, the implementation of new vaccines that could cover pneu-mococcal serotypes will be helpful.
Therefore, there exists an unmet need of the non –vaccinate serotype 15A and the present invention provides an immunogenic compositions for appropriate protection against S. pneumoniae, in particular against S. pneumoniae serogroup 15A.
In a further aspect the immunogenic compositions is 16-valent pneumococcal conjugate composition. In a further aspect the glycoconjugates of the immunogenic compositions are individually conjugated with carrier protein CRM197 and / or tetanus toxoid.

OBJECTS OF THE INVENTION
The object of the present invention is to provide an immunogenic composition compris-ing at least one glycoconjugate from Streptococcus pneumoniae serotype 15A.

Another object of the present invention is to provide a method of conjugation of 15A capsular pneumococcal polysaccharide with carrier protein.

In one object, the capsular saccharide antigens are conjugated to a carrier protein which is selected in the group consisting of: DT (Diphtheria toxin), TT (tetanus toxoid) or fragment C of TT, CRM197 (a nontoxic but antigenically identical variant of diphtheria toxin).

In another embodiment, the carrier protein of the glycoconjugates of the invention is CRM197.

The object of the present invention is to provide an immunogenic composition compris-ing at least one glycoconjugate from Streptococcus pneumoniae serotype 15 A. wherein the immunogenic compositions is a multivalent pneumococcal conjugate composition.
In a further aspect the immunogenic compositions is a 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-valent pneumococcal conjugate composition. In a further aspect, the glycoconjugates of the immunogenic compositions are individually conjugat-ed to CRM197.

In a further aspect the immunogenic compositions is a 16- valent pneumococcal conju-gate composition. In a further aspect the glycoconjugates of the immunogenic composi-tions are individually conjugated to CRM197.
In one embodiment , the above immunogenic compositions further comprise at least one glycoconjugate from Streptococcus pneumoniae 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 15C, 17F, 18C, 19A, 19F, 20, 22F, 23F, 33F and 39.
In another embodiment, the carrier protein is CRM197.
In yet another embodiment, the composition comprises an adjuvant, such as an alumi-num-based adjuvant selected from aluminum phosphate, aluminum sulphate and alumi-num hydroxide. In a particular embodiment, the adjuvant is aluminum.
In another object of the present invention, are prepared using CDAP chemistry or by re-ductive amination chemistry.
In another object of the present invention, the multivalent pneumococcal conjugate com-position further comprises an adjuvant, such as an aluminum-based adjuvant, including, but not limited to aluminum phosphate, aluminum sulphate, and aluminum hydroxide.
Yet another object is directed to a vaccine comprising the multivalent pneumococcal conjugate composition and a pharmaceutically acceptable excipient.

Another object is directed to purifying the immunogenic conjugate 15 A serotype.

In another object, the purified polysaccharides, are capsular polysaccharide from sero-types 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 15C, 17F, 18C, 19A, 19F, 20, 22F, 23F, 33F or 39 of Streptococcus pneumoniae.

Another object is directed to the use of the multivalent pneumococcal conjugate compo-sition as a vaccine to the subject.

In one object, the subject is a human who is at least 6 weeks old and the disease is pneu-monia, invasive pneumococcal disease (IPD), or acute otitis media (AOM). In some em-bodiments, the human subject is 6 weeks to 5 years of age. In other embodiments, the human subject is 2 to 15 months of age or 6 to 17 years of age.

In one object, the multivalent pneumococcal conjugate composition or vaccine is admin-istered by intramuscular injection.

In certain aspects, the multivalent pneumococcal conjugate composition or vaccine is administered as part of an immunization series.

SUMMARY OF THE INVENTION
In a general aspect the present invention provides an immunogenic composition com-prising at least one glycoconjugate from S. pneumoniae serotype 15A.

In an embodiment, the present invention provides a method of conjugation of 15A capsu-lar pneumococcal polysaccharide with carrier protein.

In an embodiment, the capsular saccharide antigens are conjugated to a carrier protein which is selected in the group consisting of: DT (Diphtheria toxin), TT (tetanus toxoid) or fragment C of TT, CRM197 (a nontoxic but antigenically identical variant of diphthe-ria toxin).

In another embodiment, the carrier protein of the glycoconjugates of the invention is CRM197.

In an embodiment, the present invention provides an immunogenic composition com-prising at least one glycoconjugate from S. pneumoniae serotype 15 A. wherein the im-munogenic composition is a multivalent pneumococcal conjugate composition.

In an embodiment, the immunogenic composition is a 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-valent pneumococcal conjugate composition.

In one embodiment , the above immunogenic compositions further comprise at least one glycoconjugate from S. pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 15C, 17F, 18C, 19A, 19F, 20, 22F, 23F, 33F and 39.

In another embodiment, the carrier protein is CRM197. In yet another embodiment, the composition comprises an adjuvant, such as an aluminum-based adjuvant selected from aluminum phosphate, aluminum sulphate and aluminum hydroxide. In a particular em-bodiment, the adjuvant is aluminum.
In an embodiment, the immunogenic compositions prepared using CDAP chemistry or by reductive amination chemistry.
In an embodiment, the multivalent pneumococcal conjugate composition further com-prises an adjuvant, such as an aluminum-based adjuvant, including, but not limited to aluminum phosphate, aluminum sulphate, and aluminum hydroxide.

In an embodiment, is directed to a vaccine comprising the multivalent pneumococcal conjugate composition and a pharmaceutically acceptable excipient.

In an embodiment, the present invention is about purifying the immunogenic conjugate 15 A serotype.
In a preferred embodiment, the purified polysaccharides, are capsular polysaccharide from serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 15C, 17F, 18C, 19A, 19F, 20, 22F, 23F, 33F or 39 of Streptococcus pneumoniae.

In an embodiment, the multivalent pneumococcal conjugate composition as a vaccine to the subject.

In an embodiment, the subject is a human who is at least 6 weeks old and the disease is pneumonia, invasive pneumococcal disease (IPD), or acute otitis media (AOM). In some embodiments, the human subject is 6 weeks to 5 years of age. In other embodiments, the human subject is 2 to 15 months of age or 6 to 17 years of age.

In an embodiment, the present invention provides the multivalent pneumococcal conju-gate composition or vaccine administered by intramuscular injection.

In certain embodiments, the multivalent pneumococcal conjugate composition or vac-cine is administered as part of an immunization series.

These and other features, aspects, become better understood with reference to the follow-ing description, appended claims, and accompanying drawings in which like characters represent like parts throughout the drawings.

DETAILED DESCRIPTION OF FIGURES
Figure 1 - Production of capsular pneumococcal polysaccharide from S. Pneumonia Serotype 15A.
Figure 2 - Conjugation of 15A capsular pneumococcal polysaccharide with CRM197 for preparation of conjugate vaccine.
Fig 3: Conjugation of pneumococcal polysaccharide (PnPs) of Streptococcus pneumonia Serotype 15A with CRM.

DETAILED DESCRIPTION OF THE INVENTION

The following is a detailed description of some of the embodiments and explanation of the present invention with some examples thereof. The embodiments are in such detail as to clearly communicate the disclosure. However, the amount of detail offered is not intended to limit the anticipated variations of embodiments; on the contrary, the inten-tion is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure as defined by the appended claims.
Unless the context requires otherwise, throughout the specification which follow, the word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be construed in an open, inclusive sense that is as “including, but not limited to.”
Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable man-ner in one or more embodiments.
As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. It should also be noted that the term “or” is generally employed in its sense including “and/or” unless the content clearly dictates otherwise.
In some embodiments, the numbers expressing quantities of ingredients, properties such as concentration, reaction conditions, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term “about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description are approximations that can vary depending upon the de-sired properties sought to be obtained by a particular embodiment. In some embodi-ments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific exam-ples are reported as precisely as practicable.
The recitation of ranges of values herein is merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.
All processes described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all ex-amples, or exemplary language (e.g, “such as”) provided with respect to certain embod-iments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specifi-cation should be construed as indicating any non-claimed element essential to the prac-tice of the invention.
The headings and abstract of the invention provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.
The following discussion provides many example embodiments of the inventive subject matter. Although each embodiment represents a single combination of inventive ele-ments, the inventive subject matter is considered to include all possible combinations of the disclosed elements. Thus, if one embodiment comprises elements A, B, and C, and a second embodiment comprises elements B and D, then the inventive subject matter is also considered to include other remaining combinations of A, B, C, or D even if not ex-plicitly disclosed.
The term ‘adjuvant’ as used herein refers to the non-antigenic component of the vaccine that enhances the immune response of the antigens comprised in the vaccine by facilitat-ing the contact between the antigen and the immune system. The adjuvant causes pro-longed immune responses against the antigens.

Immunogenic compositions of the present invention will typically comprise conjugated capsular saccharide antigens (also named glycoconjugates), wherein the saccharides are derived from serotypes of Streptococcus pneumoniae.

A component of the glycoconjugate of the invention is a carrier protein to which the sac-charide is conjugated. The terms “protein carrier” or “carrier protein” or “carrier” may be used interchangeably herein. Carrier proteins should be amenable to standard conju-gation procedures.

Reference will now be made in detail to the exemplary embodiments of the present dis-closure, examples of which are illustrated in the accompanying drawings. Wherever pos-sible, the same reference numbers are used in the drawings and the description to refer to the same or like parts.

The present invention provides an immunogenic composition comprising at least one glycoconjugate from Streptococcus pneumoniae serotype 15A.

In an embodiment, the present invention provides a method of conjugation of 15A capsu-lar pneumococcal polysaccharide with carrier protein.

In an embodiment, the capsular saccharide antigens are conjugated to a carrier proteins which is selected in the group consisting of: DT (Diphtheria toxin), TT (tetanus toxoid) or fragment C of TT, CRM197 (a nontoxic but antigenically identical variant of diphthe-ria toxin).

In another embodiment, the carrier protein of the glycoconjugates of the invention is CRM197.

In an embodiment, the present invention provides an immunogenic composition com-prising at least one glycoconjugate from Streptococcus pneumoniae serotype 15A, wherein the immunogenic compositions is a multivalent pneumococcal conjugate com-position.

In one embodiment , the above immunogenic compositions further comprise at least one glycoconjugate from Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 15C, 17F, 18C, 19A, 19F, 20, 22F, 23F, 33F and 39.

In another embodiment, the carrier protein is CRM197. In yet another embodiment, the composition comprises an adjuvant, such as an aluminum-based adjuvant selected from aluminum phosphate, aluminum sulphate and aluminum hydroxide. In a particular em-bodiment, the adjuvant is aluminum.
In an embodiment, the immunogenic compositions prepared using CDAP chemistry or by reductive amination chemistry.
In an embodiment, the multivalent pneumococcal conjugate composition further com-prises an adjuvant, such as an aluminum-based adjuvant, including, but not limited to aluminum phosphate, aluminum sulphate, and aluminum hydroxide.

In an embodiment, is directed to a vaccine comprising the multivalent pneumococcal conjugate composition and a pharmaceutically acceptable excipient.

In an embodiment, the present invention is about purifying the immunogenic conjugate 15 A serotype.

In a preferred embodiment the purified polysaccharides, are capsular polysaccharide from serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 15C, 17F, 18C, 19A, 19F, 20, 22F, 23F, 33F or 39 of Streptococcus pneumoniae.

The immunogenic composition of the present invention includes atleast one glycoconju-gate from Streptococcus pneumoniae serotype 15A with the carrier protein CRM197.

In one aspect, the above immunogenic compositions further comprise at least one gly-coconjugate from Streptococcus pneumoniae serotypes 4, 6B, 9V, 14, 18C, 19F and/or 23F.

Preferably, the number of Streptococcus pneumoniae capsular saccharides can range from 1 serotype (or "v", valences) to 25 different serotypes (25v). In one embodiment there is one serotype. In one embodiment there are 2 different serotypes. In one embodi-ment there are 3 different serotypes. In one embodiment there are 4 different serotypes. In one embodiment there are 5 different serotypes. In one embodiment there are 6 differ-ent serotypes. In one embodiment there are 7 different serotypes. In one embodiment there are 8 different serotypes. In one embodiment there are 9 different serotypes. In one embodiment there are 10 different serotypes. In one embodiment there are 11 different serotypes. In one embodiment there are 12 different serotypes. In one embodiment there are 13 different serotypes. In one embodiment there are 14 different serotypes. In one embodiment there are 15 different serotypes. In one embodiment there are 16 different serotypes. In an embodiment there are 17 different serotypes. In an embodiment there are 18 different serotypes. In an embodiment there are 19 different serotypes. In an embodi-ment there are 20 different serotypes. In an embodiment there are 21 different serotypes. In an embodiment there are 22 different serotypes. In an embodiment there are 23 differ-ent serotypes. In an embodiment there are 24 different serotypes. In an embodiment there are 25 different serotypes.
More specifically, the immunogenic composition consists of 16 different serotypes.
In a preferred embodiment though, the saccharides are each individually conjugated to different molecules of the protein carrier (each molecule of protein carrier only having one type of saccharide conjugated to it). In said embodiment, the capsular saccharides are said to be individually conjugated to the carrier protein.

The term 'glycoconjugate' indicates a capsular saccharide linked covalently to a carrier protein. In one embodiment a capsular saccharide is linked directly to a carrier protein. In a second embodiment a bacterial saccharide is linked to a protein through a spac-er/linker.

CRM197 as the carrier protein of the invention
CRM197 is a non-toxic form of the diphtheria toxin but is immunologically indistin-guishable from the diphtheria toxin. The protein CRM197 (cross-reacting material 197, 58 kDa) is a variant of the diphtheria toxin (DTx) characterized by a single mutation that reduces its toxicity, (i.e. the nucleotide variation produces a glycine-glutamic acid sub-stitution in position 52) (Uchida T. et al, 1973; Giannini G. et al, 1984). The protein CRM197 nonetheless retains the same inflammatory and immunostimulant properties as the diphtheria toxin. Like the wild-type diphtheria toxin, CRM197 comprises two do-mains, A and B, bonded together by a disulphide bridge. The A domain (21 kDa) is the catalytic domain, while the B domain (37 kDa) contains one subdomain for bonding to the cell receptor and another subdomain for the translocation (Gill D. M. et al, 1971 ; Uchida T. et al, 1973). CRM197 is produced by C. diphtheriae infected by the nontoxi-genic phase ß197tox-created by nitrosoguanidine mutagenesis of the toxigenic carynephage b (Uchida et al, Nature New Biology (1971) 233; 8-11). The CRM197 pro-tein has the same molecular weight as the diphtheria toxin but differs from it by a single base change in the structural gene. This leads to a glycine to glutamine change of amino acid at position 52 which makes fragment A unable to bind NAD and therefore non-toxic (Pappenheimer 1977, Ann Rev, Biochem. 46; 69-94, Rappuoli Applied and Environmen-tal Microbiology September 1983 p 560-564).
Accordingly, in frequent embodiments, the glycoconjugates of the invention comprise CRM197 as the carrier protein, wherein the capsular polysaccharide is covalently linked to CRM197.

In another embodiment, the carrier protein of the glycoconjugates of the invention is TT (tetanus toxoid).

Capsular Saccharide of the Invention
The term “saccharide” throughout this specification may indicate polysaccharide or oli-gosaccharide and includes both. In frequent embodiments, the saccharide is a polysac-charide, in particular a S. pneumoniae capsular polysaccharide.
In one embodiment, the immunogenic comprising at least one glycoconjugate from Streptococcus pneumoniae serotype 15A.

The serotype 15A polysaccharide may be obtained directly from the bacteria by using an isolation procedure known to those of ordinary skill in the art (including, but not limited to, the methods disclosed in (including, but not limited to, the methods disclosed in PCT Application No. PCT/US2006/012354). In addition, 15A oligosaccharides can be pro-duced using synthetic protocols.
In the present invention, capsular polysaccharides may be prepared from 15A by grow-ing Streptococcus pneumoniae serotype in a medium (e.g., in a soy-based medium), the polysaccharides are then prepared from the bacteria culture. Bacterial strains of Streptococcus pneumoniae used to make the respective polysaccharides that are used in the glycoconjugates of the invention may be obtained from established culture collec-tions or clinical specimens.
The population of the organism (each Streptococcus pneumoniae serotype) is often scaled up from a seed vial to seed bottles and passaged through one or more seed fer-mentors of increasing volume until production scale fermentation volumes are reached. At the end of the growth cycle, the cells are lysed and the lysate broth is then harvested for downstream (purification). The serotype 15A polysaccharide may be isolated from the cell lysate using purification techniques known in the art, including centrifugation, depth filtration, precipitation, ultrafiltration, treatment with activated carbon, diafiltra-tion and/or column chromatography (including, but not limited to, the methods disclosed in PCT Application No. PCT/US2006/012354).

In one aspect, the above immunogenic compositions further comprise at least one gly-coconjugate from Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 15C, 17F, 18C, 19A, 19F, 20, 22F, 23F, 33F or 39.
The glycoconjugates of the invention may also be characterized by the ratio (weight/weight) of polysaccharide to carrier protein. In some embodiments, the ratio of polysaccharide to carrier protein in the glycoconjugate (w/w) is between 0.5 and 3 (e.g., about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, or about 3.0).
In a preferred embodiment, the ratio of capsular polysaccharide to carrier protein in the conjugate is between 1:1 and 1:2. In some such embodiments, the carrier protein is CRM197
In one embodiment, the purified 15A polysaccharide is chemically activated to make the saccharide capable of reacting with the carrier protein. Once activated, 15A capsular polysaccharide is separately conjugated to a carrier protein to form a glycoconjugate. The polysaccharide conjugate may be prepared by known coupling techniques.
In one embodiment, the chemical activation of the 15A polysaccharide and subsequent conjugation to the carrier protein are achieved by means described in U.S. Pat. Nos. 4,365,170, 4,673,574 and 4,902,506. Briefly, that chemistry entails the activation of pneumococcal polysaccharide by reaction with any oxidizing agent which oxidizes a terminal hydroxyl group to an aldehyde, such as periodate (including sodium periodate, potassium periodate, or periodic acid). The reaction leads to a random oxidative cleav-age of vicinal hydroxyl groups of the carbohydrates with the formation of reactive alde-hyde groups.
Coupling to the protein carrier (e.g., CRM197) can be by reductive amination via direct amination to the lysyl groups of the protein. For example, conjugation is carried out by reacting a mixture of the activated polysaccharide and carrier protein with a reducing agent such as sodium cyanoborohydride. Unreacted aldehydes are then capped with the addition of a strong reducing agent, such as sodium borohydride.
In another embodiment, the conjugation method may rely on activation of the saccharide with 1-cyano-4-dimethylamino pyridinium tetrafluoroborate (CDAP) to form a cyanate ester. The activated saccharide may thus be coupled directly or via a spacer (linker) group to an amino group on the carrier protein. For example, the spacer could be cysta-mine or cysteamine to give a thiolated polysaccharide which could be coupled to the car-rier via a thioether linkage obtained after reaction with a maleimide-activated carrier protein (for example using GMBS) or a haloacetylated carrier protein (for example using iodoacetimide [e.g. ethyl iodoacetimide HCl] or N-succinimidyl bromoacetate or SIAB, or SIA, or SBAP). Preferably, the cyanate ester (optionally made by CDAP chemistry) is coupled with hexane diamine or adipic acid dihydrazide (ADH) and the amino-derivatised saccharide is conjugated to the carrier protein using carbodiimide (e.g. EDAC or EDC) chemistry via a carboxyl group on the protein carrier. Such conjugates are described in International Patent Application Publication Nos. WO 93/15760, WO 95/08348 and WO 96/29094; and Chu et al., 1983, Infect. Immunity 40:245-256.
After conjugation of the 15A capsular polysaccharide to the carrier protein, the polysac-charide-protein conjugate is purified (enriched with respect to the amount of polysaccha-ride-protein conjugate) by one or more of a variety of techniques.
In an embodiment, the present invention provides a method for making a multivalent immunogenic composition, further comprising purifying the immunogenic conjugate, the method comprising:
a) concentration and diafiltration of PnPs- CRM197 conjugate,
b) followed by filtration of the concentrated-diafiltered PnPs- CRM197 conjugate,
c) the mixture is incubated, filtered and followed by concentrated and subjected to diafil-tration,
d) filtered conjugate is purified using chromatography method ,
e) followed by addition of non-ionic surfactant and emulsifier,
f) non-ionic surfactant and emulsifier treated purified PnPs- CRM197 conjugated mate-rials then passed through cartridge filter, pore size 0.22-?m to get activated 15A - CRM197 conjugated protein.
g) activated 15A - CRM197 conjugated protein is further added with at least one gly-coconjugate from Streptococcus pneumoniae to produce the multivalent immunogenic composition.

The advantages of the present invention include a multivalent immunogenic conjugate composition as a vaccine to the subject. Wherein the subject is a human who is at least 6 weeks old and the disease is pneumonia, invasive pneumococcal disease (IPD), or acute otitis media (AOM). Wherein the human subject is 6 weeks to 5 years of age. In other embodiments, the human subject is 2 to 15 months of age or 6 to 17 years of age. Where-in the multivalent pneumococcal conjugate composition or vaccine administered by in-tramuscular injection.
Again, the multivalent pneumococcal conjugate composition or vaccine is administered as part of an immunization series.

This aspect of the present invention would make it more useful and advantageous espe-cially with the older and younger population who need to be vaccinated to acquire im-munity to a large number of infections and diseases caused by 15A serotype which is un-covered by the available vaccines and has a high prevalence.
This present invention also has several implications for future pneumococcal immuniza-tion programs. In particular, this study of 15A, making us aware of possible changes af-ter the introduction of extended-valency vaccines.
While the foregoing description discloses various embodiments of the disclosure, other and further embodiments of the invention may be devised without departing from the basic scope of the disclosure. The invention is not limited to the described embodiments, versions or examples, the invention is described hereinafter, with reference to the fol-lowing examples, which are to enable a person having ordinary skill in the art to make and use the invention when combined with information and knowledge available to the person having ordinary skill in the art, such examples are illustrative only and should not be construed to the limit of the scope of present invention.
Example
A. Growth test of Streptococcus pneumonia Serotype 15A
A Streptococcus pneumonia Serotype 15A strain (CCUG no.69630) was cultivated using a 100L bioreactor with a control system. This strain was identified as being a Strepto-coccus pneumonia Serotype 15A using commonly used tests, such as immune- and sero-typing, and morphology testing. The bioreactor was first filled with the basal medium (compound 1 & 2 in Table 1) before being sterilized in situ for 30 minutes at 1210 C. Just before inoculation the proper amount of stock solutions (Compound 3 to 13 in Table 1) were added to the medium. The bioreactor was inoculated using pre-culture, cultivat-ed on a 400 ml scale using the same medium and a frozen -70 0 C seed lot of the Strep-tococcus pneumonia Serotype 15A strain.

The pH was kept constant at 7.2 using 5 mol NaOH. The temperature was kept constant at 370C. The dissolved oxygen (DO) was kept constant at =10% using Airflow: 0.25 – 1.0 vvm. The stirrer speed was kept 100 rpm.

Samples were collected at an interval of 1 hr. As the growing culture reached the OD600 0.8, feeding was started. When cultivation reaches stationary phase (OD becomes con-stant and do not show any increase in value for last two consecutive samples), stop the addition of feed and stop the fermentation.
Table 1 : Composition of media
Sr. No. Reagents Concentration
1. Diapotassium hydrogen phosphate 5 g/L
2. Sodium bicarbonate 1 g/L
3. Soya Peptone

20 g/L

4. Yeast Extract 20 g/L
5. (D+) Glucose monohydrate 20 g/L
6. Magnesium sulphate heptahydrate 0.05 g/L
7. L-Glutamine 6.2 g/L
8. L-Asparagine 10 g/ L
9. Choline Chloride 1 g/L
10. Ferrous sulphate heptahydrate 5 g/L
11. Thioglycolic acid 10 ml/L
12. Zinc sulphate Heptahydrate 0.8 g/L
13. Manganese sulphate monohydrate 0.36 g/L

B.Purification of pneumococcal polysaccharide (PnPs) of Streptococcus pneumonia Serotype 15A

Purification mainly involved the operations of extraction by sodium chloride, ethanol precipitation, centrifugation, concentration – diafiltration, Chromatography, dia-filtration, filtration and drying at low temperature under vacuum to get PnPs.

Supernatant obtained from CTAB precipitation were further processed to extract PnPs.
Sodium chloride is added to the supernatant to obtain the final concentration of about 1 M (w/v). Ethanol (96%) at about 3 to about 10oC (preferably at about 4 to about 6oC) temperature was added under continuous stirring in the ratio of 1:3 (1 volume of super-natant and 3 of Ethanol). Incubate the solution at about at about 3 to about 10oC (prefer-ably at about 4 to about 6oC) for about 16 to 20 hrs. Centrifuge the solution received af-ter incubation at about 10000 ± 500 RCF for 30 ± 5 min at solution at about at about 3 to about 10oC (preferably at about 4 to about 6oC). Pellet is collected for further processing.

The pellet is dissolved in WFI (the volume of WFI is equal to initial volume of concen-trate) Add 6.0% w/v of Sodium acetate anhydrous to the suspension and dissolve. Add 0.5% w/v Sodium Deoxycholate and dissolve to the solution and adjust the pH to 6.4 ± 0.1 with 8M Acetic acid. Add ethanol about 0.5 time and incubate the solution about 3 to about 4 hrs at about 3 to about 10oC (preferably at about 4 to about 6oC). Centrifuge the solution received after incubation at about 10000 ± 500 RCF for 30 ± 5 min at solution at about at about 3 to about 10oC (preferably at about 4 to about 6oC). Supernatant is col-lected for further processing.

Add 6.0% w/v of Sodium acetate anhydrous to the suspension and dissolve. Add 0.5% w/v Sodium Deoxycholate and dissolve the solution and adjust the pH to 6.2 ± 0.1 with 8M Acetic acid. Add ethanol about 1.2 time and incubate the solution about 16 to about 24 hrs at about 3 to about 10oC (preferably at about 4 to about 6oC). Centrifuge the solu-tion received after incubation at about 10000 ± 500 RCF for 30 ± 5 min at solution at about at about 3 to about 10oC (preferably at about 4 to about 6oC). Pellet is collected for further processing. Dissolve the pellet in WFI to the original volume of concentrate. Ad-just the pH of the solution to 7.0- 7.2 using 2M NaOH.
The above solution is subjected to diafiltration using 300 kD cutoff membrane. The dia-filtration is carried out in 4 stages. In first stage, diafiltration is carried out with about 4 to about 8 volume buffer containing 50mM Tris HCl, 2mN EDTA, 0.3% DOC. The sec-ond stage is carried out with about 4 to about 8 volume buffer containing 50mM Tris HCl, 2mN EDTA. In third stage, diafiltration is carried out with about 4 to about 8 vol-ume buffer containing 150mM NaCl & in fourth stage diafiltration is carried out with about 15 to about 20 times WFI. After completion of diafiltration activity, carryout fil-tration of concentrated solution by 0.2µ capsule filter. The filtered material is subjected to Hydroxyapatite chromatography.
The Utrogel Hydroxyapatite matrix has been used to purify the polysaccharide. After completion of HA chromatography pooled fractions is subjected to diafiltration with about 10 to about 12 times WFI using 100 kD cutoff membrane on completion of diafil-tration the concentrated solution is filtered through 0.2µ filter and the filtered material is subjected to drying for 12-36 hrs under vacuum and at low temperature (-70 to -80°C). A simple lab scale freeze drier was used for the purpose. The dried material obtained was identified as purified PnPs. Purified PnPs is stored at temperature of -20°C or below. (Process flow as in Fig 1)
Fig1 PROCESS FLOW PRODUCTION OF PNEUMOCOCCAL POLYSACCHA-RIDE
Inoculum preparation from WCB

Fermentation

Inactivation using formalin followed by lysis using sodium deoxycholate

Centrifugation at 4500rpm

Ultra filtration /Diafiltration of supernatant
(Concentrate up to 1/10th volume followed by dia-filtration with of WFI)

Addition of CTAB under continuous stirring at room temperature followed by sedimentation of precipitate at 5±30C

Centrifugation at 4500rpm

Collect the soup and addition of Sodium Chloride
and stirring at room temperature

Addition of ethanol
to allow complete precipitation

Centrifugation at 4500rpm

Collect the precipitate & dissolved in WFI,
Add Sodium Acetate & Sodium Deoxycholate

Addition of ethanol to allow complete precipita-tion

Centrifugation at 4500rpm

Collect the supernatant and pass through muslin cloth

Addition of sodium acetate and adjust the pH

Addition of ethanol to allow complete precipitation

Centrifugation at 4500rpm

Collect the precipitate & dissolve in WFI

Diafiltration against different buffers followed by final diafiltration using WFI

0.22µ filtration & storage of polysaccharide solu-tion at minus 200C until further purification

Hydroxyapatite chromatography

Diafiltration of pooled fractions against WFI

0.2µ filtration of purified polysaccharide

Lyophilization and storage of the dry purified poly-saccharide below minus 20ºC

Example 2
Production of pneumococcal polysaccharide (PnPs) of Streptococcus pneumonia Serotype 15A
Pneumococcal polysaccharide (PnPs) was produced using100 L bioreactor. The inocu-lum for 100L bioreactor prepared under the conditions of Example 1 (batch fermenta-tion).
The bioreactor was first filled with the basal medium (compound 1 & 2 in Table 1) be-fore being sterilized in situ for 30 minutes at 1210 C. Just before inoculation the proper amount of stock solutions (Compound 3 to 13 in Table1) were added to the medium. The bioreactor was inoculated using inoculum, cultivated on a 100 L bioreactor scale culture.

The pH was kept constant at 7.2 using 5 mol/l NaOH. The temperature was kept constant at 370 C, Airflow: 0.15 – 0.75 vvm. The stirrer speed was kept 80 rpm.

Samples were collected at an interval of 1 hr. As the growing culture reached the OD600 0.8, feeding was started. The feed rate was monitored and manually controlled. When cultivation reaches stationary phase (OD becomes constant and do not show any increase in value for last two consecutive samples), stop the addition of feed and stop the fermen-tation.

The fermentation culture was chemically inactivated by addition of formaldehyde, there after the fermentation culture was harvested using a continuous centrifuge. The superna-tant was collected and subjected to concentration and diafiltration of the filtrate was car-ried out using 300 kD MWCO PES membrane. The precipitation preferably using CATB is carried out to obtain polysaccharide. Centrifugation is carried out preferably at 6000 – 8000 rpm. (Fig 1)

Example 3
A.Conjugation of pneumococcal polysaccharide (PnPs) of Streptococcus pneumonia Serotype 15A with CRM:

Conjugation process was carried out in 4 steps; PRP dissolution, CRM Activation, acti-vation of PnPs and conjugation of PnPs with CRM as described below.

Dissolution of pneumococcal polysaccharide:
Dried PnPs quantity was taken as per batch size preferably 10g on the basis of PnPs content. Dried purified PnPs (having 10g PnPs content) was dissolved in WFI at low temperature preferably at 5 ±3oC to get the final concentration of 10±1 mg/ml on the basis of PnPs content. (Fig-2)

Activation of pneumococcal polysaccharide:
The activation 10g of PnPs (in terms of PnPs content) was carried out with addition of
50 mg/ml of CDAP solution (75% of the weight of total PnPs weight) and the desired pH adjusted with 0.2 M TEA to get activated PnPs. (Fig-2)

Activation of CRM:
CRM is diafiltered with WFI using 10 kD cut off membrane. The CRM activation was car-ried out by mixing equal amount of 10 mg/ml EDAC solution to the Concentrated & Dia-filtered CRM. (Fig-2)

Conjugation of PnPs with CRM:
Activated CRM (CRM-EDAC mix) was added to the PnPs-CDAP complex, slowly with constant gentle stirring at low temperature preferably at 5±3oC. The reaction mixture was incubated for 16-24 hours at 5±3oC with continuous gentle stirring. Samples of the conjugate were analyzed for PnPs and free PnPs at this stage. (Fig-2)

Figure 2 - conjugation of 15A capsular pneumococcal polysaccharide with CRM197 for preparation of conjugate vaccine.

Figure 2 shows the preparation of conjugation of Pneumococcal polysaccharide (PnPs) (1) with carrier protein (1a) for preparation of conjugate vaccine such as CRM197 pro-tein. As shown PnPs is activated (3a) and CRM is Activated (3a) the modified CRM197 is added to Activated PnPs (4) the mixture is incubated (5), filtered (6) and followed by concentrated and subjected to diafiltration (7) filtered conjugate is purified using chro-matography method (8) addition of non-ionic surfactant and emulsifier (9) and finally filtered in sterile environment (10).

B.Purification of conjugate:

Concentration and diafiltration of PnPs-CRM Conjugate:
Concentration and Diafiltration of PnPs-CRM conjugate has 2 steps; Concentration and Diafiltration of PnPs-CRM conjugate followed by Filtration of the concentrated-diafiltered PnPs-CRM conjugated. PnPs-CRM conjugated is concentrated up to about 60 to about 80 % of the initial conjugated volume through 300 kD PES cassette followed by
diafiltration (of about 8 to about 20 cycle) with PBS and is filtered through 0.45 micron filter.

Size exclusion Chromatography:
Chromatography step was carried out using AKTA process/explorer or equivalent system. PnPs CRM conjugate concentrate was applied to Toyopreal HW65 matrix. Maximum up to 20% of sample (v/v of matrix volume) can be loaded on the matrix @ 20 L/hr on BPG glass column 200/500 (GE healthcare). Multiple runs can be carried out on the matrix as per the PnPs CRM conjugate concentrate and pack bed volume of matrix. Collected eluate is sampled for PnPs content and molecular size distribution.
Add 1% non-ionic surfactant and emulsifier to the purified PnPs-CRM conjugated targeting final concentration of 0.1% of non-ionic surfactant and emulsifier. The non-ionic surfactant and emulsifier treated purified PnPs-CRM conjugated materials then passed through PES capsule or cartridge filter, pore size 0.22-?m to get final active raw material. The final filtered material is stored at 5 ± 3°C as mentioned in Fig 3 and Table 2.

TABLE: 2 Composition of PnPs conjugate
Parameter Acceptance Criteria
PnPS Content To fall in the minimum limit to maintain the PnPs to pro-tein ration
Protein content To fall in the minimum limit to maintain the PnPs to pro-tein ration
PnPs to Protein ratio 0.30 - 3.00
Molecular size distribution > 80 % elution at KD = 0.5
Free polysaccharide = 25 %
Free Protein < 5 %
EDAC Content = 10ng/µg of PS
Identity Positive
CDAP Content =20ng/ µg of PS
pH 6.0-7.5
Endotoxin content < 0.75 IU/µg of Polysaccharide

Fig 3: Conjugation of pneumococcal polysaccharide (PnPs) of Streptococcus pneu-monia Serotype 15A with CRM

Example 4
Combination of 15A serotype conjugated with CRM197 with serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 18C, 19A, 19F, 22F, 23F conjugated with CRM197 protein

The present invention is used for combining of 15A serotype conjugated with CRM197 protein and other serotypes of S. pneumoniae viz. serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 18C, 19A, 19F, 22F, 23F conjugated with CRM197 protein.

Formulation and Fill Finish process
Purified Bulk of 15A conjugate is formulated along with other purified bulk conjugate into desired doses form using appropriate buffer, Aluminum compound adjuvant, suita-ble excipients and preservative. Formulation activity is performed at required tempera-ture for desired time. Once tested and approved, formulated vaccine is filled of desired dose volume of 0.5 ml containing 2.2 micrograms polysaccharide each of serotypes of 1, 3, 4, 5, 6A, 7F, 9V, 12F, 14, 15A,18C, 19A, 19F, 22F, 23F and 4.4 micrograms polysac-charide of serotype 6B.
The basic manufacturing path and most of the steps to produce all PnPs conjugates (viz. serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15A,18C, 19A, 19F, 22F, 23F conjugated individually with CRM197) remain same with variations in their fermentation strategies and purification parameters such as ethanol precipitation, chromatographic purification and centrifugation.
Process for producing adjuvant based vaccine:
Bulk conjugates of serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15A, 18C, 19A, 19F, 22F, 23F were added to Aluminum phosphate aseptically. The mixture was stirred gently at 100-400 rpm (preferably 200-300 rpm) for 30 min-3 hrs with each addition of conju-gate (preferably individually 60 min-120 min for addition of each conjugate). Once the blending part completed addition of isotonic sodium chloride solution, suitable stabilizer and preservative 2-Phenoxyethanol (2-POE) at pH ranging between 6.0 -7.5 (preferably 6.4-7.2) in a sterile and uniform suspension. All mixing and blending were carried out between 8°C -18°C (preferably 10-12°C).
The fully liquid sixteen valent conjugate vaccine formulation obtained as mentioned above, formulated, filled, stoppered, sealed and labelled in appropriate single dose and multi dose containers for individual as well as mass vaccination. The labelled vials were stored at about 2-8°C for optimum shelf life. The single dose formulation was without any preservative whereas multi dose formulation contained suitable stabilizer and 2- Phenoxyethanol (2-POE) as preservative.
Example 5
Combination of 15A serotype conjugated with tetanus toxoid with serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 18C, 19A, 19F, 22F, 23F conjugated with Tetanus Toxoid

The present invention is used for combining of 15A serotype conjugated with tetanus
toxoid and other serotypes of S. pneumoniae viz. serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 18C, 19A, 19F, 22F, 23F conjugated with tetanus toxoid protein.

Formulation and Fill Finish process
Purified Bulk of 15A conjugate is formulated along with other purified bulk conjugate into desired doses form using appropriate buffer, Aluminum compound adjuvant, suita-ble excipients and preservative. Formulation activity is performed at required tempera-ture for desired time. Once tested and approved, formulated vaccine is filled of desired dose volume of 0.5 ml containing 2.2 micrograms polysaccharide of each of serotypes of 1, 3, 4, 5, 6A, 7F, 9V, 12F, 14, 15A,18C, 19A, 19F, 22F, 23F and 4.4 micrograms poly-saccharide of serotype 6B.
The basic manufacturing path and most of the steps to produce all PnPS conjugates (viz. serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15A,18C, 19A, 19F, 22F, 23F conjugated individually with tetanus toxoid) remain same with variations in their fermentation strategies and purification parameters such as ethanol precipitation, chromatographic purification and centrifugation.
Process for producing adjuvant based vaccine:
Bulk conjugates of serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15A, 18C, 19A, 19F, 22F, 23F were added to Aluminum phosphate aseptically. The mixture was stirred gently at 100-400 rpm (preferably 200-300 rpm) for 30 min -3 hrs with each addition of conju-gate (preferably individually 60 min -120 min for addition of each conjugate). Once the blending part completed addition of isotonic sodium chloride solution, suitable stabilizer and preservative 2-Phenoxyethanol (2-POE) at pH ranging between 6.0 -7.5 (preferably 6.4-7.2) in a sterile and uniform suspension. All mixing and blending were carried out between 8°C -18°C (preferably 10-12°C).
The fully liquid sixteen valent conjugate vaccine formulation obtained as mentioned above, formulated, filled, stoppered, sealed and labeled in appropriate single dose and multi dose containers for individual as well as mass vaccination. The labeled vials were stored at about 2-8°C for optimum shelf life. The single dose formulation was without any preservative whereas multi dose formulation contained suitable stabilizer and 2- Phenoxyethanol (2-POE) as preservative.

Although the preferred embodiments of the present invention and their respective varia-tions have been described, people having ordinary skills in the art would envision vari-ous modifications of those embodiments. Accordingly, the present invention should not be limited to precise forms and manners in the above disclosure and description but should simply be taken by way of examples. Thus, the present invention can be varied and modified without departing the true scope and spirit thereof as defined in the ap-pended claims.