DESC:FIELD
The present disclosure relates to an immunogenic composition.
BACKGROUND
Streptococcus pneumoniae is a leading cause of pneumonia, meningitis and sepsis. Administration of vaccines containing polysaccharide-protein conjugates has been valuable in preventing the incidence of pneumococcal diseases.
A polysaccharide-protein conjugate can be prepared from the polysaccharides of a noxious microorganism and used in the form of a vaccine for inducing immunogenic response against the microorganism.
The pneumococcal conjugate vaccine (PCV) containing 7 polysaccharide-protein conjugates obtained from capsular polysaccharides of the most frequently isolated serotypes of Streptococcus pneumoniae (4, 6B, 9V, 14, 18C, 19F and 23F), hereinafter referred to as 7-PCV, has been useful.
However, the emergence of new serotypes led to the development of the PCVcontaining 13polysaccharide-protein conjugates, hereinafter referred to as 13-PCV.The 13-PCV containspolysaccharide-protein conjugates obtained from capsular polysaccharides of serotypes 1, 3, 5, 6A, 7F, 19A, in addition to the 7 serotypes (4, 6B, 9V, 14, 18C, 19F and 23F) of the 7-PCV.
Recently, the incidence of invasive pneumococcal disease (IPD) caused by the serotypes not included in the 13-PCV has increased. In 2012, serotypes 15A, 15B, 15C, and 24 were recognized among the Japanese children who had invasive pneumococcal disease (IPD). At Regions Hospital in St. Paul Streptococcus pneumoniae isolates were collected from adults hospitalized with IPDfrom 2002 through 2010. These cases were found to have serotypes 3, 7F, 19A, 6A, 12F and 22F.
Thus, there is felt a need to prepare compositions containing polyvalent polysaccharide-protein conjugates comprising conjugates obtained from the capsular polysaccharides of Streptococcus pneumoniae serotypes, especially those serotypes that are not included in the 13-valent pneumococcal conjugate vaccine.
OBJECTS
Some of the objects of the present disclosure, which at least one embodiment herein satisfies, are as follows.
It is an object of the present disclosure to ameliorate one or more problems of the prior art or to at least provide a useful alternative.
An object of the present disclosure is to provide a polyvalent polysaccharide-protein conjugatescomposition.
Other objects and advantages of the present disclosure will be more apparent from the following description, which is not intended to limit the scope of the present disclosure.
SUMMARY
The present disclosure relates to polyvalent polysaccharide-protein conjugates composition comprising a plurality of capsular polysaccharides of Streptococcus pneumoniae serotypes conjugated to at least one carrier protein. The Streptococcus pneumoniae serotypes are selected from the group consisting of 1, 2, 3, 4, 5, 6A, 6B, 7F, 9A, 9V, 9N, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, 33F and 45.
The polyvalent polysaccharide-protein conjugates composition of the present disclosure includes the polysaccharide-protein conjugates of serotypes 12F and 15B of Streptococcus pneumoniae, which are not included in the 13-valent PCV. Therefore, these compositions are useful as vaccines to elicit immunologic response against the Streptococcus pneumoniae serotypes 12F and 15B.
DETAILED DESCRIPTION
The emergence of new Streptococcus pneumoniae serotypes leading to invasive pneumococcal disease (IPD) has provided a challenge. To overcome this challenge, the present disclosure envisages polyvalent polysaccharide-protein conjugate compositions comprising conjugates obtained from the capsular polysaccharides of the new Streptococcus pneumoniae serotypes. These compositions can be useful for inducing immunogenic response against these serotypes.
In one aspect of the present disclosure, there is provided a polyvalent polysaccharide-protein conjugates composition comprising a plurality of capsular polysaccharides of Streptococcus pneumoniae serotypes conjugated to at least one carrier protein.
The relative burden and invasiveness of pneumococcal disease due to the serotypes 12F and 15B is higher. Hence, the polysaccharide-protein conjugates compositions of the present disclosure contains conjugates with capsular polysaccharides of Streptococcus pneumoniae serotypes 12F and 15B. Thus, the compositions of the present disclosure can provide wider protection against the invasive pneumococcal diseases as compared to the protection provided by the 13-valent PCV.
In accordance with the embodiments of the present disclosure, the Streptococcus pneumoniae serotypes include Streptococcus pneumoniae serotype 12F, Streptococcus pneumoniae serotype 15B and at least 10 other Streptococcus pneumoniae serotypes selected from the group of serotypes consisting of 1, 2, 3, 4, 5, 6A, 6B, 7F, 9A, 9V, 9N, 10A, 11A, 14, 17F, 18C, 19A, 19F, 20, 22F, 23F, 33F and 45.
The Streptococcus pneumoniae serotype 12F and Streptococcus pneumoniae serotype 15B were obtained from the Centers for Disease Control and prevention (CDC), USA.
In accordance with the preferred embodiments of the present disclosure, the polyvalent polysaccharide-protein conjugates composition includes conjugates of capsular polysaccharides of Streptococcus pneumoniae serotypes 12F and 15B, and at least 13 serotypes selected from a group consisting of 1, 2, 3, 4, 5, 6A, 6B, 7F, 9A, 9V, 14, 18C, 19A, 19F, 23F and 45.
In accordance with one embodiment of the present disclosure, the polyvalent polysaccharide-protein conjugates composition includes conjugates of capsular polysaccharides of Streptococcus pneumoniae serotypes 12F, 15B, 2, and at least 12 serotypes selected from a group consisting of 1, 3, 4, 5, 6A, 6B, 7F, 9A, 9V,14, 18C, 19A, 19F, 23F and 45.
In accordance with another embodiment of the present disclosure, the polyvalent polysaccharide-protein conjugates composition includes conjugates of capsular polysaccharides of Streptococcus pneumoniae serotypes 12F, 15B, 45, and at least 12 serotypes selected from a group consisting of 1, 2, 3, 4, 5, 6A, 6B, 7F, 9A, 9V, 14, 18C, 19A, 19F, and 23F.
In accordance with yet another embodiment of the present disclosure, the polyvalent polysaccharide-protein conjugates composition includes conjugates of capsular polysaccharides of Streptococcus pneumoniae serotypes 12F, 15B, 9A, 45, and at least 12 serotypes selected from a group consisting of 1, 2, 3, 4, 5, 6A, 6B,7F, 9V,14, 18C, 19A, 19F, and 23F.
In accordance with an exemplary embodiment of the present disclosure, the polyvalent polysaccharide-protein conjugates composition comprises 15 polysaccharide-protein conjugates.
The 15-valent composition of the present disclosure includes conjugates of capsular polysaccharides of Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15B, 18C, 19A, 19F, and 23F.
In accordance with a second exemplary embodiment of the present disclosure, the polyvalent polysaccharide-protein conjugates composition comprises 16 polysaccharide-protein conjugates.
The 16-valent composition of the present disclosure includes conjugates of capsular polysaccharides of Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15B, 18C, 19A, 19F, and 23F.
In accordance with a third exemplary embodiment of the present disclosure, the polyvalent polysaccharide-protein conjugates composition comprises 17 polysaccharide-protein conjugates.
The 17-valent composition of the present disclosure includes conjugates of capsular polysaccharides of Streptococcus pneumoniae serotypes1, 3, 4, 5, 6A, 6B, 7F, 9V, 9A, 12F, 14, 15B, 18C, 19A, 19F, 23F, and 45.
A carrier protein is conjugated with a polysaccharide to enhance the immunogenicity of the polysaccharide. The carrier protein is preferably non-toxic, non-reactogenic and readily available in sufficient amount and purity.
In accordance with the embodiments of the present disclosure, the carrier proteins can be selected from the following proteins;
1) CRM197, diphtheria toxoids, tetanus toxoid, fragment C of tetanus toxoid, pertussis toxoid, cholera toxoid, E. coli LT, E. coli ST, and exotoxin A from Pseudomonas aeruginosa;
2) bacterial outer membrane proteins such as outer membrane complex c (OMPC), porins, transferrin binding proteins, pneumococcal surface protein A, pneumococcal adhesin protein (PsaA), C5a peptidase from Group A or Group B streptococcus, Haemophilus influenzae protein D, pneumococcal pneumolysin including ply detoxified in some fashion for example dPLY-GMBS or dPLY-formol, PhtX, including PhtA, PhtB, PhtD, PhtE and fusions of Pht proteins for example PhtDE fusions, PhtBE fusions;
3) other proteins such as ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or purified protein derivative of tuberculin (PPD), PorB (from N. meningitidis), PD (Haemophilus influenzae protein D); or immunologically functional equivalents thereof;
4) synthetic peptides, heat shock proteins, pertussis proteins, cytokines, lymphokines, growth factors or hormones, artificial proteins comprising multiple human CD4+ T cell epitopes from various pathogen derived antigens such as N19 protein, iron uptake proteins, toxin A or B of C, difficile, and flagellin; and
5) Other diphtheria toxoidssuch as CRM176, CRM228, CRM 45; CRM 9, CRM 45, CRM102, CRM 103 and CRM107 and other mutations; deletion or mutation of Glu-148 to Asp, Gin or Ser and/or Ala 158 to Gly and other mutations; mutation of at least one or more residues Lys 516, Lys 526, Phe 530 and/or Lys 534 and other mutations.
The polyvalent polysaccharide-protein conjugate composition of the present disclosure contains conjugates prepared by using either a single carrier protein or more than one carrier proteins.
In accordance with one embodiment of the present disclosure, the carrier protein is CRM197.
In accordance with a second aspect of the present disclosure, there is provided a formulation comprising a plurality polysaccharide-protein conjugates of the present disclosure. The formulation comprises aqueous sodium chloride solution, at least one adjuvant, at least one buffer, at least one preservative and at least one excipient.
In accordance with the embodiments of the present disclosure, the formulation comprises each polysaccharide-protein conjugate in an amount ranging from 1 µg/mL to 10 µg/mL.
In accordance with one embodiment of the present disclosure, the amount of each polysaccharide-protein conjugate in the formulation is 4 µg/mL.
In accordance with the embodiments of the present disclosure, the formulation comprises at least one adjuvant selected from the group consisting aluminum phosphate, aluminum sulfate, aluminum hydroxide, calcium salts, magnesium salts, iron salts and zinc salts. Apart from these an insoluble suspension of acylated tyrosine or acylated sugars, cationically derivatized saccharides, anionically derivatized saccharides and polyphosphazenes can be used as an adjuvant.
In accordance with one embodiment of the present disclosure, the adjuvant is an aluminum salt.
In accordance with the embodiments of the present disclosure, the amount of the aluminium salt in the formulation ranges from 40 µg/mL to 3000 µg/mL.
In accordance with one exemplary embodiment of the present disclosure, the adjuvant is aluminum phosphate. The amount of aluminium phosphate in the formulation ranges from 2000 µg/mL to 3000 µg/mL.
In accordance with one embodiment of the present disclosure, the amount of the aluminium phosphate in the composition is 2500 µg/mL.
In accordance with the embodiments of the present disclosure, the preservative is at least one selected from a group consisting of thiomersal and 2-phenoxyethanol.
In accordance with one embodiment of the present disclosure, the preservative is thiomersal.
In accordance with one embodiment of the present disclosure, the buffer is histidine-succcinic acid buffer.
In accordance with one embodiment of the present disclosure, the excipient is polysorbate 20.
In accordance with the embodiments of the present disclosure, the polyvalentpolysaccharide-protein conjugates composition further comprises at least one protein obtained from Streptococcus pneumoniae.
In accordance with the embodiments of the present disclosure, the polyvalent polysaccharide-protein conjugates composition further comprises at least one protein obtained from Neisseria meningitidis type B.
In accordance with the embodiments of the present disclosure, the polyvalent polysaccharide-protein composition is stored in a form selected from the group consisting of a solution and lyophilized solid.
In accordance with the embodiments of the present disclosure, the formulation is useful as a vaccine.
In accordance with the embodiments of the present disclosure, the formulation is useful for inducing an immune response to a Streptococcus pneumoniae serotypes.
In accordance with a fourth aspect of the present disclosure, there is provided a method for potentiating an immune response in a mammal comprising parenterally administering to said mammal a formulation comprising the polysaccharide-protein conjugates composition of claim 1.
In accordance with a third aspect of the present disclosure, there is provided a method for the preparation of the polysaccharide-protein conjugates. The method comprises the following steps.
First, capsular polysaccharides from Streptococcus pneumoniae serotypes are isolated and sized.
The isolation and sizing of the capsular polysaccharides from Streptococcus pneumoniae of the capsular polysaccharides can be carried out by employing techniques such as homogenization, microfluidization, high pressure cell disruption, acid hydrolysis, alkaline degradation, oxidation by periodate, ozonolysis, enzymatic hydrolysis, sonication, and electron beam fragmentation.
The isolated and sized capsular polysaccharides are chemically activated to facilitate conjugation with the carrier protein. The activated capsular polysaccharide is conjugated to a carrier protein to form a polysaccharide-protein conjugate. This method step of activation and conjugation can be carried out by coupling techniques such as cyanation method and hydrazine method.
In accordance with one embodiment of the present disclosure, the capsular polysaccharides from the Streptococcus pneumoniae serotypes are activated and conjugated with the carrier protein by cyanation method using at least one cyanation reagent.
The cyanation reagent is selected from the group of reagents consisting of 1-cyano-4-(dimethylamino)-pyridinium tetrafluoroborate (CDAP), p-nitrophenylcyanate, N- cyanotriethylammonium tetrafluoroborate (CTEA), l-cyano-4-pyrrolidinopyridinium tetrafluoroborate (CPPT), 1-cyano-imidazole (1-CI), 1-cyanobenzotriazole (1-CBT), 2-cyanopyridazine-3(2H)one (2-CPO), and a functional derivative or modification thereof.
In accordance with one embodiment of the present disclosure, the capsular polysaccharides from the Streptococcus pneumoniae serotypes are activated and conjugated to the carrier protein byhydrazine method using at least one hydrazine reagent.
The hydrazine reagent is at least one selected from a group of reagents consisting of hydrazine, carbohydrazide, hydrazine chloride, dihydrazide, and adipic acid dihydrazide.
In accordance with the embodiments of the present disclosure, the ratio of the amount of the capsular polysaccharide and the amount of the carrier protein, used during the activation and conjugation method step, is in the range from 8:10 and 15:10 on mass basis.
After conjugation, the polysaccharide-protein conjugate is purified from unreacted protein and polysaccharide by standard purification techniques such as size exclusion chromatography, density gradient centrifugation, ultrafiltration, hydrophobic interaction chromatography and ammonium sulfate fractionation.
The present disclosure is further described in light of the following experiments which are set forth for illustration purpose only and not to be construed for limiting the scope of the present disclosure.
The laboratory scale experiments provided herein can be scaled up to industrial or commercial scale.
EXPERIMENTS:
The Streptococcus pneumoniae serotypes 12F, 15B, 1, 2, 3, 4, 5, 6A, 6B, 7F, 9A, 9V, 9N, 10A, 11A, 14,17F, 18C, 19A, 19F, 20, 22F,23F,33F and 45 were obtained from the Centers for Disease Control and prevention (CDC), USA.
Experiment 1: Preparation of polysaccharide-protein conjugates
The preparation of polysaccharide-protein conjugates involves the following steps.

Step 1: Fermentation of Streptococcus pneumoniae serotypes
Fermetation of a Streptococcus pneumoniae serotype was carried out in a fermenter containing media comprising glucose-100 g/l, MgSO4- 5g/l, yeast extract- 15 g/l and Hy-soya 50 g/l at 36.5 ?C ±0.5 with stirring at rpm 100 and airflow (surface aeration) of 0.5vvm. The pH of the fermentation broth was maintained at 7.1±0.2 using alkali (sodium carbonate: sodium hydroxide; 1:3). A pH dependent feeding method was used to provide batch fermentation feeding to obtain a desired volumetric yield of the pneumococcal polysaccharide. The fermentation broth was clarified.
Step 2: Purification of S. pneumoniae capsular polysaccharide serotype
Purification of S. Pneumoniae capsular polysaccharide serotype was carried out by the procedure described herein below. The purification procedure involves hydrophobic interaction chromatography (HIC) followed by ion exchange chromatography (IEC). A similar procedure was used for all the other S. Pneumoniae capsular polysaccharide serotypes.
5 liters of clarified broth from the fermenter cultures of S. pneumoniae serotype (obtained from step-1) was concentrated and diafiltered to 500 ml using a 100 kDa molecular weight cut-off (MWCO) membrane. Diafiltration was carried out using 25 mM sodium phosphate buffer at neutral pH followed by diafiltration with water for injection (WFI) to obtain a polysaccharide solution.
Nuclease was added to the polysaccharide solution to obtain a final concentration of 8 U/ml in the solution. The enzyme treatment was carried out at 370C for 10 ± 2 hrs with stirring.
Ammonium sulphate was added to the nuclease treated polysaccharide solution to 50% saturation and incubated at 2 – 8 0C for 12 ± 2 hours. The mixture was centrifuged and the pellet (precipitate) was discarded. The supernatant (~500 ml) is subjected to 100 kD diafiltration using NaCl followed by chilled WFI. The diafiltered solution containing polysaccharide with a buffer and high salt concentration was loaded on a hydrophobic interaction chromatography (HIC) column.
The HIC column (300 ml) was equilibrated with 50% saturated ammonium sulphate buffer and the polysaccharide solution (500 ml) was loaded onto the column at a pH in the range of 6 to 7. The column was then washed with the buffer containing 50% saturated ammonium sulphate. Under these conditions, the polysaccharide was recovered in the flow-through and equilibration wash from the column. The polysaccharide solution was then concentrated using a 100 kDa MWCO filter and then diafiltered with NaCl and WFI.
The ion exchange chromatography column (300 ml) (strong anion exchanger) was equilibrated with 20 mM sodium phosphate buffer. The polysaccharide solution (500 ml) obtained hereinabove was loaded onto the equilibrated column at a pH in the range of 6.5 to 7.5. The column was further washed with a buffer. The adsorbed polysaccharides were eluted with step gradient elution using 1.0 M NaCl. Various polysaccharides were eluted at different ionic strengths of NaCl. The polysaccharide solution was then concentrated using a 100 kDa MWCO filter and then diafiltered with WFI.
The diafiltered polysaccharide solution was filtered through a 0.22 µ membrane filter into polypropylene bottles. The purified polysaccharide was stored frozen at -20±50C.

Step-3: Sizing of Pneumococcal polysaccharides
Sizing of the Pneumococcal polysaccharides was carried out using a homogenizer. The size reduction was carried out a pressure in the range from 20 to 35 Kpsi and 1-3 passes. The sized polysaccharide was diafiltered and concentrated followed by 0.22 µ filtration. The sized polysaccharide was then subjected to HPSEC-RI for estimation of average molecular weight.
Step 4: Conjugation of Pneumococcal polysaccharides with protein
Conjugation of polysaccharide to carrier protein was carried out using general CDAP the conjugation method provided herein below. Details of the reaction conditions for preparation of conjugation of serotypes 12F and 15B are provided after the general procedure.
General procedure
Mechanically size reduced polysaccharides from the Streptococcus pneumoniae serotypes mentioned herein above (except for 6A which was used in native form or sized depending on the size of 6A) were added to a 2M NaCl solution. CDAP (in acetonitrile) from a 100 mg/mL stock solution was added to the polysaccharide solution as per a predetermined ratio of the polysaccharide to CDAP.
Approximately 1 minute later, 2M NaOH was added to achieve the specific activation pH. The polysaccharides were activated at this pH by standing for 4-10 minutes at 220C.
Predetermined amount of CRM 197 (the quantity depends on initial polysaccharide/Protein ratio) was added to the activated polysaccharide and the coupling reaction was performed at the specific pH from 3-8 hours depending on the serotype.
The reaction was then quenched by addition of glycine and incubating for 1 hour at 220C, and overnight at 120C. The conjugates were then purified by 300kDa diafiltration followed by 100kDa diafiltration. The polysaccharide and protein content of the purified 0.22um filtered conjugates were determined.
The conjugation of serotypes 12F and 15B is provided herein below.
Conjugation of serotypes 12F and 15B
i) For preparing 12F conjugate, using a polysaccharide to CDAP ratio of 1:1 at 22oC with a period of activation of 4 min and using a polysaccharide to protein ratio of 1:1.
ii) For preparing 15B conjugate, using a polysaccharide to CDAP ratio of 1:0.8 at 22oC with a period of activation of 9 to 10 min and a polysaccharide to protein ratio of 1:1.
Other reaction parameters are provided in Table 1.
Table 1: Reaction parameters

Conjugates 12F conjugate 15B conjugate
Polysaccharide Conc. (mg/mL) 9.5 9.5
Polysaccharide dissolution 2M NaCl 2M NaCl
Activation time (min) 4 9 to 10
Ratio Polysaccharide/CDAP 1.0:1.0 1.0:0.8
CRM Concentration (mg/mL) 20 20
Initial Ratio Polysaccharide/CRM197 1.0:1.0 1.0:1.0
pH 9.0 9.5

The conjugates obtained from the above reactions were characterized and the details of characterization are provided in Table 2.
Table 2: Conjugate characteristics
Conjugates 12F conjugate 15B conjugate
Final Ratio Polysaccharide/CRM197 0.86 0.96
CRM197/Polysaccharide 1.16 1.04
Free Polysaccharide (%) 1.5 1.07
Free CRM197 (%) ND 2.2
Avarage Mol. size (SEC-HP-RI) (kDa) 853 847
Avarage Mol. size (UV/RI/MALS) (kDa) 6012 4773

Example 2: Formulation comprising a 16-valent Pneumococcal Conjugate Composition
For the preparation of 16 valent posaccharide-protein conjugate composition, conjugates from serotype of 6A, 9V, 23F were individually adsorbed as a separate blend and then added to another blend comprising of a mixture of conjugates of serotypes 1, 2, 3, 4, 5, 6B, 7F, 12F, 14, 15B, 18C ,19A , and 19F that have been adsorbed in a combined mode.The composition of the formulation comprising 16-valent polysaccharide-protein composition is provided herein below in Table-3.
Table 3: Composition of PCV-16 formulation

S. No. Component Quantity/dose
1 Serotype 1 NLT 2.0 µg
2 Serotype 2 NLT 2.0 µg
3 Serotype 3 NLT 2.0 µg
4 Serotype 4 NLT 2.0 µg
5 Serotype 5 NLT 2.0 µg
6 Serotype 6A NLT 2.0 µg
7 Serotype 6B NLT 4.0 µg
8 Serotype 7F NLT 2.0 µg
9 Serotype 9V NLT 2.0 µg
10 Serotype 12F NLT 2.0 µg
11 Serotype 14 NLT 2.0 µg
12 Serotype 15B NLT 2.0 µg
13 Serotype 18C NLT 2.0 µg
14 Serotype 19A NLT 2.0 µg
15 Serotype 19F NLT 2.0 µg
16 Serotype 23F NLT 2.0 µg
17 Aluminium phosphate NMT 1.25 mg of Al3+
18 Histidine NLT 1.55 mg
19 CRM-197 11.3 – 113.3 µg
20 Succinic acid NLT 1.18 mg
21 Sodium Chloride NLT 4.5 mg
22 Polysorbate 20 NLT 50 µg
23 Thiomersal** 25 µg
24 WFI q.s.
* The active ingredients of the vaccine are conjugated to a carrier protein CRM197.
** Added only in multi-dose presentation.
TECHNICAL ADVANCES AND ECONOMICAL SIGNIFICANCE
The composition of the present disclosure described herein above has several technical advantages including but not limited to the realization of:
? a polyvalent polysaccharide-protein conjugates composition comprising 12 to 20 polysaccharide-protein conjugates; and
? a polyvalent polysaccharide-protein conjugates composition comprising conjugates of Streptococcus pneumoniaeserotype 12F and Streptococcus pneumoniaeserotype 15B.
Throughout this specification the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
The use of the expression “at least” or “at least one” suggests the use of one or more elements or ingredients or quantities, as the use may be in the embodiment of the disclosure to achieve one or more of the desired objects or results.
Any discussion of documents, acts, materials, devices, articles or the like that has been included in this specification is solely for the purpose of providing a context for the disclosure. It is not to be taken as an admission that any or all of these matters form a part of the prior art base or were common general knowledge in the field relevant to the disclosure as it existed anywhere before the priority date of this application.
The numerical values mentioned for the various physical parameters, dimensions or quantities are only approximations and it is envisaged that the values higher/lower than the numerical values assigned to the parameters, dimensions or quantities fall within the scope of the disclosure, unless there is a statement in the specification specific to the contrary.
While considerable emphasis has been placed herein on the components and component parts of the preferred embodiments, it will be appreciated that many embodiments can be made and that many changes can be made in the preferred embodiments without departing from the principles of the disclosure. These and other changes in the preferred embodiment as well as other embodiments of the disclosure will be apparent to those skilled in the art from the disclosure herein, whereby it is to be distinctly understood that the foregoing descriptive matter is to be interpreted merely as illustrative of the disclosure and not as a limitation. ,CLAIMS:1. A polyvalent polysaccharide-protein conjugates composition, said composition comprising a plurality of capsular polysaccharides of Streptococcus pneumoniae serotypes conjugated to at least one carrier protein; wherein said Streptococcus pneumoniae serotypes include Streptococcus pneumoniae serotype 12F, Streptococcus pneumoniae serotype 15B and at least 10 other Streptococcus pneumoniae serotypes selected from the group of serotypes consisting of 1, 2, 3, 4, 5, 6A, 6B, 7F, 9A, 9V, 9N, 10A, 11A, 14, 17F, 18C, 19A, 19F, 20, 22F, 23F, 33F and 45.
2. The composition as claimed in claim 1, wherein said composition comprises conjugates of capsular polysaccharides of 15 Streptococcus pneumoniae serotypes namely 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15B, 18C, 19A, 19F, and 23F.
3. The composition as claimed in claim 1, wherein said composition comprises conjugates of capsular polysaccharides of 16 Streptococcus pneumoniae serotypes namely 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 12F,14, 15B, 18C, 19A, 19F, and 23F.
4. The composition as claimed in claim 1, wherein said composition comprises conjugates of capsular polysaccharides of 17 Streptococcus pneumoniae serotypes namely 1, 3, 4, 5, 6A, 6B, 7F, 9V, 9A, 12F,14,15B, 18C, 19A, 19F, 23F, and 45.
5. The composition as claimed in claim 1, wherein said carrier protein is at least one selected from the group of proteins consisting of diphtheria toxoid, tetanus toxoid, pertussis toxoid, cholera toxoid, CRM197, diphtheria toxoid mutants, Haemophilus influenzae protein D and immunologically functional equivalents thereof.
6. The composition as claimed in claim 1, wherein said carrier protein is CRM197.
7. Aformulation comprising the composition as claimed in claim 1, wherein said formulation includes aqueous sodium chloride solution, at least one adjuvant, at least one buffer, at least one preservative and at least one excipient.
8. The formulation as claimed in claim 7, wherein the amount of each polysaccharide-protein conjugate is in the range from 1 µg/mL to 10 µg/mL of the formulation.
9. The formulation as claimed in claim 7, wherein said adjuvant is selected from the group consisting of aluminum phosphate, aluminum sulfate, aluminum hydroxide, calcium salts, magnesium salts, iron salts and zinc salts.
10. The formulationas claimed in claim 7,wherein said adjuvant is aluminum phosphate, and the amount of aluminium phosphate ranges from 2000 µg/mL to 3000 µg/mL of the formulation.
11. The formulation as claimed in claim 7, wherein said formulation further comprises at least one protein obtained from Streptococcus pneumoniaeand/or Neisseria meningitidis type B.
12. A method for preparing said polysaccharide-protein conjugates of claim 1, said method comprising the following steps:
- isolating and sizing capsular polysaccharides from Streptococcus pneumoniae serotypes;
- activating the isolated and sized capsular polysaccharide and conjugating the activated polysaccharide with at least one carrier protein with the help of at least one conjugation reagent; and
- purifying the polysaccharide-protein conjugate.
13. The method of claim 12, wherein the ratio of the amount of said capsular polysaccharide and the amount of said carrier protein is in the range from 8:10 and 15:10 on mass basis.
14. The formulation of claim 8 for use as a vaccine.
15. A method for potentiating an immune response in a mammal comprising parenterally administering to said mammal a formulation comprising the polysaccharide-protein conjugates composition of claim 1.