FORM-2
THE PATENT ACT, 1970
(39 OF 1970)
AND
THE PATENT RULES, 2003
(As Amended)
COMPLETE SPECIFICATION (See section 10; rule 13)
“IMPROVED IMMUNOGENIC COMPOSITIONS AGAINST ENTERIC DISEASES AND METHODS FOR ITS PREPARATION THEREOF”
SERUM INSTITUTE OF INDIA PVT. LIMITED
AN INDIAN COMPANY
OF 212/2, SOLI POONAWALLA ROAD,
HADAPSAR, PUNE-411028,
MAHARASHTRA, INDIA
The following specification particularly describes the invention and the manner in which it is to be performed.

CROSS-REFERENCE TO RELATED APPLICATION
The Present Application is a Patent of Addition filed under Section 54 of the Indian Patents Act, 1970, claiming priority to Indian patent application Ser. No. IN201921035435, filed on September 03, 2019. The invention is an improvement or modification of the invention claimed in the complete specification of the main application.
FIELD
The present disclosure relates to the field of biotechnology, more particularly to, it relates to an improved immunogenic composition for prophylaxis against infections caused by Salmonella and Non-typhoidal Salmonella infections and processes for its preparation.
BACKGROUND
All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following 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 specifically or implicitly referenced is prior art.
Upstream, Downstream, conjugation and formulation development can often be the rate-limiting step in early introduction of biopharmaceuticals into the market and in meeting the demands of the population.
Physical and covalent stabilization upon long-term storage of polysaccharide-protein conjugate is of fundamental concern because of the potential impact of degradation on immunogenicity, toxicity, and efficacy of the polysaccharide-protein conjugate.
Polysaccharide -protein conjugate vaccines, in particular, appear to have a stronger tendency to aggregate than the carrier protein alone (See Berti et al, 2004, Biophys J 86:3-9).
There are various factors affecting stability of polysaccharide-protein conjugate which include zeta potential, viscosity, solute concentration, pH changes, and temperature that contribute to storage stability.

Zeta potential is one such physical property which is essential to optimize the formulations of suspensions, emulsions and protein solutions, predict interactions with surfaces, and optimise the formation of films and coatings. Zeta potential is exhibited by any particle in suspension, macromolecule or material surface. It can be used to optimize the formulations of suspensions, emulsions and protein solutions, predict interactions with surfaces, and optimise the formation of films and coatings. Knowledge of the zeta potential can reduce the time needed to produce trial formulations. It can also be used as an aid in predicting long-term stability. In certain circumstances, the particles in dispersion may adhere to one another and form aggregates of successively increasing size, which may settle out under the influence of gravity. Further it can be seen that the zeta potential depends on the nature of the buffer used.
There are various factors affecting zeta potential which include type of components used in formulation, Concentration of a formulation component, pH and conductivity.
The variation in pH influences the electrostatic force, whereby the van der Waals forces remain constant for a given system. At a pH near the isoelectric point charge-charge repulsion is minimal between neutral molecules and attractive forces dominate resulting in a flocculated system, high viscosity and where the aggregation is most likely. This pH-effect was observed by Liu et al. and Chari et al. in antibody solutions.
The potency of conjugate vaccines relies on the effective conjugation of oligo- or polysaccharide to carrier protein(s) and the integrity of the vaccine molecules throughout their shelf-life. Factors adversely affecting of the stability of conjugates may reduce vaccine potency through reducing the amount, accessibility and solubility of conjugated saccharide and carrier protein epitopes, potentially reducing their protective efficacy.
Previous studies have shown that CRM197 conjugate vaccines including MenC-CRM197 and Hib-CRM197 are conformationally stable when stored at their recommended temperatures, but evidence of less well-folded conformation of the carrier protein with hydrolysis or depolymerisation of the oligosaccharide chains was observed at elevated temperature. While tetanus toxoid and its conjugated forms are more resistant to secondary and tertiary conformational changes due to formaldehyde-induced intramolecular cross-linking, changes in its self-association have been observed under some conditions.
The impact of pH and temperature on the stability and activity of polysaccharide-protein conjugate is factored by possible changes in the characteristics of polysaccharide-protein

conjugate that include gradual depolymerization of polysaccharide affecting both physical and chemical stability. Bulk conjugates showed some degree of degradation or apparent aggregation when stored at elevated temperatures (+37 °C and +56 °C) for 28 day. Polysaccharide-protein conjugate are often stable within a narrow pH range and temperature. It was found that at extreme pH values and temperature the polysaccharide component of polysaccharide-protein conjugate vaccines undergoes gradual depolymerization at a rate that depends on the type of conjugate, formulation components used and storage conditions which results in an increase in free polysaccharide which may adversely affect stability of product. Polysaccharide-carrier protein conjugates are known to release free polysaccharide after conjugation while further processing, lyophilization or storage in liquid as well as solid formulations. Only the polysaccharide that is covalently bound to the carrier protein i.e. Conjugated polysaccharide is immunologically important for clinical protection and excessive levels of unbound polysaccharide could potentially result in immunological hypo responsiveness to polysaccharide (Refer WHO/TRS/924 Page No. 14, A.3.3.5). Particularly, the Salmonella typhi conjugates reported in literature have a high free polysaccharide content upto 34% and high free protein content above 5% which indicates lower conjugation efficiency and lower stability of conjugates that is not desirable.
Further, a multivalent vaccine(s) comprising of Salmonella typhoid conjugate combined with atleast one additional conjugates from Salmonella paratyphi (S. paratyphi A, B, C), and Non-typhoidal Salmonella enterica serovars typhimurium (S. typhimurium) and enteritidis (S. enteritidis) into a single shot can provide immunogenicity against multiple diseases and is always advantageous over the monovalent vaccine since it reduces the number of shots given, reduced complications associated with multiple injections, reduces the administration and production costs, decreased costs of stocking, reduced risk of delayed or missed vaccinations and improves the patient compliance by reducing the number of separate vaccinations.
However, there are multiple technical challenges in maintaining immunogenicity and safety when combining vaccines. Main challenge in combination vaccine development is the risk that the efficacy or safety of the combination would be less than that seen with the administration of the vaccines separately (individually). New combinations cannot be less immunogenic, less efficacious, or more reactogenic than the previously licensed uncombined individual vaccines. Immunological, physical, and/or chemical interactions between the combined components have the potential to alter the immune response to specific components. Finally, and ideally, the many advantages of combination vaccines should not

be achieved at the cost of reduced product stability. Hence, the formulation development becomes altogether more important for long-term storage of polysaccharide-protein conjugate. Accordingly there is a need for polysaccharide-protein conjugate vaccine demonstrating free polysaccharide content less than 10%, free protein content less than 5%, and further the vaccine formulation showing low viscosity, devoid of aggregation; showing long-term stability across wide temperature ranges.
Therefore, different stabilizers are normally added to the formulations to protect polysaccharide-carrier protein conjugates against degradation during processing and storage. The choice of formulation for a polysaccharide -protein conjugate vaccine can greatly affect protein aggregation. See Ho et al., 2001, Vaccine 19:716-725. Further, formation of visible particles leading to precipitation was a concern associated with these antigens during early process development. Aggregates are widely studied as product-related impurities in biopharmaceutical drug candidates because they can be associated with potential immunogenic characteristics known to reduce the efficacy, for example, by generating anti-drug antibody responses.
In the case of vaccine bulk protein antigens, large aggregates and particles can lead to protein loss during process development (e.g., during filtration) thus affecting the productivity and cost of vaccine production.
The role of a disaccharide, particularly of sucrose, which is an amorphous sugar, is to provide an environment where the protein containing conjugates remain protected and thus stable. However in case of liquid formulations an enhanced rate of antibody aggregation was observed in sucrose containing formulations which was likely due to protein glycation following sucrose hydrolysis under accelerated conditions. (Ref: December 2009 Journal of Pharmaceutical Sciences 98(12):4501-10).
V.M. Toprani et al (2017) identified Sucrose, phosphate, sodium chloride, methionine and polysorbate-80 as potential stabilizers that protected dmLT against conformational destabilization, aggregation/particle formation or chemical degradation. Sodium chloride (table salt) is the most common ionic compound used. Ionic compounds adjust tonicity and maintain osmolarity.
The typical non-ionic surfactants used in pharmaceutical formulations include Triton™ X-100, Pluronic® F-68, F-88, and F-127 (poloxamers), Brij 35 (polyoxy-ethylene alkyl ether),

polyoxyl stearate 40, Cremophor® EL, and alpha-tocopherol TPGS. Each of these surfactants have a common fact, in that they all contain polyoxyethylene moieties and thus to a greater or lesser extent, exhibit a similar problem, in that the polyoxyethylene moiety auto oxidizes to produce reactive peroxides, which causes an increase in unwanted protein immunogenicity. (Refer Edward T. Maggio et al; Polysorbates, peroxides, protein aggregation, immunogenicity - a growing concern; Journal of Excipients and Food Chemicals 3(2):46-53; 2012). Polysorbates are prone to degradation by oxidation and hydrolysis with hydrolysis being induced either chemically or enzymatically. Polysorbate may also be auto-oxidized by temperature, light or transition trace metals, and the resulting peroxide formation may induce protein oxidation, whereas the acid produced may lead to a decrease in solution pH. PS80 degraded via hydrolysis led to slower surface adsorption rate, and the free fatty acid release from hydrolysis also forms insoluble particles, negatively impacting protein quality and stability. Polysorbate 80 has also been causally linked with an increased risk of blood clots, stroke, heart attack, heart failure, and of tumor growth or recurrence in patients with certain types of cancer.
As aggregation increases, the effective concentration of available immunogen decreases. Therefore, a need exists for compositions and methods which overcome the problem of aggregation by stabilizing biological molecules especially polysaccharide-protein conjugate vaccines against aggregation or particulate matter.
Thus, there is a need for an efficient platform process for manufacturing an effective vaccine against Salmonella serovar strains S. typhi; S. paratyphi A; S. typhimurium and S. enteritidis that meets multiple criterion including good immunogenicity, safety and affordability, in particular an improved formulation showing low viscosity, devoid of aggregation; showing long-term stability across wide temperature ranges.
To overcome the aforementioned limitations of prior art, applicant proposes improved, formulation(s) for preparing a bivalent Salmonella typhoid conjugate as well as multivalent vaccine(s) comprising of atleast one additional conjugates from Salmonella paratyphi (S. paratyphi A, B, C), and Non-typhoidal Salmonella enterica serovars typhimurium (S. typhimurium) and enteritidis (S. enteritidis)

OBJECTS
Some of the objects of the present disclosure, which at least one embodiment herein satisfies, are as follows:
An object of the present disclosure is to ameliorate one or more problems of the prior art or to at least provide a useful alternative.
It is an object of the present disclosure to develop an improved liquid vaccine formulation: i) is devoid of salt & detergents, ii) devoid of aggregates and particle formation, higher osmolality, optimized Zeta potential, low viscosity, ii) preserves the desired physicochemical & immunogenic characteristics of the saccharide-protein conjugate free polysaccharide content less than 10%, free protein content less than 5%, stability and immunogenicity for 12 months at 2-8°C, for 6 month at 25°C, for 30 Days at 40°C.
Another object of the present disclosure is to develop a multivalent polysaccharide – protein conjugate vaccine formulation for prophylaxis and treatment of infections caused by Salmonella serovar strains S. typhi; S. paratyphi A; S. typhimurium and S. enteritidis in humans combined together to be given in a single shot and which meets the criterion for the seroprotection for each of the said immunogenic components.
Yet another object of the present disclosure is to provide a multivalent polysaccharide – protein conjugate vaccine comprising polysaccharide derived from Salmonella serovar strains S. typhi; S. paratyphi A; S. typhimurium and S. enteritidis in any combination thereof, wherein the composition preserves the desired characteristics of the multiple conjugates, including low free polysaccharide content, low free protein content and high stability and immunogenicity.
Still another object of the present disclosure is to provide a method of vaccinating a host comprising parenteral immunization.

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:
Applicant has found a need to develop an improved vaccine formulation for prophylaxis and treatment of infections caused by Salmonella serovar strains S. typhi; S. paratyphi A; S. typhimurium and S. enteritidis in humans combined together to be given in a single shot and which meets the criterion for the seroprotection for each of the said immunogenic components when administered parenterally. Applicant has found that the formulation preferably is a liquid formulation comprising of multivalent polysaccharide-protein conjugate wherein the polysaccharide is derived from Salmonella serovar strains S. typhi; S. paratyphi A; S. typhimurium and S. enteritidis in any combination thereof including buffering agent such as TRIS buffer, excipients such as mannitol, preservative such as 2-Phenoxyethanol and water for injection. The new formulation overcomes the limitations of the previous formulation in the prior-art and said new formulation/composition i) is devoid of salt & detergents, ii) devoid of aggregates and particle formation, higher osmolality, optimized Zeta potential, low viscosity, ii) preserves the desired physicochemical & immunogenic characteristics of the saccharide-protein conjugate free polysaccharide content less than 10%, free protein content less than 5%, stability and immunogenicity for 12 months at 2-8°C, for 6 month at 25°C, for 30 Days at 40°C.
Description of Figure-1: Process Flow Diagram for Formulation & Development

DESCRIPTION
Although the present disclosure may be susceptible to different embodiments, and following detailed discussion, with the understanding that the present disclosure can be considered an exemplification of the principles of the disclosure and is not intended to limit the scope of disclosure to that which is illustrated and disclosed in this description.
Embodiments are provided so as to thoroughly and fully convey the scope of the present disclosure to the person skilled in the art. Numerous details are set forth, relating to specific components, and processes, to provide a complete understanding of embodiments of the present disclosure. It will be apparent to the person skilled in the art that the details provided in the embodiments should not be construed to limit the scope of the present disclosure. In some embodiments, well-known composition, well-known processes, and well-known techniques are not described in detail.
The terminology used, in the present disclosure, is only for the purpose of explaining a particular embodiment and such terminology shall not be considered to limit the scope of the present disclosure. As used in the present disclosure, the forms "a,” "an," and "the" may be intended to include the plural forms as well, unless the context clearly suggests otherwise.
The terms "comprises," "comprising," “including,” and “having,” are open ended transitional phrases and therefore specify the presence of stated features, integers, steps, operations, elements, modules, units and/or components, but do not forbid the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. The particular order of steps disclosed in the process of the present disclosure is not to be construed as necessarily requiring their performance as described or illustrated. It is also to be understood that additional or alternative steps may be employed.
The terms first, second, third, etc., should not be construed to limit the scope of the present disclosure as the aforementioned terms may be only used to distinguish one element, component, region, layer or section from another component, region, layer or section. Terms such as first, second, third etc., when used herein do not imply a specific sequence or order unless clearly suggested by the present disclosure. The present disclosure provides a vaccine composition and a process for preparing the same.

It is understood that each feature or embodiment, or combination, described herein is a non-limiting, illustrative example of any of the aspects of the invention and, as such, is meant to be combinable with any other feature or embodiment, or combination, described herein. For example, where features are described with language such as “one embodiment”, “some embodiments”, “certain embodiments”, “further embodiment”, “specific exemplary embodiments”, and/or “another embodiment”, each of these types of embodiments is a non-limiting example of a feature that is intended to be combined with any other feature, or combination of features, described herein without having to list every possible combination. Such features or combinations of features apply to any of the aspects of the invention.
The term "vaccine" is optionally substitutable with the term "immunogenic" and vice versa.
The term "dose" herein is typically one administration of the vaccine of the invention, which is typically one injection. A typical human dose is 0.5mL. However various doses may be administered in a vaccine administration schedule.
The term "saccharide" throughout this specification may indicate polysaccharide or oligosaccharide and includes both. The capsular saccharide antigen may be a full length polysaccharide or it may be extended to bacterial 'sized-saccharides' and 'oligosaccharides' (which naturally have a low number of repeat units, or which are polysaccharides reduced in size for manageability, but are still capable of inducing a protective immune response in a host.
According to a first embodiment of the present disclosure, the immunogenic composition may comprise one or more of the polysaccharide-protein conjugate, wherein the polysaccharide is derived from Salmonella serovar strains S. typhi; S. paratyphi A; S. typhimurium and S. enteritidis.
According to a second embodiment of the present disclosure, the method of obtaining the polysaccharide derived from Salmonella serovar strains S. typhi; S. paratyphi A; S. typhimurium and S. enteritidis, by fed-batch process may comprise any subset or all of the following steps:

1. Inoculation, cultivation and Harvesting of bacteria in a Fermentation medium compositions,
2. Inactivation,
3. Separation,
4. Clarification, and
5. Sterilized Filtration.
According to a first aspect of second embodiment, the fermentation medium compositions may comprise a carbon source, a magnesium salt, a phosphate source, yeast extract and soy hydrolysate. The carbon source can be selected from the group consisting of glucose, mannitol, sucrose, lactose, fructose, and trehalose, preferably Glucose. The magnesium salt may be selected from magnesium chloride, magnesium sulfate, preferably Magnesium sulfate heptahydrate. The potassium source may be selected from Di-sodium hydrogen phosphate heptahydrate, sodium di-hydrogen phosphate monohydrate, potassium phosphate, and dipotassium phosphate. Preferably the potassium source is a combination of Di-sodium hydrogen phosphate heptahydrate, sodium di-hydrogen phosphate. Preferably, the soy hydrolysate is hysoy.
Yet accordingly the fermentation medium may additionally comprise an anti-foam agent selected from the group of Antifoam 204, Antifoam C, SE-15, Y-30, Antifoam EX-Cell, S184 (pure silicon oil), SLM54474 (polypropylene glycol: PPG), VP1133 (silicon oil/PPG mixture), BREOX (polyalkylene glycol), J673 STRUKTOL (Alkoxylated fatty acid esters on vegetable base) and SE9 (aqueous emulsion with 10% silicon oil component) of Wacker-Chemie Co. The anti-foam agent in combination with soy hydrolysate and yeast extract may aid in improved yield of the polysaccharides. Yet preferably the anti-foam agent may be Antifoam C or J673 STRUKTOL.
In accordance with the embodiments of the present disclosure, the yeast extract may be a yeast autolysate, an ultrafiltered yeast extract, or a synthetic yeast extract. The yeast extract may be selected from BD BBL™, BD BACTO™, Difco™ and the like. In a preferred embodiment, the yeast extract may be an ultrafiltered yeast extract, such as Difco™ Yeast Extract, UF. The soy hydrolysate may be selected from, but not limited to, soybean meal, soy

peptone, and soy flour. In one embodiment, the soy hydrolysate may be Difco™ Select Phytone™ UF. In another embodiment, the soy hydrolysate may be Hy-Soy.
The combination of antifoam, soya peptone and yeast extract results in improved harvest yield as compared to other media.
According to a second aspect of second embodiment, the process may follow a two shot strategy by incorporating the feed contents at a fixed proportion at particular fixed time intervals during when the fermentation is already undergoing and/or allowing continuous feed throughout the fed-batch mode of fermentation comprising multiple stages with the proportionate increase in the batch size at every stage.
According to a third aspect of second embodiment, the fermentation parameters may comprise of:
Temperature: 36.0 ± 2° C
Agitation: 150 to 600 rpm
pH: 7.0 ± 0.5
Dissolved Oxygen: 30% to 90%
Air (nl/min) : 2 to 10
Gas flow: 60 – 600 nl/min
Osmolality: 400 – 600 mOsm/kg
According to a fourth aspect of second embodiment, the inactivation of the bacterial culture may be carried out using formaldehyde.
Yet preferably the inactivation of the bacterial culture may be carried out by using formaldehyde in the range of 0.1 to 2 % v/v, preferably 0.5 % v/; incubated at 34 to 38 deg C , preferably 36 deg C; for 5-12 hrs, preferably 8 to 12 hrs.

According to a fifth aspect of second embodiment, the separation may be carried out by centrifugation. Yet preferably the separation may be carried out by centrifugation with parameters set at temperature 2-8 deg C; RPM – 7000-8000; Centrifuge time 40 -60 min.
According to a sixth aspect of second embodiment, the clarification may be carried out through depth filtration.
According to a seventh aspect of second embodiment, the clarified harvest may be sterilized through filtration using 0.2 µM sterile filters.
According to eighth aspect of second embodiment, the crude Salmonella enterica serovar typhi Vi-polysaccharide (ViPs) yield at the fermentation stage may be at least 40% and the average Vi-polysaccharide yield could be in the range of 100 mg/L to 5000 mg/L; more preferably 100-700 mg/L.
According to a third embodiment of the present disclosure, the fermentation harvest may be subjected to any subset or in any order or all of the following downstream purification steps to obtain desired quality of polysaccharide from Salmonella serovar strains S. typhi; S. paratyphi A; S. typhimurium and S. enteritidis:
a) Clarification of a bacterial capsular polysaccharide harvest by direct flow filtration (DFF) through at least one membrane having a pore size of about 0.2 micrometers;
b) concentration by tangential flow ultrafiltration (TFF) and buffer exchange by diafiltration (DF) using a membrane having 10 – 300 kDa or kD molecular weight cut off (MWCO);
c) treatment with anionic or cationic detergent, ethylene diamine tetra-acetic acid (EDTA) (4 to 10mM) and Sodium acetate (5% to 10%) for denaturation of proteins, nucleic acids and lipopolysaccharide;
d) alcohol precipitation (40% to 70%);
e) Centrifugation and filtration by direct flow filtration (DFF) through at least one clarification filter having a pore size of about 0.2 µM;
f) treatment with akali salt for removal of excess detergent followed by Centrifugation and filtration by direct flow filtration (DFF) through at least one clarification filter having a pore size of about 0.2 µM;

g) concentration by tangential flow filtration (TFF) and buffer exchange by diafiltration (DF)
using a membrane having 10 - 300 kDa or kD molecular weight cut off (MWCO); h) Treatment with anionic or cationic detergent; i) centrifugation and filtration by direct flow filtration (DFF) through at least one
clarification filter having a pore size of about 0.45 micrometers to about 0.2 micrometers; j) removal of protein and nucleic acid impurities by washing pellet with alcohol (50% to
70%) in presence of sodium chloride (0.1M to 2M); k) selective precipitation of polysaccharide by utilizing alcohol (<75% OR>95% ); l) dissolving polysaccharide in WFI and subjecting to concentration by tangential flow
filtration (TFF) and buffer exchange by diafiltration (DF) using a membrane having 10 -
300kDa or kD molecular weight cut off (MWCO); and m) Sterile Filtration through at least one sterile filter having a pore size of about 0.2
micrometers under sterile conditions.
According to a first aspect of third embodiment, the purification process may be devoid of any chromatography step.
According to a second aspect of third embodiment, the purification process may result in significant recovery of about 40% to 65% with the desired O-acetyl levels ( greater than 2.0 mmol/g polysaccharide), purified Vi polysaccharide yield could be in the range of 1000 to 4000 mg/L, average molecular weight could be in the range of 40 to 400 kDa, contains less than 1% proteins/peptides, less than 2% nucleic acids, less than 100 EU of endotoxins per µg of polysaccharide (PS), Molecular size distribution (greater than 50% of PS is eluted before a distribution coefficient (KD) of 0.25 is reached)
Yet preferably the average molecular weight of the purified Vi polysaccharide could be in the range of 40 to 400 kDa.
According to a third aspect of third embodiment, the anionic detergent may be selected from the group comprising of alkyl sulfates, sodium dodecyl sulfate (SDS), sodium deoxycholate, sodium dodecyl sulfonate, sodium s-alkyl sulfates, sodium fatty alcohol polyoxyethylene ether sulfates, sodium oleyl sulfate, N-oleoyl poly (amino acid) sodium, sodium alkylbenzene sulfonates, sodium alpha olefin suffocate, sodium alkyl sulfonates, alpha-sulfo

monocarboxylic acid esters, fatty acid sulfoalkyl esters, succinate sulfonate, alkyl naphthalene sulfonates, sodium alkane sulfonates, sodium ligninsulfonate , and sodium alkyl glyceryl ether sulfonates.
Yet preferably, said anionic detergent could be alkyl sulphate, more preferably sodium dodecyl sulphate at a final concentration in the range of 0.1% to 20%, more preferably in the range of 1-20% may be added to the retentate and stirred at 25°C - 30°C for 2 hour.
According to a fourth aspect of third embodiment, the alcohol precipitation may be carried out using methanol, ethanol, n-propyl alcohol, isopropyl alcohol, acetone or t-butyl alcohol; or a combination thereof.
Yet preferably the said alcohol could be ethanol.
According to a fifth aspect of third embodiment, the alkali salt may be selected from the group of sodium, potassium, calcium and magnesium salt. More preferably the alkali salt may be potassium salt selected from the group consisting of potassium chloride, potassium acetate, potassium sulfate, potassium carbonate, potassium bicarbonate, potassium phosphate, potassium hydrogen phosphate, potassium dihydrogen phosphate, potassium nitrate, and other potassium salts, or a combination of two or more thereof.
Yet preferably, the said potassium salt could be potassium chloride at a final concentration in the range of 0.1M to 2M mixed with the supernatant, and upon its dissolution the mixture was incubated at 2-8°C for >3 hours.
According to a sixth aspect of third embodiment, the cationic detergent may be selected from the group comprising of cetyltrimethylammonium salt, tetrabutylammonium salt, myristyltrimethylammonium salt and hexadimethrine bromide; or a combination thereof.
Yet preferably, said cationic detergent could be Cetyl trimethylammonium bromide (CTAB) at a final concentration in the range of 0.1% to 12%; preferably at 2% - 3% may be added to the retentate and stirred at 25°C - 30°C for 1 -2 hour.
According to a seventh aspect of third embodiment, the final purified polysaccharide bulk may be stored at less than or equal to -20°C.

According to a fourth embodiment of the present disclosure, the fermentation harvest may be subjected to any subset or in any order or all of the following downstream purification steps to obtain desired quality of O-specific polysaccharide from Salmonella Paratyphi A lipopolysaccharide (LPS):
a) Centrifugation and separation
b) concentration by tangential flow ultrafiltration (TFF) and buffer exchange by diafiltration (DF) using a membrane having 10 - 300kDa molecular weight cut off (MWCO);
c) Acid hydrolysis of LPS
d) Centrifugation and separation
e) Neutralization
f) Clarification of a LPS by direct flow filtration (DFF) through at least one membrane having a pore size of about 0.45 and 0.2 micrometers;
g) treatment with anionic or cationic detergent,
h) Centrifugation and separation
i) Direct flow filtration (DFF) through at least one membrane having a pore size of about
0.45 and 0.2 micrometers; j) concentration by tangential flow ultrafiltration (TFF) and buffer exchange by diafiltration
(DF) using a membrane having 10 - 300kDa molecular weight cut off (MWCO); k) alcohol precipitation (40% to 70%); l) Centrifugation and filtration by direct flow filtration (DFF) through at least one
clarification filter having a pore size of about 0.2 µM; m) treatment with akali salt for removal of excess detergent followed by Centrifugation and
filtration by direct flow filtration (DFF) through at least one clarification filter having a
pore size of about 0.2 µM; n) concentration by tangential flow filtration (TFF) and buffer exchange by diafiltration (DF)
using a membrane having 10 - 300kDa molecular weight cut off (MWCO); o) removal of protein and nucleic acid impurities by washing pellet with alcohol (50% to
70%) in presence of sodium chloride (0.1M to 2M);

p) dissolving polysaccharide in WFI and subjecting to concentration by tangential flow filtration (TFF) and buffer exchange by diafiltration (DF) using a membrane having 10 -300kDa molecular weight cut off (MWCO); and
q) Sterile Filtration through at least one sterile filters having a pore size of about 0.2 micrometers under sterile conditions.
According to a first aspect of fourth embodiment, the Acid hydrolysis of LPS may be carried out preferably using acetic acid (final concentration 0.5 -5%) pH~2.0 - 3.0; temperature 30 to 90 deg Celsius and time for about 100 to 200 minutes.
According to a second aspect of fourth embodiment, Acid hydrolysis neutralization may be carried out preferably using liquor ammonia to achieve final pH of 7.0.
According to a third aspect of fourth embodiment, the anionic detergent may be selected from the group comprising of alkyl sulfates, sodium dodecyl sulfate, sodium deoxycholate, sodium dodecyl sulfonate, sodium s-alkyl sulfates, sodium fatty alcohol polyoxyethylene ether sulfates, sodium oleyl sulfate, N-oleoyl poly (amino acid) sodium, sodium alkylbenzene sulfonates, sodium alpha olefin sulfonate , sodium alkyl sulfonates, alpha-sulfo monocarboxylic acid esters, fatty acid sulfoalkyl esters, succinate sulfonate, alkyl naphthalene sulfonates, sodium alkane sulfonates, sodium ligninsulfonate , and sodium alkyl glyceryl ether sulfonates.
Yet preferably, said anionic detergent could be sodium deoxycholate at a final concentration in the range of 0.1% to 20%, more preferably in the range of 1-2% may be added to the retentate and stirred at 25°C - 30°C for 10 – 120 minutes.
According to a fourth aspect of fourth embodiment, the alcohol precipitation may be carried out using methanol, ethanol, n-propyl alcohol, isopropyl alcohol, acetone or t-butyl alcohol; or a combination thereof.
Yet preferably the said alcohol could be ethanol.
According to a fifth aspect of fourth embodiment, the alkali salt may be selected from the group of sodium, potassium, calcium and magnesium salt. More preferably the alkali salt may

be potassium salt selected from the group consisting of potassium chloride, potassium acetate, potassium sulfate, potassium carbonate, potassium bicarbonate, potassium phosphate, potassium hydrogen phosphate, potassium dihydrogen phosphate, potassium nitrate, and other potassium salts, or a combination of two or more thereof.
Yet preferably, the said potassium salt could be potassium chloride at a final concentration in the range of 0.1M to 2M mixed with the supernatant, and upon its dissolution the mixture was incubated at 2-8°C for >3 hours.
According to a sixth aspect of fourth embodiment, the cationic detergent may be selected from the group comprising of cetyltrimethylammonium salt, tetrabutylammonium salt, myristyltrimethylammonium salt and hexadimethrine bromide; or a combination thereof.
According to a seventh aspect of fourth embodiment, the purification process may be devoid of any chromatography step.
According to an eighth aspect of fourth embodiment, the process results in significant reduction of endotoxin (< 100EU of endotoxin per µg of PS), protein (< 1%) and nucleic acid (< 2%) impurities, higher recovery of capsular polysaccharide suitably in the range of 40% to 65%, with the desired O-acetyl levels (> 2.0 mmol/g polysaccharide), Molecular size distribution (>50% of PS is eluted before a distribution coefficient (KD) of 0.25 is reached) and average molecular weight of the purified O-specific polysaccharide could be in the range of 40 to 200 kDa.
According to a ninth aspect of fourth embodiment, the final purified polysaccharide bulk may be stored at less than or equal to -20°C.
According to a fifth embodiment of the present disclosure, the purified Salmonella enterica serovar strains S. typhi; S. paratyphi A; S. typhimurium and S. enteritidis, polysaccharide may be covalently bound to carrier protein (CP) using a carbodiimide, reductive amination, or cyanylation conjugation reaction.
According to a first aspect of fifth embodiment, the purified Salmonella enterica serovar strains S. typhi; S. paratyphi A; S. typhimurium and S. enteritidis, polysaccharide may be covalently bound to carrier protein (CP) selected from the group comprising of tetanus toxin,

tetanus toxoid (TT), diphtheria toxoid (DT), CRM197, Pseudomonas aeruginosa toxoid, Bordetella pertussis toxoid, Fragment C of tetanus toxoid, recombinant full-length tetanus toxin with eight individual amino acid mutations (8MTT), Clostridium perfringens toxoid, E.coli LT, E. coli ST, Escherichia coli heat-labile toxin - B subunit, Neisseria meningitidis outer membrane complex, rEPA, protein D of H. influenzae, Flagellin FliC, Horseshoe crab Haemocyanin, exotoxin A from Pseudomonas aeruginosa, outer membrane complex c (OMPC), porins, transferrin binding proteins, pneumolysin, pneumococcal surface protein A (PspA), pneumococcal surface adhesin A (PsaA), pneumococcal PhtD, pneumococcal surface proteins BVH-3 and BVH-11, protective antigen (PA) of Bacillus anthracis and detoxified edema factor (EF) and lethal factor (LF) of Bacillus anthracis, ovalbumin, keyhole limpet hemocyanin (KLH), human serum albumin, bovine serum albumin (BSA), purified protein derivative of tuberculin (PPD), synthetic peptides, heat shock proteins, pertussis proteins, cytokines, lymphokines, hormones, growth factors, artificial proteins comprising multiple human CD4+ T cell epitopes from various pathogen-derived antigens such as N 19, iron-uptake proteins, toxin A or B from C. difficile and S.agalactiae proteins with or without linker and fragments, derivatives, modifications thereof.
Yet preferably the carrier protein used for conjugation with Salmonella typhi Vi polysaccharide may be tetanus toxoid.
Yet preferably the carrier protein used for conjugation with S. paratyphi A OSP may be DT.
Yet preferably the carrier protein used for conjugation with Salmonella
typhimurium polysaccharide may be TT or DT or CRM197.
Yet preferably the carrier protein used for conjugation with Salmonella
enteritidis polysaccharide may be TT or DT or CRM197.
In accordance with the present disclosure, CRM197 is procured from Recombinant Strain CS463-003 (MB101) of Pseudomonas fluorescens from Pfenex USA.
In accordance with the present disclosure, TT is procured from Clostridium Tetani (Harvard No 49205) obtained from Central research Institute (CRI), National Control Authority,

Kasauli, Himachal Pradesh, India. Central research Institute (CRI) procured this strain from NVI, Netherland.
In accordance with the present disclosure, DT is produced from cultures of Corynebacterium diphtheriae Park-Williams Number 8 strain, the strain is obtained from Central Research Institute (CRI), Kasauli, Himachal Pradesh, India.
According to a second aspect of fifth embodiment, before conjugation the purified Salmonella enterica serovar strains S. typhi; S. paratyphi A; S. typhimurium and S. enteritidis, polysaccharide may be subjected to depolymerization/sizing by chemical means selected from the group of FeCl3, H2O2, sodium metaperiodate and sodium acetate or mechanical means consisting of ultra-sonication.
Yet preferably, the purified Salmonella enterica serovar typhi polysaccharide (ViPs) may be subjected to depolymerization/sizing by sodium acetate (5% to 10%) wherein the average molecular weight of the ViPs could be in the range of 40 to 400 kDa.
Most preferably, the purified Salmonella enterica serovar typhi polysaccharide (ViPs) may be subjected to depolymerization/sizing by sodium acetate (5% to 10%) wherein the average molecular weight of the ViPs could be in the range of 180- 220 kDa.
Yet preferably, the purified Salmonella enterica serovar paratyphi A, B & C OSP may be subjected to depolymerization/sizing by sodium acetate (5% to 10%) wherein the average molecular weight of the OSP could be in the range of 40 to 200 kDa.
Most preferably, the purified Salmonella enterica serovar paratyphi A, B & C OSP may be subjected to depolymerization/sizing by sodium acetate (5% to 10%) wherein the average molecular weight of the OSP could be in the range of 40 to 150 kDa.
Yet preferably, the purified Salmonella enterica serovar Salmonella enterica serovar typhimurium and S. enteritidis saccharide may be subjected to depolymerization/sizing by sodium acetate (5% to 10%) wherein the average molecular weight of the saccharide could be in the range of 40 to 400 kDa.

Most preferably, the purified Salmonella enterica serovar Salmonella enterica serovar typhimurium and S enteritidis saccharide may be subjected to depolymerization/sizing by sodium acetate (5% to 10%) wherein the average molecular weight of the saccharide could be in the range of 40 to 200 kDa.
Applicant has found that the conjugation efficiency/conjugate yield, immunogenicity of conjugates prepared from using partially size reduced polysaccharides was higher as compared to conjugates prepared from full length polysaccharides Also Size reduction of polysaccharides decrease the viscosity of the solution and increases the number of reactive end groups, both factors contribute to an increased frequency of covalent bond formation.
Yet alternatively the purified Salmonella enterica serovar strains S typhi; S paratyphi A; S typhimurium and S enteritidis, polysaccharide may not be subjected to depolymerization/sizing.
According to a third aspect of fifth embodiment, before conjugation the carrier protein (CP) may be derivatized to comprise, amino and/or carboxyl groups via a hetero or homo-bifunctional linker selected from the group consisting of hydrazine, carbohydrazide, hydrazine chloride, a dihydrazide, ε-aminohexanoic acid, chlorohexanol dimethyl acetal, D-glucuronolactone, cystamine and p-nitrophenylethyl amine, hexanediamine, ethylenediamine, 1,6-diaminooxyhexane or β-propinamido, nitrophenyl ethylamine, haloalkyl halide, 6-amino caproic acid, and combinations thereof using a carbodiimide, reductive amination or cyanylation reaction.
Yet preferably the hetero or homo-bifunctional linker may be dihydrazide, more preferably adipic acid dihydrazide.
Hydrazide groups can be introduced into proteins through the carboxyl groups of aspartic acid and glutamic acid residues on the protein using a carbodiimide, reductive amination, cyanylation, reaction, for example, by reaction with hydrazine, carbohydrazide, succinyl dihydrazide, adipic acid dihydrazide, hydrazine chloride (e.g., hydrazine dihydrochloride) or any other dihydrazides in the presence of carbodiimide, such as 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC). EDC is employed as a catalyst to activate and modify the protein reactant with hydrazine or the dihydrazide.

Yet preferably, reaction of carrier protein (CP) with adipic acid dihydrazide (ADH) in the presence of carbodiimide, such as 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC) may be carried out at a pH of 5 to 7; more preferably 6.
Accordingly the reaction of carrier protein (CP) with adipic acid dihydrazide (ADH) in the presence of carbodiimide, such as 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC) may be stopped by raising the pH from about 6 to about 7 to 8.
Alternatively, after derivatization of carrier protein with ADH, the method may additionally comprise the step of buffer exchange by diafiltration (DF) using a membrane either of 10 kDa or 30 kDa or 50 kDa molecular weight cut off (MWCO) and sterile filtration using 0.2 u filter; whereby the ADH derivatized carrier protein is either buffer exchanged at least 10 volumes or passed through a suitable gel filtration column and substantially all unreacted compounds, residual ADH and residual EDC are removed, yielding a purified ADH derivatized carrier protein.
Yet alternatively before conjugation the carrier protein may not be derivatized to comprise, amino and/or carboxyl groups via a hetero or homo-bifunctional linker.
According to a third aspect of fifth embodiment, before conjugation the purified Salmonella enterica serovar strains S typhi; S paratyphi A; S typhimurium and S enteritidis, polysaccharide may be derivatized to comprise, amino and/or carboxyl groups via a hetero or homo-bifunctional linker selected from the group consisting of hydrazine, carbohydrazide, hydrazine chloride, a dihydrazide, a mixture thereof, ε-aminohexanoic acid, chlorohexanol dimethyl acetal, D-glucuronolactone, cystamine and p-nitrophenylethyl amine, hexanediamine, ethylenediamine, 1,6-diaminooxyhexane or β-propinamido, nitrophenyl ethylamine, haloalkyl halide, 6-amino caproic acid, and combinations thereof using a carbodiimide, reductive amination or cyanylation reaction.
Yet preferably the hetero or homo-bifunctional linker may be dihydrazide more preferably adipic acid dihydrazide.
Hydrazide groups can be introduced into the Salmonella enterica serovar strains S typhi; S paratyphi A; S typhimurium and S enteritidis, polysaccharide by using a carbodiimide,

reductive amination, cyanylation reaction for example reaction of polysaccharide with hydrazine, carbohydrazide, succinyl dihydrazide, adipic acid dihydrazide, hydrazine chloride (e.g., hydrazine dihydrochloride) or any other dihydrazides in the presence of cyanogen bromide (CNBr) may form hydrazide derivatives of Vi polysaccharide.
Yet preferably the Salmonella enterica serovar Paratyphi A OSP is derivatized with an adipic acid dihydrazide (ADH) linker using cyanylation conjugation chemistry wherein the cyanylation reagent is selected from a group of 1-cyano- 4-pyrrolidinopyridinium tetrafluoroborate (CPPT) (CPIP), 1- cyano- imidazole (1-CI), 1-cyanobenzotriazole (1-CBT), 1-cyano-4-(dimethylamino)-pyridinium tetrafluoroborate (‘CDAP’), p-nitrophenylcyanate and N-cyanotriethylammonium tetrafluoroborate (‘CTEA’) or 2-cyanopyridazine -3(2H) one (2-CPO).
Yet preferably the Salmonella enterica serovar Paratyphi A OSP is derivatized with an adipic acid dihydrazide (ADH) linker using cyanylation conjugation chemistry wherein the cyanylation reagent is 1-cyano- 4-pyrrolidinopyridinium tetrafluoroborate (CPPT) (CPIP) mixed in a ratio of 1:1 to 1:10 by weight of OSP: ADH and the ratio of OSP: CPPT may be in between 0.5 and 1.5, reaction carried out at a pH in range of 7-10, reaction duration for 1 – 2 hour, temperature in range of 2°C to 30°C.
Yet another aspect of fifth embodiment, before conjugation the polysaccharide may not be derivatized to comprise, amino and/or carboxyl groups via a hetero or homo-bifunctional linker.
A preferred aspect of fifth embodiment wherein, Salmonella enterica serovar typhi polysaccharide (ViPs) may be covalently bound to carrier protein using carbodiimide conjugation chemistry wherein any water-soluble carbodiimide more preferably 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC) can be used as a catalyst.
More preferably the Vi polysaccharide may be covalently bound to ADH derivatized tetanus toxoid using carbodiimide conjugation chemistry wherein any water-soluble carbodiimide more preferably 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC) can be used as a catalyst.

Yet alternatively the ADH derivatized Vi polysaccharide may be covalently bound to ADH derivatized tetanus toxoid using carbodiimide conjugation chemistry wherein any water-soluble carbodiimide more preferably 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC) can be used as a catalyst.
Yet preferably the Purified ViPs is covalently bound to tetanus toxoid (TT) using carbodiimide conjugation reaction in presence of 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (ED AC) mixed in a ratio of 1:0.5 to 1:2 by weight of ViPs: ED AC and the ratio of Vi polysaccharide and tetanus toxoid (TT) may be in between 0.5 and 1.5, reaction carried out at a pH in range of 5-7, temperature in range of 2°C to 30°C.
Yet alternatively, the Purified Vi polysaccharide (ViPs) may be covalently bound to ADH derivatized tetanus toxoid (TT) in presence of 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC) wherein the ratio by weight of ViPs:TT:EDC could be 1:1:2 and the concentration of Vi polysaccharide and tetanus toxoid may be between 0.1 mg/mL - 10.0 mg/mL and the ratio of Vi polysaccharide and tetanus toxoid may be in between 0.5 and 1.5.
Yet another aspect of fifth embodiment, the reaction of Vi polysaccharide (ViPs) with derivatized tetanus toxoid (TT) in presence of 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC) may be carried out at a pH in range of 5 to 7; more preferably 6; temperature in range of 2°C to 30°C; more preferably 10 to 25°C and the conjugation conversion efficiency is ≥70% more preferably ≥90% and molecular size of conjugate is preferably between 1000 to 1600 kDa
Further the reaction of derivatized tetanus toxoid (TT) and Vi polysaccharide (ViPs) in the presence of carbodiimide, such as 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC) may be stopped by raising the pH from about 6 to about 7 to 8.
According to one aspect of the fifth embodiment, S. paratyphi A polysaccharide (OSP) may be conjugated to a carrier protein using cyanylation conjugation chemistry wherein the cyanylation reagent is selected from a group of 1-cyano- 4-pyrrolidinopyridinium tetrafluoroborate (CPPT), 1- cyano- imidazole (1-CI), 1-cyanobenzotriazole (1-CBT), 1-cyano-4-(dimethylamino)-pyridinium tetrafluoroborate (‘CDAP’), p-nitrophenylcyanate and

N-cyanotriethylammonium tetrafluoroborate (‘CTEA’) or 2-cyanopyridazine -3(2H) one (2-CPO).
More preferably S. paratyphi A OSP may be conjugated to a carrier protein using cyanylation conjugation chemistry wherein the cyanylation reagent is 1-cyano- 4-pyrrolidinopyridinium tetrafluoroborate (CPPT) or (CPIP).
Yet preferably the purified S. paratyphi A OSP is covalently bound to carrier protein (CP) using cyanylation conjugation chemistry wherein the cyanylation reagent is 1-cyano- 4-pyrrolidinopyridinium tetrafluoroborate (CPPT) mixed in a ratio of 1:0.5 to 1:2 by weight of OSP: CPPT and the ratio of OSP and carrier protein may be in between 0.5 and 1.5, reaction carried out at a pH in range of 5-7, temperature in range of 2°C to 30°C.
Yet preferably the carrier protein used for conjugation with S. paratyphi A polysaccharide OSP may be Diphtheria toxoid (DT).
Yet another aspect of the fifth embodiment, S. typhimurium polysaccharide may be conjugated to a carrier protein using cyanylation conjugation chemistry wherein the cyanylation reagent is selected from a group of 1-cyano- 4-pyrrolidinopyridinium tetrafluoroborate (CPPT), 1- cyano- imidazole (1-CI), 1-cyanobenzotriazole (1-CBT), 1-cyano-4-(dimethylamino)-pyridinium tetrafluoroborate (‘CDAP’), p-nitrophenylcyanate and N-cyanotriethylammonium tetrafluoroborate (‘CTEA’) or 2-cyanopyridazine -3(2H) one (2-CPO).
More preferably S. typhimurium polysaccharide may be conjugated to a carrier protein using cyanylation conjugation chemistry wherein the cyanylation reagent is 1-cyano- 4-pyrrolidinopyridinium tetrafluoroborate (CPPT).
Yet the carrier protein used for conjugation with S. typhimurium polysaccharide may be Tetanus toxoid OR Diphtheria toxoid OR CRM 197.
Yet another aspect of the fifth embodiment, S. enteritidis polysaccharide may be conjugated to a carrier protein using cyanylation conjugation chemistry wherein the cyanylation reagent is selected from a group of 1-cyano- 4-pyrrolidinopyridinium tetrafluoroborate (CPPT), 1-cyano- imidazole (1-CI), 1-cyanobenzotriazole (1-CBT), 1-cyano-4-(dimethylamino)-

pyridinium tetrafluoroborate (‘CDAP’), p-nitrophenylcyanate and N-cyanotriethylammonium tetrafluoroborate (‘CTEA’) or 2-cyanopyridazine -3(2H) one (2-CPO).
More preferably S. enteritidis polysaccharide may be conjugated to a carrier protein using cyanylation conjugation chemistry wherein the cyanylation reagent is 1-cyano- 4-pyrrolidinopyridinium tetrafluoroborate (CPPT).
Yet preferably the carrier protein used for conjugation with S. enteritidis polysaccharide may be Tetanus toxoid OR Diphtheria toxoid OR CRM 197.
Applicant has found methods for stabilizing final polysaccharide – protein conjugate by utilizing the alternative methods of conjugation, ratio of polysaccharide to protein, ratio of polysaccharide to coupling agents, using appropriate linkers, appropriate size of the polysaccharide. The same can result in improvement in ratio of polysaccharide – protein conjugate in the vaccine which in turn can reduce the number of free saccharide and free protein in the conjugate, reduced carrier protein suppression, improved sterile filterability of the conjugate, better control of the conjugation, and greater intra-moiety cross-links indirectly provides a good immune response.
Applicant has found that various factors influence the coupling of polysaccharides and proteins which depend upon molecular weight of the ViPs, type of carrier protein selected, the ratio of the amount of polysaccharide: carrier protein used, activation of the functional groups, use of spacers and conjugation chemistry.
Yet another aspect of fifth embodiment, after conjugation reaction the method may comprise the step of concentration by tangential flow ultrafiltration (TFF) and buffer exchange by diafiltration (DF) using a membrane having either of 100 kDa or 300 kDa molecular weight cut off (MWCO); whereby the conjugate bulk is concentrated at least 3 fold and substantially all unreacted compounds, unconjugated polysaccharide, unconjugated protein and residual EDC are removed, yielding a purified Vi polysaccharide conjugate vaccine.
Additionally the method may comprise of Gel filtration chromatography whereby the conjugate bulk is concentrated at least 3 fold and substantially all unreacted compounds, unconjugated polysaccharides, unconjugated proteins and residual EDC are removed, yielding

a purified S typhi; S paratyphi A; S typhimurium and S enteritidis, polysaccharide conjugate vaccine wherein the conjugate yield is ≥ 50%.
Further method of purification may comprise of combination of ultrafiltration and gel filtration chromatography.
According to a sixth embodiment, the immunogenic composition may be a monovalent vaccine comprising either of S typhi Vi polysaccharide conjugated to a carrier protein or S paratyphi A OSP conjugated to a carrier protein or S typhimurium polysaccharide conjugated to a carrier protein or S enteritidis polysaccharide conjugated to a carrier protein.
Yet preferably 0.5 ml of the composition comprises of Salmonella enterica serovar typhi
ViPs-TT conjugate antigen in a dose range of 1- 50 µg; more preferably 25 - 30 µg per 0.5
ml;
Yet preferably 0.5 ml of the composition comprises of Salmonella enterica serovar Paratyphi
A OSP-DT conjugate antigen in a dose range of 1 - 50 µg; more preferably 25 - 30 µg per 0.5
ml;
Yet preferably 0.5 ml of the composition comprises of Salmonella enterica serovar
typhimurium saccharide-CP conjugate antigen in a dose range of 1 - 50 µg; more preferably
25 - 30 µg per 0.5 ml; wherein the CP is either TT or DT or CRM197
Yet preferably 0.5 ml of the composition comprises of Salmonella enterica serovar enteritidis
saccharide-CP conjugate antigen in a dose range of 1 - 50 µg; more preferably 25 - 30 µg per
0.5 ml; wherein the CP is either TT or DT or CRM197
According to a seventh embodiment of the present disclosure, wherein the immunogenic composition may comprise of atleast one bivalent combination:
a) Salmonella enterica serovar typhi saccharide-carrier protein (CP) conjugate antigen;
b) Salmonella enterica serovar Paratyphi A saccharide- carrier protein (CP) conjugate
antigen;
or a) Salmonella enterica serovar typhi saccharide-carrier protein (CP) conjugate antigen;

b) Salmonella enterica serovar typhimurium saccharide-carrier protein (CP) conjugate antigen; or
a) Salmonella enterica serovar typhi saccharide-carrier protein conjugate antigen;
b) Salmonella enterica serovar enteritidis saccharide-carrier protein conjugate antigen; or

a) Salmonella enterica serovar Paratyphi A saccharide-carrier protein conjugate antigen;
b) Salmonella enterica serovar typhimurium saccharide-carrier protein conjugate antigen; or

a) Salmonella enterica serovar Paratyphi A saccharide-carrier protein conjugate antigen;
b) Salmonella enterica serovar enteritidis saccharide-carrier protein conjugate antigen; or

a) Salmonella enterica serovar typhimurium saccharide-carrier protein conjugate antigen;
b) Salmonella enterica serovar enteritidis saccharide-carrier protein conjugate antigen;
Yet preferably 0.5 ml of the composition comprises of Salmonella enterica serovar typhi
ViPs-TT conjugate antigen in a dose range of 1 – 50 µg; more preferably 25 - 30 µg per 0.5
ml;
Yet preferably 0.5 ml of the composition comprises of Salmonella enterica serovar Paratyphi
A OSP-DT conjugate antigen in a dose range of 1 – 50 µg; more preferably 25 - 30 µg per 0.5
ml;
Yet preferably 0.5 ml of the composition comprises of Salmonella enterica serovar
typhimurium saccharide-CP conjugate antigen in a dose range of 1 – 50 µg; more preferably
25 - 30 µg per 0.5 ml; wherein the CP is either TT or DT or CRM197
Yet preferably 0.5 ml of the composition comprises of Salmonella enterica serovar enteritidis
saccharide-CP conjugate antigen in a dose range of 1 – 50 µg; more preferably 25 - 30 µg per
0.5 ml; wherein the CP is either TT or DT or CRM197
According to an eighth embodiment of the present disclosure, wherein the immunogenic composition may comprise of atleast one trivalent combination:
a) Salmonella enterica serovar typhi saccharide-carrier protein (CP) conjugate antigen;

b) Salmonella enterica serovar Paratyphi A saccharide- carrier protein conjugate antigen;
c) Salmonella enterica serovar typhimurium saccharide-carrier protein conjugate antigen; or

a) Salmonella enterica serovar typhi saccharide-carrier protein conjugate antigen;
b) Salmonella enterica serovar typhimurium saccharide-carrier protein conjugate antigen;
c) Salmonella enterica serovar enteritidis saccharide-carrier protein conjugate antigen; or

a) Salmonella enterica serovar Paratyphi A saccharide- carrier protein conjugate antigen;
b) Salmonella enterica serovar enteritidis saccharide-carrier protein conjugate antigen;
c) Salmonella enterica serovar typhimurium saccharide-carrier protein conjugate antigen;
Yet preferably 0.5 ml of the composition comprises of Salmonella enterica serovar typhi
ViPs-TT conjugate antigen in a dose range of 1 – 50 µg; more preferably 25 - 30 µg per 0.5
ml;
Yet preferably 0.5 ml of the composition comprises of Salmonella enterica serovar Paratyphi
A OSP-DT conjugate antigen in a dose range of 1 – 50 µg; more preferably 25 - 30 µg per 0.5
ml;
Yet preferably 0.5 ml of the composition comprises of Salmonella enterica serovar
typhimurium saccharide-CP conjugate antigen in a dose range of 1 – 50 µg; more preferably
25 - 30 µg per 0.5 ml; wherein the CP is either TT or DT or CRM197
Yet preferably 0.5 ml of the composition comprises of Salmonella enterica serovar enteritidis
saccharide-CP conjugate antigen in a dose range of 1 – 50 µg; more preferably 25 - 30 µg per
0.5 ml; wherein the CP is either TT or DT or CRM197
According to a ninth embodiment of the present disclosure, wherein the tetravalent
immunogenic composition may comprise of: i) S. typhi Vi polysaccharide conjugated to a
carrier protein (CP), ii) S. paratyphi A polysaccharide conjugated to a carrier protein, iii) S.
enteritidis polysaccharide conjugated to a carrier protein, and iv) S.
typhimurium polysaccharide conjugated to a carrier protein.

Yet preferably 0.5 ml of the composition comprises of Salmonella enterica serovar typhi
ViPs-TT conjugate antigen in a dose range of 1 – 50 µg; more preferably 25 - 30 µg per 0.5
ml;
Yet preferably 0.5 ml of the composition comprises of Salmonella enterica serovar Paratyphi
A OSP-DT conjugate antigen in a dose range of 1 – 50 µg; more preferably 25 - 30 µg per 0.5
ml;
Yet preferably 0.5 ml of the composition comprises of Salmonella enterica serovar
typhimurium saccharide-CP conjugate antigen in a dose range of 1 – 50 µg; more preferably
25 - 30 µg per 0.5 ml; wherein the CP is either TT or DT or CRM197;
Yet preferably 0.5 ml of the composition comprises of Salmonella enterica serovar enteritidis
saccharide-CP conjugate antigen in a dose range of 1 – 50 µg; more preferably 25 - 30 µg per
0.5 ml; wherein the CP is either TT or DT or CRM197.
According to a tenth embodiment of the present disclosure, the immunogenic composition may further comprise one or more antigen selected from the group consisting of but not limited to Salmonella paratyphi B, Salmonella paratyphi C, Salmonella antigens such as Outer membrane vesicles, Outer membrane proteins (eg, OmpC, OmpD, OmpF), siderophores (enterobactin), type III secretion system proteins (eg, SipB, SipD, SseB, SseC, and PrgI), flagellin, Non-typhoidal Salmonella spp. Shigella, Shigella sonnei, Shigella dysenteriae, Shigella flexneri, Shigella boydii, Escherichia coli, Enterobacter species, Yersinia species, Pseudomonas species, Pseudomonas aeruginosa, Haemophilus influenzae (a, c, d, e, f serotypes and the unencapsulated strains), Hepatitis (A, C, D, E, F and G strains), , Influenza, Staphylococcus spp., Staphylococcus aureus, Staphylococcus aureus type 5, Staphylococcus aureus type 8, Streptococcus spp , Streptococcus pneumoniae (1, 2, 3, 4, 5,6A, 6B, 6C, 6D, 6E, 6G, 6H, 7A, 7B, 7C, 7F, 8, 9A,9L,9F,9N, 9V, 10F, 10B,10C, 10A, 11 A,11F,11B, 11C,11D,11E,12A,12B, 12F,13, 14, 15A,15C ,15B,15F,16A, 16F, 17A,17F, 18C,18F,18A,18B, 19A, 19B, 19C, 19F, 20, 20A,20B,21,22A, 22F, 23A,23B, 23F, 24A, 24B,24F , 25F, 25A,27,28F, 28A, 29, 31,32F, 32A,33A, 33C, 33D, 33E, 33F,33B, 34, 45,38,35A,35B,35C,35F,36,37,38, 39, 40,41F,41A,42,43,44,45,46,47F,47A,48), Group A Streptococcus, Group B Streptococcus(group Ia, Ib, II, III, IV, V, Vl, VII VII, VIII, and IX.), Neisseria meningitidis, Haemophilus pneumonia, Helicobacter pylori, Chlamydia

pneumoniae, Chlamydia trachomatis, Ureaplasma urealyticum, Mycoplasma pneumoniae,
Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus viridans,
Enterococcusfaecalis, Enterococcus faecium, Enterococcus faecalis Neisseria gonorrhoeae, Bacillus anthracis, Vibrio cholerae, Pasteurella pestis, Campylobacter spp., Campylobacter jejuni, Clostridium spp., Clostridium tetani, Clostridium difficile, Mycobacterium spp., Mycobacterium tuberculosis, M. catarrhalis , Klebsiella pneumoniae ,Treponema spp., Borrelia spp., Borrelia burgdorferi, Leptospira spp., Hemophilus ducreyi, Corynebacterium diphtheria, Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiseptica, Shigella spp., Ehrlichia spp., Rickettsia spp and N. meningitidis polysaccharide (A, B, C, D, W135, X, Y, Z and 29E)acellular pertussis antigen, modified adenylate cyclase, Malaria Antigen (RTS, S), anthrax, dengue, malaria, measles, mumps, rubella, BCG, Human papilloma virus, Japanese encephalitis, Dengue, Zika, Ebola, Chikungunya, Poliovirus, Rotavirus, smallpox, yellow fever, Flavivirus, Enteroviruses, Norovirus, Shingles, and Varicella virus antigens.
According to a eleventh embodiment of the present disclosure, wherein the composition comprises of atleast one combination:
a) Salmonella enterica serovar typhi saccharide-carrier protein conjugate antigen;
b) Neisseria meningitidis A saccharide – carrier protein conjugate antigen;
c) Neisseria meningitidis C saccharide – carrier protein conjugate antigen;

d) Neisseria meningitidis Y saccharide – carrier protein conjugate antigen;
e) Neisseria meningitidis W -135 saccharide – carrier protein conjugate antigen;
f) Neisseria meningitidis X saccharide – carrier protein conjugate antigen; or

a) Salmonella enterica serovar typhi saccharide-carrier protein conjugate antigen;
b) Salmonella enterica serovar Paratyphi A saccharide- carrier protein conjugate antigen;
c) Neisseria meningitidis A saccharide – carrier protein conjugate antigen;
d) Neisseria meningitidis C saccharide – carrier protein conjugate antigen;
e) Neisseria meningitidis Y saccharide – carrier protein conjugate antigen;
f) Neisseria meningitidis W -135 saccharide – carrier protein conjugate antigen;
g) Neisseria meningitidis X saccharide – carrier protein conjugate antigen;

or
a) Salmonella enterica serovar typhi saccharide-carrier protein conjugate antigen;
b) Salmonella enterica serovar Paratyphi A saccharide- carrier protein conjugate antigen;
c) Salmonella enterica serovar typhimurium saccharide-carrier protein conjugate antigen;
d) Neisseria meningitidis A saccharide – carrier protein conjugate antigen;
e) Neisseria meningitidis C saccharide – carrier protein conjugate antigen;
f) Neisseria meningitidis Y saccharide – carrier protein conjugate antigen;
g) Neisseria meningitidis W -135 saccharide – carrier protein conjugate antigen;
h) Neisseria meningitidis X saccharide – carrier protein conjugate antigen;
or
a) Salmonella enterica serovar typhi saccharide-carrier protein conjugate antigen;
b) Salmonella enterica serovar Paratyphi A saccharide- carrier protein conjugate antigen;
c) Salmonella enterica serovar typhimurium saccharide-carrier protein conjugate antigen;
d) Salmonella enterica serovar enteritidis saccharide-carrier protein conjugate antigen;
e) Neisseria meningitidis A saccharide – carrier protein conjugate antigen;
f) Neisseria meningitidis C saccharide – carrier protein conjugate antigen;
g) Neisseria meningitidis Y saccharide – carrier protein conjugate antigen;
h) Neisseria meningitidis W -135 saccharide – carrier protein conjugate antigen; i) Neisseria meningitidis X saccharide – carrier protein conjugate antigen; or
a) Salmonella enterica serovar typhimurium saccharide-carrier protein conjugate antigen;
b) Salmonella enterica serovar enteritidis saccharide-carrier protein conjugate antigen;
c) Neisseria meningitidis A saccharide – carrier protein conjugate antigen;
d) Neisseria meningitidis C saccharide – carrier protein conjugate antigen;
e) Neisseria meningitidis Y saccharide – carrier protein conjugate antigen;
f) Neisseria meningitidis W -135 saccharide – carrier protein conjugate antigen;
g) Neisseria meningitidis X saccharide – carrier protein conjugate antigen
or
a) Salmonella enterica serovar typhi saccharide-carrier protein conjugate antigen;
b) Neisseria meningitidis A saccharide – carrier protein conjugate antigen;
c) Neisseria meningitidis C saccharide – carrier protein conjugate antigen;

d) Neisseria meningitidis Y saccharide – carrier protein conjugate antigen;
e) Neisseria meningitidis W -135 saccharide – carrier protein conjugate antigen or

a) Salmonella enterica serovar typhi saccharide-carrier protein conjugate antigen;
b) Salmonella enterica serovar Paratyphi A saccharide- carrier protein conjugate antigen;
c) Neisseria meningitidis A saccharide – carrier protein conjugate antigen;
d) Neisseria meningitidis C saccharide – carrier protein conjugate antigen;
e) Neisseria meningitidis Y saccharide – carrier protein conjugate antigen;
f) Neisseria meningitidis W -135 saccharide – carrier protein conjugate antigen or

a) Salmonella enterica serovar typhimurium saccharide-carrier protein conjugate antigen;
b) Salmonella enterica serovar enteritidis saccharide-carrier protein conjugate antigen;
c) Neisseria meningitidis A saccharide – carrier protein conjugate antigen;
d) Neisseria meningitidis C saccharide – carrier protein conjugate antigen;
e) Neisseria meningitidis Y saccharide – carrier protein conjugate antigen;
f) Neisseria meningitidis W -135 saccharide – carrier protein conjugate antigen; or

a) Salmonella enterica serovar typhi saccharide-carrier protein conjugate antigen;
b) Neisseria meningitidis A saccharide – carrier protein conjugate antigen;
c) Neisseria meningitidis C saccharide – carrier protein conjugate antigen; or

a) Salmonella enterica serovar typhi saccharide-carrier protein conjugate antigen;
b) Salmonella enterica serovar Paratyphi A saccharide- carrier protein conjugate antigen;
c) Neisseria meningitidis A saccharide – carrier protein conjugate antigen;
d) Neisseria meningitidis C saccharide – carrier protein conjugate antigen; or

a) Salmonella enterica serovar typhimurium saccharide-carrier protein conjugate antigen;
b) Salmonella enterica serovar enteritidis saccharide-carrier protein conjugate antigen;
c) Neisseria meningitidis A saccharide – carrier protein conjugate antigen;
d) Neisseria meningitidis C saccharide – carrier protein conjugate antigen;

Yet preferably 0.5 ml of the composition comprises of Salmonella enterica serovar typhi
ViPs-TT conjugate antigen in a dose range of 1 - 50 µg; more preferably 25 - 30 µg per 0.5
ml;
Yet preferably 0.5 ml of the composition comprises of Salmonella enterica serovar Paratyphi
A OSP-DT conjugate antigen in a dose range of 1 - 50 µg; more preferably 25 - 30 µg per 0.5
ml;
Yet preferably 0.5 ml of the composition comprises of Salmonella enterica serovar
typhimurium saccharide-CP conjugate antigen in a dose range of 1 - 50 µg; more preferably
25 - 30 µg per 0.5 ml; wherein the CP is either TT or DT or CRM197;
Yet preferably 0.5 ml of the composition comprises of Salmonella enterica serovar enteritidis
saccharide-CP conjugate antigen in a dose range of 1 - 50 µg; more preferably 25 - 30 µg per
0.5 ml;wherein the CP is either TT or DT or CRM197.
Yet preferably 0.5 ml of the composition comprises of Neisseria meningitidis A saccharide -
TT conjugate antigen in a dose of 5 µg
Yet preferably 0.5 ml of the composition comprises of Neisseria meningitidis C saccharide -
CRM197 conjugate antigen in a dose of 5 µg
Yet preferably 0.5 ml of the composition comprises of Neisseria meningitidis Y saccharide -
CRM197 conjugate antigen in a dose of 5 µg
Yet preferably 0.5 ml of the composition comprises of Neisseria meningitidis W saccharide -
CRM197 conjugate antigen in a dose of 5 µg
Yet preferably 0.5 ml of the composition comprises of Neisseria meningitidis X saccharide -
TT conjugate antigen in a dose of 5 µg
According to a twelfth embodiment of the present disclosure, wherein the immunogenic
composition comprises of atleast one combination:
a) Salmonella enterica serovar typhi saccharide-carrier protein conjugate antigen;
b) an inactivated polio virus (IPV) antigen, selected from Salk or Sabin strain
c) a diphtheria toxoid, (D) antigen
d) a tetanus toxoid, (T) antigen
e) a whole cell pertussis, (wP) antigen or acellular pertussis, (aP);
f) a hepatitis B virus surface antigen, (HBsAg) and
g) a Haemophilus influenzae type b antigen, (Ηib);

or
a) Salmonella enterica serovar typhi saccharide-carrier protein conjugate antigen;
b) an inactivated polio virus (IPV) antigen, selected from Salk or Sabin strain
c) a rotavirus antigen;
d) a diphtheria toxoid, (D) antigen
e) a tetanus toxoid, (T) antigen
f) a whole cell pertussis, (wP) antigen or acellular pertussis, (aP);
g) a hepatitis B virus surface antigen, (HBsAg) and h) a Haemophilus influenzae type b antigen, (Ηib);
or
a) Salmonella enterica serovar typhi saccharide-carrier protein conjugate antigen;
b) Salmonella enterica serovar Paratyphi A saccharide- carrier protein conjugate antigen;
c) an inactivated polio virus (IPV) antigen, selected from Salk or Sabin strain
d) a diphtheria toxoid, (D) antigen
e) a tetanus toxoid, (T) antigen
f) a whole cell pertussis, (wP) antigen or acellular pertussis, (aP);
g) a hepatitis B virus surface antigen, (HBsAg) and h) a Haemophilus influenzae type b antigen, (Ηib);
or
a) Salmonella enterica serovar typhi saccharide-carrier protein conjugate antigen;
b) Salmonella enterica serovar Paratyphi A saccharide- carrier protein conjugate antigen;
c) a rotavirus antigen;
d) an inactivated polio virus (IPV) antigen, selected from Salk or Sabin strain
e) a diphtheria toxoid, (D) antigen
f) a tetanus toxoid, (T) antigen
g) a whole cell pertussis, (wP) antigen or acellular pertussis, (aP); h) a hepatitis B virus surface antigen, (HBsAg) and
i) a Haemophilus influenzae type b antigen, (Ηib); or
a) Salmonella enterica serovar typhi saccharide-carrier protein conjugate antigen;
b) Salmonella enterica serovar Paratyphi A saccharide- carrier protein conjugate antigen;

c) Salmonella enterica serovar typhimurium saccharide-carrier protein conjugate antigen;
d) an inactivated polio virus (IPV) antigen, selected from Salk or Sabin strain
e) a diphtheria toxoid, (D) antigen
f) a tetanus toxoid, (T) antigen
g) a whole cell pertussis, (wP) antigen or acellular pertussis, (aP); h) a hepatitis B virus surface antigen, (HBsAg) and
i) a Haemophilus influenzae type b antigen, (Ηib); or
a) Salmonella enterica serovar typhi saccharide-carrier protein conjugate antigen;
b) Salmonella enterica serovar Paratyphi A saccharide- carrier protein conjugate antigen;
c) Salmonella enterica serovar typhimurium saccharide-carrier protein conjugate antigen;
d) a rotavirus antigen;
e) an inactivated polio virus (IPV) antigen, selected from Salk or Sabin strain
f) a diphtheria toxoid, (D) antigen
g) a tetanus toxoid, (T) antigen
h) a whole cell pertussis, (wP) antigen or acellular pertussis, (aP); i) a hepatitis B virus surface antigen, (HBsAg) and j) a Haemophilus influenzae type b antigen, (Ηib); or
a) Salmonella enterica serovar typhi saccharide-carrier protein conjugate antigen;
b) Salmonella enterica serovar Paratyphi A saccharide- carrier protein conjugate antigen;
c) Salmonella enterica serovar typhimurium saccharide-carrier protein conjugate antigen;
d) Salmonella enterica serovar enteritidis saccharide-carrier protein conjugate antigen;
e) an inactivated polio virus (IPV) antigen, selected from Salk or Sabin strain
f) a diphtheria toxoid, (D) antigen
g) a tetanus toxoid, (T) antigen
h) a whole cell pertussis, (wP) antigen or acellular pertussis, (aP); i) a hepatitis B virus surface antigen, (HBsAg) and j) a Haemophilus influenzae type b antigen, (Ηib); or
a) Salmonella enterica serovar typhi saccharide-carrier protein conjugate antigen;

b) Salmonella enterica serovar Paratyphi A saccharide- carrier protein conjugate antigen;
c) Salmonella enterica serovar typhimurium saccharide-carrier protein conjugate antigen;
d) Salmonella enterica serovar enteritidis saccharide-carrier protein conjugate antigen;
e) a rotavirus antigen;
f) an inactivated polio virus (IPV) antigen, selected from Salk or Sabin strain
g) a diphtheria toxoid, (D) antigen h) a tetanus toxoid, (T) antigen
i) a whole cell pertussis, (wP) antigen or acellular pertussis, (aP);
j) a hepatitis B virus surface antigen, (HBsAg) and
k) a Haemophilus influenzae type b antigen, (Ηib); Yet preferably 0.5 ml of the composition comprises of Salmonella enterica serovar typhi ViPs-TT conjugate antigen in a dose range of 1 - 50 µg; more preferably 25 - 30 µg per 0.5 ml;
Yet preferably 0.5 ml of the composition comprises of Salmonella enterica serovar Paratyphi A OSP-DT conjugate antigen in a dose range of 1 - 50 µg; more preferably 25 - 30 µg per 0.5 ml;
Yet preferably 0.5 ml of the composition comprises of Salmonella enterica serovar typhimurium saccharide-CP conjugate antigen in a dose range of 1 - 50 µg; more preferably 25 - 30 µg per 0.5 ml; wherein the CP is either TT or DT or CRM197;
Yet preferably 0.5 ml of the composition comprises of Salmonella enterica serovar enteritidis saccharide-CP conjugate antigen in a dose range of 1 - 50 µg; more preferably 25 - 30 µg per 0.5 ml; wherein the CP is either TT or DT or CRM197.
Yet preferably the composition comprises of diphtheria toxoid, (D) antigen in an amount of 1 to 50 Lf per 0.5 ml
Yet preferably the composition comprises of a tetanus toxoid, (T) in an amount of 1 to 30 Lf per 0.5 ml
Yet preferably the composition comprises of a whole cell pertussis, (wP) antigen in an amount of 1 to 50 IOU per 0.5 ml or acellular pertussis, (aP) antigen comprising one or more of modified adenylate cyclase, Pertussis toxoid (PT) 1-50µg, Filamentous hemagglutinin (FHA) 1-50µg, Pertactin (P69 or PRN) 1-20µg or Fimbrial proteins (FIM 1 , 2 and 3) 2-25µg; per 0.5ml;

Yet preferably the composition comprises of a hepatitis B virus surface antigen, (HBsAg) in
an amount of 1 to 20 µg per 0.5 ml;
Yet preferably the composition comprises of a Haemophilus influenzae type b antigen, (Ηib)
in an amount of 1 to 20 µg per 0.5 ml;
Yet preferably the composition comprises of an inactivated rotavirus antigen selected from
CDC-9, CDC-66 or any other inactivated rotavirus strains present in an amount in the range
of 1 to 50 µg per 0.5 ml;
Yet preferably the composition comprises of an inactivated polio virus (IPV) antigen selected from Sabin or Salk Strain; wherein IPV Type 1 at a dose 1-50 D-antigen units (DU), IPV Type 2 at a dose of 1-50 D-antigen unit (DU) or IPV Type 3 at a dose of 1-50 D-antigen unit (DU), per 0.5 ml;
According to a thirteenth embodiment of the present disclosure, wherein the immunogenic composition comprises of atleast one combination:
a) Salmonella enterica serovar typhi saccharide-carrier protein conjugate antigen;
b) a rotavirus antigen;
c) a diarrheogenic Escherichia coli spp. (enterotoxigenic and enterohemorragic) antigen;
d) a Shigella spp. antigen;
e) a Campylobacter jejuni antigen;
f) a Vibrio cholerae antigen; or

a) Salmonella enterica serovar typhi saccharide-carrier protein conjugate antigen;
b) Salmonella enterica serovar Paratyphi A saccharide- carrier protein conjugate antigen;
c) a rotavirus antigen;
d) a diarrheogenic Escherichia coli spp. (enterotoxigenic and enterohemorragic) antigen;
e) a Shigella spp. antigen;
f) a Campylobacter jejuni antigen;
g) a Vibrio cholerae antigen; or

a) Salmonella enterica serovar typhi saccharide-carrier protein conjugate antigen;
b) Salmonella enterica serovar Paratyphi A saccharide- carrier protein conjugate antigen;

c) a rotavirus antigen;
d) a diarrheogenic Escherichia coli spp. (enterotoxigenic and enterohemorragic) antigen;
e) a Shigella spp. antigen;
f) a Campylobacter jejuni antigen or

a) Salmonella enterica serovar typhi saccharide-carrier protein conjugate antigen;
b) Salmonella enterica serovar Paratyphi A saccharide- carrier protein conjugate antigen;
c) a rotavirus antigen;
d) a diarrheogenic Escherichia coli spp. (enterotoxigenic and enterohemorragic) antigen;
e) a Shigella spp. antigen; or

a) Salmonella enterica serovar typhi saccharide-carrier protein conjugate antigen;
b) Salmonella enterica serovar Paratyphi A saccharide- carrier protein conjugate antigen;
c) a rotavirus antigen;
d) a Shigella spp. antigen; or

a) Salmonella enterica serovar typhimurium saccharide-carrier protein conjugate antigen;
b) Salmonella enterica serovar enteritidis saccharide-carrier protein conjugate antigen;
c) a rotavirus antigen;
d) a diarrheogenic Escherichia coli spp. (enterotoxigenic and enterohemorragic) antigen;
e) a Shigella spp. antigen;
f) a Campylobacter jejuni antigen;
g) a Vibrio cholerae antigen;
or
a) Salmonella enterica serovar typhi saccharide-carrier protein conjugate antigen;
b) Salmonella enterica serovar Paratyphi A saccharide- carrier protein conjugate antigen;
c) Salmonella enterica serovar typhimurium saccharide-carrier protein conjugate antigen;
d) a rotavirus antigen;
e) a diarrheogenic Escherichia coli spp. (enterotoxigenic and enterohemorragic) antigen;
f) a Shigella spp. antigen;
g) a Campylobacter jejuni antigen;

h) a Vibrio cholerae antigen; or
a) Salmonella enterica serovar typhi saccharide-carrier protein conjugate antigen;
b) Salmonella enterica serovar Paratyphi A saccharide- carrier protein conjugate antigen;
c) Salmonella enterica serovar typhimurium saccharide-carrier protein conjugate antigen;
d) Salmonella enterica serovar enteritidis saccharide-carrier protein conjugate antigen;
e) a rotavirus antigen;
f) a diarrheogenic Escherichia coli spp. (enterotoxigenic and enterohemorragic) antigen;
g) a Shigella spp. antigen;
h) a Campylobacter jejuni antigen; i) a Vibrio cholerae antigen;
Yet preferably 0.5 ml of the composition comprises of Salmonella enterica serovar typhi
ViPs-TT conjugate antigen in a dose range of 1 – 50 µg; more preferably 25 - 30 µg per 0.5
ml;
Yet preferably 0.5 ml of the composition comprises of Salmonella enterica serovar Paratyphi
A OSP-DT conjugate antigen in a dose range of 1 – 50 µg; more preferably 25 - 30 µg per 0.5
ml;
Yet preferably 0.5 ml of the composition comprises of Salmonella enterica serovar
typhimurium saccharide-CP conjugate antigen in a dose range of 1 – 50 µg; more preferably
25 - 30 µg per 0.5 ml; wherein the CP is either TT or DT or CRM197;
Yet preferably 0.5 ml of the composition comprises of Salmonella enterica serovar enteritidis
saccharide-CP conjugate antigen in a dose range of 1 – 50 µg; more preferably 25 - 30 µg per
0.5 ml;wherein the CP is either TT or DT or CRM197.
Yet preferably the composition comprises of an inactivated rotavirus antigen selected from
CDC-9, CDC-66 or any other inactivated rotavirus strains present in an amount in the range
of 1 to 50 µg per 0.5 ml;
According to one aspect of the embodiment, the immunogenic composition may comprise of a buffering agent selected from the group consisting of carbonate, phosphate, acetate, HEPES, Succinate, Histidine, TRIS, borate, citrate, lactate, gluconate and tartrate, as well as more complex organic buffering agents including a phosphate buffering agent that contains sodium

phosphate and/or potassium phosphate in a ratio selected to achieve the desired pH. In another example, the buffering agent contains Tris (hydroxymethyl) aminomethane, or "Tris", formulated to achieve the desired pH. Yet in another example, the buffering agent could be the minimum essential medium with Hanks salts. Other buffers, such as HEPES, piperazine-N, N′-bis (PIPES), and 2-ethanesulfonic acid (MES) are also envisaged by the present disclosure. The buffer aids in stabilizing the immunogenic composition of the present disclosure. The amount of the buffer may be in the range of 0.1 mM to 100 mM, preferably selected from 2mM, 2.5mM, 3mM, 4mM, 5mM, 6mM, 7mM, 22 mM, 23mM, 24mM, 25mM, 26 mM, 27 mM, 28 mM, 29 mM and 30 mM.
Citrate buffer may be prepared by dissolving citric acid monohydrate (CAM) in the range of 1.05 to 2.63 mg and trisodium citrate dehydrate (TCD) in the range of 1.47-3.68 mg
Preferably the immunogenic composition may comprise of TRIS or Citrate buffer or Histidine buffer or Succinate Buffer in the range of 0.1 mg - 2.0 mg per 0.5 ml of the composition.
Preferably the immunogenic composition may comprise of TRIS Buffer in the range of 0.1 mg - 1.52 mg per 0.5 ml of the composition; more preferably 0.30 mg per 0.5 ml.
Yet another aspect of the embodiment, the immunogenic composition may comprise of pharmaceutically acceptable excipients selected from the group consisting of sugar, sugar alcohol or polyol, surfactants, polymers, salts, aminoacids or pH modifiers.
Examples of the polymers may include dextran, carboxymethylcellulose, hyaluronic acid, cyclodextrin, etc.
Examples of the salts may include NaCl, KCl, KH2PO4, Na2HPO4.2H2O, CaC12, MgC12, etc. Preferably, the salt may be NaCl. Typically the amount of the salt may be in the range of 100 mM to 200 mM.
Examples of the aminoacids as excipient selected from the group of L-Histidine, Lysine, Isoleucine, Methionine, Glycine, Aspartic acid. Tricine, arginine, leucine, glutamine, alanine, peptide, hydrolysed protein or protein such as serum albumin.

Examples of the sugars as excipient selected from the group of sucrose, trehalose, mannose, raffinose, lactobionic acid, glucose, maltulose, iso- maltulose, maltose, lactose, dextrose, fructose or a combination thereof.
Examples of the sugar alcohol or polyol as excipient selected from the group of mannitol, lactitol, sorbitol, glycerol, xylitol, maltitol, lactitol, erythritol, isomalt and hydrogenated starch hydrolysates or a combination thereof
Preferably the immunogenic composition may comprise of Mannitol present at a concentration range of 1-50 mg per 0.5 ml of the composition; more preferably 25 mg per 0.5 ml of the composition.
Examples of the polymers as excipient selected from the group of dextran, carboxymethylcellulose, hyaluronic acid, cyclodextrin.
Yet preferably the single dose composition is free of preservative.
Yet preferably the multi-dose immunogenic composition may additionally comprise of preservative selected from the group consisting of 2-phenoxyethanol, Benzethonium chloride (Phemerol), Phenol, m-cresol, Thiomersal, Formaldehyde, paraben esters (e.g. methyl-, ethyl-, propyl- or butyl- paraben), benzalkonium chloride, benzyl alcohol, chlorobutanol, p-chlor-m-cresol, or benzyl alcohol or a combination thereof. A vaccine composition may include material for a single immunization, or may include material for multiple immunizations (i.e. a ‘multidose’ kit). The inclusion of a preservative is preferred in multidose arrangements. As an alternative (or in addition) to including a preservative in multidose compositions, the compositions may be contained in a container having an aseptic adaptor for removal of material. Preferably, the preservative may be 2-phenoxyethanol in the range of 0.1 mg to 50 mg; more preferably 2.5 mg per 0.5 ml of the composition.
Yet another aspect of the embodiment, the immunogenic composition may additionally comprise of auxiliary substances such as wetting or emulsifying agents, diluent pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired.

Yet preferable aspect of the embodiment, the immunogenic composition may comprise of water for injection as diluent.
Yet another aspect of the embodiment, the immunogenic composition may comprise of an adjuvant selected from the group of aluminum salt, aluminum hydroxide, aluminum phosphate, aluminum hydroxyphosphate, and potassium aluminum sulfate.
Yet another aspect of the embodiment, the immunogenic composition may additionally comprise of an immunostimulatory component selected from the group consisting of an oil and water emulsion, MF-59, a liposome, a lipopolysaccharide, a saponin, lipid A, lipid A derivatives, Monophosphoryl lipid A, 3–deacylated monophosphoryl lipid A, AS01, AS03, an oligonucleotide, an oligonucleotide comprising at least one unmethylated CpG and/or a liposome, Freund’s adjuvant, Freund’s complete adjuvant, Freund’s incomplete adjuvant, polymers, co-polymers such as polyoxyethylene-polyoxypropylene copolymers, including block co-polymers, polymer p 1005, CRL-8300 adjuvant, muramyl dipeptide, TLR-4 agonists, flagellin, flagellins derived from gram negative bacteria, TLR-5 agonists, fragments of flagellins capable of binding to TLR-5 receptors, Alpha-C-galactosylceramide, Chitosan, Interleukin-2, QS-21, ISCOMS, squalene mixtures (SAF-1), Quil A, cholera toxin B subunit, polyphosphazene and derivatives, mycobacterium cell wall preparations, mycolic acid derivatives, non-ionic block copolymer surfactants, OMV, fHbp, saponin combination with sterols and lipids.
Yet another aspect of the embodiment, the immunogenic composition may be fully liquid. Suitable forms of liquid preparation may include solutions, suspensions, emulsions, syrups, isotonic aqueous solutions, viscous compositions and elixirs that are buffered to a selected pH.
Yet preferably the immunogenic composition may be fully liquid and wherein the final pH of the composition may be in the range of pH 6.0 to pH 8.0; more preferably in the range of pH 6.5 to pH 7.5; still more preferably in the range of pH 7.2 to pH 7.5; and most preferably pH 7.2.

Yet preferably the immunogenic composition may be fully liquid and i) is devoid of salt & detergents, ii) devoid of aggregates and particle formation, higher osmolality, optimized Zeta potential, low viscosity.
Yet preferably the immunogenic composition may be fully liquid and may be stable for 12 months at 2-8°C, for 6 month at 25°C and for 30 Days at 40°C for over a period of six months
Yet preferably the immunogenic composition may be fully liquid and free polysaccharide after 6 months not more than 7.5% for 180-220 kDa polysaccharide and free polysaccharide after 6 months not more than 10.5% for 388/80/45 kDa polysaccharide.
Yet alternative aspect of the embodiment, the immunogenic composition could be lyophilized or freeze dried composition.
As used herein the terms "Freeze-drying" or “lyophilize” or "lyophilization” involves lyophilization and refers to the process by which a suspension/solution is frozen, after which the water is removed by sublimation at low pressure. As used herein, the term "sublimation" refers to a change in the physical properties of a composition, wherein the composition changes directly from a solid state to a gaseous state without becoming a liquid.
Accordingly, the lyophilized immunogenic composition may be stable at 2-8 deg C from 12 to 36 months; at 25 deg C from 2 to 6 months; at 37 deg C from 1 week to 4 weeks, at 42 deg C for 2-7 days, and at 55 deg C for 2-7 days.
According to one aspect of the embodiment, the method for reconstituting a lyophilized immunogenic composition may comprise the step of reconstituting the lyophilized immunogenic composition with an aqueous solution optionally saline or water for injection (WFI) wherein, the final pH of the immunogenic composition after reconstitution is in the range of pH 6.0 to pH 8.0; more preferably in the range of pH 6.5 to pH 7.5; still more preferably in the range of pH 7.2 to pH 7.5; and most preferably pH 7.2.
According to an embodiment of the present disclosure, the method of manufacturing immunogenic composition may comprise of:
a. Diluting atleast one antigen concentrated bulk selected from a group consisting of Salmonella enterica serovar typhi saccharide-carrier protein conjugate; Salmonella

enterica serovar paratyphi A saccharide- carrier protein conjugate; Salmonella enterica serovar paratyphi B saccharide- carrier protein conjugate; Salmonella enterica serovar paratyphi C saccharide- carrier protein conjugate; Salmonella enterica serovar typhimurium saccharide- carrier protein conjugate; Salmonella enterica serovar enteritidis saccharide- carrier protein conjugate; Salmonella enterica serovar choleraesuis saccharide- carrier protein conjugate; and Salmonella enterica serovar dublin saccharide-carrier protein conjugate; with a stabilizer comprising at least one sugar alcohol, at least one buffer and preservative;
b. Adding WFI to components of step (a) to make up the volume;
c. Adding of Components obtained in step (b) in a blending vessel / container with agitation
at room temperature;
d. Sterilizing the Components obtained in step (c) by passing it through a 0.2 µ - 0.45µ
filters;
e. Filling into individual sterile glass vials and stoppering the glass vials.
According to a preferred aspect of the embodiment, the antigen concentrated bulk may
comprise of Salmonella enterica serovar typhi ViPs-TT conjugate antigen 1 – 50 µg per 0.5
ml and Salmonella enterica serovar paratyphi A OSP – DT conjugate antigen 1 – 50 µg per
0.5 ml.
Most preferably the Salmonella enterica serovar typhi ViPs-TT conjugate antigen 25 - 30 µg
per 0.5 ml
Most preferably the Salmonella enterica serovar paratyphi A OSP – DT conjugate antigen
25-30 µg per 0.5 ml
According to another preferred aspect of the embodiment, the stabilizer may comprise of sugar alcohol consisting of Mannitol 1 – 50 mg per 0.5 ml, buffer consisting of TRIS 0.05 – 0.5 mg per 0.5 ml and preservative consisting of 2-phenoxyethanol 1 – 10 mg per 0.5 ml.
Most preferably the stabilizer may comprise of sugar alcohol consisting of Mannitol 25 mg per 0.5 ml, buffer consisting of TRIS 0.30 mg per 0.5 ml and preservative consisting of 2-phenoxyethanol 2.5 mg per 0.5 ml.

According to a ninth embodiment of the present disclosure, the immunogenic composition may be formulated for use in a method for reducing the onset of or preventing a health condition comprising Salmonella serovar strains S typhi; S paratyphi A; S typhimurium and S enteritidis infection involving administration of an immunologically effective amount of the immunogenic composition to a human subject via parenteral or subcutaneous or intradermal, intramuscular or intraperitoneal or intravenous administration or injectable administration or sustained release from implants or administration by eye drops or nasal or rectal or buccal or vaginal, peroral or intragastric or mucosal or perlinqual, alveolar or gingival or olfactory or respiratory mucosa administration or any other routes of immunization.
The immunogenic composition of the present disclosure may be in the form of transdermal preparations including lotions, gels, sprays, ointments or other suitable techniques. If nasal or respiratory (mucosal) administration is desired (e.g., aerosol inhalation or insufflation), compositions can be in a form and dispensed by a squeeze spray dispenser, pump dispenser or aerosol dispenser. Aerosols are usually under pressure by means of a hydrocarbon. Pump dispensers can preferably dispense a metered dose or a dose having a particular particle size. When in the form of solutions, suspensions and gels, in some embodiments, the immunogenic compositions contain a major amount of water (preferably purified water) in addition to the active ingredient(s).
According to the preferred aspect of the embodiment, the immunogenic composition may be administered to a human subject via intramuscular route or subcutaneous.
According to the preferred aspect of the embodiment, an immunologically-effective amount of the immunogenic composition comprising the polysaccharide - protein conjugate for vaccination against Salmonella serovar strains S typhi; S paratyphi A; S typhimurium and S enteritidis infection bacterial infection could be from about 1 μg/0.5ml of the Polysaccharide conjugate of Salmonella serovar strains S typhi; S paratyphi A; S typhimurium or S enteritidis to about 100 μg/0.5ml of the Polysaccharide conjugate of Salmonella serovar strains S typhi; S paratyphi A; S typhimurium or S enteritidis or more. In some other aspects, it could be from about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45 or 50 μg to

about 55, 60, 65, 70, 75, 80, 85, 90, or 95 μg per 0.5ml single dose. In some embodiments, the immunologically-effective amount for vaccination against Salmonella serovar strains S typhi; S paratyphi A; S typhimurium and S enteritidis infection bacterial infection is from 1 μg/0.5ml to 50 μg/0.5ml; more preferably Salmonella enterica serovar typhi saccharide-carrier protein conjugate antigen; Salmonella enterica serovar Paratyphi A saccharide- carrier protein conjugate antigen; Salmonella enterica serovar typhimurium saccharide-carrier protein conjugate antigen; Salmonella enterica serovar enteritidis saccharide-carrier protein conjugate antigen; is present in a dose range of about 5 ug/0.5ml to about 30 ug/0.5ml; yet more preferably Salmonella enterica serovar typhi saccharide-carrier protein conjugate antigen; Salmonella enterica serovar Paratyphi A saccharide- carrier protein conjugate antigen; Salmonella enterica serovar typhimurium saccharide-carrier protein conjugate antigen; Salmonella enterica serovar enteritidis saccharide-carrier protein conjugate antigen; is present in a dose of about 25-30 µg/0.5ml
According to tenth embodiment of the present disclosure, the immunogenic composition may be administered intramuscularly or subcutaneous in a dose effective for the production of neutralizing antibody and protection. The vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be prophylactically and/or therapeutically effective against Typhoidal and NTS infection. The immunogenic composition of the present disclosure can be administered as primary prophylactic agents in elders, adolescents, adults or children at the risk of infection, or can be used as secondary agents for treating infected patients. For example, the immunogenic composition as disclosed herein can be used in elders, adolescents, adults or children less than 2 years of age or more than 2 years of age at risk of Salmonella serovar strains S typhi; S paratyphi A; S typhimurium and S enteritidis infection, or can be used as secondary agents for treating Salmonella serovar strains S typhi; S paratyphi A; S typhimurium and S enteritidis infected patients.
Yet alternatively the vaccines for NTS may be administered in infants between the ages of two and four months old, before peak incidence occurs around the age of 12 months. In addition, vaccine implementation would likely also include populations infected with HIV, as they are at heightened risk of infection with NTS. In developed countries, NTS vaccines may target the elderly who experience very high case-fatality rates (up to 50 percent). It has been

proposed that, in children, programmatic field implementation would integrate directly with existing Expanded Programme on Immunization schedules, perhaps at 6, 10, and 14 weeks.
More preferably the immunogenic composition may be administered intramuscularly or subcutaneous in a dosage volume of about 0.5ml or 1ml.
According to eleventh embodiment of the present disclosure, the immunogenic composition could be formulated as single dose vials or multidose vials (2 Dose or 5 Dose or 10 Dose vials) or multidose kit or as pre-filled syringes wherein the said immunogenic composition may be given in a single dose schedule, or preferably a multiple dose schedule in which a primary course of vaccination is followed by 1-3 separate doses given at subsequent time intervals after 1-3 years if needed. The dosage regimen will also, at least in part, be determined on the need of a booster dose required to confer protective immunity.
Yet preferably the immunogenic composition may be formulated for administration to a human subject elders, adolescents, adults or children less than 2 years of age or more than 2 years of age according to a one dose or two dose regimens or 3 dose regimens consisting of a first dose and/or a second dose to be administered between 3 months to 2 years after the first dose and/or a third dose to be administered between 3 months to 2 years after the second dose.
According to an embodiment of the present disclosure, the immunogenic composition may be formulated for inducing an immune response to Salmonella enterica serovar paratyphi B; and Salmonella enterica serovar paratyphi C in a subject, comprising administering to the subject an immunologically effective amount of an immunogenic composition not covering Paratyphi B and C serotypes.
According to an embodiment of the present disclosure, the immunogenic composition may be administered in the form of a single-dose or multi-dose vaccine kit.
Yet according to an aspect of eleventh embodiment, wherein a liquid single dose vaccine kit may comprise of:
a) Salmonella enterica serovar typhi ViPs-TT conjugate antigen 1 – 50 µg per 0.5 ml, more preferably 25 - 30 µg per 0.5 ml;
b) Tris Buffer 0.05 – 0.5 mg per 0.5 ml; more preferably 0.30 mg per 0.5 ml;

c) Mannitol 1 – 50 mg per 0.5 ml; more preferably 25 mg per 0.5 ml;
d) Water for Injection (WFI) q.s.;
wherein the said vaccine formulation is sufficient to elicit the required T- dependent immune response against Salmonella enterica serovar typhi and Salmonella enterica serovar paratyphi A including in children below 2 years of age, adolescent adult, and elders through only one injection to comprise a complete vaccination schedule.
Yet according to an aspect of eleventh embodiment, wherein a liquid single dose vaccine kit may comprise of:
a) Salmonella enterica serovar paratyphi A OSP – DT conjugate antigen 1 – 50 µg per 0.5 ml, more preferably 25 - 30 µg per 0.5 ml;
b) Tris Buffer 0.05 – 0.5 mg per 0.5 ml; more preferably 0.30 mg per 0.5 ml;
c) Mannitol 1 – 50 mg per 0.5 ml; more preferably 25 mg per 0.5 ml;
d) Water for Injection (WFI) q.s.;
wherein the said vaccine formulation is sufficient to elicit the required T- dependent immune response against Salmonella enterica serovar typhi and Salmonella enterica serovar paratyphi A, B and C including in children below 2 years of age, adolescent adult, and elders through only one injection to comprise a complete vaccination schedule.
Yet according to an aspect of eleventh embodiment, wherein a liquid single dose vaccine kit may comprise of:
a) Salmonella enterica serovar typhi ViPs-TT conjugate antigen 1 – 50 µg per 0.5 ml; more preferably 25 - 30 µg per 0.5 ml;
b) Salmonella enterica serovar paratyphi A OSP – DT conjugate antigen 1 – 50 µg per 0.5 ml; more preferably 25 - 30 µg per 0.5 ml;
c) Tris Buffer 0.05 – 0.5 mg per 0.5 ml; more preferably 0.30 mg per 0.5 ml;
d) Mannitol 1 – 50 mg per 0.5 ml; more preferably 25 mg per 0.5 ml;
e) Water for Injection (WFI) q.s.;
wherein the said vaccine formulation is sufficient to elicit the required T- dependent immune response against Salmonella enterica serovar typhi and Salmonella enterica serovar paratyphi A, B and C including in children below 2 years of age, adolescent adult, and elders through only one injection to comprise a complete vaccination schedule.

Yet according to an aspect of eleventh embodiment, wherein a liquid multi-dose vaccine kit may comprise of:
a) Salmonella enterica serovar typhi ViPs-TT conjugate antigen 1 – 50 µg per 0.5 ml; more preferably 25 - 30 µg per 0.5 ml;
b) Tris Buffer 0.05 – 0.5 mg per 0.5 ml; more preferably 0.30 mg per 0.5 ml;
c) Mannitol 1 – 50 mg per 0.5 ml; more preferably 25 mg per 0.5 ml;
d) 2-phenoxyethanol 1 – 10 mg per 0.5 ml, more preferably 2.5 mg per 0.5 ml;
e) Water for Injection (WFI) q.s.;
wherein the said vaccine formulation is sufficient to elicit the required T- dependent immune response against Salmonella enterica serovar typhi and Salmonella enterica serovar paratyphi A including in children below 2 years of age, adolescent adult, and elders through only one injection to comprise a complete vaccination schedule.
Yet according to an aspect of eleventh embodiment, wherein a liquid multi-dose vaccine kit may comprise of:
a) Salmonella enterica serovar typhi ViPs-TT conjugate antigen 1 – 50 µg per 0.5 ml; more preferably 25 - 30 µg per 0.5 ml;
b) Salmonella enterica serovar paratyphi A OSP – DT conjugate antigen 1 – 50 µg per 0.5 ml, more preferably 25 - 30 µg per 0.5 ml;
c) Tris Buffer 0.05 – 0.5 mg per 0.5 ml, more preferably 0.30 mg per 0.5 ml;
d) Mannitol 1 – 50 mg per 0.5 ml, more preferably 25 mg per 0.5 ml;
e) 2-phenoxyethanol 1 – 10 mg per 0.5 ml, more preferably 2.5 mg per 0.5 ml;
f) Water for Injection (WFI) q.s.;
wherein the said vaccine formulation is sufficient to elicit the required T- dependent immune response against Salmonella enterica serovar typhi and Salmonella enterica serovar paratyphi A, B and C including in children below 2 years of age, adolescent adult, and elders through only one injection to comprise a complete vaccination schedule.
Salmonella serovar strains S. typhi; S. paratyphi A; S. typhimurium and S. enteritidis used for developing the immunogenic composition of the present disclosure may include: Salmonella enterica serovar typhi TY2 strain with "tviB" gene specific for Vi polysaccharide (Identified by Geneombio Technologies Private Limited, Pune and isolated from stool sample of typhoid

confirmed patient at Villoo Poonawalla Memorial Hospital, Pune); S. typhi: ATCC 19430; C6524 (NICED, Kolkata, India); S. paratyphi A: ATCC 9150 procured from Chromachemie Laboratory Private Limited, Bangalore, CMCC50073, CMCC50973; S. enteritidis: ATCC 4931; ATCC 13076; S. enteritidis R11; S. enteritidis D24359; S. enteritidis 618; S. enteritidis 502; S. enteritidis IV3453219;S. typhimurium: S. typhimurium 2192; ATCC 14208; S. typhimurium 2189; S. typhimurium D23580; ATCC 19585; ATCC 700408; (LT2/SL134 (ST19)); S.typhimurium 177(ST19) CDC 6516-60; ATCC 700720. Further any attenuated Salmonella serovar strain (S. typhi, S. paratyphi A, S. enteritidis and S. typhimurium) may be used for the preparation of the immunogenic composition of the present disclosure.
Salmonella enterica serovar typhi deposited at NCMR-NCCS; Pune, an International Depositary Authority having assigned Accession No. MCC 0193; Strain designation- PDL-1.
Other embodiments disclosed herein also encompasses vaccine kit comprising a first container containing a lyophilized (freeze-dried) immunogenic composition and a second container containing an aqueous solution optionally saline or WFI (water for injection) for the reconstitution of the lyophilized (freeze-dried) immunogenic composition.
The foregoing description of the specific embodiments fully reveals the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the embodiments herein has been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the embodiments as described herein.
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 “one or more” 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 invention 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 given for various physical parameters, dimensions and quantities are only approximate values and it is envisaged that the values higher than the numerical value assigned to the physical parameters, dimensions and quantities fall within the scope of the invention unless there is a statement in the specification to the contrary.
While considerable emphasis has been placed herein on the specific features of the preferred embodiment, it will be appreciated that many additional features can be added and that many changes can be made in the preferred embodiment without departing from the principles of the disclosure. These and other changes in the preferred embodiment 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 illustration of the disclosure and not as a limitation.

TECHNICAL ADVANTAGES:
1. The improved formulation overcomes the limitations of the previous formulation in the prior-art and shows low viscosity, devoid of aggregation; showing long-term stability across wide temperature ranges indirectly preserving the desired characteristics of the multiple conjugates, including low free polysaccharide content, low free protein content and high stability and immunogenicity.
2. Applicant’s new formulation/composition i) is devoid of salt & detergents, ii) devoid of aggregates and particle formation, higher osmolality, optimized Zeta potential, low viscosity iii) preserves the desired physichochemical & immunogenic characteristics of the saccharide-protein conjugate including free polysaccharide content less than 10%, free protein content less than 5%, stability and immunogenicity for 12 months at 2-8°C , for 6 month at 25°, for 30 Days at 40°C
3. Absolute zeta potential values were below than - 20 mV which is considered stable for long term storage.
4. Osmolality of a solution was found to be in optimum range between 250 to 350 mOsm/kg.
5. Viscosity Range of a solution was found to be in between 1 centipoise to 10 centipoise preferably below 5 centipoise.

EXAMPLES
The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the compositions and techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
A) STRAINS:
Salmonella serovar strains S. typhi; S. paratyphi A; S. typhimurium and S. enteritidis used for developing the immunogenic composition of the present disclosure may include: Salmonella enterica serovar typhi TY2 strain with "tviB" gene specific for Vi polysaccharide (Identified by Geneombio Technologies Private Limited, Pune and isolated from stool sample of typhoid confirmed patient at Villoo Poonawalla Memorial Hospital, Pune); Salmonella enterica serovar typhi deposited at NCMR-NCCS; Pune, an International Depositary Authority having assigned Accession No. MCC 0193; Strain designation- PDL-1, S. typhi: ATCC 19430; C6524 (NICED, Kolkata, India); S. paratyphi A: ATCC 9150 procured from Chromachemie Laboratory Private Limited, Bangalore, CMCC50073, CMCC50973; S. enteritidis: ATCC 4931; ATCC 13076; S. enteritidis R11; S. enteritidis D24359; S. enteritidis 618; S. enteritidis 502; S. enteritidis IV3453219;S. typhimurium: S. typhimurium 2192; ATCC 14208; S. typhimurium 2189; S. typhimurium D23580; ATCC 19585; ATCC 700408; (LT2/SL134 (ST19)); S.typhimurium 177(ST19) CDC 6516-60; ATCC 700720. Further any attenuated Salmonella serovar strain (S. typhi, S. paratyphi A, S. enteritidis and S. typhimurium) may be used for the preparation of the immunogenic composition of the present disclosure.
WO2018037365, WO2019016654 and WO2020075184 are being incorporated with reference to the preparation of diphtheria toxoid (DT), tetanus toxoid (TT), inactivated whole cell pertussis, (wP), acellular pertussis, (aP), hepatitis B virus surface antigen (HBsAg),

Haemophilus influenzae type b antigen (Hib) and inactivated poliovirus (standard dose and dose reduced poliovirus).
WO2018037365 is being incorporated with reference to the preparation of inactivated poliovirus and inactivated rotavirus.
WO2013114268 is being incorporated with reference to the preparation of meningococcal polysaccharide antigens from serotypes A, C, W, X and Y.
EXAMPLE 1:
1. Description of the Manufacturing Process
The manufacturing process for the Typhoid Paratyphoid Bivalent Conjugate Vaccine (Liquid) was developed and finalized based on previous experience with conjugate vaccine (MenFive), subject knowledge and extensive pre-formulation studies. The stabilizer concentrations were finalized based on the principle of minimum number of excipients and that to minimum levels that serve the purpose of additions, for small volume injections.
Based on results of experimental development studies, formulation is optimized and finalized. The initial development was mainly dedicated to achieve desired solution properties using different buffer and tonicity enhancers. Once this was achieved, other quality attributes were considered namely polysaccharide content and free polysaccharide content in the formulation.
A. Process pH optimization for final formulation:
The polysaccharide (PS) part of the conjugates is the most liable for hydrolysis hence initially pH stability of polysaccharide at different pH and temperature conditions were assessed. Polysaccharides were exposed to different temperature conditions (-20°C, 2-8°C, RT, and 37°C for 16 days), also kept in different pH conditions (4.0, 7.2, and 9.2).
Stability of Vi-TT conjugate was also assessed in Tris buffer at 5 mM, 10 mM and 20 mM concentration at Room Temperature (RT), 2-8°C, 37°C and -20°C. Experiment was conducted for 16 days and any change in terms of molecular weight was assessed.

B. Process of Buffer finalization:
Buffer suitability was checked against the active ingredients of formulation. Various buffers with different pH ranges and strengths were evaluated for liquid formulation.
PS was kept in 3 different buffers 10mM Tris pH 7.4, 10mM Tris pH 6.0, 10mM Tris pH 9.5 at -20°C, 2-8°C, RT, and 37°C for 14 days. pH was monitored daily.
Carbonate Bicarbonate buffer was unable to maintain pH for 14 days while Tris buffer maintained pH in the desired range. The buffer suitability order was observed as Tris>Citrate buffer>Carbonate Bicarbonate buffer.
At 2-8°C, the conjugate stability was Tris>Citrate>Carbonate>Bicarbonate; Hence storage temperature for conjugates and formulation was finalized at 2-8°C in Tris buffer.
C. Selection of excipients:
The different excipient such as Sugars, Polyols, Preservatives and Isotonic agents were checked in the final formulation e.g. Sucrose, Sorbitol, Mannitol, Sodium Chloride, Magnesium Chloride, Calcium Chloride, Polysorbate 80, and 2-Phenoxyethanol (2-PE). It was observed that commonly used salt NaCl induced aggregation especially OSP-DT conjugate. A number of affluent countries are moving to eliminate thiomersal, from vaccines as a precautionary measure because of concerns about the potential adverse effects of mercury. hence 2-Phenoxyethanol is used in place of Thiomersal.
The selection and finalization of excipients was based on physiochemical properties (Zeta potential, Particle size, pH, and Osmolality) and stability at 2-8⁰C and 25⁰C.

Viscosity, Osmolality and Zeta Potential optimization for final formulation
The following formulation experiments were performed to obtain clear and aggregate free (physical or chemical) bivalent formulation by visual method. All formulations were analysed for Zeta potential, pH and observed visually on daily basis, recorded observations.

Table 1: Trial 1
Sr. No. Formulation Concentration
1 Vi-TT Conjugate 100 µg/mL
2 OSP-DT Conjugate 100 µg/mL
3 Tris buffer (pH 7.2) 10 mM
4 Sodium Chloride 154 mM
5 Polysorbate 20 0.02% v/v
6 Sucrose 3% v/v
7 Sodium Citrate 0.5% v/v
8 Water for Injection q.s.
Results
Sr. No Zeta Potential pH Visual Inspection
1 -12.5 mV 7.20 Clear solution
Expt. Conclusion:- The above formulation is visibly clear and free from particulate matter on day-0; however samples were monitored daily basis and aggregation observed on day-2, hence requires further optimization for stability of the product.

Table 2: Trial 2
Sr. No. Formulation Concentration
1 Vi-TT Conjugate 100 µg/mL
2 OSP-DT Conjugate 100 µg/mL
3 Tris buffer (pH 7.2) 10 mM
4 Sodium Chloride 75 mM
5 Polysorbate 20 0.05% v/v
6 Sucrose 3% v/v
7 2-PE 0.5% w/v
8 Glycine 0.25% v/v
9 Water for Injection q.s.
Results
Sr. No Zeta Potential pH Visual Inspection
1 -13.8 mV 7.22 Clear solution
Expt. Conclusion:- The above formulation is visibly clear and free from particulate matter; however addition of Glycine and 2-PE did not improved Zeta potential value, hence requires further optimization for stability of the product.

Table 3: Trial 3
Sr. No. Formulation Concentration
1 Vi-TT Conjugate 100 µg/mL
2 OSP-DT Conjugate 100 µg/mL
3 Tris buffer 10 mM
4 Sodium Chloride 100 mM
5 Polysorbate 20 0.05% v/v
6 Sucrose 3% v/v
7 2-PE 0.5% w/v
8 Magnesium Chloride 1 mM
9 Water for Injection q.s.
Results:
Sr. No Zeta Potential pH Visual Inspection
1 -10.9 mV 7.20 Clear solution
Expt. Conclusion:- The above formulation is visibly clear and free from particulate matter; however addition of Magnesium chloride did not improved Zeta potential value, hence requires further optimization for stability of the product.

Table 4: Trial 4
Sr. No. Formulation Concentration
1 Vi-TT Conjugate 100 µg/mL
2 OSP-DT Conjugate 100 µg/mL
3 Tris buffer (pH 7.2) 10 mM
4 Sodium Chloride 40 mM
5 Polysorbate 20 0.05% v/v
6 Sucrose 2% v/v
7 2-PE 0.5% w/v
8 Histidine 25 mM
9 Arginine 75 mM
10 Water for Injection q.s.
Results:
Sr. No Zeta Potential pH Visual Inspection
1 Zeta potential (Day 0) -13.2 mV 6.50 Clear solution
2 Zeta potential (Day 1) @ 2-8°C -10.7 mV 6.48 Clear solution
3 Zeta potential (Day 3) @ 2-8°C -9.7 mV 6.51 Aggregation observed
4 Zeta potential (Day 1) @ 25°C -9.5 mV 6.49 Aggregation observed
5 Zeta potential (Day 3) @ 25°C -9.3 mV 6.49 Aggregation observed

Expt. Conclusion:- The above formulation is visibly clear and free from particulate matter; however obtained Zeta potential value is not in the expected range upon storage at different temperatures. Addition of Histidine and Arginine did not improved zeta potential as expected. Hence requires further expts for stability of the product. Formulation kept at 2-8°C shown aggregates after 3 days whereas 25 °C formulation shown after 1 day.

Table 5: Trial 5
Sr. No. Formulation Concentration
1 Vi-TT Conjugate 100 µg/mL
2 OSP-DT Conjugate 100 µg/mL
3 Tris buffer (pH 7.2) 10 mM
4 Sodium Chloride 7.5 mM
5 Polysorbate 20 0.05% v/v
6 Sucrose 2% v/v
7 2-PE 0.5% w/v
8 Histidine 50 mM
9 Mannitol 2% v/v
10 Water for Injection q.s.
Result
Sr. No Zeta Potential pH Visual Inspection
1 Zeta potential day 0 -20.8 mV 6.50 Clear solution
2 Zeta potential (Day 1) @ 2-8°C -19.1 mV 6.50 Aggregation observed
3 Zeta potential (Day 1) @ 25°C -15.2 mV 6.48 Aggregation observed
Expt. Conclusion:- The above formulation is visibly clear and free from particulate matter; upon addition of 2% Mannitol, and slight reduction in NaCl conc. zeta potential decreased below -20mV which is in the expected range however after day 1 aggregates observed might be due to slightly acidic pH.

7 Sodium Chloride 30 mM
8 2-PE 0.5% w/v
9 Water for Injection q.s.
Results:
Sr. No Zeta Potential (2-8°C) pH Visual Inspection
1 Zeta potential day 0 -16.7 mV 6.90 Clear solution
2 Zeta potential – Day 6 -16.3 mV 7.0 Aggregation Observed
Expt. Conclusion:-• Few aggregate like particles were observed in the above formulation
• Hence it was decided to discontinue with sodium chloride as osmotic agent and histidine as buffering agent.
• Instead, further trials were planned with Mannitol as osmotic agent and Tris as buffering agent as zeta potential values were in expected range when Mannitol was used.

Table 7: Trial 7 (Batch no.090221)
Sr. No. Formulation Concentration
1 Vi-TT Conjugate 100 µg/mL
2 OSP-DT Conjugate 100 µg/mL
3 Tris Buffer 10mM
4 NaCl 50mM
5 Mannitol 5%
6 2-PE 0.5% w/v
7 Water for Injection q.s.
Results:
Sr. No Zeta Potential pH Visual Inspection
1 -12.6 mV 7.10 Clear solution
Expt. Conclusion:- The above formulation is visibly clear and free from particulate matter. Combination of Mannitol, Tris and NaCl imparted optimum pH and Osmolality. However obtained Zeta potential value is not as expected hence requires further optimization for stability of the product.

From above experimental results it was observed that the presences of Sodium chloride, or Buffering agent (acidic pH), detergents (Polysorbate 20 or 80) with active ingredients are not stable. Hence the next formulation was designed with the removal of complete salt and detergents.
However to ensure the Osmolality, pH and Zeta potential of the formulation without salt and excipients following experiments were performed to achieve the optimum Mannitol and Tris concentration into final formulation.

Table 8: Trial 8
Sr. No Solution/Mixture Zeta Potential Osmolality mOsm/kG pH
Vi-TT Conjugate Bulk (100µg/ml) + OSP-
1 DT Conjugate Bulk (100µg/ml)+ 10mM -23.1 141 7.19

Tris buffer containing 2% Mannitol

+0.5% 2Phenoxyethanol
Vi-TT Conjugate Bulk (100µg/ml) + OSP-
2 DT Conjugate Bulk (100µg/ml)+ 10mM -17.6 265 7.22

Tris buffer containing 4% Mannitol

+0.5% 2Phenoxyethanol
Vi-TT Conjugate Bulk (100µg/ml) + OSP-
3 DT Conjugate Bulk (100µg/ml)+ 10mM -18.0 323 7.20

Tris buffer containing 5% Mannitol

+0.5% 2Phenoxyethanol
Vi-TT Conjugate Bulk (100µg/ml) + OSP-
4 DT Conjugate Bulk (100µg/ml)+ 5mM Tris -18.6 130 7.23

buffer containing 2% Mannitol +0.5%

2Phenoxyethanol
Vi-TT Conjugate Bulk (100µg/ml) + OSP-
5 DT Conjugate Bulk (100µg/ml)+ 5mM Tris -18.5 258 7.18

buffer containing 4% Mannitol +0.5%

2Phenoxyethanol
Vi-TT Conjugate Bulk (100µg/ml) + OSP-
6 DT Conjugate Bulk (100µg/ml)+ 5mM Tris -22 317 7.19

buffer containing 5% Mannitol +0.5%

2Phenoxyethanol

Summary and Conclusions:
From above study following conclusions were drawn;
➢ The increased concentrations of Mannitol gives 2% (141), 4% (265), 5% (323) with 5mM Tris, whereas 2% (130), 4% (258), 5% (317) with 10mM Tris buffer in osmolality values.
➢ The increased Mannitol increases the osmolality value since 250 to 350 mOsm/kg is a safety range, hence 5% concentration of Mannitol freezed in formulation.
➢ The 5mM and 10mM tris buffer concentrations were used with different Mannitol concentrations in which observed that the zeta values not differed significantly. Based on experimental data it was found that increased conc. of Tris/Salt results into aggregation or having impact on clarity of formulation, hence 5mM Tris buffer freezed for the formulation.
➢ The pH of the formulation were checked for 3weeks and observed that formulation is stable at pH 7.0 to 7.5.

Table 9: Trial 9 (Batch no. 090221)
Sr. No. Formulation Concentration
1 OSP-DT Conjugate 100 µg/mL
2 Vi-TT Conjugate 100 µg/mL
3 Tris 5mM
4 Mannitol 5%
6 2-PE 0.5% w/v
7 Water for Injection q.s.
Results:
Sr. No Zeta Potential pH Visual Inspection
1 -13.7mV 7.15 Clear solution
Expt. Conclusion:- The above formulation is visibly clear and free from particulate matter; however removed NaCl content and slightly improved Zeta potential value hence requires further optimization for stability of the product.

Table 10: Trial 10 (Batch no. 110221)
Sr. No. Formulation Concentration
1 Vi-TT Conjugate 100 µg/mL
2 OSP-DT Conjugate 100 µg/mL
3 Tris 5mM
4 Mannitol 5%
6 2-PE 0.5%
7 Water for Injection q.s.
Results:
Sr. No Zeta Potential pH Visual Inspection
1 -25.3 mV 7.20 Clear solution
Expt. Conclusion:- In this trial, sequence of addition was optimized (sequence - WFI, Tris, ViTT, OSPDT, Mannitol, 2PE). The Tris concentration slightly reduced. The above formulation is visibly clear and free from particulate matter. Optimizing sequence of addition, and Tris Conc. improved zeta potential greatly. Zeta potential value is in the expected range. Need to check consistency in further trials.

Table 11: Trial 11 (Batch no. 130221)
Sr. No. Formulation Concentration
1 Vi-TT Conjugate 100 µg/mL
2 OSP-DT Conjugate 100 µg/mL
3 Tris 5mM
4 Mannitol 5%
6 2-PE 0.5%
7 Water for Injection q.s.
Results:
Sr. No Zeta Potential pH Visual Inspection
1 Zeta potential - Initial -25.3 7.23 Clear solution
2 Zeta potential – Day 2 -23.9 7.20 Clear solution
3 Zeta potential – Day 6 -24.7 7.18 Clear solution
4 Zeta potential – Day 14 -23.2 7.25 Clear solution
5 Z eta potential – Week 3 -24.6 7.22 Clear solution
Expt. Conclusion:- The above formulation yielded optimum pH, Zeta and Osmolality results. It remained visibly clear and free from particulate matter up to 3 weeks and it is under continuous monitoring also pH and Zeta potential value remained stable up to 3 weeks at storage of 2-8°C

Table 12: Viscosity Results
Sr. No Solution/Mixture Zeta Potential Osmolality mOsm/kG pH Viscosity cP(centipoise)
Vi-TT Conjugate Bulk (100µg/ml) +
1 OSP-DT Conjugate Bulk (100µg/ml)+ 10mM Tris buffer containing 2% Mannitol +0.5% 2Phenoxyethanol -23.1 141 7.19 1.38
Vi-TT Conjugate Bulk (100µg/ml) +
2 OSP-DT Conjugate Bulk (100µg/ml)+ 10mM Tris buffer containing 4% Mannitol +0.5% 2Phenoxyethanol -17.6 265 7.22 1.61
Vi-TT Conjugate Bulk (100µg/ml) +
3 OSP-DT Conjugate Bulk (100µg/ml)+ 10mM Tris buffer containing 5% Mannitol +0.5% 2Phenoxyethanol -18.0 323 7.20 1.71
Vi-TT Conjugate Bulk (100µg/ml) +
4 OSP-DT Conjugate Bulk (100µg/ml)+ 5mM Tris buffer containing 2% Mannitol +0.5% 2Phenoxyethanol -18.6 130 7.23 1.40
Vi-TT Conjugate Bulk (100µg/ml) +
5 OSP-DT Conjugate Bulk (100µg/ml)+ 5mM Tris buffer containing 4% Mannitol +0.5% 2Phenoxyethanol -18.5 258 7.18 1.65
Vi-TT Conjugate Bulk (100µg/ml) +
6 OSP-DT Conjugate Bulk (100µg/ml)+ 5mM Tris buffer containing 5% Mannitol +0.5% 2Phenoxyethanol -22 317 7.19 1.73
Interpretation:
➢ Viscosity is the result of intermolecular forces between particles within a fluid, this is important parameter for liquid parenteral formulations. We found that viscosity of formulations increases on storage hence the viscosity of trial formulations, lead formulations and final formulation compositions was checked with and without conjugates at 2-8°C and 25°C.
• Tris (5mM, 10mM, 30mM, 50mM and 75mM),
• 0.9% NaCl

• 5mM Tris+5% Mannitol+2-Phenoxyethanol
It was observed that in all the formulation compositions significant rise in viscosity was not observed. The required viscosity range is below 10 cP preferably less than 5 cP.
➢ Osmolality of a solution refers to the concentration of osmotically active ingredients in the solution and it is critical parameter considered for formulation design. The optimum range is between 250 to 350 mOsm/kg and safety range for intramuscular injection is between 240 to 600 mOsm/kg. The osmolality was checked and optimized in the final formulation i.e. conjugates + Mannitol + 2-Phenoxyethanol + Tris buffer.
➢ Zeta potential is a measure of the magnitude of the electrostatic or charge repulsion/attraction between particles and is one of the fundamental parameters known to affect stability for colloidal solutions such as conjugates. Empirically, it is considered that absolute zeta potential values below than - 20 mV are considered stable for long term storage. Only the magnitude of the zeta potential indicates the stability of the sample, whereas the sign of the zeta potential shows whether positive or negative charges are dominant at the surface. The Zeta potential was checked and optimized in the final formulation i.e Conjugates + Mannitol + 2-Phenoxyethanol + Tris buffer. It was found below -20mV to finalize the formulation.
➢ Further the outcome from literature it was observed that Polyols increases the isotonicity of protein formulations. Since addition of sodium chloride resulting into increase in Zeta potential value towards zero, hence different concentrations of mannitol (2%, 4%, 5% w/v) with Tris buffer (5mM, 10mM, 20mM) was evaluated for osmolality of final formulation.
➢ However optimum stability with respect to viscosity, osmolality and zeta potential was observed in below concentrations of excipients:
- Tris buffer = 5mM (0.60 mg/ml)
- Mannitol = 5%w/v (50 mg/ml)
- 2-Phenoxyethanol = 0.5%w/v (5 mg/ml)
➢ Presence of Sodium chloride, Histidine, Arginine or buffers with lower pH resulted in small aggregate formation upon storage and hence was discontinued from further trials.

➢ Addition of Mannitol with combination of optimum tris buffer concentration, removal of
sodium chloride drastically improved clarity of the formulation and thereby stability of
product. ➢ From above trials, it can be concluded that TCV- Bivalent conjugate vaccine remains
stable in presence of Mannitol, Tris buffer and 2PE. ➢ Mannitol imparts required isotonicity, Tris is used as buffering agent and 2-Phenoxy
ethanol is used as preservative ➢ The proposed TCV bivalent formulation dose is 50µg/mL and hence the development
studies were carried out on 2X scale (i.e. 100 µg/mL) dose and results found were
satisfactory.

EXAMPLE 2: Finalization of excipient quantities on the basis of stability of the representative conjugates:
The experimental formulation batches were manufactured with finalized formulation composition (5mM Tris buffer, 5% Mannitol, 0.5% 2-PE and Vi-TT and OSP-DT conjugates each 100µg/ml). A short term stability study was performed at 2-8°C and accelerated temperature conditions; total PS and % free PS analysis was performed. The analytical results showed satisfactory results, the final formulation is finalized.

Table 13: TCV-BIVALENT FORMULATION STABILITY STUDY AT (2-8°C) AND IMMUNOGENICITY DATA
Test Limits/ Specification Initial 3 months 6 months 12 months
pH 6.0-8.0 7.1 7.0 7.0 6.9
Description (Appearance) Clear, colorless to pale yellowish liquid Clear, colorless to pale yellowish liquid Clear, colorless to pale yellowish liquid Clear, colorless to pale yellowish liquid Clear, colorless to pale yellowish liquid
Identity Vi-TT Positive Response PASS PASS PASS PASS

OSP-DT Positive Response PASS PASS PASS PASS
Total (PS) Polysaccharide Vi-TT Report value (µg/mL) 62.0 61.0 57.0 56.0

OSP-DT Report value (µg/mL) 54.1 59.7 56.0 55.3
Free PS Vi-TT Report value (%) 13 12 10 11

OSP-DT Report value (%) 9 13 13 10
Molecular Size Distribution Report value [% MSD should be >50% before distribution coefficient (KD) of 0.25] 100.0 99.3 99.2 99.3
Total Protein Report value (mg/mL) 0.18 0.18 0.17 0.17
Free Protein Report value (%) BQL BQL BQL BQL
2-Phenoxyethanol Content 3.50 to 6.50 mg/mL 3.83 4.01 4.14 3.94
Immunogenicity Titer Geometric Mean Titer (GMT) 383567 430539 574701 383567

fold increase over placebo (>4 ) 1208 1709 1810 1613

Geometric Mean Titer (GMT) of Old Formulation 11403 21526 18101 13561

Fold difference in GMT of New Versus Old Formulation 34 20 32 28
BQL: Below Quantitation Limit

Conclusion: TCV- Bivalent Drug Product is stable and immunogenic for 12 months at 2-8°C as per above mentioned specifications.

Table 14: TCV-BIVALENT FORMULATION STABILITY STUDY AT 25 °C± 2ºC 60 % Relative humidity (RH) ± 5% RH
Test Limits/ Specification Initial 1 month 3 months 6 months
pH 6.0-8.0 7.0 6.9 7.0 7.0
Description (Appearance) Clear, colorless to pale yellowish liquid Clear, colorless to
pale yellowish
liquid Clear, colorless to
pale yellowish
liquid Clear, colorless to
pale yellowish
liquid Clear, colorless to
pale yellowish
liquid
Identity Vi-TT Positive Response PASS PASS PASS PASS

OSP-DT Positive Response PASS PASS PASS PASS
Total Polysaccharide Content Vi-TT Report value (µg/mL) 33.0 29.0 30.0 32.0

OSP-DT Report value (µg/mL) 24.0 22.7 25.7 23.6
Unbound (Free) Polysaccharide Content Vi-TT Report value (%) 6 11 8 10

OSP-DT Report value (%) BQL BQL BQL BQL
Molecular Size Distribution Report value (%) Before
distribution coefficient (KD) of
0.25 100.0 98.9 98.9 99.8
Total Protein Report value (mg/mL) 0.06 0.06 0.05 0.06
Free Protein Report value (%) BQL BQL BQL BQL
2-Phenoxyethanol Content 3.50 to 6.50 mg/mL 4.01 3.95 4.17 3.57
BQL: Below Quantitation Limit
Conclusion: TCV- Bivalent Drug Product is stable for 6 months at 25°C (Stress) as per above mentioned specifications.

Table 15: TCV-BIVALENT FORMULATION STABILITY STUDY AT 40 ± 2ºC/75 %RH ± 5% RH
Test Limits/ Specification Initial 3 Days 7 Days 15 Days 30 Days
pH 6.0-8.0 7.0 7.0 6.9 7.1 7.0
Description (Appearance) Clear, colorless to pale yellowish liquid Clear, colorless
to pale yellowish liquid Clear, colorless
to pale yellowish liquid Clear, colorless
to pale yellowish liquid Clear, colorless
to pale yellowish liquid Clear, colorless
to pale yellowish liquid
Identity Vi-TT Positive Response PASS PASS PASS PASS PASS

OSP-DT Positive Response PASS PASS PASS PASS PASS
Total Polysaccharide Content Vi-TT Report value (µg/mL) 33.0 35.0 32.0 26.0 25.0

OSP-DT Report value (µg/mL) 24.0 23.9 22.1 24.1 24.6
Unbound (Free) Polysaccharide Content Vi-TT Report value (%) 6 11 8 13 14

OSP-DT Report value (%) BQL BQL BQL BQL BQL
Molecular Size Distribution Report value (%) Before
distribution coefficient
(KD) of 0.25 100.0 98.3 98.6 99.5 96.0
Total Protein Report value (mg/mL) 0.06 0.05 0.05 0.06 0.07
Free Protein Report value (%) BQL BQL BQL BQL BQL
2-Phenoxyethanol Content 3.50 to 6.50 mg/mL 4.01 4.11 4.07 3.74 3.95
BQL: Below Quantitation Limit Conclusion: TCV-BV Drug Product is stable for 30 Days at 40°C (A ccelerated) as per above mentioned sp ecifications.

EXAMPLE 3: IMMUNOGENICITY DATA OF THE BIVALENT VACCINE
Assessment of immunogenicity of the vaccine: The immunogenicity of final vaccine formulation was assessed in Balb/C mice model; a licensed conjugate vaccine was also used in the study. The study concluded that IgG titre of SIIPL bivalent study vaccine was comparable to licensed vaccine for Vi component and the study also concluded that the vaccine raised promising immune response against O-specific polysaccharide.
Following formulation of TCV bivalent formulation was finalized for clinical studies.

Table 16: Typhoid Conjugate Vaccine (Bivalent)-BTCV Formulation Details Multidose presentation- Primary Container: Vial
Ingredients Quantity (per Dose 0.5mL) Function Range as per SOP if any
Purified Vi polysaccharide from Salmonella Typhi conjugated to Tetanus Toxoid (Vi - TT Purified Bulk Conjugate) 25 µg Immunogen
(Active
Ingredient) 25 µg to 30 µg
Purified O-Specific Polysaccharide from Salmonella paratyphi A is conjugated to Diphtheria Toxoid (OSP-DT Purified Bulk Conjugate) 25 µg Immunogen
(Active
Ingredient) 25 µg to 30 µg
Tris Buffer 0.30 mg (5mM) Buffering agent -
Mannitol 25 mg (5% w/v) Tonicity enhancer -
2 Phenoxyethanol 2.5 mg (0.5%w/v) Preservative -
Water for Injection q.s. Solvent -
pH: Adjusted 7.2 (Range 6.0 to 8.0)
Osmolality: not less than 240 mOsmol/kg (Observed values are between 300 to 360
mOsmol/kg)

Table 17: Typhoid Conjugate Vaccine (Bivalent)-BTCV Formulation Details Single dose presentation- Primary Container: Vial & Pre Filled Syringes
Ingredients Quantity
(per Dose 0.5mL) Function Range as per SOP if any
Purified Vi polysaccharide from Salmonella Typhi conjugated to Tetanus Toxoid (Vi - TT Purified Bulk Conjugate) 25 µg Immunogen
(Active
Ingredient) 25 µg to 30 µg

Purified O-Specific Polysaccharide from Salmonella paratyphi A is conjugated to Diphtheria Toxoid (OSP-DT Purified Bulk Conjugate) 25 µg Immunogen
(Active
Ingredient) 25 µg to 30 µg
Tris Buffer 0.30 mg (5mM) Buffering agent -
Mannitol 25 mg (5% w/v) Tonicity enhancer -
Water for Injection q.s. Solvent -
pH: Adjusted 7.2 (Range 6.0 to 8.0)
Osmolality: not less than 240 mOsmol/kg (Observed values are between 300 to 360
mOsmol/kg)

Table 18: Rat (Sprague Dawley strain) Immunogenicity Study: SINGLE Dose;
Intramuscular Route
Formulation Anti-Vi IgG (GMT) Anti-OSP IgG (GMT)

D14 D28 D42 D14 D28 D42
GMT 0µg
(TCVB-Formulation 224 317 317 76 80 53
Buffer)
GMT 50µg
(TCV-1; TCV-Bivalent 912280 767133 574701 3592 5080 3020
Formulation-Multi-Dose)
Fold Rise 4064 2416 1810 48 63 57
Conclusions: BTCV drug product (new formulation), when administered at SINGLE dose, exhibited immunogenicity (> 4 fold increase in GMT) in rat model.

Table 19: Rat (Sprague Dawley strain) REPEATED Dose; Intramuscular Route Immunogenicity Study:
Formulation Anti-Vi IgG (GMT) Anti-OSP IgG (GMT)

D14 D28 D42 D14 D28 D42
GMT 0µg
(TCVB-Formulation Buffer) 159 159 238 113 95 135
GMT 1X Dose (25.0µg) 1149401 161270 383567 7184 16127 34172

(TCV-2; TCV-Bivalent Formulation- Multi-Dose)
Fold Rise 7241 1016 1613 63 170 254
Conclusions: BTCV drug product (new formulation), when administered at repeated doses, exhibited immunogenicity (> 4 fold increase in GMT) in rat model.

Table 20: Rabbit (New Zealand White strain) Immunogenicity Study: SINGLE Dose; Intramuscular Route
Formulation Anti-Vi IgG (GMT) Anti-OSP IgG (GMT)

D14 D28 D42 D14 D28 D42
GMT 0µg
(TCVB-Formulation Buffer) 252 283 283 113 101 101
GMT 50µg
(TCV-1; TCV-Bivalent Formulation- Multi-Dose) 28509 25398 50797 6350 8000 4490
Fold Rise 113 90 179 56 79 44
Conclusions: BTCV drug product (new formulation), when administered at SINGLE dose, exhibited immunogenicity (> 4 fold increase in GMT) in rabbit model.
Table 21: Rabbit (New Zealand White strain) Immunogenicity Study: REPEATED Dose; Intramuscular Route
Formulation Anti-Vi IgG (GMT) Anti-OSP IgG (GMT)

D14 D28 D42 D14 D28 D42
GMT 0µg
(TCVB-Formulation Buffer) 200 200 200 143 202 160
GMT 1X Dose (25.0µg)
(TCV-2; TCV-Bivalent Formulation- Multi-Dose) 161270 203187 228070 3564 12699 25398
Fold Rise 806 1016 1140 25 63 159
Conclusions: BTCV drug product (new formulation), when administered at REPEATED doses, exhibited immunogenicity (> 4 fold increase in GMT) in rabbit model.

Table 22: Immunogenicity Titer of Old Vs New Formulation Study AT (2-8°C) in Rat (Sprague Dawley strain) using IM route and repeated dose
Time Period Initial 3 months 6 months 12 months
Geometric Mean Titer (GMT) 383567 430539 574701 383567
fold increase over placebo (>4 ) 1208 1709 1810 1613
Geometric Mean Titer (GMT) of Old Formulation 11403 21526 18101 13561
Fold difference in GMT of New Versus Old Formulation 34 20 32 28
Conclusions: The BTCV drug product (new formulation), when administered at REPEATED doses in SD rats, exhibited immunogenicity (IgG > 4 fold increase in GMT over drug product of old formulation) in rabbit model. This suggests that the BTCV formulated using new formulation is superior, to old formulation, in terms of eliciting the immunogenicity.

We Claim:
1. A liquid based vaccine composition comprising:
a) at least one antigen selected from a group consisting of Salmonella enterica serovar typhi saccharide-carrier protein conjugate; Salmonella enterica serovar paratyphi A saccharide-carrier protein conjugate; Salmonella enterica serovar paratyphi B saccharide- carrier protein conjugate; Salmonella enterica serovar paratyphi C saccharide- carrier protein conjugate; Salmonella enterica serovar typhimurium saccharide- carrier protein conjugate; Salmonella enterica serovar enteritidis saccharide- carrier protein conjugate; Salmonella enterica serovar choleraesuis saccharide- carrier protein conjugate; and Salmonella enterica serovar dublin saccharide- carrier protein conjugate;
b) stabilizer comprising at least one sugar alcohol and at least one buffer;
c) diluent;

2. The vaccine composition according to claim 1, wherein the composition is i) is devoid of salt & detergents ii) devoid of aggregates and particle formation iii) preserves the desired physicochemical & immunogenic characteristics of the saccharide-carrier protein conjugate including free polysaccharide content less than 10%, free protein content less than 5%, stability and immunogenicity for 12 months at 2-8°C, for 6 month at 25°, for 30 Days at 40°C.
3. The vaccine composition according to claim 1, wherein the carrier protein (CP) is selected from the group comprising of tetanus toxin, tetanus toxoid (TT), fragment of tetanus toxoid, diphtheria toxoid (DT), CRM197, Pseudomonas aeruginosa toxoid, Bordetella pertussis toxoid, Clostridium perfringens toxoid, E.coli LT, E. coli ST, Escherichia coli heat-labile toxin - B subunit, Neisseria meningitidis outer membrane complex, rEPA, protein D of H. influenzae, Flagellin FliC, Horseshoe crab Haemocyanin, exotoxin A from Pseudomonas aeruginosa, outer membrane complex c (OMPC), porins, transferrin binding proteins, pneumolysin, pneumococcal surface protein A (PspA), pneumococcal surface adhesin A (PsaA), pneumococcal PhtD, pneumococcal surface proteins BVH-3 and BVH-11, protective antigen (PA) of Bacillus anthracis and detoxified edema factor (EF) and lethal factor (LF) of Bacillus anthracis, ovalbumin, keyhole limpet hemocyanin (KLH), human serum albumin, bovine serum albumin (BSA), purified protein derivative of tuberculin (PPD), synthetic peptides, heat shock proteins, pertussis proteins, cytokines, lymphokines, hormones, growth factors, artificial proteins comprising multiple human CD4+ T cell epitopes from various pathogen-derived

antigens such as N 19, iron-uptake proteins, toxin A or B from C. difficile and S.agalactiae proteins with or without linker and fragments, derivatives, modifications thereof.
4. The vaccine composition according to claim 1, wherein the antigen is present in a dose range of 1 - 50 µg per 0.5 ml dose.
5. The vaccine composition according to claim 1, wherein at least one sugar alcohol is selected from a group consisting of mannitol, lactitol, sorbitol, glycerol, xylitol, maltitol, lactitol, erythritol, isomalt and hydrogenated starch hydrolysates or a combination thereof.
6. The vaccine composition according to claim 5, wherein at least one sugar alcohol is mannitol present at a concentration of 1 – 50 mg per 0.5 ml dose.
7. The vaccine composition according to claim 1, wherein at least one buffer is selected from a group consisting of sodium chloride, acetate, carbonate, citrate, lactate, gluconate, tartrate, phosphate buffer saline, borate, Histidine buffer, Succinate buffer, HEPES, TRIS or Citrate-phosphate.
8. The vaccine composition according to claim 7, wherein at least one buffer is TRIS present at a concentration of 0.05 – 0.5 mg per 0.5 ml dose.
9. The vaccine composition according to claim 1, wherein the diluent is selected from a group consisting of saline, buffer and water for injection (WFI).
10. The vaccine composition according to claim 1, wherein the single dose composition is free of preservative.
11. The vaccine composition according to claim 1, wherein the multi-dose composition comprises of atleast one preservative selected from a group consisting of 2-phenoxyethanol, Benzethonium chloride (Phemerol), Phenol, Thiomersal, Formaldehyde, paraben esters (e.g. methyl-, ethyl-, propyl- or butyl- paraben), benzyl alcohol, m-cresol, benzalkonium chloride, benzyl alcohol, chlorobutanol, p-chlor-m-cresol.
12. The vaccine composition according to claim 11, wherein the multi-dose composition comprises of 2-phenoxyethanol present at a concentration of 1 – 10 mg per 0.5 ml dose.
13. The vaccine composition according to claim 1, wherein the composition comprises of an adjuvant selected from a group consisting of aluminum hydroxide, aluminum phosphate, aluminum hydroxyphosphate, and potassium aluminum sulfate or a mixture thereof.
14. The vaccine composition according to claim 1, wherein the composition comprises of an immunostimulatory component selected from a group consisting of an oil and water emulsion, MF-59, a liposome, a lipopolysaccharide, a saponin, lipid A, lipid A

derivatives, Monophosphoryl lipid A, 3–deacylated monophosphoryl lipid A, AS01, AS03, an oligonucleotide, an oligonucleotide comprising at least one unmethylated CpG and/or a liposome, Freund’s adjuvant, Freund’s complete adjuvant, Freund’s incomplete adjuvant, CRL-8300 adjuvant, muramyl dipeptide, TLR-4 agonists, flagellin, flagellins derived from gram negative bacteria, TLR-5 agonists, fragments of flagellins capable of binding to TLR-5 receptors, QS-21, ISCOMS, Chitosan, saponin combination with sterols and lipids.
15. The vaccine composition according to claim 1, wherein the composition comprises of a pharmaceutically acceptable excipient selected from a group consisting of sugar including sucrose, trehalose, mannose, raffinose, lactobionic acid, glucose, maltulose, iso-maltulose, maltose, lactose, dextrose, fructose; salt including NaCl, KCl, KH2PO4, Na2HPO4.2H2O, CaC12, and MgCl2; non-ionic surfactant including polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxethanol, octylphenoxypolyethoxethanol, oxtoxynol 40, nonoxynol- 9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene- 660 hydroxystearate, polyoxyethylene- 35 ricinoleate, soy lecithin and a poloxamer - 0.001%-0.05%; polymers including dextran, carboxymethylcellulose, hyaluronic acid, cyclodextrin; aminoacids including tricine, leucine, iso-leucine, L-histidine, glycine, glutamine, L-arginine, L-arginine hydrochloride, lysine, L-alanine, Tryptophan, Phenylalanine, Tyrosine, Valine, Cysteine, Glycine, Histidine, Methionine, Proline, Serine, Threonine; peptide; hydrolyzed protein including gelatin, lactalbumin hydrolysate, monosodium glutamate, collagen hydrolysate, keratin hydrolysate, peptides, Casein hydrolysate and whey protein hydrolysate, serum albumin.
16. The vaccine composition according to claim 1, wherein the final pH of the composition is in the range of pH 6.5 to 7.5.
17. The vaccine composition according to claim 1, wherein the composition is lyophilized.
18. The vaccine composition according to claim 1, wherein the saccharide is subjected to depolymerization/sizing by chemical means selected from the group consisting of FeCl3, H2O2, sodium metaperiodate and sodium acetate or mechanical means consisting of ultra-sonication and post depolymerization/sizing the average molecular weight of the Salmonella enterica serovar typhi saccharide is in the range of 180 to 220 kDa and of Salmonella enterica serovar paratyphi A , B & C, S. typhimurium and S. enteritidis is in the range of 40 to 400 kDa.
19. The vaccine composition according to claim 1, wherein the composition comprising:

a) antigen consisting of Salmonella enterica serovar typhi ViPs-TT conjugate antigen 1 – 50 µg per 0.5 ml and Salmonella enterica serovar paratyphi A OSP – DT conjugate antigen 1 – 50 µg per 0.5 ml;
b) stabilizer comprising sugar alcohol consisting of Mannitol 1 – 50 mg per 0.5 ml and buffer consisting of TRIS 0.05 – 0.5 mg per 0.5 ml;
c) Diluent consisting of WFI;
wherein the composition is i) is devoid of salt & detergents ii) devoid of aggregates and particle formation iii) preserves the desired physicochemical & immunogenic characteristics of the saccharide-carrier protein conjugate including free polysaccharide content less than 10%, free protein content less than 5%, stability and immunogenicity for 12 months at 2-8°C, for 6 month at 25°, for 30 Days at 40°C.
20. The vaccine composition according to claim 1, wherein the composition comprising:
a) antigen consisting of Salmonella enterica serovar typhi ViPs-TT conjugate antigen 1 – 50 µg per 0.5 ml and Salmonella enterica serovar paratyphi A OSP – DT conjugate antigen 1 – 50 µg per 0.5 ml;
b) stabilizer comprising sugar alcohol consisting of Mannitol 1 – 50 mg per 0.5 ml, buffer consisting of TRIS 0.05 – 0.5 mg per 0.5 ml and preservative consisting of 2-phenoxyethanol 1 – 10 mg per 0.5 ml
c) Diluent consisting of WFI;
wherein the composition is i) is devoid of salt & detergents ii) devoid of aggregates and particle formation iii) preserves the desired physicochemical & immunogenic characteristics of the saccharide-carrier protein conjugate including free polysaccharide content less than 10%, free protein content less than 5%, stability and immunogenicity for 12 months at 2-8°C, for 6 month at 25°, for 30 Days at 40°C.
21. A method of manufacturing vaccine composition according to any one of the preceding
claims, the method comprising:
a) Diluting atleast one antigen concentrated bulk with a stabilizer comprising at least one sugar, at least one buffer and preservative;
b) Adding WFI to components of step (a) to make up the volume;
c) Adding of Components obtained in step (b) in a blending vessel / container with agitation at room temperature;
d) Sterilizing the Components obtained in step (c) by passing it through a 0.2 µ - 0.45µ filters;

e) Filling into individual sterile glass vials and stoppering the glass vials. 22. The method according to claim 21, the stabilizer comprising sugar consisting of Mannitol 1 – 50 mg per 0.5 ml, buffer consisting of TRIS 0.05 – 0.5 mg per 0.5 ml and preservative consisting of 2-phenoxyethanol 1 – 10 mg per 0.5 ml.