FIELD OF THE INVENTION
The present invention relates to novel polysaccharide - protein conjugate vaccine composition. More particularly, the present invention relates to a tetravalent conjugate vaccine formulation of Neisseria meningitidis serogroup A, C, Y, W capsular polysaccharides (Men A, C, Y, W) conjugated to non-toxic mutant of diphtheria toxin called cross reacting material (CRM197) as carrier protein along with pharmaceutically acceptable components/excipients. The tetravalent conjugate vaccine of the present invention is a fully liquid or lyophilized or liquid-lyo formulation with or without adjuvant.
BACKGROUND OF THE INVENTION
Vaccine manufacture and composition is complex and tightly regulated to ensure safety of the individuals and to maximize efficacy and stability. All vaccines contain an active component which generates the protective immune response. The vaccines also contain pharmaceutically acceptable additional components to enhance the stability of formulation and/or immunogenicity for strong protective immune response.
The World Health Organization recommends that countries with a moderate or high rate of disease or with frequent outbreaks of a vaccine preventable disease should routinely vaccinate. In countries with a low risk of disease, they recommend that high risk groups should be immunized.
Meningococcal disease is an acute, potentially severe illness caused by the bacterium Neisseria meningitidis. N meningitidis (Meningococcus) is an aerobic gram-negative bacterium that has been serologically classified mainly into 13 serogroups A, B, C, D, 29E, H, I, K, L, W135, X, Y and Z. The grouping system is based on the capsular polysaccharides of the organisms.
It has been mentioned on the official website of the WHO that iV. meningitidis is one of the most common causes of bacterial meningitis in the world and the only

bacterium capable of generating large epidemics of meningitis. Major epidemics with incidence rates of up to 1000 cases per 100,000 inhabitants have been reported, particularly in sub-Saharan Africa.
iV. meningitidis is transmitted by aerosol or direct contact with respiratory secretions of patients or healthy human carriers. The endemic disease occurs primarily in children and adolescents, with highest attack rates in infants aged 3-12 months, whereas in epidemics older children and young adults may be more involved. However, the rapid progression of meningococcal disease frequently results in death within 1-2 days after onset, the iV. meningitidis infections can be prevented by vaccination.
Immunization is the only rational approach to control the meningitis disease. The most effective vaccines for iV. meningitidis are polysaccharide-protein conjugate vaccines which are formed by the covalent attachment of activated protein to active polysaccharide. Polysaccharide undergoes some chemical modification which is known as "activation" process prior to attachment to a carrier protein because most of the native polysaccharides cannot be chemically efficiently linked to a carrier protein without activation.
There are many conjugation reactions that have been employed for covalently linking polysaccharides to proteins. Four of the commonly employed methods include:
1) reductive amination, wherein the aldehyde or ketone group on one component of the reaction reacts with the amino or hydrazide group on the other component, and the C=N double bond so formed is subsequently reduced to C-N single bond by a reducing agent;
2) cyanylation conjugation, wherein the polysaccharide is activated either by cyanogen bromide (CNBr) or by 1 -cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) to introduce a cyanate group to the hydroxyl group,

which forms a covalent bond to the amino or hydrazide group upon addition of the protein component; and
3) a carbodiimide reaction, wherein carbodiimide activates the carboxyl group on
one component of the conjugation reaction, and the activated carboxyl group
reacts with the amino or hydrazide group on the other component. These reactions
are also frequently employed to activate the components of the conjugate prior to
the conjugation reaction.
4) thio-ether conjugation chemistry using thiolating agent for covalent
modification of primary amines and addition of a protected yet exposable
sulfhydryl group, enabling heterobifunctional crosslinking strategies.
According to one publication, historically Serogroup A (MenA) has been the most common cause of epidemic disease in sub-Saharan Africa. Serogroups B and C are responsible for the majority of cases in developed—countries, with the remaining cases being caused by serogroups W135 (or simply W) and Y. Serogroup C cases have been observed recently in sub-Saharan Africa where the serogroup was not so prevalent historically.
Based on the above facts, a tetravalent meningococcal ACYW polysaccharide-protein conjugate vaccine could offer a good coverage against meningococcal disease due to most prevalent serogroups except B for which a conjugate vaccine is remote possibility due to structural similarity of MenB capsular polysaccharide with the molecules found in human neuronal cells. Currently, various monovalent or multivalent vaccines including serogroup A, C, Y and W polysaccharide conjugates are licensed for sale in market. The existing state of art discloses vaccines for the prevention of various serogroups including iV. meningitidis serogroup A, B, C, W and Y such as the US patent application no. PCT/EP2006/006188 teaches an immunogenic composition comprising meningococcal capsular polysaccharide from at least one of the groups A, C, W and Y conjugated to a carrier protein.

The available multivalent meningococcal conjugate vaccines are of higher cost and have complex formulations.
There is an urgent need of meningococcal conjugate vaccine which has higher homogeneity, higher immunogenicity, lower cost and is simple to formulate and administer.
OBJECT OF THE INVENTION
In order to obviate the drawbacks in the existing state of art, the main object of present invention is to provide a novel polysaccharide - protein conjugate vaccine composition.
Yet another object of the present invention is to provide novel polysaccharide -protein conjugate vaccine composition in a fully liquid or lyophilized or liquid-lyo vaccine formulation.
Yet another object of the present invention is to provide novel polysaccharide -protein conjugate vaccine composition wherein said composition is capable of being used in production of a tetravalent meningococcal ACYW-CRM197 combination vaccine containing serogroup ACYW polysaccharides each conjugated to non-toxic mutant of diphtheria toxin called cross reacting material (CRM197) as carrier protein.
Yet another object of the present invention is to provide a novel vaccine formulation of polysaccharide - protein conjugate vaccine composition comprising of polysaccharide - protein conjugates along with pharmaceutically acceptable components/ excipients.
Yet another object of the present invention is to provide a novel vaccine formulation of polysaccharide - protein conjugate vaccine composition comprising of tetravalent meningococcal ACYW-CRM197 polysaccharide -

protein conjugates along with pharmaceutical^ acceptable components/excipients.
Yet another object of the present invention is to provide novel polysaccharide -protein conjugate vaccine composition with or without adjuvant.
Yet another object of the present invention is to provide the optimum dosage of each of the conjugates in the vaccine composition and formulation.
Yet another object of the present invention is to provide a tetravalent vaccine formulation which is stable at high temperatures and shows high immunogenicity and antigenicity.
SUMMARY OF THE INVENTION
Accordingly, the present invention provides a novel polysaccharide - protein conjugate vaccine composition and formulation thereof. More particularly, the present invention relates to a conjugate vaccine formulation comprising of polysaccharide - protein conjugates produced using conjugation chemistry. The conjugation chemistry used includes known cyanylation chemistry. The formulation of present invention is capable of being used in production of tetravalent meningococcal combination vaccines.
The polysaccharides used for preparing conjugates of the present invention are obtained through optimized fermentation process.
The novel vaccine formulation of polysaccharide - protein conjugate vaccine composition comprises of polysaccharide - protein conjugates along with pharmaceutically acceptable components/excipients. All the conjugates in vaccine composition and formulation have same carrier protein.

The novel polysaccharide - protein conjugate vaccine formulation of present invention comprises of four polysaccharide-protein conjugates. The polysaccharide is selected from gram negative bacteria, belonging to Neisseria meningitidis capsular serogroup A, C, Y and W polysaccharides. CRM197 is used as the carrier protein in preparing all the conjugates.
The capsular polysaccharide is degraded to smaller sizes suitable for conjugation with carrier protein to obtain conjugates with high antigenicity and high immunogenicity. The capsular polysaccharide is degraded in the size range of average mol. weight from 20kDa to 250kDa preferably of average 20kDa, 50kDa, lOOkDa and 200kDa when tested for molecular weight estimation using Pullulan standards on HPLC PWXL4000 and 5000 columns in series. The polysaccharides are activated but carrier protein may or may not be activated before conjugation. The individual conjugates are obtained with linker or without any linker.
The size degraded capsular polysaccharide (sized capsular polysaccharide) is conjugated with carrier protein. The formulation contains the conjugates prepared by optimized cyanylation chemistry for each of serogroup A, C, Y and W polysaccharides. The monovalent bulk conjugates are purified by gel filtration chromatography or salt precipitation method and stored in suitable buffered saline.
The pharmaceutically acceptable excipients can be buffer, preservative, stabilizer, surfactant, either alone or in combination with or without adjuvant. The formulation of present invention is liquid or lyophilized or liquid-lyo formulation with mono- or multi-dose regimen with or without a preservative. The formulation of present invention is preferably a wholly liquid formulation.
The present invention also provides the optimum dosage of each of the conjugates in the vaccine composition and formulation.

DETAILED DESCRIPTION OF INVENTION WITH NON-LIMITING EXAMPLES AND ILLUSTRATIONS
It should be noted that the particular description and embodiments set forth in the specification below are merely exemplary of the wide variety and arrangement of instructions which can be employed with the present invention. The present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. All the features disclosed in this specification may be replaced by similar other or alternative features performing similar or same or equivalent purposes. Thus, unless expressly stated otherwise, they all are within the scope of present invention. Various modifications or substitutions are also possible without departing from the scope or spirit of the present invention. Therefore, it is to be understood that this specification has been described by way of the most preferred embodiments and for the purposes of illustration and not limitation
Accordingly, the present invention provides a novel polysaccharide - protein conjugate vaccine composition and formulation thereof. More particularly, the present invention relates to a conjugate vaccine composition comprising of polysaccharide - protein conjugates produced using conjugation chemistry. The conjugation chemistry used is known cyanylation chemistry. The composition of present invention is capable of being used in production of an adjuvanted or non-adjuvanted tetravalent combination vaccine. The vaccine formulation is a fully liquid or lyophilized or liquid-lyo formulation.
The novel vaccine formulation of present invention comprises of polysaccharide -protein conjugates along with pharmaceutically acceptable components/excipients. All the conjugates in vaccine composition and formulation have same carrier protein. The present invention provides a tetravalent vaccine formulation.

The novel polysaccharide - protein conjugate vaccine composition and formulation of present invention comprises of four individual polysaccharide-protein conjugates. The polysaccharide is selected from gram negative bacteria Neisseria meningitidis serogroup A, C, Y and W capsular polysaccharides. The carrier protein used for preparing all the conjugates is CRM197.
The capsular polysaccharide is degraded to smaller sizes suitable for conjugation with carrier protein to obtain conjugates with high antigenicity, high immunogenicity and high stability. The capsular polysaccharide is degraded in the size range of average Molecular weight from 20kDa to 250kDa, preferably 50, 100 and 200kDa when tested for molecular weight using Pullulan standards on HPLC PWXL4000 and 5000 columns in series. The polysaccharide is activated but carrier protein may or may not be activated before conjugation. The conjugates are produced with or without linker arm between polysaccharide and protein moieties. The conjugates with the linker arm are produced by attaching the linker to the carrier protein. The conjugates are purified by salt precipitation or by Gel filtration chromatography. The monovalent bulk conjugates are stored in phosphate buffered saline for MenC, Y and W-CRM197 conjugates and in MES buffer for MenA-CRM197 conjugate.
The novel multivalent polysaccharide-protein conjugate vaccine formulation of the present invention comprises of meningococcal serogroups A, C, Y, W polysaccharides each individually conjugated to CRM197 (Men ACYW-CRM197) wherein serogroup A, C, Y and W polysaccharides are conjugated using optimized cyanylation chemistry. Each said conjugate has the carrier protein to polysaccharide ratio between 0.25-1.1.
The novel multivalent polysaccharide-protein conjugate vaccine formulation of the present invention comprises of meningococcal serogroups A, C, Y, W polysaccharides each individually conjugated to CRM197 (Men ACYW-

CRM197) mixed with one or more buffer and one or more pharmaceutically acceptable excipients with or without adjuvant.
The tetravalent polysaccharide-protein conjugate vaccine formulation is preferably a wholly liquid tetravalent conjugate vaccine comprises of meningococcal serogroups A, C, Y, W polysaccharides each individually conjugated to CRM197 (Men ACYW-CRM197) mixed with one or more buffer and one or more pharmaceutically acceptable excipients with or without adjuvant. The osmolality of formulation ranges between 240-330 mOsmol/Kg.
The pharmaceutically acceptable excipients can be buffer, preservative, stabilizer, surfactant, either alone or in combination with or without any adjuvant. The stabilizers may be selected from sucrose, lactose, arabinose, maltose or a combination thereof. The formulation of present invention is a liquid or lyophilized or liquid-lyo combination formulation with mono- or multi-dose regimen with or without a preservative.
In one of the best embodiments, the novel tetravalent liquid polysaccharide-
protein conjugate vaccine formulation of the present invention comprises of
Ingredient Quantity/Concentration
Men ACYW-CRM197 8-20 ug polysaccharide/ serogroup/ml
Buffer 5-30 mM Phosphate buffered saline (pH 7.0±0.2)
Excipient 0-150 mM NaCl sufficient to maintain osmolality
between 240-330 mOsmol/Kg
5-30mMHistidine
Water (MQW) Qs
The ingredients are mixed by stirring at room temperature for 0.5-2 hours and followed by filling in vials and storage at 2-8°C.

In another embodiment, the novel tetravalent liquid polysaccharide-protein conjugate vaccine formulation of the present invention comprises of
Ingredient Quantity/Concentration
Men ACYW-CRM197 8-20 ug polysaccharide/ serogroup/ml
Buffer 10-25 mM Phosphate buffered saline (pH
7.0±0.2)
Excipient 0-150mMNaCl
Water (MQW) Qs
The ingredients are mixed by stirring at room temperature for 0.5-2 hours and followed by filling in vials and storage at 2-8°C.
One of the best embodiment of the non-adjuvanted liquid formulation providing desired osmolality and high stability and desired immunogenicity comprises of
Ingredient Quantity/Concentration
Men ACYW-CRM197 10-20 ug polysaccharide/serogroup/ml
Buffer: 10-25 mM Phosphate buffered saline (pH 7.0 ±
0.2) + 5-25 mM Histidine (pH 6.8 ± 0.2)
Excipient: 0-150mMNaCl
Water (MQW) Qs
The ingredients are mixed by stirring at room temperature for 0.5-2 hours and followed by filling in vials and storage at 2-8°C.
Another embodiment of the liquid formulation without adjuvant providing desired osmolality, high stability and desired immunogenicity comprises of
Ingredient Quantity/Concentration
Men ACYWX-CRM197 10 ug polysaccharide/serogroup/ml

Buffer: Excipients: Water (MQW)

10 mM Phosphate buffered saline (pH 7.0±0.2)
0-150 mMNaCl
Qs

The ingredients are mixed by stirring at room temperature for 0.5-2 hours and followed by filling in vials and storage at 2-8°C.
Another embodiment of the lyophilized formulation without adjuvant providing desired osmolality, high stability and desired immunogenicity comprises of

Ingredient
Men ACYWX-CRM197
Buffer:
Excipients:
Water (MQW)

Quantity/Concentration
10 ug polysaccharide/serogroup/ml
10 mM Phosphate buffered saline (pH 7.0±0.2)
0-150 mMNaCl, 1-3% Sucrose, 1-3% Maltose,
1-3%) Lactose
Qs

The MenACYW-CRM197 conjugates are lyophilized with one or more of the stabilizers including Sucrose, Maltose or Lactose and the lyophilized ingredients are mixed with buffer components stored in another vial before use. The vials containing lyophilized components and diluent buffer are stored at 2-8°C.
Another embodiment of the liquid formulation with adjuvant providing desired osmolality, high stability and desired immunogenicity comprises of

Ingredient
Men ACYWX-CRM197
Buffer:
Excipient
Adjuvant
Water (MQW)

Quantity/Concentration
10 ug polysaccharide/serogroup/ml
10 mM Phosphate buffered saline (pH 7.0±0.2)
0-150mMNaCl
Aluminum phosphate 600-1500 ug Al+++/ml
Qs

The ingredients are mixed by stirring at room temperature for 0.5-2 hours followed by keeping at 2-8°C for overnight and followed by filling in vials and storage at 2-8°C.
The formulations of the present invention without adjuvant have been tested for osmolality, stability and immunogenicity. The formulations have been exposed to high temperatures of 25±2 °C for 6 months to check the rise in the free polysaccharide content. The free polysaccharide content in the vaccine formulation has been separated by deoxycholate (DOC) precipitation and filtration method and estimated by High performance anion exchange chromatography with pulsed amperometric detector (HPAEC-PAD) method.
The formulation is stable at high temperatures. Considering 40% free PS as the maximum target, the formulation shows stability at the high temperature of 25±2°C for at least 5 months and at 5±3°C for at least 6 months in ongoing stability studies.
The formulation of the present invention is preferably in liquid form. The present invention also provides the optimum dosage of each of the conjugates in the vaccine composition and formulation. The optimum dose is 5-10 ug of serogroup A polysaccharide and 5 ug of serogroup C, Y and W polysaccharide per human dose with or without adjuvant.
Example 1: Preparation of different tetravalent Meningococcal liquid formulations to establish the product stability in the presence of different excipients and buffers:
Various tetravalent Meningococcal ACYW-CRM197 formulations have been prepared by mixing the antigen with the buffers like PBS buffer and Histidine or a combination thereof. The formulations have been prepared as per the Table 1 below:

Table 1: Matrix for the formulation of liquid tetravalent Meningococcal ACYW-CRM197 conjugate vaccine prepared with PS of average size 110
kDa.

Formulation Code Buffer NaCl Histidine PBS Antigen ug/ml of each
serogroup PS

Total 130mM Total 145mM 10m M 10m M

F-A (Base formulation) PBS (pH 7.0) V - - V ACYW=10
F-B Histidine (pH 6.8) - V V - ACYW=10
F-C PBS +
Histidine (pH
7.0) V - V V ACYW=10
F-D PBS (pH 7.0) V - - V A=20, CYW=10
The above formulations have been analyzed for osmolality and pH to confirm the 5 basic characteristics of the formulation.
Table 2: Results of the liquid tetravalent meningococcal conjugate vaccine
formulations without adjuvant

Formulation Code Osmolality (mOsmol/Kg) pH
F-A lot 1 306 6.97
F-Blot 1 287 7.46
F-Clot 1 311 7.22
F-Dlot 1 305 7.00
F-A lot 2 303 7.03
F-Blot 2 288 7.41
F-Clot 2 292 7.16

The results obtained meet the desired values of Osmolality and pH
Example 2: Preparation of different tetravalent Meningococcal ACYW-CRM197 liquid formulations with different bulk conjugate characteristics:
The tetravalent Meningococcal formulations have been prepared with or without any adjuvant and different characteristics of monovalent bulk conjugates. All the formulations have been prepared in PBS. The following matrix has been used to prepare the different formulations:
Table 3: Matrix for the preparation of liquid tetravalent meningococcal ACYW-CRM197 conjugate vaccine formulations

The above formulations have been analyzed for osmolality and pH to confirm the basic characteristics of the formulation.
Table 4: Results of the liquid tetravalent meningococcal conjugate vaccine formulations with or without adjuvant and different conjugate characteristics

The results obtained meet the desired values of Osmolality and pH.
Example 3: Stability of tetravalent Meningococcal ACYW-CRM liquid formulations at Accelerated and Real-Time conditions:
The stability of non-adjuvanted liquid tetravalent meningococcal ACYW-CRM197 conjugate vaccine is tested when exposed at various temperature conditions over the period. The test has been conducted to get the effect of temperatures over certain periods of time. The stability tests have been conducted using two temperature parameters, viz. real-time storage conditions at 5 ± 3°C and higher temperature conditions at 25 ± 2°C. The results are presented in Table 5.
a. real-time storage conditions at 5 ± 3°C - recommended storage temperature
(Real Time Stability Studies).
b. higher temperatures at 25 ± 2°C - higher than those recommended for storage
(Accelerated Stability Studies).
Samples have been stored for a period of 6 months under accelerated conditions tested on monthly interval and for 3 years at real time storage conditions tested at quarterly intervals in first year and 6 monthly in 2nd and 3rd year (Study ongoing, data available till 6 months). Samples have been withdrawn as per standard plan and analyzed for the best stability indicating parameter of free PS % content over time.
Table 5: Delta rise in free polysaccharide content during accelerated and real time stability studies for 5 and 6 months, respectively

Example 4: Immunization of mice with the liquid tetravalent Meningococcal ACYW-CRM197 Conjugate vaccine formulation
Groups of 10-12 female mice (6-9 weeks old) have been immunized at 2 week interval with novel liquid non-adjuvanted or adjuvanted tetravalent meningococcal ACYW-CRM197 conjugate vaccine F-A to F-K and a vehicle control without bulk conjugates and a licensed Men ACYW conjugate vaccine as positive control. All immunizations have been performed by administering of vaccine via subcutaneous route in mice. Each mouse has been immunized with formulation equivalent to 1-2 ug polysaccharide per serogroup. Serogroup specific anti-meningococcal functional antibody titers by serum bactericidal assay in sera collected post 2 and 3 dose. The post 3 dose results for novel liquid tetravalent meningococcal ACYW-CRM197 conjugate vaccine indicate significantly high immunogenicity titers as compared to vehicle control in both post-2 and post-3 dose responses and non-inferior titers to the licensed vaccine functional antibody (SBA) titers. Even after 2 doses, the SBA titers peaked in

many cases for novel liquid non-adjuvanted Men ACYW-CRM vaccine formulations.
Example 5: Serum Bactericidal Assay (SBA) for the serogroup specific functional antibody titration
iV. meningitidis serogroup specific bacterial stock has been grown overnight on sheep blood agar plate at 37°C with 5% CO2. Isolated colonies have been picked and incubated on the surface of another sheep blood agar plate at 37°C with 5% CO2. The bacterial growth from second plate have been resuspended in optimized SBA buffer for respective serogroup to achieve 250-1000 colony forming units per 10 ul. Quality control (QC) sera and test sera samples have been heat inactivated for 30 min at 56 °C. In micro well plate, 20 ul of serial two fold dilutions of test serum has been mixed with 10 ul of bacteria at the working dilution and 10 ul of baby rabbit complement (Pel-Freez). For negative controls bacteria have been incubated, in a separate well, with active baby rabbit complement without the test serum and with test serum and heat-inactivated baby rabbit complement. The well contents have been mixed by gently tapping the assay plate and incubated the plates for 1 hour at 37°C with 5% CO2. Ten uL sample from each well plated on blood agar plate by streak plate method. The blood agar plates have been incubated overnight at 37°C with 5% CO2 and colonies have been counted. The highest serum dilution showing > 50% decrease in colony-forming units after incubation of bacteria with reaction mixture, as compared to respective active complement control has been considered as the SBA titer. The results for representative study and formulation comparisons are presented in Table 6.
Table 6: Post 2- and 3-dose serum bactericidal geometric mean titers against different serogroups in mouse after immunization with vehicle control, licensed vaccine and non-adjuvanted liquid tetravalent Meningococcal ACYW-CRM vaccine formulations and fold rise over vehicle control titers*

*The figures in parentheses indicate the fold rise in SBA titers over those with vehicle control.

CLAIM:
A tetravalent polysaccharide - protein conjugate vaccine formulation, said
formulation comprises of:
Ingredient Quantity/Concentration
Conjugates of meningococcal 8-20 ug polysaccharide/ serogroup/ml
serogroup A, C, Y, W
polysaccharides and carrier protein
Buffer 5-30 mM Phosphate buffered saline
Excipient 0-150 mM
Diluent (MQW) Qs
wherein said formulation is a liquid or lyophilized or liquid-lyo combination of tetravalent formulation of Neisseria meningitidis serogroup A, C, Y, and W capsular polysaccharides (Men A, C, Y, W), each said polysaccharide being conjugated to carrier protein (CP) separately to obtain Men A, C, Y, W - CP conjugates, with one or more buffer with or without an adjuvant along with pharmaceutically acceptable excipients, wherein said formulation provides optimum osmolality, optimum pH, high stability and high immunogenicity.
The tetravalent vaccine formulation as claimed in claim 1, wherein said carrier protein (CP) is non-toxic mutant of diphtheria toxin called cross reacting material (CRM197).
The tetravalent vaccine formulation as claimed in claim 1, wherein said buffer is selected from phosphate buffer, Histidine, either alone or in variable combinations.
The tetravalent vaccine formulation as claimed in claim 1, wherein said pharmaceutically acceptable excipients are selected from salt, buffer, preservative, stabilizer, surfactant, either alone or in combination.

The tetravalent vaccine formulation as claimed in claim 1, wherein said diluent is selected from water, normal saline, phosphate buffered saline, Histidine buffer either alone or in variable combinations.

The tetravalent vaccine formulation as claimed in claim 1, wherein said
liquid formulation comprises of
Quantity/Concentration
8-20 ug polysaccharide/ serogroup/ml
Ingredient
Conjugates of meningococcal
serogroup A, C, Y, W polysaccharides
5-30 mM Phosphate buffered saline 0-150 mMNaCl 5-30 mM histidine qs
and carrier protein CRM197
Buffer
Excipient
Diluent (MQW)
The tetravalent vaccine formulation as claimed in claim 1, wherein said
liquid formulation comprises of:
Ingredient Quantity/Concentration
Conjugates of meningococcal serogroup A, 10-20 ug polysacc
C, Y, W polysaccharides and carrier protein haride/ serogroup/ml
CRM197
Buffer 10-25 mM Phosphate buffered
saline (pH 7.0±0.2)
Excipient 0-150 mMNaCl
Diluent (MQW) Qs
The tetravalent vaccine formulation as claimed in claim 1, wherein said liquid formulation comprises of:

Ingredient

Quantity/Concentration

Conjugates of meningococcal serogroup A, C, Y, W polysaccharides and carrier protein CRM197 Buffer
Excipient
Diluent (MQW)

10-20 ug polysaccharide/ serogroup/ml
10-25 mM Phosphate buffered saline (pH
7.0±0.2)
0-150 mM NaCl + 5-25 mM histidine (pi
6.8±0.2)
qs

The tetravalent vaccine formulation as claimed in claim 1, wherein said
liquid formulation comprises of:
Quantity/Concentration
10 ug polysaccharide/ serogroup/ml
Ingredient
Conjugates of meningococcal
serogroup A, C, Y, W polysaccharides
10 mM Phosphate buffered saline (pH
7.0±0.2)
0-150mMNaCl
qs
and carrier protein CRM197
Buffer
Excipient Water (MQW)

The tetravalent vaccine formulation as claimed in claim 1, wherein said lyophilized formulation comprises of:
Ingredient
Conjugates of meningococcal serogroup A, C, Y, W polysaccharides and carrier protein CRM197 Buffer
Excipient

Quantity/Concentration
10 ug polysaccharide/ serogroup/ml
10 mM Phosphate buffered saline (pH
7.0±0.2)
0-150mMNaCl, 1-3% sucrose, 1-3%

maltose, 1-3% lactose

Diluent (MQW)

qs

Quantity/Concentration
10-20 ug polysaccharide/serogroup/ml
11. The tetravalent vaccine formulation as claimed in claim 1, wherein said liquid formulation comprises of: Ingredient
10-25 mM PBS (pH 7.0±0.2)
0-150mMNaCl
Aluminum phosphate as 600-1500 ug
Al+++/ml
Qs
Conjugates of meningococcal serogroup A, C, Y, W polysaccharides and carrier protein CRM197 Buffer: Excipient: Adjuvant
Diluent (MQW)

Quantity/Concentration
10 ug polysaccharide/serogroup/ml
12. The tetravalent vaccine formulation as claimed in claim 1, wherein said liquid formulation comprises of: Ingredient
10mMPBS(pH7.0±0.2)
0-150mMNaCl
Aluminum phosphate as 600-1500 ug
Al+++/ml
Qs
Conjugates of meningococcal serogroup A, C, Y, W polysaccharides and carrier protein CRM197 Buffer: Excipient: Adjuvant
Water (MQW)
13. The tetravalent vaccine formulation as claimed in claim 1, wherein said lyophilized formulation comprises of:

Ingredient
Conjugates of meningococcal
serogroup A, C, Y, W polysaccharides
and carrier protein CRM197
Buffer:
Excipients:
Diluent

Quantity/Concentration
10-20 ug polysaccharide/serogrou
5-20 mM PBS (pH 6.5 -7.5)
2-10%
Qs

14. The tetravalent vaccine formulation as claimed in claim 1, wherein said Liquid-Lyo combination formulation comprises of:
Ingredient
Conjugates of meningococcal
serogroup A, C, Y, W
polysaccharides and carrier
protein CRM197
Buffer:
Excipients:
Diluent

Quantity/Concentration
10-20 ug polysaccharide/serogroup/ml
5-20 mM PBS (pH 6.5 -7.5)
2-10%
Qs

15. The tetravalent vaccine formulation as claimed in claim 1, wherein said Men A, C, Y, W - CRM197 conjugates are obtained employing optimized cyanylation conjugation chemistry.
16. The tetravalent vaccine formulation as claimed in claim 1, wherein each said capsular polysaccharide is degraded in the size range of average molecular weight from 20kDa to 250kDa, preferably 20, 50, 100 and 200 kDa before conjugation.
17. The tetravalent vaccine formulation as claimed in claim 1, wherein said conjugates are produced with or without having a linker arm between said

polysaccharide and said carrier protein wherein a linker is if present is attached to said carrier protein.
18. The tetravalent vaccine formulation as claimed in claim 17, wherein said linker is adipic acid dihydrazide.
19. The tetravalent vaccine formulation as claimed in claim 1, wherein said conjugates are produced with activated polysaccharide and activated or non-activated carrier protein.
20. The tetravalent vaccine formulation as claimed in claim 1, wherein each said conjugate has the carrier protein to polysaccharide ratio between 0.25-1.1.
21. The tetravalent vaccine formulation as claimed in claim 1, wherein said formulation is liquid or lyophilized or liquid-lyophilized formulation with mono- or multi-dose regimen with or without adjuvant and with or without a preservative.
22. The tetravalent vaccine formulation as claimed in claim 1, wherein optimum human dose of serogroups A, C, Y and W ranges between 2-10 ug polysaccharide per serogroup per dose, preferably 4-10 ug each of serogroup A polysaccharide and 5 ug each of serogroup C, Y and W polysaccharides.
23. The tetravalent vaccine formulation as claimed in claim 1, wherein said optimum osmolality of formulation is 240-330 mOsmol/Kg.
24. The tetravalent vaccine formulation as claimed in claim 1, wherein said optimum pH of formulation is 6.5-7.5.

25. The tetravalent vaccine formulation as claimed in claim 1, wherein said liquid formulation is stable at high temperature of 25±2 °C for at least 5 months showing less than 40 % free polysaccharide increase.
26. The tetravalent vaccine formulation as claimed in claim 1, wherein said liquid formulation is stable at temperature of 5±3°C for at least 6 months showing less than 40 % free polysaccharide increase.
27. The tetravalent vaccine formulation as claimed in claim 1, wherein said liquid formulation shows high immunogenicity.
28. The tetravalent vaccine formulation as claimed in claim 1, wherein said formulation and said composition is capable of being used in production of liquid tetravalent meningococcal ACYW-CRM197 combination vaccine.
29. The tetravalent vaccine formulation as claimed in claim 1 wherein said formulation is a wholly liquid formulation without adjuvant.
30. The tetravalent vaccine formulation as claimed in claim 1 wherein each said capsular polysaccharide conjugated to said carrier protein in bulk is stored in phosphate buffered saline for MenC-CRM197, Y- CRM197 and W-CRM197 conjugates and in MES buffer for MenA-CRM197 conjugate.
31. A method to obtain tetravalent multivalent polysaccharide - protein conjugate vaccine formulation as claimed in claim 1 wherein said method comprises the steps of:

(a) degradation of capsular polysaccharides of meningococcal ACYW in size range of average molecular weight from 20kDa to 250kDa, preferably 50, 100 and 200kDa;
(b) activation of said degraded capsular polysaccharide obtained in step (a);

(c) conjugation reaction of said activated capsular polysaccharide obtained in step (b) with a carrier protein to obtain polysaccharide-protein conjugates;
(d) the MenA bulk conjugate is stored in MES buffer and MenC, Y, W bulk conjugates are stored in PBS buffer;
(e) mixing each of the polysaccharide-protein conjugates of Men A, C, Y and W with at least one buffer, at least one excipient and at least one diluent to obtain a mixture for formulation of vaccine;
(f) stirring said mixture at room temperature for 0.5 to 2 hours to obtain formulation of vaccine;
(g) The formulation may be lyophilized or kept in liquid form or one-two of the bulk conjugates are lyophilized, while other bulk conjugates can be in liquid form;
(h) filling said formulation of vaccine in vials;
(i) storing said vials containing said formulation of vaccine at 2-