FORM-2
THE PATENTS ACT, 1970
(39 OF 1970)
AND
THE PATENTS RULES, 2003
(As Amended)
COMPLETE SPECIFICATION (See section 10; rule 13)
"Stabilizers for Vaccine Compositions"
Serum Institute of India Ltd., a corporation organized and existing under the laws of India, of 212/2, Off Soli Poonawalla Road, Hadapsar, Pune 411028, Maharashtra India.
The following specification particularly describes the nature of this invention and the manner in which it is to be performed:

Stabilizers For Vaccine Compositons
Field of Invention:
The invention pertains to multi-dose lyophilized formulations of antigenic polysaccharides comprising a paticularly advantageous and novel stabilizer composition.
Existing State Of art:
Nesseria meningitidis is a leading cause of bacterial meningitis and sepsis throughout the world. The incidence of endemic meningococcal disease during the last thirty years ranges from 1 to 5 per 100,000 in the developed world, and from 10 to 25 per 100,000 in developing countries (Reido, F. X., et al., J. Ped. Infect. Dis., 14, pp. 643-657 [19951). During epidemics the incidence of meningococcal disease approaches 1000 per 1000,000. There are approximately 2,600 cases of bacterial meningitis per year in the United States, and on average 330,000 cases in developing countries. The case fatality rate ranges between 10 and 20%.
Pathogenic meningococci are enveloped by a polysaccharide capsule that is attached to the outer membrane surface of the organism. Thirteen different serogroups of meningococci have been identified on the basis of the immunological specificity of the capsular polysaccharide (Frasch, C. E., et al., Rev. Infect. Dis., 7, pp. 504-510 [1985)). Of these thirteen serogroups, five cause the majority of meningococcal disease; these include serogroups A, B, C, W135, and Y. Serogroup A is responsible for most epidemic disease. Serogroups B, C, and Y cause the majority of endemic disease and localized outbreaks.
Vaccines based on meningococcal polysaccharide have been described which elicit an immune response against the capsular polysaccharide. These antibodies are capable of complement-mediated bacteriolysis of the serogroup specific meningococci. The meningococcal polysaccharide vaccines are shown to be

efficacious in children and adults (Peltola, H., et al., New Engl. J. Med., 297, pp. 686-691 [1997]; and Artenstein, M. S., et al., New Engl. J. Med., 282, pp. 417-420 [1970].
Stable meningococcal polysaccharide containing multi-dose formulations are viewed by the biopharmaceutical industry as particularly advantageous and commercially attractive.
As compared to single-dose vaccine vials , multi-dose vaccine vials have faster filing rate , cheaper packaging costs, are smaller and lighter for transport, occupy less cold chain capacity, requires fewer reconstitution syringes and result in smaller medical waste volume. See Drain et al " Single dose versus multi-dose vaccine vials for immunization programmes in developing countries " Bulletin of the World Health Organization ; 2003 ; 81 (10) ;pgs 726-731.
Existing Multidose formulations of meningococcal polysaccharides utilize lactose alone as a stabilizer.Menomune (Groups A, C, Y and W-135 polysaccharides Combined) is a freeze dried vaccine available in single and 10 dose vials, contains distilled water as diluent ,thimerosal as preservative ,lactose as stabilizer and isotonic sodium chloride.NmVac-4 (Groups A, C, Y and W-135 polysaccharides Combined) is a lyophilized vaccine available in single and 10 dose and 50 dose vials, contains lactose as stabilizer and sodium chloride.Mencevax (Groups A, C, Y and W-135 polysaccharides Combined) is a lyophilized vaccine available in single and multidose vials, contains saline as diluent for monodose, diluent containing phenol for multidose and lactose as excepient.Quadrimeningo ((Groups A, C, Y and W-135 polysaccharides Combined)is a lyophilized vaccine containing lactose as stabilizer.
However development of stable antigenic polysaccharide -containing multi-dose formulations with stabilizers other than lactose has proven difficult by virtue of the instability displayed by polysaccharides, particularly meningococcal polysaccharides during storage. Polysaccharide antigens readily depolymerize

and their relative molecular mass diminishes when they are exposed to ambient temperatures. The degree of depolymerization is therefore a useful indicator for assessing both immunogenicity and thermal stability.See Galazka et al, WHO/GPV,1998
The immunogenicity of Meningococcal vaccine is related to the molecular size of the protective antigens, polysaccharides A and C; the antibody response increases with the molecular weight. WHO recommends a molecular weight size distribution coefficient of 85%.
Also it is known that even in a lyophilized state, the Meningococcal-vaccines show an extraordinary instability.
US 4134214 discloses an improved process for preserving vaccine derived from Group A and Group C polysaccharides of N. meningitidis which comprises of lyophilizing an isotonic solution of said polysaccharide maintained at a temperature of from -20° C. to -30° C.
It has been suggested that replacement of sodium chloride by lactose as a menstruum for lyophilization stabilizes polysaccharide vaccines against thermal depolymerization, represented a major step in achieving more stable vaccines. See Tiesjema et al, BULL WORLD HEALTH ORG,1977, 55,43-48.However an explanation for the increase in the stability with lactose and unsatisfactory results with mannitol could not be given.
GB 2009198A describes the stabilization of Meningococcal polysaccharides under lyophilization, by combining them with various sugars including sucrose, raffinose, glucose and trehalose.This also suggests synergistic effect in improvement of meningococcal polysaccharide stability if mixtures of two of the stabililizing compounds like glycine or mannitol are used.
US 4206200 discloses use of lactose alone or in combination with sucrose,raffinose, glucose or trehalose.

US 6051238 claims combination of sorbitol and sucrose before lyophilizing mumps vaccine.
US 6616931 claims combination of sucrose,mannitol and sodium phosphate buffer for stabilizing rotavirus vaccine.
WO2001/041800 claims MenC-CRM197 vaccine wherein combination of sucrose and histidine buffer/imidazole buffer is employed before lyophilization.
There is prior art that discusses lyophilization studies and storage stability of recombinant monoclonal antibody wherein sucrose/mannitol or trehalose/mannitol combinations have been disclosed, see, Jeffrey; Journal of pharmaceutical sciences (2001). Vol-90. lssue-3. pgs 310-321 . However a capsular polysaccharide antigen is physically, chemically & immunologically distinct from a therapeutic glycoprotein (monoclonal antibody). Further Jeffrey et al does not discuss preparation of a stable, lyophilized multidose polysaccharide vaccines
There is a need for an invention to utilize an alternative stabilizer composition for formulating multi-dose, lyophilized and polyvalent meningococcal polysaccharide vaccine (Men A C Y & W-135). The present invention addresses these needs.
Summary of Invention
The present invention generally provides a novel stabilizer composition for preparation of multidose and multivalent (Men A , C, Y & W-135) meningococcal- capsular-polysaccharide vaccine formulation.
Specifically, this invention provides a stable meningococcal polysaccharide lyophilized vaccine wherein the lyophilized vaccine is obtained by using a stabilizer mixture selected from a) saccharides b) sugar alcohols and c) amino acids.

Short duration lyophilization cycle, uniformly Lyophilized product, optimal meningococcal polysaccharide weight retention and improved immunogenecity of the quadrivalent menincococcal polysaccharide formulation are some of the advantageous features of the instant invention.
In yet another aspect of the invention, the vial may include a single dose of vaccine, or it may include more than one dose (a 'multidose' vial) e.g. 2,5 and10 doses.
The present invention provides a method of protecting a human or animal susceptible to infection from N. meningitidis comprising administering an immunologically effective dose of the vaccine of the invention to the human or animal.
These and other features of the invention will be better understood through a study of the following detailed description of a specific embodiment of the invention. The scope of the invention is limited only through the claims appended hereto.
Detailed Description
The antigenic polysaccharides, particularly meningococcal polysaccharides are unstable and their properties are changed when they are present in liquid environment or exposed to high temperature. Therefore lyophilization technique is utilized. However development of stable antigenic polysaccharide -containing multi-dose formulations with stabilizers other than lactose has been difficult.
Polysaccharide antigens readily hydrolyze and the relative molecular mass gradually diminishes when kept for long-term storage this phenomenon is also observed even at 4°C. This results in an increase in the distribution of molecular

mass of the polysaccharide. The molecular size distribution provides an indication of manufacturing consistency. The distribution constant (Kd) is used to depict molecular size distribution of the polysaccharide at the main peak of the elution curve obtained by any suitable chromatographic method. High Performance Liquid Chromatography with Sepharose CL-4B columns may be used for this distribution constant determination. The Kd is calculated by the following formula.
Kd = ( Ve-Vo / Vt-Vo )
Where Ve = The volume of the eluate from start of the chromatography to elution maximum of the main component of polysaccharide
Vo = The volume of eluate of substance excluded through voids e.g. Dextran Vt = The volume of eluate of substance included through resin e.g. Acetone
The antigenic polysaccharides are unstable and their properties can change when they are present in liquid environment or exposed to high temperature. This phenomenon can also observed for meningococcal polysaccharides even if they are exposed with negligible amount of water. Therefore specialized technique called "Lyophilization" can be used for drying sensitive biologilcals. This technique can involve solidifying the aqueous content called freezing, sublimation of water under influence of reduced pressure and removal of remaining moisture by heating at relatively higher temperature. The dried mass obtained by this process is stable and can be regenerated to its closest state as before. However lyophilization generates a variety of stresses that tend to destabilize or denature the antigen.
The present invention generally provides a novel stabilizer composition for preparation of multi-dose and multivalent (Men A , C, Y & W-135) meningococcal- capsular-polysaccharide vaccines, wherein the formulation

comprises one stabilizer selected from saccharides, one stabilizer selected from the sugar alcohols and one stabilizer selected from amino acids.
Two mechanisms have been proposed for protective effects of sugars upon dehydration. First, drying of macromolecules in the presence of sugars(saccharides) results in an amorphous, glassy state of very high viscosity. This vitrification of macromolecules embeds them into a rock candy type of glassy state inhibiting chemical and biochemical reactions. Second sugars (saccharides) can replace water in dried state, thus satisfying hydrogen bond interactions thus protect macromolecules against Cryogenic and Lyophilization stresses. The combination of said sugars with sugar alcohol and amino acid is found to stabilize capsular polysaccharides, particularly meningococcal polysaccharides
As used herein saccharides means monosaccharides, disaccharides and oligosaccharides.
Suitably the saccharide is selected from dextrose, sucrose, lactose, trehalose, cellobiose, raffinose and isomaltose.
Preferably, the saccharide is selected from the group consisting of sucrose and trehalose.
As used herein a sugar alcohol means a hydrogenated form of a saccharide (suitably a monosaccharide) where a carbonyl group of the saccharide has been reduced to a primary or secondary hydroxyl group.
Suitably, the sugar alcohol is selected from the group consisting of mannitol, sorbitol, xylitol, glycerol, erythtritol, arabitol and allitol, and preferably the sugar alcohol is mannitol.
Suitably the amino acid is selected from glycine, alanine, glutamine, arginine, lysine, proline, serine and histidine and pharmaceutically acceptable salts thereof. Preferably the amino acid is glycine.
Thus, in one embodiment of the invention, the formulation comprises one stabilizer selected from saccharides, such as trehalose and sucrose, one

stabilizer selected from the sugar alcohols, such as mannitol, and one stabilizer which is an amino acid , such as glycine.
When the formulation comprises sucrose and mannitol the weight ratio of sucrose to mannitol is suitably from 1:4 to 4:1, or from 1:2 to 2:1, e.g.1:4, 3:2,2:3,4:1, 2:1, 1:2 or 1:1, and preferably the ratio is 1:4.
When the formulation comprises trehalose and mannitol, the weight ratio of trehalose to mannitol is suitably from 1:4 to 4:1, or from 1:2 to 2:1, e.g.1:4, 3:2, 2:3, 4:1, 2:1,1:2 or 1:1, and preferably the weight ratio is 1:4.
When the suspension comprises sucrose, mannitol and glycine, the weight ratio of sucrose to mannitol to glycine is 1:4:1
Another embodiment of the invention is a lyophilization cycle for formulation, that can comprise of a primary drying or sublimation at a temperature ranging from below -27°C for about 4 to 6hrs and secondary drying at a temperature range between 20 to 24°C for a duration ranging from 6 to 8 hrs. This duration is dependent on dimension of container and fill volume of the formulation.
Yet another embodiment of the present invention is that the duration of said short lyophilization cycle can range from about 18 to 22hrs.
Further said lyophililization can result in an intact homogenous pellet type cake with less than 2% residual mositure and more than 90% molecular weight retention of meningococcal polysaccharides.
By way of example, the polysaccharide may be from Gram negative bacteria selected from the group consisting of: Escherichia coli, Francisella tularensis, Haemophilus influenzae, Klebsiella, Moraxella catarrhalis, Neisseria meningitidis, Porphyromonas gingivalis, Pseudomonas aeruginosa, Burkholderia cepacia,

Salmonella typhi, Salmonella typhimurium, Salmonella paratyphi, Shigella dysenteriae, Shigella flexneri, Shegella sonnei and Vibrio cholera. The polysaccharide may be from Gram positive bacteria selected from the group consisting of: Enterococcus faecalis, Enterococcus faecium, Group A Streptococcus, Group B Streptococcus, Mycobacterium tuberculosis, Staphylococcus aureus, Staphylococcus epidermidis and Streptococcus pneumoniae.
It is preferred that the polysaccharide is from pathogenic Neisseria, preferably N. meningitidis. For example, the polysaccharide may be a Neisseria meningitidis capsular polysaccharide.
As used herein "polyvalent" refers to the N. meningitidis selected from N. meningitidis serogroup A, C, W135 and Y (e.g., A and C; or A and W135; or A and Y; or C and W135; or C and Y; or W135 and Y; or A, C and W135; or A, C and Y; or C, W135 and Y; or A, Y and W135; or A, C, W135 and Y).
The lyophilzed vaccine composition prepared by method of invention can be dissolved in isotonic sodium chloride before administration.
The following Examples further illustrate the essential features of the present invention. However, it will be apparent to those skilled in the art that the specific chemicals, apparatus, instruments, conditions and methods used in the Examples do not limit the scope of the present invention.
DESCRIPTION OF THE FIGURES
FIGS. 1-4 show certain embodiments of the present invention.

EXAMPLES
Example 1
Preparation of Nesseria meningitidis Serog roups A Capsular Polysaccharide
Media for Fermentation:
The media employed in the production of Meningococcal A polysaccharide essentially includes Glutamic acid, di-sodium phosphate, potassium chloride, ammonium chloride, cystine, casamino acid and yeast extract. The biomass is grown in a fermenter to reach a minimum OD of 5.0.
Downstream Processing:
The fermenter harvest is precipitated by addition of hexa-decyl-tri-methyl-ammoniumbromide, (CTAB) to final concentration of 1% The precipitated contents are separated by centrifugation. The CTAB paste is slowly homogenized with absolute ethanol. The content so obtained are passed through specific filters and finally precipitated by addition of calcium chloride. The precipitated mass is centrifuged to remove supernatant. Calcium salt of meningococcal polysaccharide A is dissolved in distilled water and finally diafiitered to attain purified polysaccharide.
Preparation of Nesseria meningitidis serogroups C, Y and W135 capsular polysaccharides
Media for Fermentation:
The media employed in the production of Meningococcal C polysaccharide essentially includes Ammonium sulfate, Glutamic acid, serine, Argenine, sodium chloride, yeast extract, glucose, di-sodium phosphate, cystine, magnesium chloride, calcium chloride and ferrous sulphate. The biomass is grown in a fermenter to reach a minimum OD OD of 5.0.

Downstream Processing:
The polysaccharides were purified as described in "Studies on the Group-specific Polysaccharide of Neisseria Meningitidis serogroup X and improved procedure for its isolation" in Journal of Biological Chemistry (1974), 15, 4797-4801 and "Pilot-Scale Production of Group A and Group C Meningococcal Polysaccharide Immunogen" in Infect. Immun. Vol 2 No.5 Nov 1970. 640-643.
Example 2
Preparation of Sucrose, Trehalose, Mannitol & Glycine solutions
Weigh about 1.0 gm of sucrose and dissolve it 10 ml of cool Water for Injection (WFI), after complete dissolution of the sugar make up the volume to 20 ml with cool WFI (5% sucrose solution). Filter this solution with the help of 0.2u filter in a sterile container. Similarly weigh the 2.5 gm of Mannitol and dissolve it 10 ml of cool WFI, after complete dissolution of the sugar make up the volume to 50 ml with cool WFI (5% Mannitol solution). Also weigh about 2.5 gm of Trehalose and dissolve it 20 ml of cool WFI, after complete dissolution of the sugar make up the volume to 50 ml with cool WFI (5% Trehalose solution). Weigh about 2.5 gm of Glycine and dissolve it 30 ml of cool WFI, after complete dissolution make up the volume to 50 ml with cool WFI (5% Glycine solution). Bulk Solutions: Table 1
Sr.No. Content Concentration
1 Men A purified Polysaccharide 9.99 mg/ml
2 Men C purified polysaccharide 15.92 mg/ml
3 Men Y purified polysaccharide 12.4 mg/ml
4 Men W135 purified polysaccharide 13.3 mg/ml
5 Sucrose 50 mg/ml
6 Mannitol 50 mg/ml
7 Trehalose 50 mg/ml
8 Glycine 50 mg/ml

Example 3
Formulation 1: Nesseria meningitidis Serogroups A & C Capsular Polysaccharide (Trehalose and Mannitol)
Formulation Contents:
In a clean and sterile container following solutions are added and gently mixed.
Table 2

Sr.No. Content Volume
l Men A Polysaccharide 5.005 ml
2 Men C Polysaccharide 3.140 ml
3 Trehalose (50 mg/ml) 10 ml
4 Mannitol (50 mg/ml) 40 ml
5 Water for Injection (0.2μ Syringe filtered) 41.855 ml
Total 100 ml
The bulk was fitted into 20 mm diameter and 50 mm height glass vial ((JSP type-I) with fill volume 1 ml.
Formulation 2: Nesseria meningitidis Serogroups A Capsular Polysaccharide (Sucrose, Mannitol and Glycine)
Formulation Contents:
In a clean and sterile container following solutions are added and gently mixed.
Table 3

Sr.No. Content Volume
1 Men A Polysaccharide 5.005 ml
2 Sucrose (50 mg/ml) 10 ml
3 Mannitol (50 mg/ml) 40 ml
4 Glycine 10 ml
5 Water for Injection (0.2μ Syringe filtered) 34.995 ml
Total 100 ml

The bulk was filled into 20 mm diameter and 50 mm height glass vial (USP type-I) with fill volume 1 ml.
Formulation 3: Nesseria meningitidis Serogroups A ,C,Y and W135 Capsular Polysaccharide (Sucrose and Mannitol)
Formulation Contents:
In a clean and sterile container following solutions are added and gently mixed.
Table 4:

Sr.No. Content Volume
1 Men A Polysaccharide 5.005 ml
2 Men C Polysaccharide 3.140 ml
3 Men Y Polysaccharide 4.030 ml
4 Men W135 Polysaccharide 3.750 ml
5 Sucrose (50 mg/ml) 10 ml
6 Mannitol (50 mg/ml) 40 ml
7 Water for Injection (0.2μ, Syringe filtered) 34.075 ml
Total 100 ml

Example 4
Lyophilization
The general scheme for lyophilization cycle was as follows: Table 5:

Precooling
Shelf Precooling None — —
Freezing
Shelf Temp Control Final T°C Ramp
Duration
(Min) Soak Duration (Min)
Ramp 1 & soak l -40 70 240
Sublimation
Shelf Temp Control Final T°C Ramp Duration Soak Duration (Min)
Ramp 1 & Soak l -27 30 240
Ramp 2 & Soak 2 -10 35 180
Chamber pressure control
-270 ramp and soak 200 mTorr, -100 ramp and sc ak 100μbar.
Secondary Drying
Shelf Temp Control Final T°C Ramp
Duration
(Min) Soak Duration
(Min)
Ramp 1 and soak 1 21 100 450
Chamber pressure control
210 ramp 100 mTorr, 210 50 mTorr.

Example 5
Analysis
Molecular size distribution: The samples are analyzed by High Performance Liquid Chromatography (HPLC) on Tosohaas 5000 Pw XL gel filtration column with 1X PBS as mobile phase and 1 ml flow rate.
Residual Moisture: By Karl Fischer method
Fig: 1 (Control):
HPLC profile of formulation 1 before Lyophilization. The main peak has area of 96.06% .


Retention Time (min) %Area Area (μV'sec)
19.533 3.33 126427
23.317 96.06 3650463
29.067 0.61 23186

Fig: 2(Formulation 1):
HPLC profile of formulation 2 after Lyophilization. The main peak has an area of 94.13%. There is -2% main peak broadening due to lyophilization.

Table 7

Retention Time (min) % Area Area μV*sec)
18.536 4.19 62982
23.033 94.13 1414786
29.050 1.68 25288

Fig: 3(Formulation 2):
HPLC profile of formulation 3 after Lyophilization. The main peak has an area of 94.46%. There is -1.5% main peak broadening due to lyophilization.

Table 8

Retention Time (min) % Area Area μV'sec)
18.603 4.20 62200
23.133 94.46 1399275
29.050 1.34 19793

Fig: 4(Formulation 3):
Table 9
HPLC profile of formulation 1 after Lyophilization. The main peak has area of 94.85%. There is ~1% main peak broadening due to lyophilization.

Retention Time (min) % Area Area μVsec)
18.653 3.12 50999
23.350 94.85 1550071
29.050 2.03 33118

Table 10

Sample Molecular Residual Dissolution Kd after 1 Kd after 3
Details Size Moisture time (In yr storage months
distribution
coefficient
(Kd) .Seconds) at 2-8°C storage at 37°C
Control 96.06 - - - -
F-1 94.13 1.50 30 91 89
F-2 94.46 1.75 35 90 87
F-3 94.85 1.35 25 92 90
F-1: Formulation 1; F-2: Formulation 2; F-3: Formulation 3
The study demonstrates that the lyophilized formulation of antigenic meningococcal polysaccharides obtained by utilizing the stabilizer combination comprising sucrose and mannitol has a uniform /intact pellet, has less than 2% residual mositure, shows molecular distribution coefficient (Kd) more than 85% than that of before lyophilization with less than 2% degradation and on reconstitution with sodium chloride complete dissolution of lyophilized mass in less than 40 seconds is observed. The stability(Kd) of the these formulations on storage at 2-8°C (1year) or 37°C (3months) is more than 85 %.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative examples and that the present invention may be embodied in other specific forms without departing from the essential attributes thereof, and it is therefore desired that the present embodiments and examples be considered in all respects as illustrative and not restrictive, reference being made to the appended claims, rather than to the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are/therefore intended to be embraced therein.

We claim,
1. A stable, lyophilized vaccine formulation comprising atleast one polysaccharide antigen and a stabilizer mixture of i) saccharides and ii) sugar alcohol
2. A formulation according to claim 1, wherein the stabilizer mixture further comprises of amino acids.
3. A formulation according to claim 1, wherein the stabilizer comprises of a saccharide selected from a group consisting of sucrose, trehalose, dextrose, lactose,raffinose, mannose and xylose .
4. A formulation according to claim 3, wherein the stabilizer comprises a saccharide selected from sucrose and trehalose.
5. A formulation of claim 1, wherein the formulation comprises a sugar alcohol selected from a group of mannitol, sorbitol and allitol.
6. A formulation of claim 2, wherein formulation comprises an amino acid selected from glycine,alanine, glutamine, proline, serine, arginine, lysine and histidine.
7. The formulation according to any of the preceding claims which comprises a mixture of: a) trehalose and mannitol b) sucrose and mannitol or c) sucrose, mannitol and glycine
8. The composition of claim 1, wherein lyophilization process comprises of
a) freezing at a temperature of atleast -30°C , b) a primary drying at a
temperature ranging from -20 to -30°C, for about 3 to 6hrs and c) secondary drying at a temperature ranging from about 20 to 25°C for a duration rangingfrom 5 to 10 hrs.

9. The lyophilization process according to claim 8, wherein a) freezing is
carried at a temperature of -40°C , b) primary drying at a temperature of -27 °C for about 4 hrs and c) secondary drying at a temperature of 21 °C for aduration of 7.5 hrs.
10. A lyophilization process of claim 8, wherein the lyophilization cycle duration is in the range of 15 hrs to about 25 hrs.
11. A lyophilization process of claim 10, wherein the lyophilization cycle duration is of 22 hrs.
12. A lyophilized product obtained by lyophilization process of claim 8, wherein molecular size distribution of antigenic polysaccharide post lyophilization is atleast 85% than that of before lyophilization and with less than 2% degradation.
13. A lyophilized product obtained by lyophilization process of claim 8, containing not more than 5% moisture.
14. A lyophilized product according to claim 13 , wherein said moisture content is 0 to 2%.
15. A lyophilized product according to claim 1 or 12, wherein said product is stable for atleast 3 months of storage at 37°C with a molecular size distribution coefficient of atleast 85%.
16. A lyophilized product according to claim 1 or 12, whereih said product is stable for atleast 12 months of storage at 2-8°C with a molecular size distribution coefficient of atleast 85%.

17. A lyophilized product obtained by lyophilization process of claim 8, wherein reconstitution results in rapid and complete dissolution of a lyophilized mass in less than 40 seconds.
18. A formulation of any preceding claim characterized in that the formulation is single dose or multidose.
19. A formulation according to claim 18, characterized in that the multidose formulation is selected from a group consisting of 2 dose,5 dose and 10 dose .
20. A formulation of claim 1, wherein said polysaccharide is from a bacteria selected from the group consisting of: Escherichia coli, Francisella tularensis, Haemophilus influenzae,Klebsiella, Moraxella catarrhalis, Neisseria meningitidis, Porphyromonas gingivalis, Pseudomonas aeruginosa, Burkholderia cepacia, Salmonella typhi, Salmonella typhimurium, Salmonella paratyphi, Shigella dysenteriae, Shigella flexneri, Shegella sonnei and Vibrio cholera.
21. A formulation of claim 1, wherein said polysaccharide is from a bacteria selected from the group consisting of: Enterococcus faecalis, Enterococcus faecium, Group A Streptococcus, Group B Streptococcus, Mycobacterium tuberculosis, Staphylococcus aureus, Staphylococcus epidermidis and Streptococcus pneumoniae.
22. A formulation according to claim 20, wherein said polysaccharide is from pathogenic Neisseria selected from A and C; or A and W135; or A and Y; or C and W135; or C and Y; or W135 and Y; or A, C and W135; or A, C and Y; or C, W135 and Y; or A, Y and W135; or A, C, W135 and Y.