FORM-2
THE PATENTS ACT, 1970
(39 OF 1970)
AND
THE PATENTS RULES, 2003
(As Amended)
COMPLETE SPECIFICATION
(See section 10; rule 13)
"A NOVEL METHOD FOR PREPARING BACTERIAL VACCINES"
Serum Institute of India Ltd., a corporation organized and existing under the laws of India, of 212/2, Off Soli Poonawalla Road, Hadapsar, Pune 411 028 Maharashtra India.
The following specification particularly describes the nature of this invention and the manner in which it is to be performed:

BACKGROUND OF THE INVENTION
Isolated bacterial polysaccharides from Haemophilus influenzae serotype b, Streptococcus pneumoniae, Neisseria meningitidis have been successfully used as vaccines in adults. However, vaccines based on isolated polysaccharides are ineffective in young infants due to the T-helper cell independent (TI antigens) character of the Polysaccharide antigens. Conversion of TI antigens of bacterial Polysaccharides into T-helper cell dependent (TD) antigens, can be accomplished by covalent coupling of size degraded polysaccharides to immunogenic carrier proteins. The conjugation is carried out by the reaction of single end groups of the polysaccharide, hence, using this technique several polysaccharide molecules can he covalently attached to one protein molecule, resulting in conjugates that are highly immunogenic and easy to define with regard to chemical conjugation.
It is known that size reduced polysaccharides have following advantages over plain polysaccharides when used to prepare the conjugates: (a) the conjugates prepared from using size reduced polysaccharides may be inherently more immunogenic than the corresponding conjugates prepared from full length polysaccharides; and (b) reactions used to prepare these conjugate vaccines can offer a higher degree of control, as well as more versatility in process design,

when using size reduced polysaccharides.
It is necessary to reduce the molecular mass of the polysaccharide prior to the coupling reaction in order to perform conjugation, with high yields. Size reduction of polysaccharides decreases the viscosity of the solution and increases the number of reactive end groups, both factors contribute to an increased freguency of covalent bond formation. Furthermore the optimal immunogenicity of polysaccharide components of conjugate vaccines is said to be related to their molecular size.
Several methods of partial depolymerization of bacterial and non-bacterial polysaccharides are described which yields fragments suitable for conjugation, such as acid hydrolysis, alkaline degradation, oxidation by periodate, ozonolysis (Wang et al. Carb. Res. 1999, 319, 1-4,141-147), enzymatic hydrolysis, sonication (Pawlowski et al. Vaccine, 2000, 18.18, 1873-1885), electron beam fragmentation (Pawlowski et al.Micro Lett, 1999, 174.2, 255-263) etc. However acid hydrolysis, alkaline degradation are time consuming and the resultant size reduced sample has high polydispersity.Also periodate oxidation have deleterious effects on labile antigenic epitopes of some polysaccharides, Ozonolysis can only be used with
polysaccharides containing p-Z?-aldosidic linkages, and only few endoglycanases have been isolated till date.

US 20090041802 discloses fragmentation of Meningococcal polysaccharides by Emulsiflex C-50 (conventional homogenizer) (Avestin).Homogenizers , pressurize the sample in a chamber (via a crank shaft mechanism or compressed gas and then release it through a manually or automatically controlled valve (homogenizing valve) into another chamber. Traditionally, these cannot go to extremely high pressures (40 kpsi or 2,700 Bar) and due to the homogenizing valve and pressure creation mechanism, variability over the pressure can have implications on percentage breakage and repeatability between operating intervals.
In homogenizers , energy used to produce the high pressure is released as heat due to compression and frictional forces as the fluid passes through the valve. The fluid temperature rises by 1-2 °C for every 1,000 psi to which the sample is subjected.
Conventional homogenizers operate at peak pressures for mere moments (approximately 7%) of each cycle, leading to wider deviations, less stable products and the need to run more passes or use higher pressures than should be required.
US 20090234108 describes method of partial deacetylation of Pneumococcal serotype 1 polysaccharide by chemical

treatment with Sodium carbonate buffer (pH 9.0). This process is time consuming and prone to destruction of immunogenic moieties which may affect immunogenicity of conjugates.
Further US 20090010959 discusses sizing of pneumococcal polysaccharides by microfluidization using Microfluidics (High pressure homogenizer).Microfluidization has improvements over conventional homogenizers however it has very high processing cost. Microfluidics technology platform {fixed-geometry interaction chamber) generates a uniform shear field for particle size reduction and robust cell disruption, with repeatable and scalable results, not possible with even the most effective homogenizers.
"High pressure cell disruption system"(HPCDS)(i.e. B Series cell disrupter 4.4 KW. from Constant Systems Ltd ,Northants U.K)is a type of high shear mechanical cell disruption method which uses a hydraulic mechanism that acts on a piston seal within a cylinder to force the sample through an orifice to a chamber of lower pressure. The sample is taken in and instantly pressurized up to a maximum of 40,000 PSI before being passed through a very small and fixed orifice and then returned back to atmospheric pressure. As the sample is being processed this type of cell disruptor ensures that the pressure is maintained

throughout the process, ensuring repeatability throughout the sample run.
Further the electrically controlled hydraulic system and fixed orifice guarantee that the disruption environment is repeatable between operating intervals without much affecting relative antigenicity of polysaccharide with respect to native polysaccharides. An in-built cooling jacket provides a high surface area for cooling that helps in maintaining a controlled temperature throughout the disruption cycle.
HPCDS has been previously utilized for cell disruption of Saccharomyces cerevisiae, Pichia pastoris and Escherichia coli to release soluble proteins.
The present invention is based on a surprising finding that HPCDS which was conventionally utilized for cell disruption can alternatively be employed as a cost-efficient method for reproducible size reduction as well as partial de-O-acetylation of bacterial capsular polysaccharides to obtain polysaccharide fragments of desired size that can further be conjugated to polysaccharides.
SUMMARY OF THE INVENTION

The instant invention relates to preparation of polysaccharide protein conjugates ,wherein the polysaccharide chain is initially mechanically depolymerized down to oligosaccharides of average molecular weight in the range of 10-40,000 daltons.
The method of the present invention utilizes a HPCDS as a mechanical high-pressure shear method for depolymerization of bacterial polysaccharides.
Hence, the method of the present invention provides a process whereby type specific polysaccharides can be depolymerized to obtain polysaccharide fragments of desired size based on a) number of passes & b) pressure
DESCRIPTION OF THE DRAWINGS
Fig 1: HPLC profile of Streptococcus pneumoniae
polysaccharide of serotype 1;
Fig 2: HPLC profile of Streptococcus pneumoniae
polysaccharide of serotype 1 post depolymerization (after
1st pass) ;
Fig 3:HPLC profile of Streptococcus pneumoniae
polysaccharide of serotype 1 post depolymerization (after
2nd pass) ;

Fig 4:HPLC profile of Streptococcus pneumoniae
polysaccharide of serotype 1 post depolymerization (after
3rd pass) ;
Fig 5:HPLC profile of Streptococcus pneumoniae
polysaccharide of serotype 1 post depolymerization (after
4th pass);
Fig 6: HPLC profile of Streptococcus pneumoniae
polysaccharide of serotype 5;
Fig 7: HPLC profile of Streptococcus pneumoniae
polysaccharide of serotype 5 post depolymerization (after
1st pass) ;
Fig 8: HPLC profile of Streptococcus pneumoniae
polysaccharide of serotype 5 post depolymerization (after
2nd pass) ;
Fig 9: HPLC profile of Streptococcus pneumoniae
polysaccharide of serotype 5 post depolymerization (after
3rd pass);
Fig 10: HPLC profile of Streptococcus pneumoniae
polysaccharide of serotype 5 post depolymerization (after
4th pass};
Fig 11: HPLC profile of Streptococcus pneumoniae
polysaccharide of serotype 5 post depolymerization (after
5th pass);

Fig 12: HPLC profile of Streptococcus pneumoniae
polysaccharide of serotype 5 post depolymerization {after
6th pass) ;
Fig 13: HPLC profile of Streptococcus pneumoniae
polysaccharide of serotype 5 post depolymerization (after
7th pass);
Fig 14: HPLC profile of Streptococcus pneumoniae
polysaccharide of serotype 5 post depolymerization (after
8th pass);
Fig 15: HPLC profile of Streptococcus pneumoniae
polysaccharide of serotype 5 post depolymerization (after
9th pass);
Fig 16: HPLC profile of Streptococcus pneumoniae
polysaccharide of serotype 5 post depolymerization (after
10th pass);
Fig 17: HPLC profile of Streptococcus pneumoniae
polysaccharide of serotype 6A;
Fig 18: HPLC profile of Streptococcus pneumoniae
polysaccharide of serotype 6A post depolymerization (after
1st pass) ;
Fig 19: HPLC profile of Streptococcus pneumoniae
polysaccharide of serotype 6A post depolymerization(after
2nd pass);
Fig 20: HPLC profile of Streptococcus pneumoniae
polysaccharide of serotype 6B;

Fig 21: HPLC profile of Streptococcus pneumoniae
polysaccharide of serotype 6B post depolymerization(after
1st pass) ;
Fig 22: HPLC profile of Streptococcus pneumoniae
polysaccharide of serotype 7F;
Fig 23: HPLC profile of Streptococcus pneumoniae
polysaccharide of serotype 7F post depolymerization(after
1st pass) ;
Fig 24: HPLC profile of Streptococcus pneumoniae
polysaccharide of serotype 7F post depolymerization(after
2
nd „ ^ pass);
Fig 25: HPLC profile of Streptococcus pneumoniae
polysaccharide of serotype 9V;
Fig 26: HPLC profile of Streptococcus pneumoniae
polysaccharide of serotype 9V post depolymerization (after
1st pass) ;
Fig 27: HPLC profile of Streptococcus pneumoniae
polysaccharide of serotype 14;
Fig 28: HPLC profile of Streptococcus pneumoniae
polysaccharide of serotype 14 post depolymerization (after
1st pass) ;
Fig 29: HPLC profile of Streptococcus pneumoniae
polysaccharide of serotype 14 post depolymerization (after
2nd pass) ;

Fig 30: HPLC profile of Streptococcus pneumoniae
polysaccharide of serotype 18C-
Fig 31: HPLC profile of Streptococcus pneumoniae
polysaccharide of serotype 18C post depolymerization (after
1st pass) ;
Fig 32: HPLC profile of Streptococcus pneumoniae
polysaccharide of serotype 18C post depolymerization (after
2nd pass};
Fig 33: HPLC profile of Streptococcus pneumoniae
polysaccharide of serotype 18C post depolymerization (after
3rdpass) ;
Fig 34: HPLC profile of Streptococcus pneumoniae
polysaccharide of serotype 18C post depolymerization (after
4thpass) ;
Fig 35: HPLC profile of Streptococcus pneumoniae
polysaccharide of serotype 18C post depolymerization (after
5thpass) ;
Fig 36: HPLC profile of Streptococcus pneumoniae
polysaccharide of serotype 19A ;
Fig 37: HPLC profile of Streptococcus pneumoniae
polysaccharide of serotype 19A post depolymerization (after
lstpass) ;
Fig 38: HPLC profile of Streptococcus pneumoniae
polysaccharide of serotype 19F;

Fig 39: HPLC profile of Streptococcus pneumoniae
polysaccharide of serotype 19F post depolymerization (after
lstpass) ;
Fig 40: HPLC profile of Streptococcus pneumoniae
polysaccharide of serotype 23F;
Fig 41: HPLC profile of Streptococcus pneumoniae
polysaccharide of serotype 23F post depolymerization (after
lstpass) ;
Fig 42: HPLC profile of Haemophilus influenza b
Polysaccharide (Hib);
Fig 43:HPLC profile of Haemophilus influenza b
Polysaccharide (Hib)post depolymerization (after lstpass);
Fig 44:HPLC profile of Haemophilus influenza b Polysaccharide (Hib)post depolymerization (after 2nd pass); Fig 45:HPLC profile of Haemophilus influenza b Polysaccharide (Hib)post depolymerization (after 3rd pass); Fig 46:HPLC profile of Haemophilus influenza b Polysaccharide (Hib)post depolymerization (after 4th pass); Fig 47:HPLC profile of Haemophilus influenza b Polysaccharide (Hib)post depolymerization (after 5th pass); Fig 48:HPLC profile of Meningococcal Serogroup A polysaccharide;
Fig 49:HPLC profile of Meningococcal Serogroup A polysaccharide post depolymerization (after 1st pass);

Fig 50:HPLC profile of Meningococcal Serogroup A
polysaccharide post depolymerization (after 2nd pass);
Fig 51:HPLC profile of Meningococcal Serogroup A
polysaccharide post depolymerization (after 3rd pass);
Fig 52:HPLC profile of Meningococcal Serogroup A
polysaccharide post depolymerization (after 4th pass);
Fig 53:HPLC profile of Meningococcal Serogroup A
polysaccharide post depolymerization (after 5th pass);
DETAILED DESCRIPTION OF THE INVENTION
The instant invention relates to a method for preparing a construct comprising a polysaccharide or oligosaccharide covalently attached to a protein molecule, said method comprising: a) subjecting polysaccharide and/or oligosaccharide to a mechanical size reducing treatment ; b)obtaining size reduced polysaccharide that is partially de-O-acetylated and has decreased polydispersity.
One aspect of the instant invention is that said mechanical depolymerization of the capsular polysaccharides can be carried out using fixed orifice and constant pressure processors i.e.HPCDS.

The depolymerization reaction of the instant invention yields size reduced ,less polydisperse and partially deacetylated polysaccharide.The said reaction proceeds by a random cleavage of glycosidic bonds , wherein the immunological epitopes of polysaccharides are not altered after size reduction.
In a preferred embodiment polysaccharide fragmentation by HPCDS alone can yield fragments having a} average molecular weight between 50 KDa and 400 Kda; b)polydispersity between 1 and 2 and c) de-O-acetylated to approximately 20 % by weight.
According to the instant invention the mechanical size reduction treatment by HPCDS can comprise of passing polysaccharide sample in suitable liquid medium at a preset pressure.The passes can be repeated until polysaccharide chain length is reduced to desired size.
In yet another embodiment the size reduction by HPCDS requires a pressure between 20000 and 40000 psi wherein number of passes required can be between 1 and 10.
In another embodiment of the invention the liquid medium of polysaccharide can be selected from a group of but not

limited to water for injection, sodium chloride, amino acid solution such as glycine or any suitable buffer.
The method of the instant invention comprises fragmentation of bacterial polysaccharide of both gram-positive and gram-negative bacteria. The said polysaccharide is a bacterial capsular polysaccharide selected from the group consisting of Escherichia coli, Francisella tularensis, Haemophilus influenzae,Klebsiella, Moraxella catarrhalis, Neisseria meningitidis groups A ,C , W135 Y and X, Porphyromonas gingivalis, Pseudomonas aeruginosa, Burkholderia cepacia, Salmonella typhi, Salmonella typhimurium, Salmonella paratyphi, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Vibrio cholera,Enterococcus faecalis, Enterococcus faecium, Group A Streptococcus, Group B Streptococcus, Mycobacterium tuberculosis, Staphylococcus aureus , Staphylococcus epidermidis and Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5,6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, 33F .
According to one advantageous embodiment of the invention said polysaccharide can be partially or completely de-O-acetylated during sizing by HPCDS. The instant invention can thus provide an alternative deacetylation method having following advantages a) Separate deacetylation step not

required; b)Steric features of the polysaccharide can be retained and c) It can be less time consuming than the conventional chemical deacetylation.
Yet another aspect of the invention is that the
depolymerization of polysaccharide can occur in presence of
Water for injection(WFI) , and alternatively in presence of
NaCl for polysaccharides sensitive to WFI.
Accordingly the polysaccharides depolymerized by instant method can be used for preparation of plain polysaccharide and polysaccharide-protein conjugate vaccines.
Following are some of the advantages of the instant polysaccharide fragmentation method:
a) The depolymerization method can be utilized for all polysaccharides independent of polysaccharide's chemical structure;
b) Polysaccharide sample to be depolymerized can comprise of a single type of polysaccharide or a mixture of different type of polysaccharides;
c) Immunological epitopes of polysaccharides are retained post depolymerization;
d) Size reduced polysaccharides have low polydispersity and thus better homogeneity;

e) Partial de-O-acetylation of polysaccharide can be carried simultaneously with depolymerization;
f) Reproducible fragmentation yields can be obtained once parameters like pressure and number of passes are optimized;and
g) Size reduced polysaccharides obtained do not require any additional processing and thus can be directly conjugated to carrier proteins.
Characterization of pneumococcal polysaccharide depolymerized by a novel method:
Pneumococcal polysaccharide molecular weight and
polydispersity
By measuring the weight-average molecular weight, Mw, by a diffusion, sedimentation, or chromatographic means, and the number-average-molecular weight, MN, by a colligative property such as viscosity, freezing-point-depression, or boiling-point-elevation, the polydispersity of the Pn-Ps preparation, is obtained as the ratio Mw /MN. The closer this number approaches unity, the more homogeneous the polysaccharide preparation. The polydispersity of a number of Pn-Ps preparations is given herein and a preferred process for achieving this enhanced homogeneity is also disclosed.

Following examples further demonstrate the essential feature of the invention, however it is apparent to skilled person that these examples do not limit scope of the invention.
Example 1:
Pneumococcal Polysaccharide (PnPs) Serotype 1 was obtained from pneumococcal polysaccharide production department of Serum Institute of India, Pune. The sample was passed through high pressure Homogenizer B Series, 4.4 KW Constant System UK with set pressure 40 000 psi. The collected sample was analyzed on HPLC as described previously. Multiple passes were carried out in similar manner and samples were analyzed for molecular size on GFC-HPLC.
The samples were analyzed by High Performance Liquid Chromatography (HPLC) on Shodex OHpak 805 and Shodex OHpak 804 gel filtration columns connected sequentially with OHpak SB-G guard column with 10 mM Phosphate Buffered Saline as mobile phase, 1 ml flow rate and 100μl injection volume.

Polydispersity (PDI) was calculated by formula:
PDI= Mw / Mn
where Mw: Weight Average Molecular Weight
Mn: Number Average Molecular Weight
Table 1:Summary for PnPs Type 1

Sample Name Pressure
Psi X
Passes Approximate
Average
Molecular
Weight Polydispers ity Mw / Mn
PnPS 1 Initial Sample - 851 kD 2.304
Sample 1 40000 X 1 339 kD 1.606
Sample 2 40000 X 2 303 kD 1.583
Sample 3 40000 X 3 277 kD 1.548
Sample 4 40000 X 4 264 kD 1.530
Example 2:
Pneumococcal Polysaccharide (PnPs)Serotype 5 {concentration 3.94 mg/ml} was obtained from pneumococcal polysaccharide production department of Serum Institute of India, Pune. The sample was passed through high pressure Homogenizer B Series, 4.4 KW Constant System UK with set pressure 40 000 psi. The collected sample was analyzed on HPLC as described

previously. Multiple passes carried out in similar manner and samples were analyzed for molecular size on GFC-HPLC.
Table 2:Summary for PnPS Type 5

Sample Name Pressure X Passes Approximate
Average
Molecular
Weight Polydispersity Mw / Mn
PnPS 5 Initial Sample - 638 kD 2.159
Sample 1 40- 000 X 1 359 kD 1.623
Sample 2 40 000 X 2 306 kD 1.558
Sample 3 40 000 X 3 278 kD 1.528
Sample 4 40 000 X 4 258 kD 1.495
Sample 5 40 000X5. 243 kD 1.481
Sample 6 40 000 X 6 231 kD 1.468
Sample 7 40 000 X 7 222 kD 1.458
Sample 8 40 000 X 8 214 kD 1.445
Sample 9 40 000 X 9 207 kD 1.437
Sample 10 40 000 X 10 200 kD 1.436
Example 3:
Pneumococcal Polysaccharide {PnPs) Serotype 6A (Concentration 3 mg/ml} from Good Manufacturing Practice Lot was obtained from pneumococcal polysaccharide production department of Serum Institute of India, Pune. The sample was passed through high pressure Homogenizer B Series, 4.4 KW Constant System with set pressure 25000 & 38000 psi, analyzed for molecular size on GFC-HPLC.

Table 3:Summary for PnPS Type 6A

Sample Name Pressure
Psi X
Passes Approximate
Average
Molecular
Weight Polydispersity Mw / Mn
PnPS 6A Initial Sample - 1000 kD 1.38
Sample 1 38 000 X 1 144 kD 1.41
Sample 2 25 000 X 1 169 kD 1.38
Example 4:
Pneumococcal Polysaccharide (PnPs) Serotype 6B
(Concentration 5.65 mg/ml) from Good Manufacturing Practice
Lot was obtained from pneumococcal polysaccharide
production department of Serum Institute of India, Pune.
The sample was passed through high pressure Homogenizer B
Series, 4.4 KW Constant System with set pressure 35000 psi
analyzed for molecular size on GFC-HPLC. Table 4:Summary for PnPS Type 6B

Sample Name Pressure
Psi X
Passes Approximate
Average
Molecular
Weight Polydispersity Mw / Mn
PnPS 6B Initial Sample - 1040 kD 2.759
Final Sample 35 000 X 1 225 kD 1. 632
Example 5:

Pneumococcal Polysaccharide (PnPs) Serotype 7F
(Concentration 4.7 mg/ml) from Good Manufacturing Practice
Lot was obtained from pneumococcal polysaccharide
production department of Serum Institute of India, Pune.
The sample was passed through high pressure Homogenizer B
Series, 4.4 KW Constant System with set pressure 40 000 psi
analyzed for molecular size on GFC-HPLC. Multiple passes
carried out with set pressure 40 000 psi and samples were
analyzed for size as described earlier. Table 5:Summary for PnPS Type 7F
Sample Name Pressure
Psi X
Passes Approximate
Average
Molecular
Weight Polydispersity Mw / Mn
PnPS 7F Initial Sample - 1179 kD 2.036
Sample 1 40 000 X 1 192 kD 1.642
Sample 2 40 000 X 2 147 kD 1.503
Example 6:
Pneumococcal Polysaccharide (PnPs) Serotype 9V
(Concentration 5.5 mg/ml) from Good Manufacturing Practice
Lot was obtained from pneumococcal polysaccharide
production department of Serum Institute of India, Pune.
The sample was passed through high pressure Homogenizer B
Series, 4.4 KW Constant System with set pressure 35 000
psi analyzed for molecular size on GFC-HPLC. Table 6:Summary for PnPS Type 9V

Approximate
Pressure Polydispersity
Sample Name Average
psi Molecular Mw / Mn

Weight
PnPS 9V Initial Sample - 881 kD 2.932
Final Sample 35 000 194 kD 1.501
Example 7:
Pneumococcal Polysaccharide (PnPs) Serotype 14 (Concentration 5.5 mg/ml) from Good Manufacturing Practice Lot was obtained from pneumococcal polysaccharide production department of Serum Institute of India, Pune. The sample was passed through high pressure Homogenizer B Series, 4.4 KW Constant System with set pressure 40 000 psi. Multiple passes carried out and samples were analyzed for molecular size on GFC-HPLC.
Table 7:Summary for PnPS Type 14

Sample Name Pressure
Psi X
Passes Approximate
Average
Molecular
Weight Polydispersi ty Mw / Mn
PnPS 14 Initial Sample - 537 kD 2.383
Sample 1 40 000 X 2 173 kD ' 1.558
Sample 2 40 000 X 4 111 kD 1.587
Example 8:
Pneumococcal Polysaccharide (PnPs) Serotype 18C (Concentration 3 mg/ml) from Good Manufacturing Practice

Lot was obtained from pneumococcal polysaccharide production department of Serum Institute of India, Pune.
The sample was passed through high pressure Homogenizer B Series, 4.4 KW Constant System with different set pressures and these samples were analyzed for molecular size on GFC-HPLC.
Table 8:Summary for PnPS Type 18C

Sample Name Pressure
Psi X
Passes Approximate
Average
Molecular
Weight Polydispersity Mw / Mn
PnPS 18C Initial Sample - 690 kD 3.097
Sample 1 30 000 X 1 307 kD 1.992
Sample 2 40 000 X 2 217 kD 1.701
Sample 3 40 000 X 3 173 kD 1.576
Sample 4 40 000 X 4 162 kD 1.492
Sample 5 40 000 X 5 153 kD 1.482
Example 9:
Pneumococcal Polysaccharide (PnPs) Serotype 19A
(Concentration 5.5 mg/ml) from Good Manufacturing Practice
Lot was obtained from pneumococcal polysaccharide
production department of Serum Institute of India, Pune.

The sample was passed through high pressure Homogenizer B Series,, 4.4 KW Constant System with set pressure 40 000 psi analyzed for molecular size on GFC-HPLC.
Table 9:Summary for PnPS Type 19A

Approximate
Sample Name Pressure psi Average
Molecular
Weight Polydispersity Mw / Mn
PnPS 19A Initial 48 6 kD 1.813
Sample
1st Sample 40 000 176 kD 1.614
Example 10:
Pneumococcal Polysaccharide (PnPs) Serotype 19F (Concentration ~5 mg/ml) . from Good Manufacturing Practice Lot was obtained from pneumococcal polysaccharide production department of Serum Institute of India, Pune. The sample was passed through high pressure Homogenizer B Series, 4.4 KW Constant System with set pressure 30 000 psi analyzed for molecular size on GFC-HPLC.
Table 10:Summary for PnPS Type 19F

Sample Name Pressure psi Approximate
Average
Molecular
Weight Polydispersity Mw / Mn
PnPS 19F Initial Sample - 787 kD 3.255
1st Sample 30 000 220 kD 1.133
Example 11:

Pneumococcal Polysaccharide (PnPs) Serotype 23F (Concentration 3.1 mg/ml) from Good Manufacturing Practice Lot was obtained from pneumococcal polysaccharide production department of Serum Institute of India, Pune. The sample was passed through high pressure Homogenizer B Series, 4.4 KW Constant System with set pressure 40 000 psi analyzed for molecular size on GFC-HPLC.
Table 11:Summary for PnPS Type 23F

Sample Name Pressure psi Approximate
Average
Molecular
Weight Polydispersity Mw / Mn
PnPS 23F Initial Sample - 2103 kD & 103 kD 1.353 & 1.507
1st Sample 40 000 275 kD 1.639
Example 12:
Haemophilus influenza b Polysaccharide (Hib) (Concentration 5.5 mg/ml) from Good Manufacturing Practice Lot was obtained from polysaccharide production department of Serum Institute of India, Pune. The sample was passed through high pressure Homogenizer B Series, 4.4 KW Constant System UK with different set pressure. The collected sample was analyzed on HPLC as described previously.
Table 12:Summary for Rib Polysaccharide:

Approximate
Pressure Polydispersity
Sample Name Average
Psi X pass Molecular Mw / Mn

Weight
Hib Initial Sample - 6201 & 536 kD 1.072& : L. 684
Sample 1 27 000 284 kD 1.429
Sample 2 33 400 248 kD 1.449
Sample 3 35 000 187 kD 1.401
Sample 4 40 000 176 kD 1.307
Sample 5 40 000 X 3 135 kD 1.341
Example 13:
Meningococcal polysaccharide Serogroup A (Men A)
(Concentration 13.08 mg/ml) from Good Manufacturing
Practice Lot was obtained from polysaccharide production
department of Serum Institute of India, Pune. The sample
was passed through high pressure Homogenizer B Series, 4.4
KW Constant System UK with different set pressures.
Multiple passes were also carried out. The collected sample
was analyzed on HPLC as described previously. Table 13:Summary for Men A Polysaccharide:

Sample Name Pressure Psi x pass Approximate Average Molecular Weight Polydispersity Mw / Mn
Men A Initial Sample - 321 kD 1.717
Sample 1 20 000 X 1 237 kD 1.521
Sample 2 25 000 X 1 213 kD 1.553
Sample 3 30 000 X 1 198 kD 1.566
Sample 4 40 000 X 1 171 kD 1.478
Sample 5 40 000 X 1 143 kD 1.403 (
Example 14:
Pneumococcal Polysaccharide (PnPs) Serotype 1 from Good Manufacturing Practice Lot was obtained from pneumococcal

polysaccharide production department of Serum Institute of India, Pune. The sample was passed through high pressure Homogenizer B Series, 4.4 KW Constant System UK with set pressure 40,000 psi. The initial sample and the collected sample were analyzed for acetylation as per Hestrin method.The polysaccharide obtained is found to be de-0-acetylated to approximately 20 % by weight.
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 method for preparing a size reduced polysaccharide or oligosaccharide, said method comprising: a) subjecting polysaccharide or oligosaccharide to a mechanical size reducing treatment and ; b)obtaining

size reduced polysaccharide that is partially deacetylated and has decreased polydispersity.
2. The method of claim 1 wherein said polysaccharide is a bacterial capsular polysaccharide selected from the group consisting of Escherichia coli, Francisella tularensis, Haemophilus influenzae,Klebsiella, Moraxella catarrhalis, Neisseria meningitidis groups A , C , W135 Y and X, Porphyromonas gingivalis, Pseudomonas aeruginosa, Burkholderia cepacia, Salmonella typhi, Salmonella typhimurium, Salmonella paratyphi, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Vibrio cholera,Enterococcus faecalis, Enterococcus faecium, Group A Streptococcus, Group B Streptococcus, Mycobacterium tuberculosis, Staphylococcus aureus , Staphylococcus epidermidis and Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5,6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, 33F.
3. The method according to claim 1, wherein mechanical size reduction and partial de-O-acetylation of the capsular polysaccharides is carried out simultaneously using high pressure cell disrupter .

4. The method according to claim 3,wherein the mechanical size reduction comprises of i) passing polysaccharide sample in suitable liquid medium through high pressure cell disruption system at a preset pressure ii)repeating the passes until polysaccharide chain length is reduced to desired size.
5. The method according to claim 4,wherein the polysaccharide sample can comprise of a single type of polysaccharide or a mixture of different polysaccharides.
6. The method according to claim 4,wherein the liquid medium can be selected from a group of water for injection, sodium chloride, glycine solution and buffer.
7. The method according to claim 4,wherein the pressure for sizing is in a range between 1300 and 2700 bar.
8. The method according to claim 4,wherein the number of passes is between 1 and 10.

9. The method according to claim 1, wherein the size reduced polysaccharides have an average molecular weight of 50 to 400 KDa.
10. The method according to claim 1,wherein the size reduced polysaccharide is de-O-acetylated to approximately 20 % by weight.
11. The method according to claim 1,wherein the said size reduced polysaccharide has polydispersity between 1.0 and 2.0.
12. The method according to claim 1,wherein the said size reduced polysaccharide retains the immunogenic epitopes.
13. The method according to claim l, further comprising the step of directly conjugating the size reduced capsular polysaccharide to one or more carrier proteins.
14. A vaccine composition comprising one or more polysaccharide protein conjugates prepared according to claim 13.