FORM 2
THE PATENTS ACT, 1970
(39 OF 1970)
AND
THE PATENTS RULES, 2003
(As Amended)
COMPLETE SPECIFICATION
(See section 10; rule 13)
"Purification of capsular polysaccharides"
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:

FIELD OF THE INVENTION
The present invention relates to a process for purification of polysaccharides/ specifically antigenic polysaccharides.
BRIEF DESCRIPTION:
In the preparation of conjugate vaccines directed to the prevention of invasive diseases caused by the organisms , selected serotypes are grown to supply polysaccharides needed to produce the vaccine. The cells are grown in large fermentors with lysis induced at the end of the fermentation by addition of sodium deoxycholate (DOC) or an alternate lysing agent. The lysate broth is then harvested for downstream purification and the recovery of the capsular polysaccharide which surrounds the bacterial cells. After conjugation with a carrier protein, the polysaccharide is included in the final vaccine product and confers immunity in the vaccine's target population to the selected serotypes.
Although the cellular lysate produced in this process" contains the target polysaccharide, it also contains large quantities of cellular debris including DNA, RNA, proteins, and residual media components. Traditional processing has involved a minimal pH reduction of the lysate to 6.6 by addition of acetic acid to help precipitate out the lysing agent and some of the impurities. This material is subjected to centrifugation followed by filtration to remove most of the solids down to a 0.45 urn nominal size. However, such traditional processing methods have shown minimal reduction in impurities with subsequent difficulty in removing soluble proteins to meet purified polysaccharide specifications.
For most of the serotypes for conjugate vaccine , the purification process reported in literature requires sixteen

steps involving many expensive, labor intensive and technologically demanding operations, such as chromatography and multiple membrane separations.
WO 2008129559 claims a process wherein fermentation broth is subjected to Concentration and diafiltration followed by ammonium sulphate precipitation and centrifugation.This is followed by benzonase treatment, hydroxyapatite chromatography and diafiltration in sequence.For some serotypes, polysaccharide purification by utilizing ammonium sulphate precipitation results in precipitation of protein and nucleic acid contaminants.However, it also precipitates capsular polysaccharides, resulting in polysaccharide loss of atleast 80%.
Maimoni et al discusses pneumococcal polysaccharide purification, wherein fermentation broth is subjected to centrifugation or tangential flow ultrafiltration followed by concentration and/or diafiltration. This is further subjected to Ethanol precipitation followed by centrifugation. Next step is benzonase treatment followed by concentration and diafiltration.Refer w Simple and efficient method of bacterial polysaccharides purification for vaccines production using hydrolytic enzymes and tangential flow ultrafiltration " Formatex, 2007, pg, 450-457-However alcohol based process can result in some operational problems, thus requiring long processing times.
US 2006/0228381 claims improved purification processes for S. Pneumoniae polysaccharides by pH manipulation during fermentation and recovery.It claims a process wherein fermentation broth is subjected to deoxycholate assisted lysis to obtain a lysate containing soluble proteins, cell debris, nucleic acids and polysaccharide.This is followed by

reduction in pH to less than 4.5, wherein proteins are removed. This is followed by activated carbon filtration and ultrafiltration/diafiltration. However, the removal of impurities in these processes is still spread over many labor intensive and costly steps.
It has been previously reported that solium deoxycholate (DOC) binds protein. DOC has been used as a co-precipitant {in acidic environment) . US 590037 6 claims use of DOC in combination with an acidic agent and NaCl during protein estimations.
Deoxycholate alone has also been utilized for removal of proteins,please refer Hideyo Yamaguchi;"Macromolecular Structure and Morphology of Native Glycogen Particles Isolated from Candida albicans" Journal Of Bacteriology, Oct. 1974, pg. 441-449.
The present invention is based on a surprising finding that protein contaminants can be removed from polysaccharides using sodium deoxycholate (DOC) . The instant method overcomes the drawbacks of prior art as it results in precipitation of proteins without precipitating capsular polysaccharides.
SUMMARY OF INVENTION:
The invention relates to a process for purification of antigenic polysaccharides ,wherein atleast 80% of protein impurities are removed with minimum polysaccharide loss.
The process of the instant invention comprises following steps:
a) obtaining crude bacterial polysaccharide from lysed broth;

b) subjecting the crude polysaccharide to concentration and diafiltration;
c) treatment of the solution comprising polysaccharide with nuclease;
d) treatment of nuclease treated polysaccharide solution with deoxycholate sodium;
e) subjecting the polysaccharide solution to centrifugation followed by diafiltration;
f) Processing the solution by hydrophobic interaction chromatography;
g) subjecting the polysaccharide solution to concentration and diafiltration.
DETAILED DESCRIPTION OF INVENTION:
The instant method relates to an alternative method for a) removal of protein contaminants & b) high polysaccharide recovery during purification of polysaccharides.
The process of instant invention is quite robust which can be applied for purification of any polysaccharide, specifically antigenic polysaccharide.
The method of the instant invention comprises purification 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, 19F, 19A, 20, 22F, 23F, and 33F.
One embodiment of the instant invention is that the said method is particularly applicable for removal of proteins from pneumococcal polysaccharides(PnPs) , for which ammonium sulphate precipitation results in precipitation of both polysaccharide as well as proteins with low polysaccharide yields.The said polysaccharides being pneumococcal serotype 4 & 5.
Another embodiment of the instant invention is that the said method utilizes deoxycholate sodium for removal of protein contaminants.
Further as per the instant method ,70 to 90% protein contaminants,preferably 7 5 to 85% are removed with a polysaccharide recovery of atleast 80%.
In a particular embodiment of the instant invention, concentration(%w/v) of deoxycholate can be between 0.25 and 1,preferably between 0.5 and 0.75.
According to the instant invention concentration(mM) of NaCl can be between 20 and 200,preferably between 40 and 80.
The said purified polysaccharide can be used for preparation of polysaccharide-protein conjugate vaccines.

Example-1
S. Pneumoniae Capsular Polysaccharide Serotypes 4 & 5
5L of clarified broth from the fermenter culture of S. pneumoniae serotype 4 & 5 was concentrated and diafiltered to ~ 500 ml using a 100 kDa MWCO membrane. Diafiltration was accomplished by using 140 mM NaCl followed by WFI using 100 kDa MWCO membrane till constant conductivity was achieved.
Nuclease was added to the polysaccharide solution to achieve a final concentration of 10 U/ml of solution. The enzyme treatment was carried out at 37 °C, for 10 ± 2 hrs.
Solid Ammonium sulphate to 50% saturation was added to the nuclease-treated polysaccharide solution and incubated at 2 - 6 °C for 10 ± 2 hrs. The mixture was subjected to centrifugation. The pellet (precipitate) was discarded. The centrifuged supernatant solution {- 500 ml) containing polysaccharide was diafiltered against 140 mM NaCl followed by WFI using 100 kDa MWCO membrane till constant conductivity was achieved.
The PnPs recovery was very low, although protein removal was within acceptable limit of 75 to 80 % as compared to protein content in crude PnPs. Approximately 8 0% PnPs was lost due to precipitation of PnPs along with protein when treated with 50% Ammonium sulphate saturation.
The diafiltered polysaccharide solution after hydrophobic interaction chromatography was filtered through a 0.22 u membrane filter into polypropylene bottles. The purified polysaccharide was stored frozen at -20±5°C.

Example-2:
S. Pneumoniae Capsular Polysaccharide Serotypes 4 & 5
5L clarified supernatant from lysed broth of cultures of S. pneumoniae serotype 5 was concentrated and diafiltered to 500 ml by using a 100 kDa MWCO membrane. Diafiltration was accomplished by using 140 mM followed by diafiltration with water for injection {WFI) till constant conductivity was achieved.
Nuclease was added to the polysaccharide solution to achieve a final concentration of 8 U/ml of solution. The nuclease treatment was carried out at 37°C, for 10 ± 2 hrs with stirring.
Deoxycholate and NaCl were added to the nuclease-treated polysaccharide solution to final concentration of 0.5% and 60 mM respectively and incubated at 2 - 8 °C for 12 ± 2 hrs for mild stirring for 30 mins (serotypes 4 and 5) . The mixture was subjected to centrifugation. The pellet {precipitate containing protein & other impurities) was discarded. The solution (~500 ml) is subjected to 100 kD diaf iltration using NaCl followed by chilled WFI till constant conductivity was achieved. This diafiltered solution containing polysaccharide with a buffer and high salt concentration was loaded on Hydrophobic interaction chromatography(HIC) column.
The PnPs recovery was 80% and protein removal was within acceptable limit of 75 to 80 % as compared to protein content in crude PnPs.Approximately 15 to 20 % PnPs was lost
The hydrophobic interaction chromatography column (300 ml) was equilibrated with 5 M buffered NaCl and the buffered

polysaccharide solution (500 ml} containing 5 M NaCl was then loaded onto the column in pH range 6 to 8, preferably at pH 6 to 7 pH . The column was further washed with the buffer containing 5 M NaCl. Under these conditions, the polysaccharide was recovered in the flow-through and equilibration wash from the column.
The polysaccharide solution from flowthrough and wash was concentrated using a 100 kDa MWCO filter and then diafiltered with NaCl and Water for Injection (WFI) till constant conductivity was achieved.
The diafiltered polysaccharide solution was filtered through a 0.22 u membrane filter into polypropylene bottles. The purified polysaccharide was stored frozen at -20 ± 5°C.
Example-3:
S. Pneumoniae Capsular Polysaccharide Serotype 5 & 4
5L clarified supernatant from lysed btoth of cultures of S. pneumoniae serotype 5 was concentrated and diafiltered to 500 ml using a 100 kDa MWCO membrane. Diafiltration was accomplished using 140 mM followed by diafiltration with water for injection (WFI).
Nuclease was added to the polysaccharide solution to achieve a final concentration of 8 U/ml of solution. The nuclease treatment was carried out at 37°C, for 10 ± 2 hrs with stirring.
Deoxycholate (0.5%) and NaCl (40 mM) was added to the enzyme-treated polysaccharide, pH was adjusted to 6.25 to 6.3 and incubated at 2 - 8 °C for 8-10 hrs for mild stirring for 30 mins (serotypes 5 and 4). The mixture was subjected to

centrifugation. The pellet (precipitate containing protein & other impurities) was discarded. The solution (-500 ml) is subjected to 100 kD diafiltration using NaCl followed by chilled WFI. This diafiltered solution containing polysaccharide with a buffer and high salt concentration was loaded on HlC column.
The PnPs recovery was 80% and protein removal was 75 % as compared to protein content in crude PnPs.Approximately 15 to 20 % Pn£s was lost.
The hydrophobic interaction chromatography column (300 ml) was equilibrated with 5 M buffered NaCl and the buffered polysaccharide solution (500 ml) containing 5 M NaCl was then loaded onto the column in pH range 6 to 8, preferably at pH 6 to 7 pH . The column was further washed with the buffer containing 5 M NaCl. Under these conditions, the polysaccharide was recovered in the flow-through and equilibration wash from the column.
The polysaccharide solution was then concentrated using a 100 kDa MWCO filter and diafiltered with NaCl and Water for Injection (WFI) till constant conductivity was achieved.
The diafiltered polysaccharide solution was filtered through a 0.22 u membrane filter into polypropylene bottles. The purified polysaccharide was stored frozen at -20 ± 5°C.
Exarople-4
S. Pneumonias Capsular Polysaccharide Serotype 4

5L of clarified broth from the fermenter cultures of S. pneumoniae serotype 4 was concentrated and diafiltered to ~ 500 ml and using a 100 kDa MWCO membrane. Diafiltration was accomplished using 25mM sodium phosphate buffer at neutral pH followed by diafiltration by water for injection (WFI) till constant conductivity was achieved.
Nuclease was added to the polysaccharide solution to achieve a final concentration of 10 U/ml of solution. The nuclease treatment was carried out at 37 °C, for 10 ± 2 hrs.
Deoxycholate(0.5% w/v) and NaCl (80 mM) were added to the nuclease-treated polysaccharide solution and incubated at 2 - 6 °C for 10+2 hrs. The mixture was subjected to centrifugation. The pellet (precipitate) was discarded. The solution (-400 ml) containing polysaccharide was diafiltered using 140 mM NaCl followed by WFI using 100 kDa MWCO membrane till constant conductivity was achieved.
The PnPs recovery was 80% and protein removal was 80 % as compared to protein content in crude PnPs.Approximately 20 % PnPs was lost.
The diafiltered polysaccharide solution was filtered through a 0.22 u membrane filter into polypropylene bottles. The purified polysaccharide was stored frozen at -20±5°C.
Example-5
S. Pneumoniae Capsular Polysaccharide Serotype 5
5L of clarified broth from the fermenter cultures of S. pneumoniae serotype 4 was concentrated and diafiltered to ~ 500 ml and using a 100 kDa MWCO membrane. Diaf iltration was accomplished using 140 mM NaCl followed by diafiltration by water for injection (WFI) till constant conductivity was achieved.

Nuclease was added to the polysaccharide solution to achieve a final concentration of 10 U/ml of solution. The nuclease treatment was carried out at 37 °C, for 10 ± 2 hrs.
Deoxycholate(0.75% w/v) and NaCl (60 mM) were added to the enzyme-treated polysaccharide solution to and incubated at 2 - 6 °C for 10±2 hrs. The mixture was subjected to centrifugation. The pellet (precipitate) was discarded and pellet was little loose. The solution (~ 400 ml) containing polysaccharide was 140 mM NaCl followed by WFI using 100 kDa MWCO membrane till constant conductivity was achieved.
The PnPs recovery was 80% and protein removal was 80 % as compared to protein content in crude PnPs.Approximately 30 % PnPs was lost.
The diafiltered polysaccharide solution was filtered through a 0.22 µ membrane filter into polypropylene bottles. The purified polysaccharide was stored frozen at -2 0±5°C.
Example-6
S. Pneumoniae Capsular Polysaccharide Serotype 5
5L of clarified broth from the fermenter cultures of S. ' pneumoniae serotype 4 was concentrated and diafiltered to -500 ml and using a 100 kDa MWCO membrane. Diafiltration was accomplished using 25mM sodium phosphate buffer at neutral pH followed by diafiltration by water for injection (WFI).
Nuclease was added to the polysaccharide solution to achieve a final concentration of 10 U/ml of solution. The nuclease treatment was carried out at 37 °C, for 10+2 hrs.

Deoxycholate (1% w/v) and NaCl (60 mM) was added to the enzyme-treated polysaccharide solution, pH adjusted to 6.2 ±0.1 and incubated at 2 - 6 °C for 8 -10 hrs . The mixture was subjected to centrifugation. The pellet (precipitate) was discarded. The precipitated pellet was loose. The centrifuged supernatant was not clear which blocks or chokes filter. The solution (-300 ml) containing polysaccharide was diafiltered against 140 mM NaCl followed by WFI using 100 kDa MWCO membrane till constant conductivity was achieved.
The PnPs recovery was 80% and protein removal was 7 5% as compared to protein content in crude PnPs.Approximately 50 % PnPs was lost.
The diafiltered polysaccharide solution was filtered through a 0.22 u membrane filter into polypropylene bottles. The purified polysaccharide was stored frozen at -20±5°C.
Table 1:

Pneumococca Deoxycholate NaCl Protein Polysacch aride
1 Serotype sodium concentration Removal loss(%)
concentratio n (%w/v) (mM) {%)
4 & 5 * NA NA 80 80
without HIC
step
4 & 5 with 0.5 60 80 15-20
HIC step
4 & 5 with 0.5 40 75 15-20
HIC step
4 without 0.5 80 80 20
HIC step
5 without 0.75 60 80 30
HIC step

5 without 1 60 75 50
HIC step
*:Ammonium sulphate (50% saturation) HIC :Hydrophobic interaction chromatography protein estimation by Lowry method
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 purification process for removal of protein contaminants from antigenic polysaccharide which comprises: a)obtaining crude bacterial polysaccharide from lysed broth; b)subjecting the crude polysaccharide to concentration and diafiltration ; c)treatment of the solution comprising polysaccharide with nuclease; d)treatment of nuclease treated polysaccharide solution with a mixture of detergent & saline; e) adjusting the pH between 6.1 and 6.3 and incubating mixture at 2 to 8°C for 10 to 14 hrs; f) subjecting the polysaccharide solution to centrifugation followed by diafiltration; g)processing the solution by chromatography, wherein said process results in reduction of protein

contaminants by 70 to 90% ,wherein said process does not require ammonium sulphate for protein removal.
2. A process of claim 1, wherein polysaccharide recovery is
at least 80%.
3. A process of claim 1, wherein the detergent is
deoxycholate.
4 . A process of claim 1, wherein concentration of deoxycholate is between 0.25 and 1% w/v.
5. A process of claim 4, wherein concentration of deoxycholate is between 0.5 and 0.75% w/v
6. A process according to claim 1,wherein concentration of NaCl is between 20 and 200 mM.
7. A process according to claim 6, wherein concentration of NaCl is between 40 and 80 mM.
8. A process according to claim 1 ,wherein the polysaccharide is selected from the group consisting of Escherichia coli, Haemophilus influenzae,Klebsiella, Neisseria meningitidis groups A , C , W135 Y and X, Pseudomonas aeruginosa, Salmonella typhi, Shigella dysenteriae. 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, 19F, 19A, 20, 22F, 23F, and 33F.
9. A purification process for removal of protein contaminants from pneumococcal polysaccharide of serotype 4 and 5 which comprises:
a}obtaining crude bacterial polysaccharide from lysed broth; b)subjecting the crude polysaccharide to concentration and diafiltration ; c)treatment of the

solution comprising polysaccharide with benzonase; d) treatment of nuclease treated polysaccharide solution with a mixture of 0.5% w/v deoxycholate & 60 mM NaCl; e)adjusting the pH between 6.1 and 6.3 and incubating mixture at 2 to 8°C for 12 hrs; f)subjecting the polysaccharide solution to centrifugation followed by diafiltration; g)processing the solution by hydrophobic interaction chromatography;
wherein said process results in reduction of protein contaminants by 75 to 85% ,wherein said process does not require ammonium sulphate for protein removal ,wherein polysaccharide recovery is at least 80%.