FORM-2
THE PATENTS ACT, 1970
(39 OF 1970)
AND
THE PATENTS RULES, 2003
(As Amended)
COMPLETE SPECIFICATION
(See section 10; rule 13)
"Removal of "polyose type" teichoic acid contaminants from bacterial polyoses"
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 bacterial polyoses, wherein “polyose type” teichoic acid contaminants are removed .
BACKGROUND OF THE INVENTION
The invention relates to a method for removal of contaminants, particularly “ polyose type” teichoic acids, from “capsular polyose” preparations of a Gram positive bacterial culture.
Teichoic acids are known to be constituents of the complex constituting the membrane and the wall of Gram positive bacteria. They are in close contact with the peptidoglycan of the bacterial cell wall. Covalent bonds reinforce this link. Teichoic acids, which have in their chemical structure a lipid or a glycolipid end can anchor themselves in the outer membrane via hydrophobic bonds.
There are mainly two types of teichoic acids: The first type gathers teichoic acids, which are linear polymers of ribitol phosphate or glycerol phosphate linked themselves by phosphodiester bonds. These teichoic acids are referred as “alcohol polymers”. The second type gathers teichoic acids, which are polyoses having a chain of repeating units.

Some Gram positive bacteria have a capsule and/or are responsible for serious infections. The bacterial “capsular polyoses” utilized for preparing immunogenic constructs are always associated with a substantial amount of a common impurity/contaminant, called “polyose type” teichoic acids. The methods used to purify the ”capsular polyoses” eliminates as much as possible the teichoic acids. These teichoic acids have also been classified as an impurity for immunogenic preparations by WHO and these can trigger undesirable inflammatory reactions (Infection and Immunity (2003), 71:5541)
The prior art methods utilize CTAB ,alcohol, chromatography for teichoic acids removal and have limitations. These methods are not efficient for removal of teichoic acids from certain “capsular polyoses”.
Surprisingly we have found a method for teichoic acids reduction for such “capsular polyoses” .According to the instant method, when crude “capsular polyoses“ are subjected to sodium metaperiodate it can result in a substantial reduction of group specific teichoic acid content. The said process a) Is applicable to all bacterial polyoses & b) Does not require use of alcohol & CTAB.
Further sodium metaperiodate has been utilized for a) preparing reducing end groups of polysaccharides as well

as for b) depolymerization of certain capsular polyoses. However, teichoic acid removal by utilizing sodium metaperiodate has not been reported earlier.
SUMMARY OF INVENTION:
The invention relates to a process for purification of capsular polyoses ,wherein sodium metaperiodate treatment results in reduction of atleast 80% “polyose type” teichoic acid contaminants as compared to pretreatment capsular polyoses preparation.
DESCRIPTION OF THE DRAWINGS
Figure 1:Pneumococcal serotype 5 capsular polyose structure
Figure 2: Standard NMR profile of Pneumococcal serotype 5 polysaccharide(capsular polyose)& C-Ps (“polyose type” teichoic acid).
Figure 3: NMR profile of pre- sodium metaperiodate
treatment, Pneumococcal serotype 5
polysaccharide(capsular polyose) & C-Ps (“polyose type” teichoic acid) as per Serum Institute. Mole % of C-Ps = 25 = 36 wt% [(25 x (1322/919)].C-Ps calculation using methyl signals = 9H
Figure 4 : NMR profile of post-sodium metaperiodate treatment (PnPs:SMP = 1:1 molar ratio/45 mins), Pneumococcal serotype 5 polysaccharide(capsular polyose) & C-Ps (“polyose type” teichoic acid) as per Serum Institute 8% w/w C-Ps.
Figure 5 : NMR profile of posts metaperiodate treatment
(PnPs:SMP = 1:1 molar ratio/75 mins), Pneumococcal
serotype 5 polysaccharide(capsular polyose) & C-Ps
(“polyose type” teichoic acid) as per Serum Institute 7.2%

w/w C-Ps.
Figure 6 : NMR profile of post-sodium metaperiodate treatment (PnPs: SMP = 1: 3 molar ratio/30 mins), Pneumococcal serotype 5 polysaccharide(capsular polyose) & C-Ps (“polyose type” teichoic acid) as per Serum Institute 4.2% w/w C-Ps.
Figure 7 : NMR profile of post-sodium metaperiodate treatment (PnPs:SMP = 1:5 molar ratio/30 mins), Pneumococcal serotype 5 polysaccharide(capsular polyose) & C-Ps (“polyose type” teichoic acid) as per Serum Institute, Trace amount of C-Ps.
DETAILED DESCRIPTION OF INVENTION:
The process of the instant invention comprises treatment of the crude or purified capsular polyose preparation with sodium metaperiodate.
The method of the instant invention comprises purification of bacterial capsular polyoses of gram-positive bacteria. The said capsular polyoses is from the group consisting of pneumococcus 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.
Another embodiment of the instant invention is that said method is particularly applicable for capsular polyoses purification wherein “polyose type” teichoic acids removal by prior art methods is not efficient.Such

capsular polyoses belong to 4 and 5 pneumococcus serotypes.
A preferred embodiment of the instant invention is that the teichoic acids content of sodium metaperiodate treated bacterial polyoses is between 2 to 10 (%w/w),wherein atleast 80% teichoic acid reduction is obtained.
According to the instant invention sodium metaperiodate reacts with vicinal diols present on capsular polyose and “polyose type” teichoic acids,wherein “polyose type” teichoic acids were found to be more susceptible to complete degradation by sodium metaperiodate oxidation.
Yet another embodiment of the instant invention is that the ratio of pneumococcal capsular polyose to sodium metaperiodate can ranges from 1:1 to 1:5,preferably 1:5. Further the pneumococcal capsular polyose preparation can be subjected to sodium metaperiodate treatment under controlled conditions,wherein reaction is carried out in presence of 50mM acetate buffer having pH 5.5, at a temperature ranging from 20 to 30 0C,for a duration ranging from 30 to 50 mins,with mild stirring.
According to the current invention the pneumococcal
capsular polyose preparation can be subjected to sodium
metaperiodate treatment under controlled

conditions,wherein reaction is carried out in presence of 50mM acetate buffer having pH 5.5, at 25 0C,for a duration of 30 mins,with mild stirring.
According to the instant invention sodium metaperiodate treatment of pneumococcal capsular polyoses results in 2 to 10 fold reduction of “polyose type” teichoic acids.
Also the content of “polyose type” teichoic acids in pneumococcal polyoses obtained post sodium metaperiodate treatment can be less than 10% w/w,preferably less than 2% w/w.
Examples:
Example 1:
NMR Analysis of Serotype 5 pneumococcal capsular polyose
Serotype 5 polysaccharide are not readily characterized in detail by NMR due to broad peaks that results in weak or no crosspeaks in the 2D NMR correlation experiments. Full literatures are not available although anomeric (2 α- and 3 β-linked) resonances have been reported. Degradation of the keto sugar may result in heterogeneity in the NMR spectrum. Characteristic methyl (3) and acetyl signals (3) can be discerned near 1 -2 ppm. The C-Ps structure of PnPs 5 and NMR spectrum has been published,

as well as the contamination by peptidoglycan.
Pneumococcal serotype 5 samples:
Freeze-dried samples in vials were analyzed directly after dissolution in D2O by 1D proton NMR analysis using zg3-0 and zgpr Brucker programmes). Approx. 10 mg PnPs 5 was dissolved in 0.6 ml D2O and placed in 5 mm tube when fully dissolved.
Integrate C-Ps (3.4 – 3.14 ppm, narrow range if overlap)-set at integral of 1.00 (corresponds to 18H). Integrate Acetyl signals (2.19 – 1.90 pmm). Integrate methyl signals (1.45 – 0.95 ppm) – corresponds to 9H for PnPs 5.
Mol% C-Ps = [(area of 3.23 ppm/18)] / [(area of methyl/9] x (100/1) and wt% = Mol% x (1322/919).
RU mol wt of C-Ps and serotype 5 are 1322 and 919 respectively.
Example 2:
500 ml of pneumococcal serotype 5 capsular polyose preparation (crude or purified) was reacted with sodium metaperiodate in presence of acetate buffer(pH 5.5), at a temperature of 250C .The reaction mixture was kept under mild stirring.The reaction was further stopped by addition of glycerol(15%) and then subjected to diafiltration.
Table 1

Molar Ps SMP Ps Moles SMP Moles Reaction Post SMP
Exp.No Ratio of Amount amount

duration treatment
serotype (gm) (mg)

(min) C-Ps(w/w)

5 PnPs:SMP
1 1:1 1 214 1.088 1.088 45 8
2 1:1 1 214 1.088 1.088 75 7.2
3 1:3 1 642 1.088 3.26 30 4.2
4 1:5 1 1070 1.088 5.44 30 Traces
The results indicate that molar ratio of 1:5 and reaction time of 30 minutes are the optimal conditions for removal of “polyose type” teichoic acids.
5PnPs:Pneumococcal Serotype 5 polysaccharide(capsular
polyose)
SMP: Sodium metaperiodate
C-Ps: C-polysaccharide(“polyose type” teichoic acid)
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 removal of “polyose type” teichoic acids from bacterial capsular polyoses by treating the capsular polyose preparation with an oxidizing agent.
2. The method as claimed in claim 1,wherein said oxidizing agent is sodium metaperiodate(m-periodate).
3. The method as claimed in claim 1,wherein m-periodate treatment results in “polyose type” teichoic acid reduction by 5 to 10 fold.
4. The method as claimed in claim 1 ,wherein content of “polyose type”teichoic acids in m-periodate treated capsular polyose ranges from 2 to 10% w/w.
5. The method as claimed in claim 1,wherein the capsular polyose and m-periodate are reacted in a molar ratio ranging from 1:1 to 1:5.
6. The method as claimed in claim 5,wherein the capsular polyose and m-periodate are reacted in 1:5 molar ratio .
7. The method as claimed in claim 1,wherein the capsular polyose reacts with m-periodate in presence of acetate buffer having pH 5.5.
8. The method as claimed in claim 1,in which capsular polyose preparation is treated with m-periodate at a temperature from 25 to 30° C.
9. The method as claimed in claim 8,in which capsular polyose preparation is treated with m-periodate at a temperature of 25°C.
10. The method as claimed in claim 1,in which capsular polyose preparation is treated with m-periodate for a duration ranging from 30 to 75 minutes.

11. The method as claimed in claim 1,in which capsular polyose preparation is treated with m-periodate for a duration of 30 minutes.
12. The method as claimed in claim 1,in which said bacterial capsular polyoses can be selected from pneumococcal capsular polyoses of serotype 1, 2, 3, 4, 5, 6A, 6B,7F, 8, 9N, 9V, 10A, 11A, 12F, 14 15B, F, 18C, 19A, 19F, 20, 22F,23F and 33F.
13. The method as claimed in claim 12,in which said bacterial capsular polyoses are 4 & 5.