Page:l: FORM-2
The Patent Act, 1970
(39 of 1970)
Complete Specification
(Section 10; Rule 13)
Title: - An improved process for the purification of capsular polysaccharides of Haemophilus influenza - b, Neisseria meningitis such as serotypes A,C,Y and W-135, and other similar related capsular polysaccharides produced form both gram negative and gram positive microorganisms using aluminum phosphate with alcohol,
Address,
BIOLOGICAL E LIMITED
LAKSHMI BUILDING,
3RD FLOOR, ROOM Nos.45 & 46,
SIR P.M.ROAD, FORT,
MUMBAI-400 001
The following specification describes the invention, and the manner in which it is to be performed

Field of the invention
The present invention relates an improved process for the purification of capsular polysaccharides of Haemophilus influenza -b, such as serotypes A,C,Y and W-135, and other similar related capsular polysaccharides produced form both gram negative and gram positive microorganisms using aluminum phosphate with alcohol, more particularly in the field of new vaccines development, immunology, and medicine and for the prevention of infection by bacterial pathogens having protective polysaccharide capsule, by immunization
Back Ground of the invention:-
US Patent 7588765 discloses about the polysaccharide and glycoconjugate vaccines, more particularly the published prior art relates to a conjugation process which involves activation of an endotoxin-free polysaccharide antigen to polyfunctional capsular polysaccharide through a diamino-alkyl spacer introduced. In the said conjugation process an aluminum hydroxide and aluminum phosphate had been used proportionately adjusting conjugation pH
Patent application number: 20100040647, which teaches about the method of making a vaccine (i.e. a combination vaccine) using inactivated poliovirus type 1, comprising the steps of: mixing diphtheria toxoid and tetanus toxoid, followed by the addition of inactivated poliovirus type 1 at a dose greater than 10 D-antigen units and less than 20 D-antigen units and optionally IPV type(s) 2 and/or 3 (IPV).
US Patent 699192 The said published prior art discloses about a process for the production of inactivated Hepatitis A virus substantially free of host cell contamination, comprising the following steps of:
a) a culturing Hepatitis A virus and harvesting a hepatitis A preparation;
b) treating said hepatitis A preparation with a protease, thereafter;
c) separating intact virus from protease-digested protein; and
d) inactivating said virus

S Patent 7357936 , which teaches about the process for the manufacture of a vaccine
composition comprising admixing a) an adjuvant composition containing an
immunostimulant adsorbed onto a first metallic salt particle substantially free of antigen,
and b) an antigen, wherein the antigen is adsorbed onto a second metallic salt particle
substantially free of immunostimulant, wherein the metallic salt of each of the first
metallic salt particle and the second metallic salt particle may be the same.
The following problems are associated with the prior art, that the polysaccharide vaccines have been licensed for many years and have proved valuable in preventing bacterial meningitis in high risk individuals. Despite of several studies carried out on these encapsulated bacteria, there is a lack of information in the literature related to large- scale processes of bacterial cultivation and removal of impurities from capsular polysaccharides [3, 4].
EP00024 disclose the method which comprises the use of toxic components like Phenol, organic solvents like isopropanol, ethanol, use of detergents like CTAB for polysaccharide precipitation and the use of sodium acetate for pH adjustments,
Furthermore, EPO 497525 discloses the method which involves thermal hydrolysis and sodium acetate to hydrolyze the sample.
CA 1206905 describes a method which uses toxic and organic solvents like Phenol, butanol, Toluene and chloroform and use of detergent Cetavlon (CTAB).
US 4242501 and some more studies mention a method which involves serotype specific anion exchange chromatography [5]. Classical methods described by Institute Merieux give details of the use of Cetavlon and protease [6] for purification of N. Meningitidis serogroup A, C polysaccharide purification [7], Some other methods made use of proteinase for removal of proteins and use of lectin agarose columns resulting in high costs and process which can't be scaled up easily [8, 9].
US 2006O2283S0 discloses a method which comprises Cetavlon for polysaccharide precipitation, carbon filter for nucleic acid removal and potassium iodide for precipitation of Cetavlon [10, 11],

US Patent 5,847,112 mentions a method which makes use of multiple isopropyl alcohol precipitations and Cetavlon for the precipitation of PS.
The above disclosed prior arts teach about a method which is not efficient in removing the endotoxin from capsular polysaccharides of bacteria if the initial load is high and if produced in aggregate forms. The published prior art do not teaches about the use of aluminum phosphate in combination with DOC, EDTA etc, which is an efficient process to purify the capsular polysaccharides and complete removal of endotoxin during the process.
An inventive feature that the use of aluminum phosphate along with alcohol has not been reported in any kind of published documents, and a use of Aluminum phosphate along with alcohol achieved upto the mark of purification of capsular polysaccharide which is also reduces cost of manufacturing as well. An improvement in a process using a new ingredient is an inventive over the prior art an improved process is not obvious in the filed of purification of polysaccharide technology, therefore the present invention is novel and inventive over the prior art.
Objects of the invention:-
Important object of the present invention is that an improved process is a simple, efficient and improved purification process that eliminates the impurities from the above mentioned polysaccharides meeting the specifications as per the compendial requirements. The significant finding of this invention is applicable to Haemophilus influenza- b and Neisseria such as menigitides serotypes A,C,Y and W-135, and other similar related capsular polysaccharides produced form both gram negative and gram positive microorganisms which are used in polysaccharide conjugate vaccine manufacturing.
Further important object of the present invention is that a process can purify polysaccharides robustly at commercial scale, requiring low cost materials in the process and no specialized facilities or disposal of hazardous materials, therefore an alleged invention is cost effective.

Further very important object of the present invention is that the present process is very effective in preparing a high quality product with high yields. And very much effective to remove impurities by use of aluminium phosphate from the capsular polysaccharides meeting the compendial requirements.
Summary of the invention
An improved process for purification of polysachharide using an alcohol / A1P04 method for the purification of Haemophilus and Neisseria such as serotypes A, C, Y and W - 135, other similar related capsular polysaccharides produced form both gram negative and gram positive microorganisms which are used in the preparation of polysaccharide vaccines. A1P04 in combination of alcohol and DOC buffer is used for the removal of impurities. The process is a generic process to purify Haemophilus influenzae -b, Neisseria serotypes A, C, Y and W-135, and other similar related capsular polysaccharides produced form both gram negative and gram positive microorganisms.
Description of the Invention
The present invention relates to an improved process for the purification of capsular polysaccharides of Haemophilus influenza - b, Neisseria meningitis such as serotypes A,C,Y and W-135, and other similar related capsular polysaccharides produced form both gram negative and gram positive microorganisms using aluminum phosphate with alcohol, and the use of alcohol / DOC during Haemophilus and Neisseria polysaccharides purification and the inefficiency in the prior art process in removal of endotoxin, the present invention makes use of aluminum phosphate and DOC buffer for removal of endotoxin, alcohol / DOC treatment for nucleic acid and protein removal very effectively. Impurities in all Haemophilus and Neisseria polysaccharide types are effectively adsorbed with aluminum phosphate in presence of alcohol without significant loss to the polysaccharide content. An alcohol / DOC treatment efficient for removal of all the nucleic acids and protein contaminants. An alleged invention is an alcohol based purification process along with aluminum phosphate and DOC buffer for Haemophilus and Neisseria polysaccharides which meet all the release test specifications required for the vaccination of adults Haemophilus and Neisseria type cultures, used in this invention are available worldwide from a great number of culture libraries.

The American Type Culture Collection (ATCC), 12301 Parklawn Dr., Rockville, Md„ U.S.A.
20852, lists all of the serotypes of this invention as being freely available.
Therefore, the present invention relates to a process for preparing a purified Haemophilus polysaccharide the said process comprises the following steps ras shown in flow chart - 1.
Flow chart 1:
CRUDE POLYSACCHARIDE
j I
CETAVLON PRECIPITATION
f 0.5M NaCI DISSOLUTION &.
I 0.45|J FILTRATION
A 1
72% ETHANOL PRECIPITATION
. „ __i -^ —- -,
PELLET DISSOLVED IN BUFFER
(0.3% DOC-t- 2mM EDTA+ 20mM Tris) &
Incubation for 2 Hrs
Addition of 0.5% DOC and 32% Ethanol PELLET .
I
I — . i .
Addition of Aluminium Phosphate Gel with a ratio of 1:8
(PRP : Gel) & Overnight Incubation
64% Ethanol PPT .
' ,
i 1^ _~
300KD Diafiltration & followed by 0.22 μ filtation
The primary step of the present invention for purification of polysaccharide is
Cetavlon precipitation
Taken the crude PRP in the cold room and centrifuge at 6000 RPM / 30 min / 2 - 8 °C, after centrifugation discarded the pellets formed, collected the supernatant solution and added 10% Cetavlon to a concentration of 0.65 %, the resultant mixture is allowed to incubate at room temperature under constant stirring for a period of two hours.

0.5 M NaCl dissolution:
After completion of incubation time the above mixture is allowed to centrifuge at 4000 RPM at 1 to 10c for 15 to 35 minutes to recover pellet. The said pellet is dissolved in 0.5 M NaCl (Make up the volume to initial crude volume). This solution is filtered through 0.45u, filter.
72% Ethanol precipitation:
Added 72% Ethanol to the above filtrate to get a final concentration of 72%. The addition of ethanol is carried out under constant stirring. Adjusted the pH to 6.8 ± 0.1 using 8M acetic acid, stored the precipitated solution at room temperature for 2 - 4 hrs.
DOC buffer treatment
After the completion of incubation time the above mixture is centrifuged at 4000 RPM at 2-8°C for 30 minutes to recover pellet. Pellet collected is dissolved in DOC buffer containing 0.3%v/v DOC and 2mM EDTA and 20mM TRIS pH 7.2. (Make up the volume to initial crude volume). Stir the solution for two hours at room temperature.
32% Ethanol precipitation
After completion of incubation period added 6% w/v Sodium Acetate & 0.7% vv/v DOC to the above solution the above DOC treatment was carried out under agitation for 10 to 20 minutes adjusting pH at of 6.3 ± 0.1 to this solution added Ethanol to a final concentration of 32% v/v under agitation Adjusting the pH to 6.8± 0.1 using 8M acetic acid, with or without Incubation However incubation is preferred, the solution is precipitated at (2°C-8°C)for 2-4hrs.
A1P04 adsorption
At the end of incubation time, centrifuge the solution at 6000 RPM/30 min at temperature between 2 - 8 °C, discarded pellet, and collected supernatant. To the supernatant solution added A1P04 at a ratio of 1:8 w/w of polysaccharide and A1P04 (to one gram of polysaccharide add 8g of A1P04 gel) at pH 7.4 kept the solution for 8 -12 hrs under constant stirring conditions at room temperature which forms lipid type solution. The lipid part of the LPS is effectively adsorbed through A1P04 gel under these conditions, which are separated and removed by centrifugation followed by filtration. The resultant product is ready for use.


64% Ethanol precipitation
After 24 hours or after a day the above mixture is centrifuged at 6000 RPM at 2-8°C for 30 minutes to recover supernatant. Added sodium acetate to the supernatant to a final concentration of 6% w/v & pH is adjusted to 6.3 ± 0.1 using 8M Acetic acid, then added ethanol to the solution to a final concentration of 64% v/v under agitation adjusted the pH to 6,8± 0.1 using 8M Acetic Acid, with or without incubation . However incubation is preferred, the solution is precipitated at 2°C - 8°C for 2-4hrs.
300kD Diafiltration followed by 0.2micron filtration
After the end of incubation separate the pellet from above mixture by decanting the supernatant. Dissolved the pellet in WFI (initial volume of crude taken). Clear solution is subjected to diafiltration on 300 kD ULF membrane using 10-20 volumes with WFI. This purified PRP is concentrated and harvested. Then the PRP is filtered through pre sterilized 0.22 n PES capsule filter and stored in -20°C after sampling.
The product is characterized using sup.lH-NMR data shows that the consistent with the chemical structure by the assignment of signals assigned to the protons of the polysaccharide molecule. The.lH-NMR spectrum showed a series of well-resolved signals (protons from the methyl group) for the quantification of functional groups in the polysaccharide. Size exclusion chromatography media (CL-4B) was used to profile the relative molecular size distribution of the polysaccharide. The purified polysaccharides comply with the WHO specifications for pure polysaccharides.

WHO Specifications for Purified polysaccharides:
Protein Content: - NMT 1% dry weight of the polysaccharide.
Nucleic acid Content: - NMT 1% dry weight of the polysaccharide.
Pyrogen content: - Less then 10IU/μg of polysaccharide. EXAMPLE
Taking a thawed PRP kept in the cold room. Diluted the same with PBS for 4 times centrifuged at 6000 rpm per 30 minutes at 2 to 8 degree centigrade, and discarded the pellet. The sup is filtered through 0.45 micron filter and added 10% CTAB to getO.65% final concentration. Incubate at 2 to 8 degree centigrade for 2 hours. Further centrifuged at the rate of 4000 rpm for 30 minutes at 2 to 8 degree, discarded supernatant, precipitate dissolve in 0.5 M Nacl .This solution is filtered through 0.45 micron filter and add ethanol to 72% final concentration incubated at 2 to 8 C for over night.
Centrifuge at 4000 rpm for 30 minutes at 2 to 8 centigrade discarded supernatant ppt dissolve in DOC buffer incubate at RT for 2 hours followed by addition of .0.7% DOC w/v, adjust pH 6.2 add 6% w/v sodium acetate add ethanol to 32% final concentration incubated at 2 to 8 C for 4 hours. Addition of AIP04 at the ratio of 1:8 (PRP: gel) w/w at pH 7.4 incubated for over night at room temperature. Centrifuge at the rate of 600 rpm, for 30 minutes at 2 to 8 C temperature and discarded the pellet obtained , filter the supernatant through ).22 micron filter add 6% Na-Acetate, adjust pH at 6.1 to 6.3 add ethanol to 62% for final concentration incubate it at 2 to 8° C for 2 to 4 hours. Further centrifuge at 4000 rpm, for 30 minutes and discard supernatant , dissolve the pellet in WFI dia filter on 300 kda membrane with 10 Vol. of WFI, and allow to concentrate and clarify through )0.22 Micron filter and store at -20° C.


Preparation of Haemophilus influenza ~ b, Neisseria meningitis such as serotypes
AfC, Y,W-13S and other similar related capsular polysaccharides produced form both gram
negative and gram positive microorganisms

We Claim,
1. A process for preparing a pure capsular polysaccharides of Haemophilus influenza -
b, Neisseria meningitis using aluminum phosphate with alcohol, the said process comprising the following steps,
a) precipitating the crude polysaccharide with cetavlon (CTAB - cetyl trimethyl ammonium), taken a crude polysaccharide in the cold room allowed to centrifuge at 6000 RPM for 30 minutes at 2 - 8 °C temperature , after centrifugation discarded the pellet, and collected supernatant solution, to this added 10% Cetavlon to a final concentration of 0.65 %w/v allowed to incubate at room temperature under constant stirring for a period of 2 to 4 hours,
b) centrifugated the reaction mixture obtained at step (a) at 1 to 10 C temperature for a period of 15 to 35 minutes . to recover pellet, dissolved the pellet in a sodium chloride followed by filtration,
c) added 72% Ethanol/or any other organic solvent to the filtrate obtained at step (b) , under constant stirring while maintaining pH 6.8 • 0.1 using 8 M acetic acid, stored the precipitated solution at room temperature for 2 - 4 hrs/Overnight or without incubation
d) after incubation centrifuged at 2-8°C for 30 minutes to recover pellet the said pellets are dissolved in DOC (Sodium Deoxy cholate) buffer containing 0.3%v/v and 2mM EDTA and 20mM TRIS pH 7 stirred the solution for two hours at room temperature,
e) added 6% to 8% Sodium acetate and 0.5% to 0.7% DOC (Sodium Deoxy cholate) w/v to the above solution obtained at step (d) under constant stirring condition and organic solvent is added at low concentration followed by with or without incubation, centfifuged to remove contaminants, as pellet.
f) added A1P04 while adjusting the pH between 7.0 - 7.4 to the supernatant solution obtained at step (e) and Kept the solution under constant stirring at room temperature to remove the process impurities,

g) added 5 to 7%w/v of sodium acetate to the above solution and organic
solvent to a make a higher concentration and collected the Polysaccharide as precipitate after incubation followed by centrifugation,
h) precipitate collected from the above step (g) is dissolved in WFI and filtered
with 10 -20 volumes of WFI using the 300Kd MWCO cassettes the polysaccharide solution is stored at -20 C.
2 A process as claimed in claim 1, wherein the purification of polysaccharide extended to serotypes A,C,Y,W-135 and other similar related capsular polysaccharides produced form both gram negative and gram positive microorganisms
3. A process as claimed in claim 1, wherein the DOC (Sodium Deoxy cholate) buffer containing 0.3%w/vand 2mM EDTA (Ethyl dimethyl tetra acetic acid) and 20mM TRIS are used,
4. A process as claimed in claim 2, wherein 6% Sodium acetate and 0.7% DOC w/v , organic solvent is added to a low concentration, organic solvents are selected from ethanol, methanol,
5. A process as claimed in claim 1 and 2, wherein the 0.5% DOC and 32% ethanol pellet are used,
6. A process as claimed in claim 1, wherein the A1P04 in organic solvent such as ethanol and / or methanol used for removal of an impurities,
7. A process as claimed in claim 1, and 4, wherein the impurities such as protein nucleic acid, and endotoxin have been removed,
8. A process as claimed in claim 1, wherein purified polysaccharides are useful for preparation of vaccine for Haemophilus influenza - b, Neisseria meningitis serotypes A,C, Y, W-135 and other similar related capsular polysaccharides produced form both gram negative and gram positive microorganisms
9. A process as claimed in above claims wherein the purified capsular polysaccharides are used for further conjugation with suitable carrier protein to form glyco -conjugate vaccines,

10. A process as claimed in above claims wherein the usage of aluminum phosphate for the purification of capsular polysaccharides of Haemophilus influenza - b, Neisseria meningitis serotypes A,C,Y,W-135 and other similar related capsular polysaccharides produced form both gram negative and gram positive microorgan isms
11. A process for preparing pure capsular polysaccharides of Haemophilus influenza -b, Neisseria meningitis serotypes A,C,Y,W-13S and other similar related capsular polysaccharides produced form both gram negative and gram positive
microorganisms using aluminum phosphate with alcohol, such as herein described with reference to an examples and description.