WO2019175900A1

WO2019175900A1 - Purification of neisseria meningitidis polysaccharides

Info

Publication number: WO2019175900A1
Application number: PCT/IN2019/050208
Authority: WO
Inventors: Sandeep Sharma; Nitin Kumar; Sarmad HANIF; Manoj Kumar CHHIKARA; Davinder Gill
Original assignee: MSD Wellcome Trust Hilleman Laboratories Pvt Ltd
Current assignee: MSD Wellcome Trust Hilleman Laboratories Pvt Ltd
Priority date: 2018-03-16
Filing date: 2019-03-15
Publication date: 2019-09-19
Anticipated expiration: 2020-09-16

Abstract

The present invention relates to a novel process for purification of bacterial polysaccharide specifically the polysaccharides of Neisseria meningitidis serogroup C, W and Y. It is an efficient process for eliminating impurities from Neisseria meningitidis serogroup C (Men C), W (Men W) and Y (Men Y) polysaccharides in a very short time by simple, efficient, improved and commercially scalable method. The purification process provides high quality product with better yield that meet the relevant quality specifications. The purified polysaccharides are capable of being used in the production of polysaccharide and polysaccharide-protein conjugate vaccine(s) against meningococcal infections.

Description

TITLE OF THE INVENTION:

PURIFICATION OF NEISSERIA MENINGITIDIS POLYSACCHARIDES

FIELD OF THE INVENTION:

The present invention relates to a process of purification of bacterial polysaccharide. The present invention particularly relates to purification process for purification of Neisseria meningitidis serogroup C, W and Y polysaccharides. The N. meningitidis polysaccharides of the present invention are capable of being used in the production of polysaccharide and polysaccharide-protein conjugate vaccine(s) against meningococcal infections.

BACKGROUND OF THE INVENTION:

Polysaccharides, especially antigenic polysaccharides, are used in preparation of vaccines. Monovalent, bivalent and poly (multi) valent vaccines containing one, two or more polysaccharides and their conjugates are available in the market for prevention of certain diseases or infections caused by various microorganisms such as Streptococcus pneumoniae, Haemophilus influenzae and N. meningitidis and have proved valuable in preventing the respective diseases to a significant extent. Surveillance data gathered in the years following the introduction of the vaccine Prevenar has clearly demonstrated a reduction of invasive pneumococcal disease in US infants as expected. Despite of several studies carried out on these polysaccharides and conjugates, a need for improving yields as well as quality (purity) of the polysaccharides always exist in the industry as evidenced by the continuing research.

The production of purified N. meningitidis capsular polysaccharides is the foremost requirement for an effective conjugation with the carrier protein and its development as a conjugate vaccine. The cost for the cultivation of N. meningitidis and the purification of polysaccharides is generally high and involves long working hours since it involves a series of production and purification steps.

Improvement in one or more of the steps of polysaccharide production would bring a significant change in the overall conjugate vaccine production and consequently makes the process relatively cost effective. Evidence collected through numerous research findings defines the immunogenic aspect of the polysaccharide conjugate vaccine. Furthermore, there are research articles as well as patents describing the production and purification of the capsular polysaccharide of Men C, Men Y and Men W but none of them define a purification process with extremely reduced time lines which is robust, scalable, and reproducible.

There are a number of patents, which describe the process for the purification of Men-C polysaccharides. The existing state of the art described in US patent no 7,491, 517B2 for precipitating Men-C polysaccharides with CTAB is found to involve overnight incubation at 4°C. Also, the removal of contaminants requires the use of costly enzyme Proteinase K. In addition to this, the process also requires gel filtration for the purification of the Men-C polysaccharides. The overall procedure requires significant time for purification and also the process becomes costly because of the use of enzymes.

Another Patent application no. US2009/ 0182128 Al, Paolo Costantino et al, relates to purification of Men W, A, and Y using CTAB and ethanol treatment (50% - 95%) and use of CaCh and carbon filtration. The disclosed patent application uses multiple steps for the purification of crude polysaccharides and long hours for the purification process.

In order to obviate the drawbacks of the existing state of art, the present invention discloses process that has been optimized to enable the purification of N. meningitidis serogroups in lesser time without requiring chromatography.

OBTECT OF THE INVENTION:

The main object of the present invention is to provide a process of purification of bacterial polysaccharide.

Another object of the present invention is to provide a process of purification of Neisseria meningitidis serogroup C, W and Y polysaccharides.

Yet another object of the present invention is to purify Neisseria meningitidis polysaccharides while eliminating impurities in a very short time by simple, efficient, improved and commercially scalable method. Yet another object of the present invention is to produce high quality product with better yield that meet the relevant quality specifications.

SUMMARY OF THE INVENTION:

The present invention describes a rapid, industrially scalable, cost effective process for the purification of Neisseria meningitidis polysaccharides. The said process provides a purification method for purifying N. meningitidis serogroup C, W and Y polysaccharide at a significantly reduced time. The purification process does not require any chromatography step thereby making the invention rapid and cost effective. The process of the present invention commences with collecting the fermentation broth after centrifugation of inactivated bacterial fermenter culture, diafiltration of said fermentation broth to obtain concentrated fermentation broth, adjusting pH of said concentrated fermentation broth to desired level, regular intermittent stirring of said concentrated fermentation broth of desired pH at pre-determined temperature for pre- determined time and thereafter diafiltration of the solution is carried out with specific reagents to obtain partially purified polysaccharide.

The partially purified polysaccharide so obtained is mixed with specific buffers and chemical reagents and the resultant solution is incubated at specific temperature for pre-determined duration with intermittent stirring at regular intervals. Ethanol treatment of the incubated solution thus obtained is carried out at specific temperature for pre-determined duration with intermittent stirring at regular intervals along with or after incubation with the earlier reagents. The supernatant of the ethanol treated solution is separated and diafiltered with specific reagents. The diafiltered solution is then carbon filtered using carbon filter of predefined specifications until optimum optical density of the solution is achieved. The solution is then concentrated at desired level and filtered and stored at temperature less than 0° C. The process of purification of present invention is completed in less than 11 hours. The process is rapid, cost effective, entirely scalable and purified polysaccharides meet the desired specifications with better yields.

BRIEF DESCRIPTION OF DRAWINGS:

Figure-1: Depicts the NMR spectrum of Men C Figure-2: Depicts the NMR spectrum of MenY

Figure-3: Depicts the NMR spectrum of MenW

Figure-4: Depicts the HPLC Chromatogram of Men C

Figure-5: Depicts the HPLC Chromatogram of Men Y

Figure-6: Depicts the HPLC Chromatogram of Men W

DETAILED DESCRIPTION OF THE INVENTION:

The present invention describes the purification method of Bacterial polysaccharides (PS) specifically the polysaccharides of Neisseria meningitidis. The essence of the invention is the rapid and cost- effective purification process of Men C_,, Men W and Men Y of Neisseria meningitidis. Bacterial polysaccharides are used in the preparation of vaccines against many fatal diseases and infections. PS vaccine preparation requires purified polysaccharides free from salts or solvents containing proteins, peptides, nucleic acids, and other biomolecules to enhance their sensitivity.

The invention discloses the method that has been optimized to enable the purification of MenC, MenW and Men Y polysaccharide at reduced time. Neisseria meningitidis strain from which the PS is to be extracted is inoculated in the flask and subsequently in fermenter containing appropriate media components required for bacterial growth. After achieving the maximum optical density, in the range of 8 to 10 depicting substantial bacterial growth, it is subjected to growth termination by adding prerequisite concentration of formaldehyde and the resultant Fermentation Broth (FB) is obtained.

The fermented broth as obtained above is used for purifying Men C, Men

W and Men Y Polysaccharides of Neisseria meningitides. The fermented broth (FB) is concentrated and diafiltered using lOOkDa polyether sulfone membrane (PES) with 10-12 volumes of MilliQ water (MQW) to obtain diafiltered concentrate. The pH of the diafiltered concentrate is adjusted in the range of 13±0.1 after which the resultant PS is incubated at 90°C for 1-2 hrs.

The incubated diafiltered concentrate of PS is cooled to room temperature , i.e. in the range of 25°C ±2 using ice bath. After cooling, the incubated PS is again concentrated and diafiltered using lOOkDa polyether sulfone membrane (PES) with 15-20 volumes of MilliQ water to obtain clarified PS concentrate. To this clarified PS concentrate, tri-sodium citrate (TSC) is added in the concentration range of 0.25M-0.40 M, sodium sulfate (SS) is added in the concentration range of 25% -50% v/v of 2M stock,, sodium deoxycholate (DOC) is added in the concentration range of 1.6±0.5 % w/v and 30%± 5% v/v absolute ethanol is added in a stepwise manner, and the resultant mixture incubated at 37°C for 3±1 hrs. Combination of TSC, SS, DOC and ethanol has been optimized to reduce protein impurity loads majorly and to some extent nucleic acid reduction from Men Y and Men W crude PS, and is considered to be a novel composition. To the concentrated PS, 30% ± 5% v/v absolute ethanol is added, and it is stirred at room temperature for 2 hrs. The resultant mixture is concentrated and diafiltered using lOOkDa PES with 20 volumes of MilliQ water (MQW) to obtain partially purified polysaccharide.

For intermittent and final diafiltration and concentration of Men C polysaccharide, 30 kDa PES membrane is used.

The concentrated and diafiltered resultant mixture, i.e. the partially purified polysaccharide as obtained above is carbon filtered by utilizing Millistak carbon filter to reduce the remaining nucleic acid impurity and to lower down remaining protein impurities.

For purifying PS from the concentrate, water primed millistak POD filters are used. For Men Y and Men W water primed millistak POD filters with 0.027 m² are used while for Men C water primed millistak POD filters

0.054m² are used.

The partially purified PS is then circulated through the first filter, and OD260nm of the filtrate is noted and recorded. If the OD260nm of the filtrate is less than 0.1, the material is recovered fully by giving a wash of 300-400ml milli Q water (MQW). However, if the OD260nm is greater than

0.2, then the material is recirculated for 15mins (30 mins in case of Men C) with masterflex pump speed of 40rpm (90 ± 10 ml/ min flow rate) for both first pass and recirculation. After recirculation, the material is recovered and collected from the first filter, and OD260nm of the filtrate is recorded. If the OD260nm of this filtrate is recorded to be less than 0.1, the material is recovered fully by giving a wash of 300-400ml MQW. If the OD260nm is still greater than 0.2, the first filter is kept aside without flushing, and the collected filtrate is circulated through the second filter with a pump speed of 40rpm and the material is recirculated with a masterflex pump speed of 150-160rpm (420 ± 20 ml/ min flow rate) until the OD260nm is less than 0.1.

The recirculation time on the second filter is generally between 3-5hrs for Men W PS, whereas for Men Y PS it is 2-3hrs. After the desired O.D. is achieved, the recovered material is kept aside. Both the filters are then joined in series and a wash of 300-600ml was given to recover the bound PS. The wash is mixed with the recovered material.

The process to purify Men C, Men Y and Men W PS remains same except for some difference in recirculation time which may vary depending on the initial impurity load in a particular batch. Recirculation time and pump speed are significant factors to guide the process in terms of reducing nucleic acid and protein load so as to bring the impurity levels well below the specified limits. The corroboration of the above employed procedures can be understood from Table 1 which clearly shows that the purified polysaccharide specifications meet the WHO standards.

Table 1: Purified Men C, Men Y and Men W polysaccharide specifications in accordance with WHO specification are shown below

The overall process of Men C purification is completed in 8-9 hours while for Men Y and Men W it is completed in 10-12 hours.

The identity of extracted and purified polysaccharides is verified by 1H- NMR spectroscopy. NMR yields a spectrum of magnetic sensitive nuclei as shown in Fig. 1-3. Relative Average Molecular size (Mw) of Men C, Men Y and Men W is determined using High Performance Liquid Chromatography (HPLC). The yield for Purified polysaccharide of Men C, Men Y and Men W as shown in HPLC chromatograms of Fig. 4-6 is 290±10mg/L, 344±10 mg/L and 290±10 mg/L respectively. The process to purify Men C, Men Y and Men W is unique in terms of less time taken to purify polysaccharides, novel composition of different components employed to reduce impurity load within specified limits and reproducibility.

Various aspects of the invention described in detail above are now illustrated with non-limiting examples:

Purification of Men W/Men Y Polysaccharides:

Example-1 (MenY and MenW):

MenY and MenW Polysaccharide (PS) purification using sodium sulfate, tri-sodium citrate and sodium deoxycholate (DOC):

The fermented broth (FB) is concentrated and diafiltered using lOOkDa polyether sulfone membrane (PES) with 10-12 volumes of MilliQ water (MQW). Afterwards, the pH of the partially purified PS is adjusted to 12.5 and then incubated at 90°C for 1.5 hrs. The PS is then cooled to RT (25±2°C).

Afterwards, lOOkDa concentration and diafiltration of the crude polysaccharide is performed with 15 volumes of MilliQ water. To the diafiltered PS, 0.25 M tri-sodium citrate (TSC), sodium sulfate (SS) (25% v/ v of 2M stock), 1.2 % w/ v DOC is added, and the crude PS kept at 37°C for 3 hrs. Afterwards, 30% v/v absolute ethanol added to the PS for 2 hours at RT. The resultant mixture is concentrated and diafiltered using lOOkDa PES membrane with 20 volumes of MilliQ water (MQW). Carbon filtration is performed to reduce nucleic acid and protein impurity load with millistak filter until the OD260_nm reaches below 0.2. The collected filtrate is then concentrated by lOOkDa PES followed by 0.2m filtration. The analysis revealed that there is PS loss, therefore the process is not considered for PS purification.

Example-2 (MenY and MenW):

The fermented broth (FB) is concentrated and diafiltered using lOOkDa polyether sulfone membrane (PES) with 10-12 volumes of MilliQ water (MQW). Afterwards, the pH of the partially purified PS is adjusted to 13 and then incubated at 90°C for 2 hrs. The PS is then cooled to RT (25±2°C). Afterwards, lOOkDa concentration and diafiltration of the crude polysaccharide is performed with 15 volumes of MilliQ water. To the diafiltered PS, 0.25M tri-sodium citrate (TSC), sodium sulfate (SS) (25% v/v of 2M stock), 1.3 % w/ v DOC is added, and the crude PS kept at 37°C for 4 hrs. Afterwards, 30% v/ v absolute ethanol is added to the PS for 2 hours at RT. The resultant mixture is concentrated and diafiltered using lOOkDa PES with 20 volumes of MilliQ water (MQW). Carbon filtration is performed to reduce nucleic acid and protein impurity load with millistak filter until the OD260_nm reaches below 0.2. The collected filtrate is then concentrated by lOOkDa PES followed by 0.2m filtration. The analysis revealed that there is PS loss therefore the process is not considered for PS purification. Example-3 (MenY and MenW):

The fermented broth (FB) is concentrated and diafiltered using lOOkDa polyether sulfone membrane (PES) with 10-12 volumes of MilliQ water (MQW). Afterwards, the pH of the partially purified PS is adjusted to 13 and then incubated at 90°C for 2 hours. The PS is then cooled to RT (25±2°C). Afterwards, lOOkDa concentration and diafiltration of the crude polysaccharide is performed with 15 volumes of MilliQ water. To the diafiltered PS, 0.25M tri-sodium citrate (TSC), sodium sulfate (SS) (25% v/v of 2M stock), 1.4 % w/v DOC is added, and the crude PS kept at 37°C for 3.5 hrs. Afterwards, 30% v/v absolute ethanol added to the PS for 2 hours at RT. The resultant mixture is concentrated and diafiltered using lOOkDa PES with 20 volumes of MilliQ water (MQW). Carbon filtration is performed to reduce nucleic acid and protein impurity load with millistak filter until the OD260_nm reaches below 0.2. The collected filtrate is then concentrated by lOOkDa PES followed by 0.2m filtration. The analysis revealed that there is PS loss therefore the process is not considered for PS purification.

Example-4 (MeuY and MenW):

MenY and MenW Polysaccharide (PS) purification using sodium sulfate, tri-sodium citrate and sodium deoxycholate (DOC) (best mode):

The fermented broth (FB) is concentrated and diafiltered using lOOkDa polyether sulfone membrane (PES) with 10-12 volumes of MilliQ water (MQW). Afterwards, the pH of the partially purified PS is adjusted to 13 and then PS is incubated at 90°C for 2 hrs. The incubated PS is then cooled using ice bath to RT (25±2°C). After cooling, the 100 kDa concentration and diafiltration of the partially purified polysaccharide is performed with 20 volumes of MilliQ water. To this PS concentrate, 0.25M TSC, SS (25% v/v of 2M stock) and 1.4 % w/v DOC is added in a stepwise manner, and the resultant mixture is incubated at 37°C for 3.5 hrs. Afterwards, 30% v/v absolute ethanol is added to the and same is stirred at RT for 2 hrs. The resultant mixture is concentrated and diafiltered using lOOkDa PES with 20 volumes of MilliQ water (MQW). Carbon filtration is then performed to reduce the remaining nucleic acid impurity and to lower down remaining protein impurities by utilizing Millistak carbon filter. Carbon filtration is initiated through water primed millistak POD filters (0.027m2). The partially purified PS is circulated through the first filter, and OD260nm is noted and recorded, if the OD260nm <0.2/ more preferably less than 0.1, the material is recovered fully by giving a wash of 300-400ml MQW, however, if the OD260nm >0.2, then the material is recirculated for 15mins, with pump speed of 40rpm (90 ± 10 ml/ min flow rate) for both first pass and recirculation. After 15mins of recirculation, the material is recovered and collected from the first filter, and OD260nm is recorded. If the OD260nm is recorded to be <0.2/ more preferably less than 0.1, the material is recovered fully by giving a wash of 300-400ml MQW. If the OD260nm is still >0.2, the first filter is kept aside without flushing, and the collected filtrate is circulated through the second filter with a pump speed as 40rpm and the material is recirculated with a masterflex pump speed as 150-160rpm (420 ± 20 ml/ min flow rate) until the OD260nm reaches <0.2/ more preferably less than 0.1. After the desired O.D. is achieved, the recovered material is kept aside. Both the filters are then joined in series and a wash of 300-400ml is given to recover the bound PS. The wash is mixed with the recovered material. As established, the recirculation time on the second filter is generally between 3-5hrs for Men W PS, whereas for Men Y PS it is 2-3hrs. Recirculation time and pump speed were significant factors to guide the process in terms of reducing nucleic acid and protein load so as to bring the impurity levels well below the specified limits.

Purification of Men C Polysaccharide:

Example-1 (MenC):

MenC Polysaccharide (PS) purification using sodium sulfate, tri-sodium citrate and sodium deoxycholate (DOC):

The fermented broth (FB) is concentrated and diafiltered using lOOkDa polyether sulfone membrane (PES) with 10-12 volumes of MilliQ water (MQW). Afterwards, the pH of the partially purified PS is adjusted to 13 and then the PS is incubated at 90°C for 2 hrs. The incubated PS is then cooled using ice bath to RT (25±2°C). Afterwards, 30 kDa concentration and diafiltration of the partially purified polysaccharide is performed with 15 volumes of MilliQ water. To the diafiltered PS concentrate, 0.5 M tri-sodium citrate (TSC), sodium sulfate (SS) (50% v/v of 2M stock), 1.6 % w/v DOC and 30% v/v absolute ethanol are added, and pH is again adjusted to 13 and the mixture is kept at stirring for 2 hrs at RT (25±2°C). Afterwards, the resultant material is concentrated and diafiltered using 30 kDa PES with 20 volumes of MilliQ water (MQW). Zeta carbon disc filtration is performed until the OD260_nm reaches below 0.2. The collected material is then concentrated by 30 kDa PES followed by 0.2m filtration. The analysis revealed that there was significant PS loss post carbon filtration, therefore the protocol is not considered for PS purification.

Example-2 (MenC):

The fermented broth (FB) is concentrated and diafiltered using lOOkDa polyether sulfone membrane (PES) with 10-12 volumes of MilliQ water (MQW). Afterwards, the pH of the partially purified PS is adjusted to 13 and then the PS is incubated at 90°C for 1.5 hrs. The incubated PS was then cooled using ice bath to RT (25±2°C). Afterwards, 30 kDa concentration and diafiltration of the partially purified polysaccharide is performed with 15 volumes of MilliQ water. To the diafiltered PS concentrate, 0.25 M TSC, SS (25% v/v of 2M stock), 1.3 % w/v DOC and 30% v/v absolute ethanol are added and pH is again adjusted to 13 and the mixture is kept at stirring for 2 hours at RT (25±2°C). Centrifugation is done and supernatant collected. The supernatant is concentrated and diafiltered using 30 kDa PES membrane with 20 volumes of MilliQ water (MQW). Carbon filtration using a combination of Millistak and Zeta carbon filters is performed until the OD260_nm reaches below 0.2. The collected material is then 30 kDa concentrated followed by 0.2m filtration. The analysis revealed that there is PS loss and the combination of carbon filters could not help in yield improvements, therefore the protocol was not considered for PS purification.

Example-3 (MenC):

The fermented broth (FB) is concentrated and diafiltered using lOOkDa polyether sulfone membrane (PES) with 10-12 volumes of MilliQ water (MQW). Afterwards, the pH of the partially purified PS is adjusted to 13 and then the PS is incubated at 90°C for 2 hrs. The incubated PS is then cooled using ice bath to RT (25±2°C). Afterwards, 30 kDa concentration and diafiltration of the crude polysaccharide is performed with 15 volumes of MilliQ water. To the diafiltered PS concentrate, 0.25 M TSC, SS (25% v/ v of 2M stock), 1.4 % w/v DOC, and 30% v/v absolute ethanol are added and pH is again adjusted to 13 and the mixture is kept at stirring for 2 hours at RT (25±2°C). Afterwards, the mixture is concentrated and diafiltered using 30 kDa PES membrane with 20 volumes of MilliQ water (MQW). Carbon filtration using Millistak and Zeta carbon filtration performed until the OD260_nm reaches below 0.2. The collected material is then 30 kDa concentrated followed by 0.2m filtration. The analysis revealed that there is PS loss therefore the protocol is not considered for PS purification.

Example-4 (MenC):

MenC Polysaccharide (PS) purification using sodium sulfate, tri-sodium citrate and sodium deoxycholate (DOC) (best mode):

The fermented broth (FB) is concentrated and diafiltered using lOOkDa polyether sulfone membrane (PES) with 10-12 volumes of MilliQ water (MQW). Afterwards, the pH of the partially purified PS is adjusted to 13 and then the PS is incubated at 90°C for 2 hrs. The incubated PS is then cooled using ice bath to RT (25±2°C). Afterwards, 30 kDa concentration and diafiltration of the crude polysaccharide is performed with 15 volumes of MilliQ water. To the diafiltered PS concentrate, 0.25 M TSC, SS (25% v/ v of 2M stock), 1.6 % w/v DOC and 30% v/v absolute ethanol are added and pH is again adjusted to 13 and the mixture is kept at stirring for 2 hours at RT (25±2°C). Afterwards, the mixture is concentrated and diafiltered using 30 kDa PES membrane with 20 volumes of MilliQ water (MQW). Carbon filtration is performed through water primed millistak POD filters (0.054m2). The partially purified PS is circulated through the first filter, OD260nm is noted and recorded. If the OD260nm is <0.2/ more preferably less than 0.1, the material is fully recovered by giving a wash of 500-600ml MQW. If the OD260nm was >0.2, then recirculate of the material is continued for 30mins, with pump speed of 40rpm (90 ± 10 ml/min flow rate) for both first pass and recirculation. After 30mins of recirculation, the material is recovered and collected from the first filter, OD260nm is noted and recorded. If the OD260nm is <0.2/more preferably less than 0.1, the material is fully recovered by giving a wash of 500-600ml MQW. If the OD260nm was >0.2, the first filter is kept aside without flushing, the collected filtrate is circulated through the second filter with a pump speed of 40rpm (90 ± 10 ml/ min flow rate) and the material is recirculated with a masterflex pump speed between 150-160rpm (420 ± 20 ml/ min flow rate) until the OD260nm reached <0.2/more preferably less than 0.1. After the desired O.D. is achieved, the recovered material kept aside. Both the filters are joined in series and a wash of 500-600ml is given to recover the filter bound PS, if any. The wash is then mixed with the recovered material.

Claims

We claim:

1. A process for purifying Neisseria meningitidis serogroup C, W and Y polysaccharides, wherein said process comprises the steps of:

(a) centrifugation of the inactivated bacterial fermenter culture to clarify the fermented broth to obtain fermented supernatant;

(b) concentrating the fermented supernatant of step (a) by diafiltration with PES membrane to obtain diafiltered concentrate;

(c) Adjusting the pH and incubating the diafiltered concentrate of step (b);

(d) cooling the concentrate of step (c) to room temperature and subjecting it to diafiltration and concentration to obtain clarified concentrate;

(e) mixing the clarified concentrate of step (d) with tri-sodium citrate (TSC), sodium sulfate (SS) and sodium deoxycholate (DOC) and incubating at pre-determined temperature for pre-determined duration;

(f) treating the incubated solution of step (e) with ethanol at pre- determined temperature for pre-determined duration;

(g) concentration and diafiltration of the resultant mixture of step (f) to obtain partially purified polysaccharide;

(h) carbon filtration of the partially purified polysaccharide mixture of step (g) with Millistak carbon filters to obtain purified polysaccaharide;

wherein the said purification process is completed rapidly in less than 12 hours.

2. The process as claimed in claim 1, wherein said diafiltration is carried out by 30kDa polyether sulfone (PES) membrane for Men C and 100 kDa polyether sulfone (PES) membrane for Men Y and Men W.

3. The process as claimed in claim 1, wherein the pH of the said diafiitered supernatant is adjusted in the range of 13+0.1

4. The process as claimed in claim 1, wherein said tri-sodium citrate concentration is in the range 0.25M-0.40 M, said sodium sulfate concentration is in the range of 25% -50% v/v of 2M stock and said sodium deoxycholate concentration is in the range of 1.3% -1.6 % w/v.

5. The process as claimed in claim 1, wherein incubation of the said diafiitered concentrate of step (b) is done at 90°C± 5°C for 2±0.5 hrs.

6. The process as claimed in claim 1, wherein incubation of said concentrate of step (e) is done at 37°C±2 for 3±1 hr.

7. The process as claimed in claim 1, wherein concentration of said ethanol is in the range of 30%± 5% v/ v.

8. The process as claimed in claim 1, wherein carbon filtration is carried out with water primed millistak POD filters in the range 0.027m²- 0.054m².

9. The process as claimed in claim 1, wherein each diafiltration step is carried out with Milli Q water.

10. The process as claimed in claim 1, wherein the purification process for Men C is completed in 8-9 hours.

11. The process as claimed in claim 1, wherein the purification process for Men Y and Men W is completed in 10-12 hours.

12. The process as claimed in claim 1, wherein the yield for purified polysaccharide Men C is 290±10mg/L, purified polysaccharide Men Y is 344±10 mg/L and purified polysaccharide Men W is 290±10 rng/L