FIELD OF THE INVENTION:

The present invention relates to a rapid high yielding purification process for Neisseria meningitidis serogroup X capsular polysaccharide. The N. meningitidis serogroup X bacterial capsular polysaccharide of the present invention is capable of being used in the production of economical monovalent capsular polysaccharide or polysaccharide protein conjugate vaccine or multivalent combination vaccines as well as a diagnostic tool against meningococcal serogroup X infections.

BACKGROUND OF THE INVENTION:
N. meningitidis (meningococcus or Men) is an aerobic gram-negative bacterium that has been serologically classified mainly into 13 serogroups A, B, C, D, 29E, H, I, K, L, W135, X, Y and Z. The grouping system is based on the capsular polysaccharides of the organisms.
WHO official website states that N. meningitidis is one of the most common causes of bacterial meningitis in the world and is the only bacterium capable of generating large epidemics of meningitis. Epidemics with incidence rates of up to 1000 cases per 100,000 inhabitants have been reported, particularly in sub-Saharan Africa.
N. meningitidis is transmitted by aerosol or direct contact with respiratory secretions of patients or healthy human carriers. As a rule, endemic disease occurs primarily in children and adolescents, with highest attack rates in infants aged 3-12 months, whereas in epidemics older children and young adults may be more involved. However, the rapid progression of meningococcal disease frequently results in death within 1-2 days after onset. N. meningitidis infections can be prevented by vaccination.
MenA has been the most prevalent serogroup in Africa and Asia, but is rare/ practically absent in North America. In the Europe and United States, serogroup B (MenB) is the predominant cause of disease and mortality, followed by serogroup MenC and MenW. In recent past, MenX outbreaks have started showing up in sub-Saharan Africa. According to Xie O et al, “Emergence of serogroup X meningococcal disease in Africa: need for a vaccine”, Vaccine. 2013 Jun 12;31(27):2852-6, Serogroup X meningococci have previously been considered a rare cause of sporadic meningitis, but during 2006-2010, outbreaks of serogroup X meningitis occurred in Niger, Uganda, Kenya, Togo and Burkina Faso, the latter with at least 1300 cases of serogroup X meningitis among the 6732 reported cases.

According to another NCBI publication, in Togo during 2006-2009, serogroup X meningococci accounted for 16% of the 702 confirmed bacterial meningitis cases. Kozah district experienced a serogroup X meningococci outbreak in March 2007 with a serogroup X meningococci seasonal cumulative incidence of 33/100,000. In Burkina Faso during 2007-2010, serogroup X meningococci accounted for 7% of the 778 confirmed bacterial meningitis cases, with an increase from 2009 to 2010 (4% to 35% of all confirmed cases, respectively). In 2010, serogroup X meningococci epidemics occurred in northern and central regions of Burkina Faso; the highest district cumulative incidence of serogroup X meningococci was estimated as 130/100,000 during March-April.

The multiple serogroups have hindered development of a universal vaccine for meningococcal disease. The production of the first meningitis polysaccharide vaccine was accomplished in 1978 as there was an urgent need to combat this fatal disease. Later it was observed that the plain polysaccharide vaccines were not very efficient in children below two years of age. These observations led to further research which revealed that infants have an immature immune system and cannot elicit immune response against plain polysaccharides.

The immune response may be characterized as T-cell dependent (TD) immune response and T-cell independent (TI) immune response. Proteins and peptides are known to elicit TD antigens by stimulating the helper T lymphocytes and generating memory cells. In contrast, polysaccharides belong to the TI antigens which do not induce T-cell activation and do not form any memory B cells, which is a major drawback while dealing with infants as they have an immature immune system.

Thus, there was a need for conjugating the bacterial polysaccharide to a protein carrier which induces a T-cell-dependent immune response characterized by increased immunogenicity among infants, prolonged duration of protection and in the reduction of nasopharyngeal carriage of meningococci. This need was fulfilled by ingenious research resulting in the production of polysaccharide-protein conjugate vaccines and the first meningococcal conjugate vaccine was licensed in United Kingdom in 1999.

The polysaccharides, especially antigenic capsular polysaccharides, used in preparation of vaccines may be monovalent, bivalent and poly (multi) valent vaccines containing one, two or more polysaccharides, respectively. These are readily available in the market for prevention of certain diseases or infections caused by various microorganisms. The monovalent or multivalent polysaccharide based vaccines have been used for many years and have proved valuable in preventing diseases such as Pneumococcal, Meningococcal or Haemophilus influenzae diseases. However, there is no licensed vaccine containing meningococcal serogroup X conjugate.
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 the production and purification steps would lead to formulation of efficacious and economically viable conjugate vaccines.

There are number of patents and non-patent disclosures that describe the processes of production and purification of polysaccharides. In one non-patent literature, David Bundle et al, “Studies on the Group-specific Polysaccharide of Neisseria meningitidis Serogroup X and an improved Procedure for its isolation” Journal of Biological Chemistry, 1974, discusses preparation and isolation of Men X polysaccharide from N. meningitidis strain 247 X wherein said strain was grown on a chemically defined medium (NCDM) for 18 hours and yields about 20 mg/L. Another disclosure is the US2015/0299750, Pisal Sambhaji Shankar et al, which relates to purification of Men X using novel fermentation medium, optimal feed solution including addition strategies and an improved purification process devoid of any chromatographic methods. The disclosed patent application uses multiple steps for the purification of crude polysaccharides and long hours for the purification process.

Presently, the various methods used for the purification of N. meningitidis serogroup X take relatively long purification time thereby increasing the cost of production and making the process commercially less feasible since they cannot be scaled up in a cost-effective and timely manner. Further, they include inflammable solvents at multiple stages.

It is an object of the present invention to provide improved process of purification of N. meningitidis serogroup X bacterial polysaccharide at reduced time and with high yield using the simple salts such as tri-sodium citrate and sodium sulfate and require lesser amount of inflammable solvants. Said improved process of purification of the present invention will result in manufacturing polysaccharide protein conjugate vaccine at lesser price and subsequently vaccine can be made available to children especially of developing countries at an affordable price.

OBJECT OF THE INVENTION:

The main object of the present invention is to provide a rapid high yielding purification process for Neisseria meningitidis serogroup X capsular polysaccharide.

Another object of the present invention is to provide a rapid high yielding purification process for Neisseria meningitidis serogroup X polysaccharide deploying simple salts to precipitate impurities.

Another object of the present invention is to provide a rapid high yielding purification process for Neisseria meningitidis serogroup X polysaccharide deploying reduced amount of inflammable solvants.

Yet another object of the present invention is to provide a rapid high yielding purification process for Neisseria meningitidis serogroup X polysaccharide which is simple, cost effective, time efficient and commercially scalable.

Yet another object of the present invention is to provide a rapid high yielding purification process to obtain a purified Neisseria meningitidis serogroup X polysaccharide capable of being used in the production of economical capsular polysaccharide or polysaccharide protein conjugate monovalent vaccine or multivalent combination vaccines as well as a diagnostic tool against meningococcal serogroup X infections.

Yet another object of the present invention is to produce high quality product with better yield that meets the relevant quality specifications.

SUMMARY OF THE INVENTION:

Accordingly, the present invention provides a rapid high yielding purification process for Neisseria meningitidis serogroup X capsular polysaccharides The purified N. meningitidis serogroup X capsular polysaccharides obtained from the process of present invention is capable of being used in the production of economical monovalent capsular polysaccharide or polysaccharide protein conjugate vaccine or multivalent combination vaccines as well as a diagnostic tool against meningococcal serogroup X infections.

The said purification process provides a purified N. meningitidis serogroup X (also referred to as MenX) polysaccharide at a significantly reduced time. Said purification process does not require any chromatography step.

The purification process of present invention deploys simple salt such as trisodium citrate and sodium sulphate in the optimum concentrations. The crude polysaccharide of the fermentation broth is mixed with simple salts and subjected to centrifugation and diafiltration with 100KDa MWCO polyether sulfone membrane to form a solution with reduced impurities. The said solution with reduced impurity is treated with a cationic reagent preferably quaternary ammonium surfactant (CTAB) and subjected to mixing at room temperature.

The resultant solution after mixing, is subjected to centrifugation to obtain pellets. The pellet is dissolved in salt and ethanol and subjected to incubation. The resultant incubated solution is again centrifuged to obtain pellet which is dissolved in MQW along with salts, detergent and ethanol and then subjected to mixing. The resultant solution is again subjected to centrifugation and supernatant is collected.

The collected supernatant is subjected to the carbon filtration followed by the concentration and diafiltration with Milli Q water (MQW) and finally subjected to sterile filtration and the purified bacterial polysaccharide is stored.

The purified polysaccharide is thus recovered in a significantly reduced time using a scalable, cost-effective and efficient method.

The process exhibits a number of advantages over prior art, such as providing a robust and rapid method of producing purified polysaccharides meeting the desired specifications with better yields and minimal use of inflammable solvents. An additional advantage is that this process is entirely scalable.
The purified bacterial polysaccharides of the present invention is capable of being used in the production of economical monovalent capsular polysaccharide or polysaccharide protein conjugate vaccine or multivalent combination vaccines as well as a diagnostic tool against meningococcal serogroup X infections.

BRIEF DESCRIPTION OF DRAWINGS
Figure-1 depicts the process flow for the MenX-PS purification
Figure-2 depicts the HPLC Chromatogram of purified MenX PS using RI detector
Figure-3 depicts the 1H-NMR spectrum of purified MenX PS

DETAILED DESCRIPTION OF THE INVENTION WITH NON-LIMITING EXAMPLES AND ILLUSTRATIONS

The present invention provides a rapid high yielding purification process for Neisseria meningitidis serogroup X capsular polysaccharides. The purified N. meningitidis serogroup X capsular polysaccharides obtained from the process of present invention is capable of being used in the production of economical monovalent capsular polysaccharide or polysaccharide protein conjugate vaccine or multivalent combination vaccines as well as a diagnostic tool against meningococcal serogroup X infections.

The said purification process provides a purified N. meningitidis serogroup X (also referred to as MenX) polysaccharide at a significantly reduced time. Said purification process does not require any chromatography step.

The purification process of present invention deploys simple salts such as trisodium citrate and sodium sulphate in the optimum concentrations. The crude polysaccharide of the fermentation broth is mixed with simple salts and subjected to centrifugation and diafiltration with 100KDa MWCO polyether sulfone (PES) membrane to form a solution with reduced impurities. The said solution with reduced impurity is treated with a cationic reagent preferably quaternary ammonium surfactant (CTAB) and subjected to mixing.

The resultant mixture is subjected to centrifugation to obtain pellets. The pellets are dissolved in salt and ethanol and subjected to incubation. The resultant incubated solution is again centrifuged to obtain pellet which is dissolved in MQW along with salts, detergent and ethanol and then subjected to mixing. The resultant solution is again subjected to centrifugation and supernatant is collected.

The collected supernatant is subjected to the carbon filtration followed by the concentration and diafiltration with MQW and finally subjected to sterile filtration and purified bacterial polysaccharide stored.

Figure 1 shows the Men X purification process flow. The rapid high yielding purification process for Neisseria meningitidis serogroup X bacterial polysaccharides of the present invention comprises the steps of:
(a) treating a fermentation broth of Neisseria meningitidis serogroup X by adding predetermined simple salt solution and dry salt powder of specific concentration;
(b) adding specific volume of organic solvent to the salt treated solution of step (a);
(c) mixing the solution of step (b) at room temperature for specific duration;
(d) carrying out centrifugation of solution of step (c) at specific rpm for pre-determined period;
(e) collecting the supernatant from the centrifuged solution of step (d);
(f) subjecting said supernatant of step (e) to concentration and diafiltration using a molecular weight cut off membrane to obtain partially purified bacterial polysaccharide;
(g) precipitating said partially purified bacterial polysaccharide of step (f) with at least one cationic detergent under specific condition followed by centrifugation at specific rpm for pre-determined period to obtain first pellet;
(h) dissolving said first pellet in inorganic precipitating agent;
(i) subjecting said dissolved first pellet of step (h) to mixing in at least one organic solvent at predetermined incubation conditions for enhancing precipitation;
(j) subjecting said solution of step (i) to centrifugation under specific conditions followed by collection of second pellet;
(k) dissolving said second pellet in MQW and treating with specific chemical reagents, specific organic solvent followed by incubation at specific conditions;
(l) subjecting said incubated solution of step (k) to centrifugation under specific centrifugation condition followed by collection of supernatant;
(m) subjecting said supernatant of step (l) to carbon filtration to obtain filtrate until desired optical density is achieved;
(n) filtering said carbon filtrate of step (m) using PES filtration assembly followed by concentration and diafiltration under specific conditions;
(o) filtering said diafiltered supernatant of step (n) using specific PES filtration assembly to obtain purified polysaccharide;
(p) storing said purified polysaccharide of step (o) under specific conditions.
The process yields purified polysaccharide of Neisseria meningitidis serogroup X in a range of 142-323 mg/L. The average yield is 200 mg/L. The process of purification completes in 24 ± 2 hours.

In one of the best embodiments, the fermentation broth has been treated by adding 0.25 M tri sodium citrate, 25% v/v sodium sulfate solution from 2M stock solution followed by the addition of 25% v/v of absolute ethanol. The solution has been mixed continuously for 2 hours at room temperature (RT, 25±2°C). The solution has been centrifuged at 10550 x g for 30 minutes and supernatant has been collected. The supernatant has been concentrated and diafiltered with 0.1m2 100 kDa polyether sulfone (PES) membrane with 10-12 volumes of MQW.

The concentrated and diafiltered material has been treated with 15% v/v of 10 % Hexadecyltrimethylammonium bromide (CTAB) stock solution with continuous mixing for 4 hours at RT. Centrifugation at 10550 x g for 40 minutes has been performed and the collected pellet has been dissolved in 0.15 M calcium chloride solution. After the dissolution, 90% v/v of absolute ethanol has been added and incubated overnight at 2-8°C. Centrifugation at 10550 x g for 40 minutes has been performed and the collected pellet has been dissolved properly in MQW and further treated with 10 % w/v sodium acetate, 1.1% w/v sodium deoxycholate (DOC) and 30 % v/v of absolute ethanol with incubation for 2 hours at 2-8°C. Centrifugation at 10550 x g for 30 minutes has been performed and the collected supernatant has been carbon filtered using ethanol primed millistak pod carbon filters until the optical density 260nm reaches = 0.2. The material has been filtered using 0.2µ PES filtration assembly. Afterwards, the filtrate has been concentrated and diafiltered with 0.1m2 100 kDa PES membrane with 15-20 volumes of MQW and then filtered using 0.2µ PES filtration assembly. The purified MenX PS is stored at -20±2°C.

The purification process of the present invention yields at 2.5 L fermentation scale are approximately 142-323 mg/L of purified MenX PS.

The purified polysaccharide is thus recovered in a significantly reduced time using a scalable, cost-effective and efficient method.

The process exhibits a number of advantages over prior art, such as providing a robust and rapid method of producing purified polysaccharides meeting the desired specifications with better yields and minimal use of inflammable solvents. An additional advantage is that this process is entirely scalable.

The purified bacterial polysaccharides of the present invention is capable of being used in the production of economical monovalent capsular polysaccharide or polysaccharide protein conjugate vaccine or multivalent combination vaccines as well as a diagnostic tool against meningococcal serogroup X infections.

The purified polysaccharides have been tested using battery of known analytical tests to control the quality of the polysaccharide. The different assays conducted on each polysaccharide batch have been listed in table 1 along with the results of representative purified MenX polysaccharide batches.

Table-1: Purified MenX Analytics
Parameters Batch 1 Batch 2 Batch 3 Batch 4 Average SD
Phosphorus (%) 10.3 9.73 10.3 10.1 10.1 0.3
Mol. Weight (kD) 456 569 661 554 560.0 83.9
PS content (mg/ml) 1.97 0.9 0.67 0.83 1.1 0.6
Nucleic Acid content (%) 1.34 0.9 0.8 0.7 0.9 0.3
Protein content (%) 0.44 0.44 0.42 0.47 0.4 0.0
Endotoxin (EU/µg) 7.8 7.8 7.8 7.8 7.8 0.0
Molecular size distribution (%) 93.38 99.77 99.23 98.38 97.7 2.9
PS yield (mg/L fermentation broth) 323 152 147 179 200 83
EU: Endotoxin unit; SD: Standard deviation

Fig. 2 shows HPLC-SEC chromatogram of representative purified MenX polysaccharide using RI detector. The HPLC-SEC analysis of the purified polysaccharide is done using TSK 4000 – 5000 PWXL HPLC columns in series and monitored by UV detector using sodium nitrate as running buffer. The single major peak in HPLC-SEC chromatogram and other physico-chemical analyses (Table 1) confirm the purity of the MenX polysaccharide to the desired levels.

Fig. 3 shows 1H-NMR spectrum of representative purified MenX polysaccharide. The 1H-NMR for purified MenX polysaccharide is recorded on Bruker Avance 500 MHz instrument using deuterium oxide (D2O) as a solvent. The spectral peaks confirm the identity of the MenX polysaccharide. The peak at 2 ppm in the spectrum corresponds to the three protons of CH3 group from the N-acetyl group (NH-Ac) present in the polysaccharide monomer structure. The peak at 5.5 ppm represents the anomeric alpha H-1 proton. The broad multiplet at 4.5-3.6 ppm corresponds to the all other protons on the glucosamine ring. The spectrum shows no major peak of impurity indicating the purity of the polysaccharide.

We Claim:
1. A rapid high yielding purification process for Neisseria meningitidis serogroup X capsular polysaccharide, wherein the said process comprises the steps of:
(a) treating a fermentation broth of Neisseria meningitidis serogroup X by adding predetermined simple salt solutions and dry salt powder of specific concentration;
(b) adding specific volume of organic solvent to the salt treated solution of step (a);
(c) mixing the solution of step (b) at room temperature for specific duration;
(d) carrying out centrifugation of solution of step (c) at specific rpm for pre-determined period;
(e) collecting the supernatant obtained from the centrifuged solution of step (d);
(f) subjecting said supernatant of step (e) to concentration and diafiltration using a molecular weight cut off membrane to obtain partially purified bacterial polysaccharide;
(g) precipitating said partially purified polysaccharide of step (f) with at least one cationic detergent under specific condition followed by centrifugation at specific rpm for pre-determined period to obtain first pellet;
(h) dissolving said first pellet in an inorganic precipitating agent;
(i) adding to the said dissolved first pellet of step (h) at least one organic solvent followed by incubation at predetermined conditions;
(j) subjecting said solution of step (i) to centrifugation under specific conditions followed by collection of second pellet;
(k) dissolving said second pellet in MQW and treating with specific chemical reagents and specific organic solvent followed by incubation at specific conditions;
(l) subjecting said incubated solution of step (k) to centrifugation under specific centrifugation condition followed by collection of supernatant;
(m) subjecting said supernatant of step (l) to carbon filtration to obtain carbon filtrate until desired optical density is achieved;
(n) filtering said carbon filtrate of step (m) using PES filtration assembly followed by concentration and diafiltration under specific conditions;
(o) filtering said diafiltered supernatant of step (n) using specific PES filtration assembly to obtain purified polysaccharide;
(p) storing said purified polysaccharide of step (o) under specific conditions.
wherein said process is economic, scalable and yields purified polysaccharide of Neisseria meningitidis serogroup X in a range of 147-323 mg/L of fermentation broth.

2. The process as claimed in claim 1 wherein said predetermined simple salt solution and dry salt powder of specific concentration are 0.25 ± 0.1 M tri sodium citrate and 25 ± 5% v/v sodium sulfate solution from 2M stock solution.

3. The process as claimed in claim 1 wherein said organic solvent of step (b) is 25 ± 5% v/v of absolute alcohol.

4. The process as claimed in claim 1 wherein said specific duration of step (c) for mixing is 2 ± 0.5 hours.

5. The process as claimed in claim 1 wherein said centrifugation in step (d) is carried out at 10000-11000 x g for 30 ± 5 minutes.

6. The process as claimed in claim 1 wherein said diafiltration in step (f) is carried out with 100 kDa PES membrane with 10-12 volumes of MQW.

7. The process as claimed in claim 1 wherein said cationic detergent of step (g) is 15 ± 2% v/v of 10% CTAB stock solution.

8. The process as claimed in claim 1 wherein said specific condition in step (g) for precipitation with cationic detergent is 4 ± 1 hours at room temperature.

9. The process as claimed in claim 1 wherein said centrifugation in step (g) is carried out at 10000-11000 x g for 40 ± 10 minutes.

10. The process as claimed in claim 1 wherein said inorganic precipitating agent is calcium chloride solution.

11. The process as claimed in claim 1 wherein said at least one organic solvent in step (i) is 90 ± 5% v/v of absolute ethanol.

12. The process as claimed in claim 1 wherein said pre-determined incubation condition in step (i) is overnight incubation at 2-8 ?C.

13. The process as claimed in claim 1 wherein said specific condition of centrifugation is 10000-11000 x g for 40 ± 10 minutes.

14. The process as claimed in claim 1 wherein said specific chemical reagents of step (k) includes but limited to 10 ± 2 % w/v sodium acetate and 1.1 ± 0.1 % w/v deoxycholate.

15. The process as claimed in claim 1 wherein said specific organic solvent of step (k) is 30 ± 5 % v/v of absolute ethanol.

16. The process as claimed in claim 1 wherein said specific conditions for incubation of step (k) are 2-8 ?C for 2 ± 0.5 hours.

17. The process as claimed in claim 1 wherein said specific conditions for centrifugation of step (l) is 10000-11000 x g for 30 ± 5 minutes.

18. The process as claimed in claim 1 wherein said carbon filtration of step (m) is carried out with ethanol primed millistak pod carbon filters.

19. The process as claimed in claim 1 wherein said desired optical density of step (m) is OD260nm less than or equal to 0.2.

20. The process as claimed in claim 1 wherein said specific conditions for concentration and diafiltration in step (n) is 100 kDa cutoff PES membrane with 10-20 volumes of MQW.

21. The process as claimed in claim 1 wherein said specific PES filtration assembly of step (o) is 0.2µ PES filtration assembly.

22. The process as claimed in claim 1 wherein said specific conditions for storing said purified polysaccharide is -20 ± 20 C.

23. The process as claimed in claim 1 wherein said process is in compliance with polysaccharide specific quality standards.

24. The process as claimed in claim 1 wherein yield of purified polysaccharide of Neisseria meningitidis serogroup X is up to 323 mg/L.

25. The process as claimed in claim 1 wherein said process to obtain said purified polysaccharide of Neisseria meningitidis serogroup X the purified polysaccharide completes in 24 ± 2 hours.

26. The process as claimed in claim 1 wherein said purified bacterial polysaccharide is capable of being used in the production of economical monovalent capsular polysaccharide or polysaccharide protein conjugate vaccine or multivalent combination vaccines as well as a diagnostic tool against meningococcal serogroup X infections.