FIELD OF THE INVENTION:
The present invention relates to production of bacterial polysaccharides. The present invention particularly relates to culture media composition, feed composition and fermentation conditions for production of Neisseria meningitidis polysaccharides. The N. meningitidis polysaccharides of the present invention are capable of being used in the production of economical polysaccharide protein conjugate vaccine(s) against meningococcal infections.
BACKGROUND OF THE INVENTION:
Neisseria meningitidis, often referred to as meningococcus, is a Gram-negative bacterium that can cause meningitis and other forms of meningococcal disease such as meningococcemia.
On the basis of the type of capsular polysaccharide present on N. meningitidis (Men), thirteen serogroups have been identified and among the 13 identified capsular types of N. meningitidis, six (A, B, C, W135, X, and Y) account for most meningococcal disease cases worldwide. MenA has been the most prevalent serogroup in Africa and Asia but is rare/ practically absent in North America. In 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. 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 based

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 characterised 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 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 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.

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 for production of capsular polysaccharides is generally high and involves long working hours since it involves a series of production and quality control steps. An optimized medium/process can obviate these issues.
Improvement in the polysaccharide production steps would lead to formulation of efficacious and economically viable conjugate vaccines.
There are a number of patents and non-patent disclosures that describe the processes of production and purification of polysaccharides. One such disclosure is patent application no. US 12/041,745 discloses a method of producing a meningococcal meningitis vaccine, the method, includes culturing N. meningitidis to produce capsular polysaccharides of serogroups A, C, Y and W-135 in N. meningitidis fastidious medium (NMFM), isolating the capsular polysaccharides from the culture, purifying the capsular polysaccharides of any residual cellular biomass; and depolymerizing the capsular polysaccharide mechanically. The cited art utilizes long hours for the production of purified capsular polysaccharide.
Another US patent publication no. US 20150299750 Al discloses an improved culture, fermentation and purification conditions for preparing Neisseria meningitidis polysaccharides. Another US patent publication no.: 20080318285 Al discloses Neisseria meningitidis fastidious medium designed to maximize the yield of capsular polysaccharides and

generate minimal cellular bio mass and endotoxin in a short duration of fermentation.
It is an object of the present invention to provide improved culture media and feed media, for better production of N. meningitidis polysaccharides by fermentation in reduced time and with high yields. Said improvements will result in manufacturing polysaccharide protein conjugate vaccine at lesser price and subsequently vaccine can be made available to children of developing countries at an affordable rate.
QBTECT OF THE INVENTION:
The main object of the present invention is to provide a process of production of bacterial polysaccharide.
Another object of the present invention is to provide a process of production of capsular polysaccharides of various serogroups of Neisseria meningitidis.
Yet another object of the present invention is to provide optimized culture media and feed media composition for better growth.
Yet another object of the present invention is to provide improved culture media composition for growth of fastidious Neisseria meningitidis serogroups A, C, W, and Y.
Yet another object of the present invention is to provide improved feed media composition for growth of fastidious Neisseria meningitidis serogroups A, C, W, and Y.

Yet another object of the present invention is to provide process of fermentation in reduced time with better polysaccharide yield with low impurities in a very short time by simple, efficient, improved and commercially scalable methods.
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 growth of bacteria preferably Neisseria meningitidis for production of bacterial polysaccharide.
The present invention describes culture media for N. meningitidis including but not limited to di-sodium hydrogen phosphate in the range of 3.00 ± 1.0 g/L, potassium phosphate monobasic in the range of 0.8125 ± 0.01 g/L, L-cystine in the range of 0.05 ± 0.01 g/L, Magnesium sulphate in the range of 0.3 ± 0.1 g/L, beta alanine in the range of 0.025 ± 0.01 g/L, L-glutamic acid in the range of 1.5 ± 0.25 g/L, casamino acid in the range of 12.00 ± 2 g/L, yeast extract in the range of 10.00 ± 2 g/L and dextrose in the range of 10.00 ± 2.0 g/L, The above-mentioned culture media composition provides optimal growth for N. meningitidis serogroups.
The present invention also describes feed media for N. meningitidis including but not limited to L-glutamic acid in the range of 8.00 ± 2.0g/L, dextrose in the range of 25 ± 2.0 g/L, soya peptone in the range of 20 ± 2.0 g/L and other components like ammonium chloride as per the requirement. The above-mentioned feed media composition provides optimal growth for N. meningitidis serogroups when added in the

fermentation broth during fermenter culture with the aforementioned culture media.
The present invention describes the fermentation process at predetermined temperature, pH, airflow, dissolved oxygen and rate of agitation, such that the fermentation is completed within 11 ± 3 hours.
BRIEF DESCRIPTION OF DRAWINGS:
Figure-1 depicts Shake Flask studies for media optimization using MenW
(4 media compositions)
Figure-2 PS concentration of MenW with different media compositions by
inhibition ELBA
Figure-3 depicts growth curves of MenA
Figure-4 depicts growth curves of MenC
Figure-5 depicts growth curves of MenY
Figure-6 depicts growth curves of MenW
Figure-7 depicts NMR profile of MenA
Figure-8 depicts NMR profile of MenC
Figure-9 depicts NMR profile of MenY
Figure-10 depicts NMR profile of MenW
DETAILED DESCRIPTION OF THE INVENTION:
The present invention discloses a process of production of bacterial polysaccharides. The present invention particularly relates to optimized culture media and feed media for growth of fastidious Neisseria meningitidis in lesser time. The invention also relates to fermentation conditions for production of Neisseria meningitidis polysaccharides. The N. meningitidis polysaccharides of the present invention are capable of being

used in the production of economical polysaccharide protein conjugate vaccine(s) against meningococcal infections.
Before the preferred embodiment of the present invention is described, it is understood that this invention is not limited to the particular materials described, as they may vary. It is also understood that the terminology used herein is for the purpose of describing the particular embodiment only and is not intended to limit the scope of the invention in any way.
In a preferred embodiment the present invention describes culture media for N. meningitidis comprising sodium phosphate dibasic in a concentration of 3.00 g/L, potassium phosphate monobasic in the concentration of 0.8125 g/L, L-cystine in the concentration of 0.05 g/L, magnesium sulphate in the concentration of 0.3 g/L, beta alanine in the concentration of 0.025 g/L, L-glutamic acid in the concentration of 1.5. g/L, casamino acid in the concentration of 12.0 g/L, yeast extract in the concentration of 10 g/L, dextrose in the concentration of 10 g/L and ammonium chloride in the concentration of 2.00 g/L.
All the above optimized concentrations of culture media are listed in Table 2 of the specification. The above-mentioned culture media composition provides optimal growth for N. meningitidis serogroups MenA, MenC, MenY and MenW. Ammonium chloride is added only to Serogroups W for optimal polysaccharide production.
The present invention also describes feed media for N. meningitidis including but not limited to L-glutamic acid in the range of 8.00 ± 2.0g/L, dextrose in the range of 25.00 ± 2.0 g/L and soya peptone in the range of

20.00 ± 2.0 g/L. The above-mentioned feed media composition provides optimal growth for N. meningitidis serogroups.
The above-mentioned feed media composition provides optimal growth for N. meningitidis serogroups MenA, MenC, MenY and MenW. The optimized feed composition is listed in Table 3 of the specification.
After growth of bacteria in flask with optimized culture media, the bacteria are subjected to fermentation as disclosed in Example 5 and Example 6 of the specification. The fermentation conditions are so optimized that the resultant fermentation harvest (broth) have high polysaccharide yield and low level of impurities and the fermentation process is completed within 11 ± 3 hours, preferably 10 to 12 hours.
In a preferred embodiment, the fermentation is carried out in a temperature range of 36 ± 1° C with rpm in the range of 100 to 700 rpm, the air flow of the fermenter is maintained at 0.2 to 1.2.1/m and the partial pressure of Oxygen (PO2) is maintained from 100% to 15% during the course of fermentation along with a pH of 7.3 ± 0.1.
Therefore, the present invention provides a rapid, industrially scalable, cost effective process for the production of Neisseria meningitidis serogroups MenA, MenC, MenY and MenW with optimized culture media and feed media which provides maximum growth to the Neisseria meningitidis.
Various aspects of the invention described in detailed above is now illustrated with non-limiting examples:

Example-1: Shake Flask experiments for media optimization using
MenW
Shake Flask study 1:
Four shake flasks and each having different compositions of fastidious media as disclosed in Tablel are used for media optimization of Neisseria meningitidis serogroup W (MenW). The ODssomn of flask culture is recorded after every 2 hours, until 12th hour for all the four flasks. The growth curves are presented in Figure-1. The culture samples are inactivated at the 12th hour with 1% v/v formalin and are tested for the polysaccharide (PS) concentration using inhibition ELISA for all the four flasks at the 10th and 12th hour. Based on the inhibition ELISA results (Figure 2), media compositions 2 and 3 are shortlisted as both of them produced high PS for MenW. In an endeavor to improve the media further for better growth, both media 2 and 3 were merged and a new media composition (table-2) is finally selected to conduct fermentation experiments for MenW and other serogroups (A,C and Y).

Table-1: Shake flask Study using Menl IV
Media Media 1 Media
2 Media 3 Media 4
S.no. Media Composition 8/L 8/L 8/L 8/L
1 Sodium phosphate dibasic 3.25 3.25 3.25 3.25
2 Potassium
dihydrogen
phosphate NA NA 0.8125 NA
3 Sodium dihydrogen phosphate dihydrate 1.625 1.625 NA NA
4 KC1 0.09 0.09 0.09 0.09
5 Select phytone NA NA NA 15
6 TC yeastolate NA NA NA 10
7 Mono sodium glutamate 1 2 NA 2
8 Dextrose 15 25 10 20

9 L-cystine 0.03 0.03 0.03 0.03 (L-cysteine)
10 Magnesium sulfate 0.6 0.6 0.3 0.6
11 Ammonium chloride 2.5 NA 2 (at 8 hrs of fermentation) 2 (at 4 hrs of fermentation)
12 Casamino acid 15 10 12 NA
13 Yeast extract 10 10 10 NA
14 Soyatone NA NA 20 NA
15 NAD 0.25 NA 0.25 0.25
16 Thiamine HC1 0.1 NA NA NA
17 P- alanine NA NA 0.025 NA
18 NaCl NA 2 NA NA
19 L-glutamic acid NA NA 1.5 NA
NA- Not applicable
-Example-2: Inhibition ELISA Protocol
Inhibition ELBA method is used for estimation of the polysaccharide content in the bacterial culture broth. In this the sample containing meningococcal capsular polysaccharide is incubated with the serogroup specific polyclonal antibody (primary antibody) so that complexes will be formed between the antibody and antigens in the sample. These complexes are then added to a container in which competitor homologous antigens are immobilized. Antibody which is not complexed with immunogens from the polysaccharide test sample bind to these immobilized competitor antigens. The antibody which is bound to the immobilized competitor antigens (after usual washing steps, etc.) can then be detected by adding an enzyme labelled secondary antibody which binds to the primary antibody. The label is used to identify the reaction of immobilized primary antibody to secondary antibody utilizing a chromogenic substrate. The reduction in the absorbance in test well as compared to the control well (without any test sample) confirms the presence of the specific antigen in the test sample and the percentage

inhibition of the antibody is directly proportional to the polysaccharide concentration in the test sample.
Briefly, the ELISA is performed, wherein the Plate A is coated with lOOjxl of coating solution having equal volume of in-house PS and mHSA and incubated for overnight at 2-8°C. A no-antigen-control is included as control. The coated plate is blocked at room temperature with 200(xl of blocking buffer. Quality control polysaccharide (Standard) of defined concentration are serially diluted three-fold as are the bacterial culture supernatant (test samples) and incubated in Plate B with serogroup specific polyclonal primary antibody for 1 hour at 37°C. The antigen-antibody mixture from Plate B is transferred to blocked Plate A and further incubated for two hours (1.5 hours at 37°C and half an hour at room temperature). The plate is further incubated with secondary antibody for 1 hour and reaction is developed using lOOjxl of TMB substrate and incubated for 10 min. The reaction is stopped with 50jxl of 2M H2SO4 per well before OD at 450 nm is observed with reference to 630nm. The inhibition percentage is calculated from inhibition of OD in standard or test sample dilutions in relation to OD of no-antigen control wells. The standard curve is generated from inhibition percentages for quality control dilutions which is used to extrapolate the concentration of polysaccharide in the test samples using Combistat software (Figure- 1).
Example-3: Optimized media composition
Both media compositions 2 and 3 are merged and are finally selected and taken forward for scale-up/fermentation experiments (2.5L scale) each for Men A, C, Y and W serogroups. The growth of all the serogroups is depicted in Figures 3-6. The final fermenter media composition is described in Table-2 below.

Table 2: Optimized media composition

S.No Components Concentration g/L
1 Sodium phosphate dibasic 3
2 Potassium phosphate monobasic 0.8125
3 L-cystine 0.05
4 Magnesium sulphate 0.3
5 beta-alanine 0.025
6 L-glutamic acid 1.5
7 Casamino acid 12
8 Yeast extract 10
9 Dextrose 10
Example-4: Feed composition
The Feed composition is finalized during the fermentation experiments and is described in Table-3 for the production of MenA, C, Y and W. Furthermore to achieve higher growth for MenW, ammonium chloride is added at the 10th hour of fermentation in the range of 2 ± 0.2 g/L in the feed media
Table-3: Optimized feed media

S.No Reagents Concentration (g/L)
1 L- glutamic acid 8.00
2 Dextrose 25.00
3 Soya peptone 20.00
The above-mentioned feed composition as listed in Table-3 is unique and supports better growth of all serogroups (MenA, MenC , MenY, and MenW).
The nutrient fermentation media and feed components utilized in the present invention are cost effective, simple and lead to low cellular biomass production with low levels of endotoxins and thus result in

polysaccharide which has minimal level of impurities in the harvested fermentation broth.
Example 5: Fermentation Procedure:
One WCB vial is withdrawn from deep freezer and transfered into mini cooler. The vial is thawed inside the biosafety cabinet and one modified GC agar plate is aseptically streaked with the help of inoculation loop. Afterwards, the plate is incubated at 36 ± 1°C with 5 ± 0.5% C02 for 24 to 36 hours. Colony morphology is checked on modified GC agar plate and Gram staining is done to check the purity. Gram negative, non-spore forming, non- motile, encapsulated, diplococci, appeared under the microscope. Consequently, one flask is inoculated with 4-5 colonies and incubated at 36 ± 1°C with 5 ± 0.5% C02 at 150 ± 5 rpm for 4 to 6 hours. After 4 to 6 hours OD at 550nm and purity by Gram staining is checked. When the growth reaches an ODssomn of 1.00 ± 0.1, the flask culture is aseptically inoculated into the fermenter.
Example 6: Fermentation Conditions:
After approx. 15 minutes of inoculation, p02 tends to decrease. Maintain the p02 at 30% for initial 6 hours of fermentation, followed by 20% p02 from 7th hour to 8th hour of fermentation and 15% p02 from 8th hour of fermentation till the end of fermentation run by increasing rpm and airflow. If the p02 is not controlled by rpm, compressed air is replaced with pure oxygen. OD (at 550nm) \ and purity of culture is checked after every 2 hours. As soon as the OD at 550nm of culture reaches 1 ± 0.1 the addition of feed is initiated at a flow rate of 1 ml/min.
The fermentation is carried out in optimized conditions as enumerated in Table 4 below:

Table 4: Fermenter conditions

S.No Parameters Range
1 Temperature 36 ± 1°C
2 rpm 100 rpm to 700 ± 5 rpm
3 Air flow 0.2 to 1.2 1/m
4 pH 7.3± 0.1
5 PO2 100 % to 15 %
Example 7: Inactivation and Harvesting fermentation broth
As the OD at 550nm starts to decrease or pH starts to increase or both happen simultaneouly, inactivation of fermented broth is done for 4±1 hrs and the inactivation is done by adding 1.0% (v/v) formalin solution at 36 ± 1°C. The inactivated fermented broth is collected in 1L centrifuge bottles and centrifuged at 10550 x g for 30 minutes. Supernatant is collected and pellets discarded. Purification of fermentation broth is started consequently.
Example 8: Purified PS yields for MenA, C, Y and W
The average purified PS yields for MenA, C, Y and W is given below in
table-5. Structural identity of polysaccharide purified with the
fermentation harvest as start material has been depicted in Fig. 7,8,9 and
10.
Table-5: Average purified PS yields for MenA, C, Y and W

Purified PS yields at 2.5L fermentation scale
Serogroup Yield
MenA 205±10 mg/L 20
MenC 290±10 mg/L
MenY 344±10 mg/L
MenW 290±10 mg/L

Thus, the present invention provides improved culture and feed media, for better production of N. meningitidis polysaccharides by fermentation in reduced time with high yields.

We claim:
1. A process for the production of Neisseria meningitidis polysaccharides of serogroups A, C, Y and W wherein said process comprises of providing a culture media, a feed media and specific fermentation process parameters for rapid growth of the Neisseria meningitides with enhanced yield.
2. The process as claimed in claim 1 wherein said culture media for the production of Neisseria meningitidis polysaccharides of said serogroups A, C and Y comprises of combination of two or more ingredients out of:

- sodium phosphate dibasic
- potassium phosphate monobasic
- L-cystine
- magnesium sulphate
- beta alanine
- L-glutamic acid
- casamino acid
- yeast extract
- dextrose
wherein said combination provides for rapid growth of Neisseria meningitidis polysaccharides of serogroups A, C and Y with enhanced yield.
3. The process as claimed in claim 2 wherein said culture media
comprises of the following in the concentration range of:
Ingredients Concentration (g/L)
sodium phosphate dibasic 3.00 ± 1.0

potassium phosphate monobasic 0.8125 ± 0.01
L-cystine 0.05± 0.01
magnesium sulphate 0.3 ± 0.1
beta alanine 0.025± 0.01
L-glutamic acid 1.5± 0.25
casamino acid 12.0± 2
yeast extract 10±2
dextrose 10± 2.0
4. The process as claimed in claim 1 wherein said culture media for
the production of Neisseria meningitidis polysaccharides of
serogroup W comprises of combination of two or more of:
- sodium phosphate dibasic
- potassium phosphate monobasic
- L-cystine
- magnesium sulphate
- beta alanine
- L-glutamic acid
- casamino acid
- yeast extract
- dextrose ammonium chloride
wherein said combination provides for rapid growth of Neisseria meningitidis polysaccharides of serogroup W with enhanced yield.
5. The process as claimed in claim 4 wherein said culture media
comprises of the following in the concentration range of:
Ingredients Concentration (g/L)
sodium phosphate dibasic 3.00± 1.0

potassium phosphate monobasic 0.8125± 0.01
L-cystine 0.05± 0.01
magnesium sulphate 0.3± 0.1
beta alanine 0.025± 0.01
L-glutamic acid 1.5± 0.25
casamino acid 12.0± 2
yeast extract 10±2
dextrose 10±2
Ammonium chloride 2.00 ± 0.2
6. The process as claimed in claim 1 wherein said feed media for the
production of Neisseria meningitidis polysaccharides of serogroups
A, C, Y and W comprises of combination of two or more of:
- L-glutamic acid
- dextrose
- soya peptone
wherein said combination provides for rapid growth of Neisseria meningitidis polysaccharides with enhanced yield.
7. The process as claimed in claim 6 wherein said feed media
comprises of the following in the concentration range of:
Ingredients (fermentation broth) Concentration (g/L)
L-glutamic 8.00 ± 2
Dextrose 25.00 ± 2
soya peptone 20.00 ± 2
8. The process as claimed in claim 1 wherein said culture media and
feed media provides enhanced yields of Neisseria meningitidis

polysaccharides of serogroup A, C, Y and W in the following concentrations :

Serogroup Yield
MenA 205±10 mg/L
MenC 290±10 mg/L
MenY 344±10 mg/L
MenW 290±10 mg/L
9. The process as claimed in claim 1 wherein said fermentation process process parameters are in the range of:

Parameters Range
Temperature 36 ± 1°C
Rpm 100 to 700 rpm
Air flow 0.2 to 1.2.1/m
pH 7.3 ± 0.1
PO2 100% to 15%
10. The process of ferementation as claimed in claim 9 wherein said fermentation process is completed in the time range of 11 + 3 hours.
11. The process of ferementation as claimed in claim 9 wherein said fermentation process is completed in the time range of 10 to 12 hours.
12. The process for the production of Neisseria meningitidis polysaccharides seogroups A, C, Y and W as claimed in claim 1, wherein said process yields N. meningitidis polysaccharides which

are capable of being used in the production of economical polysaccharide protein conjugate vaccine(s) against meningococcal infections.