FIELD OF THE INVENTION:
The present invention relates to a novel process for purifying bacterial
polysaccharide. More particularly the present invention relates to a rapid
process for purifying Neisseria meningitidis serogroup A (Men-A)
polysaccharide at room temperature (RT) capable of being used as such o5 r
in a derivatized form or linked to other molecules, for the preparation of
vaccines, more particularly conjugate vaccines for N. meningitidis infection.
BACKGROUND OF THE INVENTION:
10 Monovalent, divalent and poly (multi) valent vaccines containing one, two
or more polysaccharides are available in the market for prevention of
certain diseases or infections caused by various microorganisms. N.
meningitidis, often referred to as meningococcus is a gram negative bacteria,
which causes an array of diseases such as meningitis and septicaemia. On
15 the basis of the type of capsular polysaccharide present on N. meningitidis,
thirteen serogroups have been identified and the most prevalent
serogroups causing the infections are A, B, C, W, X and Y.
N. meningitidis with serogroup A (hereinafter ‘Men-A’) is the pathogen
20 most often implicated in epidemic diseases in sub-Saharan Africa,
whereas, Serogroup B and C have been found responsible for vast
majority of cases of epidemic diseases in developed countries such as
USA.
25 Mono- or multi-valent polysaccharide vaccines are readily available in the
market for prevention of certain diseases or infections caused by various
microorganisms. The multivalent polysaccharide vaccines have been used
3
for many years and have proved valuable in preventing diseases such as
Pneumococcal, Meningococcal or Haemophilus influenza.
The production of the first meningitis polysaccharide vaccine was
accomplished in 1978 as there was an urgent need to combat this 5 fatal
disease. Later it was observed that the 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.
10 The immune response may be characterized as T-cell dependent (TD)
immune response and T-cell independent (TI) immune response. Proteins
and peptides are TD antigens which stimulate helper T lymphocytes to
elicit immune response. TD antigens generate long lasting immune
response due to formation of plasma and memory B cells. In contrast,
15 polysaccharides are TI antigens which do not involve 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
20 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 in 1999 as United Kingdom became
the first country to introduce the meningococcal serogroup C conjugate
25 vaccine.
Conjugate meningococcal vaccines are available as monovalent serogroup
A and serogroup C vaccines; bivalent serogroups A, C and tetravalent
serogroups A, C, Y, W-135. Evidence collected through numerous
4
research findings defines the immunogenic aspect of the polysaccharide
conjugate vaccine. However, there is very little information to aid the
rapid purification of the capsular polysaccharide of Men-A.
The production of purified Men-A is the foremost requirement for 5 an
effective conjugation with the carrier protein and its development as a
conjugate vaccine. The cost for the cultivation and the purification of
Men-A is generally high and involves long working hours (hrs) since it
involves a series of production and purification steps. Improvement in one
10 or more of the steps of cultivation and purification may bring a significant
change in the overall conjugate vaccine production and consequently
makes the process relatively cost effective.
There are a number of patents, which describe the process for the
15 purification of Men-A polysaccharides. The existing state of the art
described in US patent no 7,491,517B2 for precipitating Men-A
polysaccharides with CTAB is found to involve overnight incubation at
4°C. In addition to this, the process also requires gel filtration for the
purification of the Men-A polysaccharides. The overall procedure requires
20 significant time for purification and also the process becomes costly
because of the use of chromatography.
European Patent application no. 2,277,539A2 discloses 12 hours of
incubation for the precipitation of Men-A polysaccharides with CTAB.
25 The patent also describes 16-20 hours of ethanol extraction after CTAB
precipitation. The problem with the invention is the significantly high
time (26-28 hrs) required to purify the polysaccharide.
5
Also the International patent publication no. WO 2011/148382 A1
describes the method of preparing pure capsular polysaccharide using
aluminium phosphate and alcohol for the purification of capsular
polysaccharides of H. influenzae b, N. meningitidis such as serogroups A, C,
Y, W-135 and other similar related capsular polysaccharides produc5 ed
from both gram negative and gram positive microorganisms. The said
published prior art discloses a time of 16-20 hrs for the purification of the
polysaccharides.
10 Presently, the various methods used for the production and purification of
Men-A take relatively long cultivation and purification time of
approximately 24 to 40 hrs 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.
15
Further the above disclosed prior arts teach a method which involves the
treatment of polysaccharides with enzymes which results to the addition
of costs in research and production. Moreover, some of the current
methods discuss the use of chromatography steps which leads to
20 production of a polysaccharide with low yields and may increase the cost
of production at large scales or during scale-up. These methods further
increase the total process time to a great extent.
Despite several studies and research carried out on these polysaccharides,
25 there is a long felt need to develop an industrial process which can be
completed in short span of time. The present process provides the
polysaccharides with better yield and enhanced quality.
6
OBJECT OF THE INVENTION:
The main object of the present invention is to provide a novel process for
purifying bacterial polysaccharide.
Another object of the invention is to provide a rapid process for purifyin5 g
bacterial polysaccharide while eliminating the impurities in a very short
time at room temperature by simple and efficient process.
Yet another significant object of the present invention is to carry out the
10 purification of the polysaccharide without enzymatic treatment resulting
in a cost effective and commercially scalable process.
Yet another object of the present invention is to produce high quality
polysaccharide with higher yield that meets the WHO specifications.
15
SUMMARY OF THE INVENTION:
Accordingly, the present invention discloses a novel process for the
purification of bacterial polysaccharide more specifically the “Men-A
polysaccharide”. The process relates to a rapid, cost effective and scalable
20 method, for purification of the bacterial Men-A polysaccharide in a
significantly reduced time and is performed at RT.
The process of the instant invention involves use of fermented broth of
suitable bacterial strain which in this case is N. meningitidis serogroup A.
25 This fermented broth is prepared by known process which involves steps
such as preparation of media, inoculating said bacterial strain in glycerol
stock culture and incubating at predefined temperature for an optimal
time period at predefined revolutions per minute (rpm). The selected
bacterial strain is cultivated on optimized cultivation media in the
7
fermenter to obtain the fermented broth which is then used in the novel
process of purification.
The fermented broth so obtained is subjected to centrifugation in the range
of 4000rpm/3000 x g to 6000rpm/6750 x g to clarify the fermented brot5 h
for 30 mins followed by concentrating the fermented supernatant by
ultrafiltration using molecular weight cut off membranes.
The ultra-filtered concentrated supernatant having the crude Men-A
10 polysaccharide (MenA-PS) is precipitated by mixing it with a cationic
detergent of high concentration. The preferred cationic detergent but
limited to Hexa Cetyl trimethylammonium bromide (CTAB) is used to
carry out the purification process. The precipitated CTAB pellet with the
crude MenA-PS is liquefied in ethanol solution of very high concentration
15 preferably in the range of 76 % (v/v) to 96% (v/v) followed by carbon
filtration. The supernatant obtained from the carbon filtration is
precipitated with CaCl2. This precipitate is dissolved in MilliQ water
(MQW) and then diafiltrated and concentrated with CaCl2 and
subsequently with MQW.
20
The process exhibits a number of advantages over prior art, such as
providing a robust and rapid process of preparing Men A polysaccharide
having phosphorous backbone. The process is also cost effective as it
reduces the time for purification and can be carried out at RT. An
25 additional advantage is that this process is completely scalable.
8
BRIEF DESCRIPTION OF DRAWINGS
Figure-1 Depicts the process flow for the MenA-PS purification and
recovery.
Figure-2 Depicts the HPLC Chromatogram of MenA-PS
Figure-3 Depicts the NMR spectrum of MenA-5 PS.
DETAILED DESCRIPTION OF THE INVENTION WITH REFERENCE
TO DRAWINGS AND EXAMPLES
The invention discloses a series of steps that have been optimized to
10 enable the purification of bacterial polysaccharide in lesser time and at RT.
The strain of bacterial polysaccharide is inoculated in the fermenter
containing appropriate media components required for bacterial growth.
After achieving a certain pre-determined optical density the bacterial
15 growth is subjected to termination by adding formaldehyde and the
resultant fermentation broth (FB) is obtained.
The fermentation broth obtained from the fermenter is collected in the
bottles and the FB is subjected to centrifugation to obtain the supernatant.
20 The resultant supernatant is concentrated and diafiltrated using molecular
weight cut off membrane preferably but limited to 100 KDa 0.1m2 slice
cassettes. After the concentration and diafiltration the recovered material
is then processed for purification.
25 The diafiltered and concentrated FB having the crude polysaccharide is
treated with cationic detergent and is incubated at RT for predetermined
time period resulting in formation of precipitated pellet of polysaccharide,
i.e. first pellet. The first pellet so obtained is homogenized at predetermined
rpm for pre-determined time. The homogenized first pellet is
9
then liquefied in organic solvent, more preferably ethanol followed by
carbon filtration. The filtered supernatant is treated with precipitating
agent resulting in precipitates. The precipitates so obtained are
centrifuged to obtain second pellet. The second pellets are then dissolved
in MQW. The dissolved pellets are then subjected to concentration 5 n and
diafiltration with predetermined volumes of CaCl2 and MilliQ water
(MQW). Consequently sterile filtration is performed to obtain the purified
bacterial polysaccharide.
10 The purified bacterial polysaccharide so obtained is analysed for
phosphorus, O-actyl, protein, nucleic acid, endotoxin and average relative
molecular weight.
In a preferred embodiment, MenA (Neisseria meningitidis (Albrecht and
Ghon) Murray (ATCC® 13077™15 ) is obtained from the ATCC (American Type
Culture Collection), USA and is further inoculated in the fermenter
containing appropriate media components required for bacterial growth.
After the completion of growth phase which is determined by start of
increase in pH and decrease in the optical density (as compared to the
20 maximum OD achieved), fermentation is terminated by the addition of
0.2% formaldehyde (HCHO) and the temperature of fermenter is
maintained at 35°C for 15 minutes (mins).
The fermentation broth obtained from the fermenter is collected in the
25 bottles and the fermented broth is subjected to centrifugation to obtain the
supernatant. The resultant supernatant is concentrated and diafiltrated.
After concentration and diafiltration the recovered material is then
processed for purification.
10
The purification scheme as shown in Figure 1 is largely dependent on the
physico-chemical properties of polysaccharide, amount of impurities,
which are required to be further removed, and the time taken to complete
the whole process.
5
The diafiltered and concentrated broth having the crude polysaccharide of
Men A is treated with cationic detergent CTAB and is incubated at room
temperature for a time period of 1 hr to 6 hrs, more preferably for 1 hr
resulting in formation of precipitated first pellet i.e. CTAB pellet with the
10 crude Men-A polysaccharide. The CTAB pellet so obtained is rubbery in
nature and highly viscous. The CTAB pellet is liquefied in ethanol at a
concentration range of 76% to 96%, but more preferably homogenized for
20 mins at 1000 rpm in minimal volume of 96% ethanol and thereafter
some more volume of 96% ethanol is added to the homogeneous CTAB
15 pellet under continuous stirring for 40 mins. This process results in
reducing the viscosity of the CTAB pellet to a great extent. This reduction
in viscosity is sufficient for the CTAB pellet to get dissolved in ethanol to a
greater extent leading to rapid purification of MenA-PS. This homogenous
solution is then subjected to centrifugation to separate out any
20 undissolved material. The homogenized solution is carbon filtered more
preferably with zeta carbon filters. The filtered supernatant is treated with
CaCl2 in the range of 0.05M to 0.2 M and kept aside for precipitation for
predetermined time at room temperature resulting in precipitates. The
resultant precipitates are subjected to centrifugation at 4000rpm/3000 x g
25 to 6000rpm/6750 x g to obtain second pellet. The second pellet is then
dissolved in MQW. The dissolved pellets are then subjected to
concentration and diafiltration with predetermined volumes of CaCl2 and
MilliQ water (MQW). Consequently sterile filtration is performed to
obtain the purified MenA-PS.
11
The MenA-PS is analyzed by a series of analytical tests and the results are
presented in Table-1
Table-1: Purified Men-A polysaccharide specifications in accordance
with WHO specification are shown below5 :
Tests Purified MenA
polysaccharide
Results
WHO Specifications
Phosphorus content % 11.19% Not less than 8%
O-acetyl content 2.37mmol/g Not less than 2mmol/g
Protein % 0.26% Not more than 1%
Nucleic Acid % 0.04 % Not more than 1%
Endotoxin <10 EU/μg Less than 100EU/μg
Average relative Mol wt 673KD Actual value from HPLC
Identity Positive Positive agglutination reaction
The purified bacterial polysaccharide is then stored at -20ºC and is capable
of being used as such or in a derivatized form or linked to other
molecules, for the preparation of vaccines, more particularly conjugate
10 vaccines for N. meningitidis infection.
Therefore, the present invention provides a novel , rapid and scalable
process for purifying the Men-A polysaccharides in significantly reduced
time as compared to those disclosed in the prior arts which can be
15 completed in less than 9 hours and is in compliance with the WHO set
standard.
12
Analytical Procedures:
Phosphorus concentration for Men-A is determined by the colorimetric
assay. In this assay, samples are initially acidified, then oxidized with
perchloric acid (Chen method) (Chen Jr et al., 1956) or magnesium nitrate
(Ames method) (Ames, 1966) to activate and hydrolyse the polysacc5 haride
prior to ashing of the material at high temperature. In the second part of
the assay, ascorbic acid acts to reduce a phosphomolybdate complex to a
blue-coloured compound. The absorbance is taken at 820nm with Dribose-
5-phosphate disodium dihydrate as a standard. O-acetyl content of
10 MenA polysaccharide is determined by colorimetric method (Hestrin,
1949). The assay is based on the finding that hydroxylamine at an alkaline
pH in water rapidly converts acetylcholine stoichiometrically
hydroxyamic acid throughout a wide range of ester concentration. The
density of the purple-brown colour is determined at 540 nm.
15
Lipopolysaccharide (LPS) is determined using compact and simple
Endosafe®-PTS™. Protein impurity is determined by Lowry’s method
(Lowry et al., 1951) using bovine serum albumin (BSA) as a standard and
the absorbance is taken at 750 nm. Nucleic acids (NA) is estimated at 260
20 nm and the amount is calculated assuming an absorbance of 1.0 A = 50
μg/mL (Frasch, 1990).
Relative Average molecular size (Mw) is determined using HPLC
(Alliance, Waters) as shown in Figure 2. The columns used are PWXL-4000
25 and PWXL-5000 in series for Men-A. Furthermore, a range of 5 kD to 800
kD Pullulans (Shodex) are used as standards for MenA. Also the buffer
used is 0.1 M Sodium Nitrate with a run time of 30 min at a flow rate of 1
ml/m. The identity of MenA-PS is verified by 1H-NMR spectroscopy as
shown in Figure 3. NMR yields a spectrum of magnetic sensitive nuclei (e.
13
g. 1H, 13C and 31P). Also the MenA-PS is identified serologically by
combining with the specific antisera against the polysaccharide. As the
WHO specs [WHO, 2006] to determine the purity and to characterize the
polysaccharide is based on dry weight basis, the polysaccharide is first
lyophilized and then tested. The moisture content is thus subtracted to ge5 t
the exact dry weight. Moisture content of lyophilized cake is determined
by Thermo gravimetric Analyzer (TGA) from Perkin Elmer.
Various aspects of the invention described in detailed above is now
10 illustrated with non-limiting examples:
Example-1
The clarified fermentation broth (FB) of MenA culture is concentrated and
diafiltered with MilliQ water (MQW) using 100 KDa 0.1m2 slice cassettes.
15 The cationic detergent namely Hexa Cetyl trimethylammonium bromide
(CTAB) preferably in final concentration of 0.5-2% (w/v) is added in the
FB and left for overnight precipitation at 2-8°C. The centrifugation is done
at 5000rpm/4690 x g for 30mins in the end of CTAB precipitation to obtain
the pellet. The CTAB pellet is liquefied in minimal volume of Ethanol
20 solution (preferably in a concentration range of 76-96%). The dissolution is
performed for 8hrs with incubation at 2-8°C. Centrifugation is again
performed at 5000rpm/4690 x g for 30min. Consequently the supernatant
obtained was further processed using carbon filtration with zeta carbon
filters until the OD260nm reaches 0.2 or less. This is followed by 0.22μm
25 filtration. Furthermore, CaCl2 (2M stock solution) in a working
concentration between 0.05-0.2M is added into the filtered supernatant
and kept for precipitation for 4hrs min at 2-8°C. Centrifugation is done at
5000rpm/4690g for 30 min to obtain pellet. Finally the pellet is dissolved
in MQW, and 300KDa diafiltration is done with 10-15 volumes of 50mM
14
CaCl2 and then with 10-15 volumes of MQW. The Men A polysaccharide
obtained is filtered with 0.22 μm filter, and stored at -20°C or less for
further use.
Example-5 2
The clarified FB of MenA culture is concentrated and diafiltered with
MQW using 100 KDa 0.1m2 slice cassettes. The cationic detergent, CTAB
preferably in final concentration of 0.8-1.6% (w/v) is added in the FB and
left for overnight precipitation at 2-8°C. The centrifugation is done at
10 5000rpm/4690 x g for 30mins in the end of CTAB precipitation to obtain
the pellet. The CTAB pellet is liquefied in minimal volume of Ethanol
solution (preferably in a concentration range of 86-96%). The dissolution is
performed for 4hrs with incubation at RT. Centrifugation is again
performed at 5000rpm/4690 x g for 30min. Consequently the supernatant
15 obtained is further processed using carbon filtration with zeta carbon
filters until the OD260nm reaches 0.2 or less. This is followed by 0.22μm
filtration. Furthermore, CaCl2 (2M stock solution) in a working
concentration between 0.09-0.18M is added into the filtered supernatant
and kept for precipitation for 3hrs min at 2-8°C. Centrifugation is done at
20 5000rpm/4690g for 30 min to obtain pellet. Finally the pellet is dissolved
in MQW, and 300KDa diafiltration is done with 10-15 volumes of 50mM
CaCl2 and then with 10-15 volumes of MQW. The Men A polysaccharide
obtained is filtered with 0.22 μm filter, and stored at -20°C or less for
further use.
25
Example-3
The clarified FB of MenA culture is concentrated and diafiltered with
MQW using 100 KDa 0.1m2 slice cassettes. The cationic detergent CTAB
preferably in final concentration of 1-1.5% (w/v) is added in the FB and
15
left for 6hrs at RT. The centrifugation is done at 5000rpm/4690 x g for
30mins in the end of CTAB precipitation to obtain the pellet. The CTAB
pellet is liquefied in minimal volume of Ethanol solution (preferably in a
concentration of 90-96%). The dissolution is performed for 3hrs with
incubation at RT. Centrifugation is again performed at 5000rpm/4690 x 5 g
for 30min. Consequently the supernatant obtained is further processed
using carbon filtration with zeta carbon filters until the OD260nm reaches
0.2 or less. This is followed by 0.22μm filtration. After the filtration, CaCl2
(2M stock solution) in a working concentration between 0.10-0.15M is
10 added into the filtered supernatant and kept for precipitation for 2hrs at
RT. Centrifugation is done at 5000rpm/4690g for 30 min to obtain pellet.
Finally the pellet is dissolved in MQW, and 300KDa diafiltration is done
with 10-15 volumes of 50mM CaCl2 and then with 10-15 volumes of
MQW. The Men A polysaccharide obtained is filtered with 0.22 μm filter,
15 and stored at -20°C or less for further use.
Example-4
The clarified FB of MenA culture is concentrated and diafiltered with
MQW using 100 KDa 0.1m2 slice cassettes. The cationic detergent, CTAB,
20 preferably in final concentration of 1.2% (w/v) is added in the FB and left
for precipitation for 1hr at RT followed by centrifugation at 5000
rpm/4690 x g for 30 mins to obtain the pellet. The CTAB pellet is
homogenized for 20mins at 1000rpm in minimal volume of 96%ethanol
and thereafter some more volume of 96% ethanol is added to the
25 homogeneous CTAB pellet under continuous stirring for 40 mins. The
total dissolution is performed for 1hr with incubation at RT. CTAB pellet,
which is rubbery in nature and highly viscous, loses its viscosity to a
greater extent during homogenization thereby allowing the proper and
rapid mixing of ethanol within an hour. Centrifugation is again performed
16
at 5000rpm/4690 x g for 30min. Consequently the supernatant obtained is
further processed using carbon filtration with zeta carbon filters until the
OD260nm reaches 0.2 or less. This is followed by 0.22μm filtration. After the
filtration , 0.15M CaCl2 (using 2M stock solution) is added into the filtered
supernatant and kept for precipitation for 30 m at RT. Centrifugation 5 ion is
done at 5000rpm/4690 x g for 30 min to obtain pellet. Finally the pellet is
dissolved in MQW, and 300KDa diafiltration is done with 8-10 volumes of
50mM CaCl2 and then with 8-10 volumes of MQW. The Men A
polysaccharide so obtained is filtered with 0.22 μm filter, and stored at -
10 20°C for further use.
17

We Claim:
1. A novel process for purifying bacterial polysaccharide of a bacterial
strain at room temperature in sterile environment, wherein the said
process comprises the steps of5 :
(a) clarifying the fermentation broth of said bacterial strain by
centrifugation to obtain fermented supernatant ;
(b) subjecting said fermented supernatant of step (a) to
concentration and diafiltration using a molecular weight cut
10 off membrane to obtain crude bacterial polysaccharide;
(c) precipitating said crude bacterial polysaccharide of step (b)
with at least one cationic detergent followed by
centrifugation to obtain first pellet;
(d) subjecting said first pellet of step (c) to homogenization at
15 predetermined rpm for predetermined incubation time in at
least one organic solvent to obtain homogenized first pellet.
(e) subjecting said homogenized first pellet of step (d) to
centrifugation followed by carbon filtration to obtain filtered
supernatant.
20 (f) precipitating said filtered supernatant of step (e) with
precipitating agent to obtain precipitate and subjecting said
precipitate to centrifugation to obtain second pellet;
(g) subjecting said second pellet of step (f) to dissolution in
MQW and then subjecting the dissolved pellets to
25 diafiltration and concentration
to obtain purified polysaccharide in less than 9 hours wherein the
purity of said purified polysaccharide is in compliance with the
WHO standards.
18
2. The process as claimed in claim 1 wherein said bacterial strain is
Neisseria meningitidis and said bacterial polysaccharide is Neisseria
meningitidis serogroup A (Men-A) polysaccharide.
3. The process as claimed in claim 1 wherein the purified polysaccharid5 e
is obtained preferably in 6 to 9 hours.
4. The process as claimed in claim 1 wherein said centrifugation of the
fermented broth in step (a) is carried out in range of 4000rpm/3000 x g
10 to 6000rpm/6750 x g.
5. The process as claimed in claim 1 wherein said molecular weight cut
off membrane is 100 KDa 0.1 m2 slice cassettes.
6. The process as claimed in claim 1 wherein said cationic detergent is
15 Hexa Cetyl trimethylammonium bromide (CTAB) used in
concentration range of 0.5%(w/v) to 2 %(w/v).
7. The process as claimed in claim 1 wherein said first pellet of step (c) is
CTAB pellet obtained by centrifugation at 4000rpm/3000 x g to
20 6000rpm/6750 x g for 30 min.
8. The process as claimed in claim 1 wherein said first pellet of step (d) is
homogenized at 1000 rpm for said incubation time of 20 mins using
said organic solvent.
25
9. The process as claimed in claim 1 wherein said homogenized first
pellet of step (d) is further dissolved under continuous stirring for 40
mins using an organic solvent.
19
10. The process as claimed in claim 1 wherein the said organic solvent is
ethanol at concentration range of 76% (v/v) to 96% (v/v).
11. The process as claimed in claim 1 wherein said carbon filtration of step
(e) is carried out with zeta carbon filters attaining the OD260nm be5 low
0.2 followed by 0.22μm filtration to obtain said supernatant.
12. The process as claimed in claim 1 wherein said precipitating agent is
CaCl2 in the range of 0.05M to 0.2M which is added to the filtered
10 supernatant to obtain precipitate in 30 mins to 2hrs.
13. The process as claimed in claim 1 wherein said centrifugation of
precipitate in step (f) is carried out at 5000rpm/4690g for 30 mins.
15 14. The process as claimed in claim 1 wherein said second pellet is
dissolved in MQW.
15. The process as claimed in claim 1 wherein said second pellet is
diafiltered and concentrated with 8-15 volumes of 50 mM CaCl2
20 followed by 8-15 volumes of MQW and further filtered using 0.22 μm
filter to yield purified MenA-PS.