purification of meningococcal group c polysaccharide by a procedure suitable for scale-up
TRANSCRIPT
7/27/2019 Purification of Meningococcal Group C Polysaccharide by a Procedure Suitable for Scale-up
http://slidepdf.com/reader/full/purification-of-meningococcal-group-c-polysaccharide-by-a-procedure-suitable 1/5
ELSEVIER
JournalofMicrobiological
Journal of Microbiological Methods 27 (1996) 19-23Methods
Purification of meningococcal group C polysaccharide by a
procedure suitable for scale-up
Martha M. Tanizaki”, Ligiane R. Garcia, Julia B. Ramos, Luciana C.C. Leite,
Harold0 Hiss, Joana A. Furuta, Joaquin Cabrera-Crespo, Isaias Raw
Centro de Bi otecnologi a, I nstit ute Butant an, Av enida Vi tal Brasil 1500, CEP 05503-900, Go Paula, Brazil
Received 12 January 1996; revised 6 June 1996; accepted 7 June 1996
Abstract
Neisseriu mertingitidis group C capsular polysaccharide is the antigen for the vaccine. An easier method has been
developed for the purification of N. meningitidis group C capsular polysaccharide. In this method, two steps of the traditional
procedure have been modified: the removal of protein and lipopolysaccharide. The phenol extraction for removal of
contaminant protein was substituted by proteinases digestion using three different proteinases: proteinase K, nagarse and
trypsin. Tangential ultrafiltration in hollowfiber 100 kDa cutoff was used instead of ultracentrifugation. Extensive
diafiltration on a 100 kDa cutoff in Tris-HCl buffer containing 0.5% of deoxycholate was able to eliminate lipopolysac-
charide as well as low molecular weight protein. The resultant purified polysaccharide contained around 2% of protein, 1.5%of nucleic acid, and ir passed the pyrogen test for lipopolysaccharide in rabbit.
Keywords: Neisseria Jneningitidis; Capsular polysaccharide; Purification; Tangential ultrafiltration
1. Introduction
During the past decade, polysaccharide (PS) vac-
cines of Neisseria meningitidis group A, C, W135, Y
and Z [1,2] were developed. These vaccines are
composed of purified polysaccharides, which are the
main components of the bacterial capsule. Polysac-
chat-ides groups A and C have been used successfully
to stop epidemics [3,4]. Since 1971, two great
epidemics have occurred in Brazil, reaching up to
200 cases per 100 000 inhabitants. The first epidemic
was caused by N. meningitidis group C and the
second one by group A. The last outbreak began in
April, 1974, at same the time as the first one, with a
*Correspondingauthor. Fax: +55 11 8511505.
higher incidence. Both epidemics have been repelled
by polysaccharide vaccines produced by Institut
Merieux (France) and by Instituto Oswald0 Cruz
(Brazil) which received the French technology.
The N. meningitidis capsular PS purification meth-
od, which is the method of the French technology [6]
currently in use in Brazil, was first described in 1969
by Gotschlich [5] acquired from Institut Merieux.
This method includes the following steps: (1) the
negative charged PS is precipitated along with the
cells by the addition of Cetavlon to the bacterial
culture to a final concentration of 0.1% (w/v). (2)
The Cetavlon precipitate is recovered by centrifuga-
tion and the paste is resuspended in 1 M calcium
chloride; nucleic acids, lipopolysaccharide (LPS) and
some proteins will also be dissolved, and the cell
0167-7012/96/$15.00 Q9 1996 Elsevier Science Ireland Ltd. All rights reserved
PI Z SO167-7612(96)00921-9
7/27/2019 Purification of Meningococcal Group C Polysaccharide by a Procedure Suitable for Scale-up
http://slidepdf.com/reader/full/purification-of-meningococcal-group-c-polysaccharide-by-a-procedure-suitable 2/5
20 M .M. Tani zaki et al. I Journal of M icrobiological M ethods 27 (1996) 19-23
debris is removed by centrifugation. (3) The nucleic
acid is then removed by fractional ethyl alcohol
precipitation at a final alcohol concentration of 25%
(v/v) and the precipitate is removed by centrifuga-
tion. (4),The PS is precipitated by adjusting the ethyl
alcohol concentration to 80% (v/v). (5) The PS is
recovered by centrifugation and the residual Cetav-
lon and calcium chloride are removed by washing
the pellet three times with absolute ethyl alcohol; at
this stage, PS contains large amounts of protein and
LPS. (6) The contaminating protein is removed from
crude PS by cold phenol extraction. One volume of
phenol solution is added to a 10 mg/ml solution of
PS in 10% sodium acetate and shaken vigorously.
The aqueous phase is recovered and reextracted
twice more with phenol. The pooled aqueous phases
are dialyzed against distilled water to remove the
phenol. (7) The LPS is separated from the PS
solution by ultracentrifugation at 100 OOOXg. This
process has two inconvenient steps for large scale
production: step 6 and 7. Phenol is a very corrosive
reagent. Therefore, contaminant protein elimination
by phenol extraction should be substituted. The
ultracentrifugation step is expensive for large scale
production since many ultracentrifuges are needed.
Besides, this step should also be eliminated. We
developed an alternative process to substitute the
phenol extraction and ultracentrifugation steps, in
order to have a process which would be more
convenient for scale up.
2. Materials and methods
2. I. Analyticat procedures
Polysaccharide content was determined using re-
sorcinol as reagent as described by Svennerholm [7].
Protein was determined by the method of Lowry et.
al. [8]. Lipopolysaccharide (LPS) was determined as
KDO (2-keto-3-deoxyoctonate) by the method of
Osbom [9] and by the pyrogenity test in rabbits
using 0.025 ,uglkg of rabbit [lo]. Nucleic acids were
estimated at 260 nm and the amount of nucleic acid
was calculated assuming an absorbance of 1=50 ,ug
[lo]. PS molecular size was determined in a column
of Sepharose 4B and the Kd (Ve-Vo/Vf) was de-
termined using Blue Dextran and riboflavin as void
volume and final volume, respectively. Pyrogen test
in rabbits was performed according to the United
States Pharmacopeia, USP XXII (1989), using three
rabbits for each test and injecting 0.025 pg in 10 ml
per Kg of body weight.
2.2. PS fermentation
N. meningitidis was fermented in a New Brun-
swick model MPP 80 fermentor (40 L) in Frantz
medium [ 1 l] and the fermentation protocol was done
according to description of Ramos et al. [12]. The
inoculum was prepared in 250 ml of the Frantz
medium. The flasks were inoculated at 35°C for 5 h
on a rotary shaker at 120 rpm. This culture was then
transferred to an 2000 ml Erlenmeyer flask con-
taining 400 ml of the same medium (50 ml for 400
ml medium) and cultivated using the same con-
ditions. The contents of two of these Erlenmeyer
flask were used as inoculum for the fermentor with
40 L of Frantz medium. The fermentation conditions
were: temperature of 35.0+0.5”C, air flow rate 5
l/min, agitation frequency 120 rpm (2x16.5 cm
Rushton impellers), 6 psi. vessel pressure. After 10 h
fermentation, a glucose syrup (5 1 containing 250 g
of glucose) was added and the fermentation con-
tinued for another 10 h. The pH was controlled at
6.5.
2.3. PS purijcation
The initial steps of PS purification were made as
already described [5]. The fermentor content was
precipitated with 0.1% Cetavlon. Cetavlon precipi-
tated was recovered with centrifugation at 15 OOOXg
for 30 min and ressuspended in 500 ml of 1M CaCl,.
The cell debris was discarded after centrifugation at
15 OOOXg for 30 min. The nucleic acid was then
removed by fractional ethyl alcohol (25% v/v)
precipitation. After sitting for 1 h, the liquid was
centrifuged at 15 OOOXg for 30 min to pellet the
nucleic acid. PS was precipitated adjusting the ethyl
alcohol concentration to 80%. The mixture was left
overnight to permit complete precipitation of the PS.
The PS was recovered by centrifugation at 15 OOOXg
for 30 min. The precipitate PS was ressuspended in
Tris-HCl buffer, 20 mM, pH 8.5, in a final volume of
250 ml. In order to eliminate protein contaminants,
7/27/2019 Purification of Meningococcal Group C Polysaccharide by a Procedure Suitable for Scale-up
http://slidepdf.com/reader/full/purification-of-meningococcal-group-c-polysaccharide-by-a-procedure-suitable 3/5
M .M. Tani zaki et al. I Joumal of M icrobi ologi cal M ethods 27 (1996) 19-23 21
Cell culture
4 Cetavlon 0.1%
PSI precipitated
J CaCl2
PS2 suspension
cell debris c -1 centrifugatlon
PS3
nucleic acids c 1 ethyl alcohol 25%
PS4
& ethyl alcohol 80% and centrlfugation and
resuspension
PS5 suspension
& proteinax treatment
PS6 with low mol.wgt. protein
LPS c 1 0.5% DOC and tangential ultrafiltration
PS7 detoxified
& sterile filtration
Bulk PS
Scheme 1. Neisseria meningit idi s polysaccharide purification
this solution was incubated overnight at room tem-
perature with 5 mg of proteinase K (Sigma), 5 mg of
trypsin (Sigma) and 5 mg of nagarse (Sigma) and
this treatment was repeated for further 4 h. Elimina-
tion of LPS and low molecular weight proteins were
obtained by ultrafiltration in 100 kDa cutoff hollow-
fiber (AMICON, model HlP30-43, 0.03 m’) in the
presence of 0.5% of deoxycholate (DOC). The
conditions of ultrafiltration were: an inlet pressure of
10 psi and an average ultrafiltration flux of 0.33
Wmin10.03 m2. DOC was added to the protein
digested solution which was then incubated for 30
min at 60°C and sonicated for 15 min. The solution
was washed in the hollow-fiber with five separate
volumes of 1 1 of ‘Tris-HCl buffer 20 mM, 2 mM
EDTA and 0.5% DOC, five separate volumes (1 1) of
Table 1Purification of Neisseria meiai ngiti dis group C polysaccharide
the same buffer without DOC and three separate
volumes of water.
3. Results and discussion
The process used for the modified purification
method is shown in Scheme 1. The initial steps used
in the Merieux process [6] were maintained and only
the protein and LPS elimination steps were modified.
The process started in a 40 1 fermentation in Frantz
medium and the purification procedure is described
in the Materials and methods. Table I shows a
typical PS purification data. The PS content in 40 1
fermented medium was 2.62 g with high level of
protein contaminants (861.5 mg) and 134.3 mg of
LPS (measured as KDO).
In order to eliminate these contaminant proteins,
proteinase digestion was tested. For this purpose,
proteinase K, nagarse and trypsin were tested using
overnight incubation at room temperature with 5 mg
of each proteinase followed by 4 h incubation with
an additional 5 mg of proteinase. The contaminant
protein was determined after tangential ultrafiltration,
as described in the Materials and methods. By testing
protease K alone, the remaining protein contaminant
was 9%. Using protease K plus nagarse, the remain-
ing protein contaminant was 5%. The digestion was
improved when trypsin was added. As showed in the
Table 1, proteinase digestion using a mixture of
protease K, nagarse and trypsin followed by tangen-
tial ultrafiltration were able to eliminate almost 97%
of the total protein contaminant.
LPS forms a high molecular weight complex
which is efficiently pelleted by ultracentrifugation
[5]. Detergents such as DOC are able to disaggregate
the complexed LPS to a molecular weight, low
enough to be included in Sephadex G-75 [ 131 or
Sephacryl S-300 columns [ 141. Tangential ultrafiltra-
Sample Volume
(ml)
P (total)
k)
LPS ( KDO)
(total) (mg)
Protein
(total) (mg)
Nucleic acid
(total) (mg)
PS Recovery
(%)
After CaCl, 600 2.62 134.3 861.5 ND 100
Before ultrafiltration 250 1.52 61.7 614.6 ND 58.0
Final product 250 1.38 3.5 30 19.7 52.6
7/27/2019 Purification of Meningococcal Group C Polysaccharide by a Procedure Suitable for Scale-up
http://slidepdf.com/reader/full/purification-of-meningococcal-group-c-polysaccharide-by-a-procedure-suitable 4/5
22 MM . Tarti zaki et al. I Journal of M icrobiological M ethods 27 (1996) 19-23
tion on a hollow fiber 100 kDa cutoff was used
instead of gel filtration for LPS separation. Ultrafil-
tration was performed in 20 mM Tris- HCl con-
taining 2mM EDTA and 0.5% DOC. Before diafiltra-
tion, the sample was incubated for 30 min at 60°C
with the same buffer and sonicated in order to make
LPS disaggregation proceed easily. Extensive diafil-
tration with the buffer, followed with buffer without
DOC, and then water, was able to eliminate almost
95% of LPS as shown in Table 1. Tangential
ultrafiltration has been used since the 1970’s in
industry, making it easier to have operations like
remotion of undesirable contaminants during a purifi-
cation process instead of traditional processing which
involved extration, centrifugation or several types of
columns [ 151. In microbiology, tangential microfil-
tration has been currently employed for harvesting
and for concentrating fermentation medium [151. In
our process, tangential ultrafiltration was used not
only to eliminate low molecular weight proteins or
peptides, but mainly to eliminate LPS in a very
simple way.
In four experiments, the yield of polysaccharide
recovery was around 50% (48-55). The final product
obtained by this process contains near 1.3% (0.5-
1.9) of protein, l-2% of nucleic acid contaminant
related to total PS, and was appoved in pyrogen test
in rabbit. Despite sonication and 60°C incubation, the
obtained PS still has a high molecular weight with a
Kd around 0.30.
Capsular polysaccharide from Klebsiella sp was
purified from the Gotschlich [5] method [16] and the
total PS obtained was 70-750 mg/L of culture with
0.8-2.5% of residual protein and 12% of nucleic
acids. Therefore, although the quality of the polysac-
charide obtained by this process agrees with a
capsular PS from Klebsiella sp., the quantity should
be improved at the fermentation yield.
The main favourable point for the use of this
process in large scale production is the economic
reason since the cost of a hollowfiber is around
hundred times cheaper than a ultracentrifuge.
Another advantage of the hollowfiber use is the fact
that larger volumes, for instance, a scale of 40-400 I,
of fermentation can be processed in the same time
and by the same way, instead of having the necessity
of many ultracentrifugations as in the Merieux
process.
Acknowledgments
This work was supported by grant of FAPESP and
Secretaria do Estado da SaGde de Slo Paulo. M.M.
Tanizaki received a reseach fellowship from CNPq.
The authors thanks Dr Carl Frasch for his helpful
suggestions.
References
[l] Farquhar, J.D., Hatins, W.A., Desanctis, A.N., Demeio, J.L.and Metzgar.D.P. (1977) Clinical and serological evaluation
of purified polysaccharide vaccines prepared from Neisseria
rneningi t id is group Y (39828). Proc. Sot. Exp. Biol. Med.
155, 53-455.
[2] Farquhar, J.D., Hankins, W.A., Desanctis, A.N., Demeio.A.N. and Metzgar, D.P (1978) Clinical and serological
evaluation of a meningococcal polysaccharide vaccine
groups A, C, and Y (39995). Proc. Sot. Exp. Biol. Med. 155,
79-82.
[3] Peltola, H.P., Makela, P.H., Kayhty, H., Jousimies, J.. Herva,
E., Hllstrom. K., Sivonen. O.V., Renkonen, O.V., Pettay, 0..
Karanko, V., Ahvonen, P. and Sama, S. (1977) Clinical
efficacy of meningococcus group A capsular polysaccharide
vaccine in children three mouths to five years of age. N.
Engl. J. Med. 297, 686-691.
[4] Taunay. A.E., Galvb, EA., Morais, J.S., Gotschlich, E.C.
and Feldman, R.A. (1974) Disease prevention by meningo-
coccal serougroup C. polysaccharide vaccine in preschool
children: results after eleven mouths in SBo Paulo, Brazil.
Pediatr. Res. 8, 429-235.
[5] Gotschlich, E.C., Liu, T.Y. and Artenstein, MS. (1969)
Human immunity to the meningococcus. J. Exp. Med. 129,
1349-l 365.
[6] Ayne G., Donikian, R., Mynard, M.C. and Lagrandeur, G.
( 1973) Production and controls of serogroup A Neisseiria
meningi t id is polysaccharide vaccine. In: Table ronde ssur
immunoprofilaxie de la meningite cerebra-spinale (Ed. R.
Triav). Lyon Fondation Merieux.. pp. 4-30.
[7] Svennerholm, L. (1957) Quantitative estimation of sialic
acids. II. A calorimetric resorcinol-hydrochloric acid meth-
od. Biochim Biophys. Acta 241, 604-611.
[8] Lowry, O.H., Rosenbrough, N.J. and Farr, R H. (1951)
Protein measurement with the Folin phenol reagent. J. Biol.Chem. 193, 165-275.
[9] Osbom. M.J. (1963) Studies on the gram negative cell wall.
Evidence for the role of 2-keto-3-deoxyoctanate in the
lipopolysaccharide of Salmonella typhimurium. Proc. Natl.
Acad. Sci. 50, 499-506.
[lo] Frasch, C.E. (1990) Production and control of Neisseria
meningi t id is vaccines. In: Bacterial vaccines (Ed. Alan R.
Liss). Inc. New York, pp. 123-145.
[l l] Fran@ I.D. Jr. (1942) Growth requirements of the meningo-
coccus. J. Bacterial. 43. 757-761.
7/27/2019 Purification of Meningococcal Group C Polysaccharide by a Procedure Suitable for Scale-up
http://slidepdf.com/reader/full/purification-of-meningococcal-group-c-polysaccharide-by-a-procedure-suitable 5/5
M .M. Tanizaki et al. I Journal of Mi crobiol ogical M ethods 27 (1996) 19-23 23
[12] Ramos, J.B., Hiss, H., Vicentin, M.A., Paz, M.F., Peixoto,
A., Leal, M.B.B., Goncalves, P.C. and Raw, I. (1996) Batch
cultivation kinetics of Neisseria meningit idi s (Serogroup C)
in Frantz medium. I. Growth and polysaccharide production.
Arq. Biol. Tecnol. 39, 215-220.
[13] Wu, L., Tsai, C.M. and Frasch, C.E. (1987) A method for
purification of bacterial R - type lipopolysaccharides
(lipooligosaccharides). Anal. Biochem. 160, 281-289.
[14] Gu, X.X. and Tsai, C.M. (1991) Purification of rough type
lipopolysaccharides of Neisseria meningit idi s from cells and
outer membrane vesicles in spent media. Anal. Biochem.
196, 311-318.
[15] Cheryan, M. (1986).Ultrafiltration applications. In: Ultrafil-
tration Handbook. (Ed. Alan S. Michaels). Technomic
Publishing Co. Inc. Pennsylvania, pp. 23 l-340.
[16] Cryz, S.J., Furer, E. and Germanier, R. (1985). Purification
and vaccine potential of Klebsiella capsular polysaccharides.
Infect. Immun. 50, 225-230.