Purification of Meningococcal Group C Polysaccharide by a Procedure Suitable for Scale-up

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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



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,



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



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.

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Purification of Meningococcal Group C Polysaccharide by a Procedure Suitable for Scale-up

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