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ELSEVIER

Journal of Microbiological

Methods

27

( 1996) 19-23

Journal ofMicrobiological Methods

Purification

of meningococcal group C polysaccharide procedure suitable for scale-up

by a

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 Biotecnologia, Institute Butantan, Avenida Vital 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 lipopolysaccharide 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) vaccines 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. Polysacchat-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 method, 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 centrifugation 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 PIZ SO167-7612(96)00921-9

20

M.M. Tanizaki et al. I Journal of Microbiological Methods 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 centrifugation. (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 Cetavlon 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.

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 Brunswick 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 35C for 5 h on a rotary shaker at 120 rpm. This culture was then transferred to an 2000 ml Erlenmeyer flask containing 400 ml of the same medium (50 ml for 400 ml medium) and cultivated using the same conditions. 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+0.5C, 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 continued 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 precipitated 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,

2. Materials 2. I. Analyticat

and methods procedures

Polysaccharide content was determined using resorcinol 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 determined using Blue Dextran and riboflavin as void

M.M. Tanizaki et al. I Joumal of Microbiological Methods 27 (1996) 19-23

21

Cell culture

Cetavlon 0.1%

the same buffer without volumes of water.

DOC and three separate

PSI precipitated J PS2


cell

CaCl2

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 remaining 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 tangential 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-

suspension

debris

c PS3

-1

centrifugatlon

nucleic acids c PS4

ethyl alcohol 25%

&

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 & Bulk PS Scheme 1. Neisseria meningitidis polysaccharide purification sterile filtration

this solution was incubated overnight at room temperature 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. Elimination of LPS and low molecular weight proteins were obtained by ultrafiltration in 100 kDa cutoff hollowfiber (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 60C 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 1 Purification Sample

of Neisseria meiaingitidis group C polysaccharide Volume (ml) P (total) k) 2.62 1.52 1.38 LPS ( KDO) Protein Nucleic acid PS Recovery

(total) (mg)
134.3 61.7 3.5

(total) (mg)
861.5 614.6 30

(total) (mg)
ND ND 19.7

(%)
100 58.0 52.6

After CaCl, Before ultrafiltration Final product

600 250 250

22

MM. Tartizaki et al. I Journal of Microbiological Methods 27 (1996) 19-23

tion on a hollow fiber 100 kDa cutoff was used instead of gel filtration for LPS separation. Ultrafiltration was performed in 20 mM Tris- HCl containing 2mM EDTA and 0.5% DOC. Before diafiltration, the sample was incubated for 30 min at 60C with the same buffer and sonicated in order to make LPS disaggregation proceed easily. Extensive diafiltration 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 1970s in industry, making it easier to have operations like remotion of undesirable contaminants during a purification process instead of traditional processing which involved extration, centrifugation or several types of columns [ 151. In microbiology, tangential microfiltration 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% (0.51.9) of protein, l-2% of nucleic acid contaminant related to total PS, and was appoved in pyrogen test in rabbit. Despite sonication and 60C 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 polysaccharide 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
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