CA1137901A - Isolation of capsular polysaccharide of haemophilus influenzae - Google Patents

Abstract

ABSTRACT
A process for the isolation and purification of immunologically active polyribosyl ribital phosphate (PRP), the capsular polysaccharide of Haemophilus influenzae type b. The PRP has been purified with ethanol, Cetavlon (hexadecyltrimethyl ammonium bromide) and a phosphate containing adsorbent, hydroxylapatite. The contaminants (nucleic acid, proteins and endotoxins) are removed to the minimum level by a treatment with hydroxyl-apatite. Also described is a process for the preparation of a combined vac-cine containing the PRP (prepared as forementioned) and B. pertussis anti-gens. The vaccine elicits anti-PRP antibody and antipertussis antibody (as measured by microagglutination) formations in young animals. This sera with anti-PRP antibody exhibits a strong bactericidal activity.

Description

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This invention is in the field of vaccines for immunization against Haelllopll us influenzae type b infections, such as meningitis. More specifi-cally, this invention relates to a method for the isolation of antigenic poly-saccharide polyribosyl ribitol phosphate (PRP) from Haemophilus influenza type b cultures. The PRP may be used for the preparation of a combined vacc-ine containing PRP and B. pertussis antigens. The PRP of this invention should also be effective when used in conjunction with other non-pa~hogenic strains of bacteria, such as E. coli, B. subtilis, S. aureus, B. punilus and L.
plantarum, to trigger an antibody response in warm-blooded animals.
The purified polyribitol phosphate (PRP) from Haemophilus influen-zae type b is being investigated as a protective immunogen. However, an active antigenic response in young animals and infants has not been achieved.
The prior art method for the purification employed ethanol and Cetavlon (hex-adecyltrimethyl ammonium bromide). The contaminants, endotoxins, and pyro-genic substances were removed by the use of cold phenol or chloroform and t-butanol which may result in the loss of the antigenic nature of this poly-saccharide.
A new process for the isolation and purification of immunologically active PRP from Haemophilus influenzae type b has now been discovered.
~0 The process to be described has distinct advantages over the prior art procedures in that all contaminants (nucleic acids, proteins and endoto-~ins) are removed to the minimum levPl by a treatment with hydroxylapatite.
The PRP prepared by the process described here gives a higher molecular weight polysaccharide than those reported previously.
More importantly, the PRP prepared by this new procedure, as well as a combined PRP and pertussis vaccine, is highly immunogenic in warm-blood-ed animals as opposed to the PRP produced by prior art procedures.

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: According ~o the invention, in the method of isolating and purify-ing immunologically active polyribosyl ribitol phosphate (PRP), the capsular polysaccharide of Haemophilus influenzae type b, fermenting strains of the organism Haemophilus influenzae type b under art recognized conditions, iso-lating the PRP by conventional ethanol precipitation, further isolating the PRP as a hexadecyltrimethyl ammonium bromide complex by conventional proced-ures, there is provided the improvement comprising:
purifying the PRP by adding hydroxylapatite [3.Ca3(PO4)2.Ca(OH)2] in a 0~02M sodium phosp]late buffer at a pH from about 6.7 to about 6.9, mixing at a 1~) tcmperature of about 4C., centrifuging, removing the supernatant and repeating the foregoing procedure at least two more times, filtering the supernatant through suitable filters, dialyzing against pyrogen-free distilled water, and then lyophilizing.
According to another aspect of the invention, in the method of isolating and purifying immunologically active polyribosyl ribitol phosphate (PRP), the capsular polysaccharide of Haemophilus influenzae type b, ferment-- ing strains of the organism Haemophilus influenzae type b under art recognized conditions, isolating the PRP by conventional ethanol precipitation, further isolating the PRP as a hexadecyltrimethyl ammonium bromide complex by conven-tional procedures, there is provided the improvement comprising:
purifying the PRP by column chromatography in a 0.02M sodium phosphata buffer at a pH of about 5.8 through hydroxylapatite [3.Ca3(P0~)2.Ca(OH)2], eluting with a stepwise gradient of 0.02M and 0.05M sodium phosphate buffer at a pH of about 5.S, isolating fractions by analysis for PRP, dialyzing said fractions against pyrogen-free distilled water and then lyophilizing.
The process of the invention removes contaminants (proteins, nucleic acids and endotoxins) from the polysaccharide PRP by the above defined treat-~3~

ment of the partially purified polysaccharide with thc phosphate containingadsorbent ~hydroxylapatite) which does not absorb the polysaccharide under designated conditions.
(I) Isolation and purification of polyribosyl ribitol phosphate, the capsul r polysaccharide of Haemophilus influenzae type b.
Organisms, Growth Medium and Culture Two strains of Haemophilus influenzae type b are used. The Rab strain is obtained from Grace Leidy, Babies Hospital, Columbia University, New York City, New York. The CK strain was isolated from a patient at Water-bury ~lospital ~aterbury, Conn. The organisms are passed through mice several times to insure their virulence. The organisms are isolated at autopsy from the brain tissue of mice~ subcultured on either 3.7% Brain Heart Infusion (BHI) (Difco Lab., Detroit Mich.) medium or on 5% BHI agar supplemented with 0.01% nicotinamide adenine dinucleotide ~NAD) ~P-L Biochemicals, Milwaukee, Wis.) and 1% (v/v) defibrinated horse blood (Animal Blood Center, Syracuse, N.Y.) and then distributed in one ml portions in ampules, lyophilized and stored at -70~C.
Basal medium used for the growth of the organisms is 3.7% (B~II).
Tlle basal medil~l is supplemented with 10 mg of NAD and 20 mg of hemin (East-_~ man Kodak, Rochester, N.Y.) per liter. One percent (v/v) defibrinated horseblood is added per 50 ml of seed culture. The supplements are freshly pre-pared and filtered through a 0.45~ Nalgene filter unit(N~lge Sybron Corp., Rochester, N. Y.) before use. For growth of the organism in a 14 liter fer-mentor, the medium is further supplemented with 0.5% glucose.
To prepare a flask of seed organisms, one ml of frozen stock cul-ture is thawed and transferred to 50 ml of the enriched BHI medium supplemen-ted with defibrinated horse blood and NAD. The culture is grown for 8 hours , ; ~ . : , . .. .

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at 37C on a gyrotory shaker at 150 rpm. The organisms are added as a 1%inoculum to 500 ml portions of the enriched BHI broth in 2 liter flasks which are then incubated at 37C with moderate shaking on a gyrotory shaker for 8 hours (for isolation of PRP) or 14 hours (as seed cultures).
Th~ fermentation of each batch in a 14 liter fermentor is initiated by aseptically transferring 700 ml of the seed culture to 7 liters of the enriched BHI broth supplemented with hemin instead of defibrinated horse blood~ The culture is continuously maintained at 37~1C. The tank is stirred at a rate of 150 rpm and an air flow of 0.25 liter of air per liter of mash per minute is maintained. During the growth 0.001~ of a silicone antiform (FD-82, Hodag Chemical Corp.) is added as needed. Cultures are grown to the late logarithmic of growth (8 to 10 x 109 viable cells/ml), usu-ally about 8 hours and then growth is terminated by adding 0.4~ formaldehyde and standing overnight at 4C.
Isolation of Polyribosyl Ribitol Phosphate (PRP) Culture broth prepared as described above is centrifuged at 27,000 x g for 30 minutes at 4C. The cell free supernatant is collected and treated as follows:
Step l, Ethanol Precipitation -~0 To the culture supernatant is added sodium acetate (final concen-tration 4~). The solution is adjusted to pH 6.0-6.2 and 44 liters of 3A
ethanol are added slowly with vigorous stirring at 4C. The mixture is adjus-ted to p~l 6.8 with glacial acetic acid and then allowed to stand for 12 hours at 3C. The resulting precipitate is collected by decantation and then cen-trifuged to afford crude PRP.
St æ 2, Cetavlon`(hexadecyltrimethyl ammonium bromide) Treatment The crude PRP from Step l is dissolved in pyrogen-free distilled . .
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water and centrifuged to remove the residue. The clear brown solution is then slowly added to lOO ml of a lO~ aqueous solution of Cetavlon with mixing ~final conc. 0.5%). The mixture is stirred for one hour and then centrifuged.
The precipitate of nucleic acids and PRP-Cetavlon complex is mixed with 2 liters of 0.3M sodium chloride. The cloudy solution is centrifuged to elim-inate insoluble materials such as nucleic-Cetavlon salt.
The supernatant is diluted with an equal volume of water causing the PRP-Cetavlon salt to precipitate. The mixture is stirred Eor one hour, the precipitate is collected by centrifugation and is then dissolved in 2 liters 1~ of 0.3M sodium chloride.
Step 3,_Ethanol Precipitation Cetavlon and the contaminants of nucleic acids and proteins are further removed by ethanol precipitation (at least 2 times). The PRP is pre-cipitated as described in Step 1 while Cetavlon is dissolved in the alcoholic solution. The PRP is recovered by centrifugation, dissolved in 2 liters of pyrogen-free distilled water and then reprecipitated as desecribed above.
The final PRP precipitate is solubilized in 20mM sodium phosphate, pH 6.8.
Step 4, Hydroxylapatite Treatment Contaminants ~e.g. nucleic acids, proteins and endotoxins) in the i2~ partial purified PRP preparations are selectively removed by adsorption on a speciic calcium phosphate containing adsorbent, namely, hydroxylapatite [3.Ca3tPo~)2~ca(OH)2]~
This invention is based on the discovery that the polysaccharide PRP is not adsorbed by the calcium phosphate adsorbent containing phosphate buffer ~20mM) having a pH of about 6.7-6.9; or a phosphate buffer (SOmM) having a pH of about 5.8; however, the contaminants (such as nucleic acidsJ
proteins and endotoxins) are adsorbed under these conditions. The process , ~ ,, ,~, . . .

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of the present invention may be carried out in batch-wise or column opera-tion. In batch process, the hydroxylapatite is added to the partially puri-fied PRP preparation ~in 20mM phosphate; p~l 6.9). The mixture is mixed well at about 4C and centrifuged to remove non-desired solids ~adsorbent and the contaminants adsorbed by the adsorbent). The supernatant fluid is subjected to the foregoing procedure at least 2 more times. The resulting solution is filtered through millipore filters, dialyzed against pyrogen-free distilled ~ater, and lyophilized.
In column operation, the partially purified PRP in 20mM phosphate la buffer (pH of at least 5.8) is applied to a column containing the adsorbent hydro.~ylapatite which has been equilibrated with 20mM phosphate buffer pH
5.S, and then the PRP is eluted with a stepwise gradient of sodium phosphate buffer ~pH 5.8) from 20n~ to lOOmM. Fractions are collected and assayed for pentose ~for polyribosyl ribitol phosphate). Those fractions which are posi-tive for pentose are dialyzed against pyrogen-free distilled water and lyophilized.
(II) A process for preparlng a combination vaccine consisting of PRP
from H. influenzae type b and B. pertussis antigens.
To prepare a PRP vaccine, lyophilized PRP ~prepared as aforemen-0 tioned) is dissolved at a concentration of 20jug/ml in phosphate buffered saline (PBS) ~0.113 g potassium diphosphate, 0.83 g disodium phosphate, and 8.5 g sodium chloride per liter, pH 7.0, containing 0.01~ thimerosal). The ~accine is sterile filtered through 0.45 ~ millipore filter units, dispensed into glass vials, and stored at 4C.
A concentrated solution of ~he PRP is prepared by weighing pre-determined amounts of the polysaccharide and dissolving it in phosphate buff-ered saline (0.113 g potassium diphosphate, 0.83 g disodium phosphate, and 7~

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S.5 g sodium chloride per liter, pH 7.0, containing 0.01% thimerosal). This concentrated solution of PRP is then mixed with an appropriate volume of fresh B. pertussis cell suspensions to prepare the stock solution. The characteristics of B. pertussis strain 138 are described in sergey15 Manual of Determinative Bacteriology, 8, suchanan and Gibbons, editors, Willams and l~ilkins Co., Maryland U.S.A. 1974, on page 283. This strain is available from the American Type Culture Collection, Maryland, U.S.A. under deposit No.
103S0. The combined vaccine in this stock solution contains 200~ g of PRP
and approximately 70 opacity units ~op) of cells per ml. The stock solution is kept at 4C for 90 days to allow for detoxification of pertussis antigens before the final product (vaccine) is prepared ~which contains 10~ g of PRP
and 3.5 op units of pertussis cells/0.5 ml dose).
Example l Hydroxylapatite (e.g. Bio. gel HTP* Bio-Rad Laboratories, Richmond, Calif.), 2.5 g is added to 250 ml partially purified PRP preparations ~after Cetavlon and ethanol treatments) (containing approximately 1.0 mg PRP/ml) - in 20m~1 sodium phosphate buffer, pH 6.9 and mixed at ice-cold water bath ~1-4C) for one hour. The mixture is centrifuged in the Sorvall RC2-B for 30 millutes at 16,000 x g. The supernatant fluid is then passed through 0.65JU
~0 millipore filter and subjected to the foregoing procedure (each time treated ith 2~5 g hydroxylapatite) 2 more times. The resulting solution is filtered through 0.65~ and 0.~5~ millipore filters, dialyzed against pyrogen-free dis-tilled water and lyophilized. The lyophilized product exhibits strong immu-nogenic activity (see below combined vaccine and animal experiment) and con-tains very low contaminants (such as nucleic acids, proteins and endotoxins) ~see Table I below). Approximately 170 mg of the lyophilized PRP is obtained.
The recovery of PRP by this process is approximately 70~ of the starting ~Trademark 7_ :, , ~ :

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polysaccharide. The PRP should be stored under sui~able conditions, such as at 4C in a desiccator over phosphorus pentoxide and silica gel.
Example 2 A partially purified PRP ~300 mg PRP) is dissolved in 100 ml of 20m~1 sodium phosphate buffer, p~l 5.8 (i.e. 3 mg PRP/ml). This solution is applied to a column at ambient temperature (5.0 x 45 cm) of hydroxylapatite ~approximately 250 ml bed volume which has been equilibrated with 20mM sodium phosphate buffer, pll 5.8 and eluted with a stepwise gradient of 20mM, 50mM, lOOn~ sodium phosphate buffer, pH 5.8. The individual fractions (200 ml) eluted with 20mhl and 50mhl sodium phosphate buffer (pH 5.8), positive for pentose ~pentose determination by the orcinal method) are collected, dialyzed against pyrogen-free distilled water and lyophilized. Approxinnately 206 mg of the lyophilized product, PRP, is obtained.
The purity of the polysaccharide, PRP, is assayed by estimating to what extent it is contaminated with nucleic acids, proteins and endotoxins.
Protein concentration is determined by the method of Lowry, et al. in J. Biol. Chem. 193:265 ~1951) with bovine serum albumin as standard. Nucleic acid is measured by the absorption of the PRP solution at 260nm. The absorb-ance of 50 pg of nucleic acid in one ml of water in a cell of l-cm light path ~0 is assumed to be equal to 1Ø Molecular size is estimated by means of Sepharose ~B or 2B gel filtration on columns 1.5 x 90 cm (Pharmacia-Fine Chemicals, Piscataway, N. J.). Partition coefficient ~Kd) values are cal-culated from the elution pattern developed by the orcinal reaction ~Herbert, et al., Methods in Microbiology, Vol. 5B, 285-291.

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Exam~le 3 A combination vaccine con~aining PRP from H. influenzae type b and B. p_rtussis antigens is prepared as follows: 140 mg PRP ~as prepared in - Example 2) is dissolved in 350 ml sterile phosphate buffered saline (PBS) (0.113 8 potassium diphosphate, 0.83 g disodium phosphate, and 8.5 g sodium chloride per liter, pH 7.0, containing 0.01% thimerosal), and filter through 0.45~ millipore filter This concentrated PRP solution (400 ~g/ml) is used to prepare a combination vaccine which consists of PRP and B. pertussis antigens .
To 150 ml of the concentrated PRP solution ~400 ~g/ ml) add 150 ml of fresh B. pertussis (strain 138) cell suspension in PBS, pH 7.0 (contain-ing 149 opacity; op, unit cells/ml) to prepare the stock solution of the combined vaccine. This stock solution (300 ml) is kept at 4C until th0 endotoxin level of the pertussis is decreased (at least 90 days). The sterility of the stock solution is tested with thioglycollate media (at 32-33~C). The pH of the stock solution after 90 days incubation is checked and adjusted to pH 7.0 -1. The final product (vaccine) used for animal experiments is prepared by diluting the stock combined solution with PBS and it contains 10 ~g of PRP and 3.5 op units of pertussis cells/0.5 ml (dose).
The results of the antibody response to this combined vaccine in youn~ rats appears in Figures 1 and 2 as shown below.
E~ample 4 Tlle stock solution of PRP (400 ~g/ml) is prepared by dissolving 30 mg PRP (as prepared in Example 1) in 75 ml sterile phosphate buffered saline ~PBS) and filtered through 0.45 ~ millipore filter. To 25 ml of this con-centrated PRP is added an equal volume (i.e. 25 ml) of fresh B. pertussis (strain 138) cell suspension in PBS, pH 7.0 (containin~ 149 op units cells/-: ' ml) to prepare the stock solution of the combined vaccine. This stock solu-tion (50 ml) is kept at 4C for ~at least) 90 days. The sterility of the stock solution is tested as aforemen~ioned with thioglycollate media. The pH of the stock solution is pH 7.0+1. The final product (vaccine) used for animal experiments is prepared by diluting the stock combined solution with PBS, and it contains 10 ~g of PRP and 3.7 op units of pertussis cells/0.5 ml (dose).
Example 5 The PRP stock solution ~200 ~g/ml in PBS (pH 7.0~ is prepared as 10in Example 4. The pertussis stock solution (containing 75 op units/ml) is prepared by diluting 25 ml of fresh B. pertussis cell suspension (149 op units/ml) with an equal volume of PBS. Both stock solutions are incubated ; separately at 4C for 90 days or slightly longer. The combined vaccine is made by taking 14 ml of PRP stock solution ~200 ~g/ml), plus 14 ml (detoxi-fied) stock solution of pertussis antigens (75 op units/ml) and diluting it with ll~ ml PBS ~final val. 140 ml). The final vaccine used for animal experiments contains 10 ~g PRP and 3.7 op units pertussis/0.5 ml ~dose).
` The results of the antibody response to this combined vaccine in young rats appears in ~igure 3 and in Table II.

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Claims (2)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. In the method of isolating and purifying immunologically active polyribosyl ribitol phosphate (PRP), the capsular polysaccharide of Haemophilus influenzae type b, fermenting strains of the organism Haemophilus influenzae type b under art recognized conditions, isolating the PRP by conventional ethanol precipitation, further isolating the PRP as a hexadecyltrimethyl ammonium bromide complex by conventional procedures, the improvement comprising:
purifying the PRP by adding hydroxylapatite [3-Ca3(PO4)2'Ca(OH)2] in a 0.02M sodium phosphate buffer at a pH from about 6.7 to about 6.9, mixing at a temperature of about 4°C., centrifuging, removing the supernatant and repeating the foregoing procedure at least two more times, filtering the supernatant through suitable filters, dialyzing against pyrogen-free distilled water, and then lyophilizing.

2. In the method of isolating and purifying immunologically active polyribosyl ribitol phosphate (PRP), the capsular polysaccharide of Haemophilus inf1uenzae type b, fermenting strains of the organism Haemophilus influenzae type b under art recognized conditions, isolating the PRP by conventional ethanol precipitation, further isolating the PRP as a hexadecyltrimethyl ammonium bromide complex by conventional procedures, the improvement comprising:
purifying the PRP by column chromatography in a 0.02M sodium phosphate buffer at a pH of about 5.8 through hydroxylapatite [3-Ca3(PO4)2'Ca(OH)2], eluting with a stepwise gradient of 0.02M and 0.05M sodium phosphate buffer at a pH of about 5.8, isolating fractions by analysis for PRP, dialyzing said fractions against pyrogen-free distilled water and then lyophilizing.