CH649781A5 - METHOD FOR PRODUCING A POLYRIBOSYL RIBITOL PHOSPHATE AND A COMBINATION VACCINE CONTAINING THIS. - Google Patents

Description

The invention relates to methods for isolating and purifying the antigenic polysaccharide polyribosyl-ribotol phosphate (PRP) from Haemophilus influenzae type b cultures and a method for producing a combination vaccine, the PRP and Bordetella pertussis - Contains antigens. This vaccine can be used to immunize against Haemophilus influenzae type b infections, such as meningitis. The PRP is also effective in conjunction with other non-pathogenic strains of bacteria such as Escherichia coli, Bacillus subtilis, Staphylococcus aureus, B. punilus and L. plantarum to trigger an antibody response in warm-blooded animals.

The purified polyribitol phosphate (PRP) from Haemophilus influenzae type b is investigated as a protective immunogen. However, an effective antigen response has not yet been achieved in young animals and children. In the known method for cleaning, ethanol and hexadecyltrimethylammonium bromide (cetavlon) are used. The contaminants, endotoxins and pyrogenic substances are removed by using cold phenol or chloroform and t-butanol, which can lead to the loss of the antigenic nature of this polysaccharide.

New methods for the isolation and purification of immunologically active PRP from Haemophilus influenzae type b have now been found.

The processes still to be described have clear advantages over the known working methods, since all impurities (nucleic acids, proteins and endotoxins) are removed by treatment with hydroxyapatite. The processes according to the invention for the production of PRP lead to a polysaccharide with a higher molecular weight than the previously known processes.

The PRP produced by the new processes is, more importantly, in contrast to the PRP produced according to known procedures, highly immunogenic for warm-blooded animals.

The removal of impurities (proteins, nucleic acids and endotoxins) of the polysaccharide PRP is achieved by treating the partially purified polysaccharide with a phosphate-containing adsorbent which does not adsorb the polysaccharide under the specified conditions.

The further object of the invention is to provide a method for producing a combined PRP and pertussis vaccine which has a highly immunogenic effect in young animals.

The isolation and purification of polyribosyl ribitol phosphate, the shell polysaccharide of Haemophilus influenzae type b, is carried out according to the invention by the method of claims 1 and 4. Preferred embodiments of the invention are described below.

Organisms, culture medium and culture

There are preferably two strains of Haemophilus

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influenzae type b used. The Rab tribe has been known for a long time and is freely accessible, e.g. from The Journal of Immunology 107 (1971), 1071-1080 and Infection and Immunity 13 (1976), 581-589, in particular from the reference to previous, published studies with the same strain. He has also been deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852 (USA) under the number ATCC 31512. In the present case, the strain was obtained from Grace Leidy, Babies Hospital, Columbia University, 630 West 168 Street, New York, NY 10032 (USA). The CK strain was deposited on October 3, 1978 with the American Type Culture Collection under the number ATCC 31441. This strain has been isolated from a patient at Waterbury Hospital, Waterbury (CT, USA).

The organisms are subjected to several passages through mice to ensure their virulence. They are isolated from mouse brain tissue at autopsy and used to grow them using one of the following media: 3.7% Brain Cardiac Infusion (BHI) (Difco Lab., Detroit, Mich.) Medium or 5% BHI agar supplemented Horse blood defibrinated by 0.01% nicotinamide adenine dinucleotide (NAD) (PL Biochemicals, Milwaukee, Wis.) and 1% (volume / volume) (Animal Blood Center, Syracuse, NY). For further cultivation, 1 ml portions are distributed to ampoules, lyophilized and stored at -70 ° C.

The basic medium used for the cultivation of the organisms is 3.7 percent BHI. The base medium is supplemented with 10 mg NAD and 20 mg hemin (Eastman Kodak, Rochester, N.Y.) per liter. 1% (volume / volume) defibrinated horse blood is added to each 50 ml inoculum. The supplements are freshly prepared and a 0.45 | j. Nalgene filter unit (Nalge Sybron Corp., Rochester, N.Y.) filtered before use. The medium is also supplemented with 0.5% glucose for growing the organism in a 14-1 fermentation vessel.

To prepare organisms for vaccination, 1 ml of frozen stock culture is thawed and added to 50 ml of the enriched BHI medium, which is supplemented with defibrinated horse blood and NAD. The culture is grown for 8 hours at 37 ° C on a rotary shaker at 150 revolutions / minute. The organisms are added as a 1% inoculum to 500 ml portions of the enriched BHI medium in 2-1 flasks, which are then at 37 ° C with moderate shaking on a rotary shaker for 8 hours (to isolate PRP) or 14 hours (as seed cultures) are incubated. The fermentation of each portion in a 14-1 fermentation vessel is initiated by aseptically transferring 700 ml of the seed culture to 71 of the enriched BHI medium supplemented with hemin instead of defibrinated horse blood. The culture is kept constantly at 37+ I ° C. An air flow of 0.25 l of air per liter of medium and minute is maintained with stirring at 150 revolutions / minute. During growth, 0.001% of a silicone defoamer (FD-82, Hodag Chemical Corp.) is added as needed. Culturing continues until only exponential growth (8 to 10 x 109 living cells / ml) occurs, usually about 8 hours, and then cultivation is accomplished by adding 0.4% formaldehyde and allowing to stand overnight at 4 ° C ended.

Isolation of polyribosyl ribitol phosphate (PRP)

The mash obtained as described above is centrifuged at 4 ° C. for 30 minutes at 27,000 g. The cell-free supernatant liquid is separated off and treated as follows:

Level 1, ethanol precipitation

The supernatant liquid of the culture is mixed with sodium acetate to a final concentration of 4%. After adjusting the pH of the solution to 6.0 to 6.2, 44 1 3A ethanol are slowly added with vigorous stirring at 4 ° C. The mixture is adjusted to a pH of 6.8 with glacial acetic acid and then left to stand at 3 ° C. for 12 hours. The precipitate formed is obtained by decanting and centrifuging, whereby crude PRP is obtained.

Stage 2, treatment with hexadecyltrimethylammonium bromide (Cetavlon)

The precipitate obtained after step 1 is dissolved in pyrogen-free distilled water and centrifuged to remove the residue. The clear brown solution is then slowly added to 100 ml of a 10% aqueous solution of hexadecyltrimethylammonium bromide to a final concentration of 0.5% with mixing. The mixture is stirred for 1 hour and then centrifuged. The precipitate of nucleic acids and PRP-hexadecyltrimethylammonium bromide complex is mixed with 2 1300 mM sodium chloride. The cloudy solution is centrifuged to remove insoluble substances such as the nuclein-hexadecyltrimethylam monium bromide salts.

The supernatant liquid is diluted with an equal volume of water, causing the PRP-hexadecyltrimethylammonium bromide salt to precipitate. The mixture is stirred for 1 hour and the precipitate is collected by centrifugation and then dissolved in 2 1300 mM sodium chloride solution.

Stage 3, release of the PRP from the complex

Hexadecyltrimethylammonium bromide and the contaminating nucleic acids and proteins are further removed by ethanol precipitation at least twice. The PRP is precipitated as described in Step 1, while hexadecyl trimethylammonium bromide is dissolved in the alcoholic solution. The PRP is centrifuged off, dissolved in 21 pyrogen-free distilled water and then precipitated again as described above. The PRP precipitate finally obtained is dissolved in 20 millimolar sodium phosphate, pH 6.8.

Stage 4, hydroxyapatite treatment

The impurities (for example nucleic acids, proteins and endotoxins) in the partially purified PRP preparations are selectively removed by adsorption on an adsorbent containing calcium phosphate, namely hydroxylapatite 3Ca3 (P04) 2-Ca (0H) 2.

The improvement according to the invention is defined in claims 1 and 4.

The invention is thus based on the perception that the polysaccharide PRP contains calcium phosphate adsorbent, which contains phosphate buffer (20 mM) with a pH of 6.5 to 7.0 or 5.8, in contrast to the impurities, such as nucleic acid, proteins and endotoxins, is not adsorbed under these conditions. The method according to the invention can be carried out batchwise or in column operation.

When working in batches, the hydroxyapatite is added to the partially cleaned PRP preparation (in 20 mM phosphate, pH e.g. 6.9). After thorough mixing, centrifugation is carried out to remove unwanted solids (adsorbent and the impurities adsorbed thereon). The supernatant fluid is subjected to the procedure described above at least two more times. The solution obtained is filtered through Millipor filter, dialyzed against pyrogen-free distilled water and lyophilized.

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During column operation, the partially purified PRP in 20 mM phosphate buffer (pH 5.8) is applied to a column containing the hydroxylapatite adsorbent, which has been equilibrated with 20 mM phosphate buffer, pH 5.8, and with a step-wise modified sodium phosphate buffer ( pH 5.8) from 20 mM to 100 mM. The collected fractions are checked for pentose (polyribosyl ribitol phosphate). Those fractions which are positive for pentose are dialyzed against pyrogen-free distilled water and lyophilized.

To prepare a PRP vaccine, lyophilized PRP prepared as described above at a concentration of 20 lig / 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 vaccine is sterile filtered through 0.45-ji Millipor filter units, placed in glass vials and stored at 4 ° C.

The methods for producing a combination vaccine from PRP from Haemophilus influenzae type b and Borde-tella pertussis antigens are defined in claims 9 and 10. Preferably, a concentrated solution of the PRP is obtained by dissolving weighed predetermined amounts of the polysaccharide 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 ) produced. This concentrated solution of PRP is then mixed with fresh Bordetella pertussis cell suspensions, whereby the stock solution is obtained. The characteristics of B. pertussis strain 138 are given in Bergey's Manual of Determinative Bacteriology, VIII, editor Buchanan and Gibbons, WMS and Wilkins Co., Maryland, USA, 1974, on page 283. This strain is from the American Type Culture Collection, Maryland, V.St.A. available under the accession number ATCC 10380. The combination vaccine in this stock solution contains 200 µg PRP and about 70 opacity units (op) cells / ml. The stock solution is left to detoxify pertussis antigens at 4 ° C for 90 days before the finished product (vaccine) is prepared, which contains 10 (ig PRP and 3.5 op units of pertussis cells / 0.5 ml dose) .

The invention is further illustrated by the following examples.

example 1

2.5 g of hydroxyapatite (e.g. Bio.Gel HTP, Bio-Rad Laboratories, Richmond, Calif.) Are made into 250 ml partially cleaned PRP preparations (according to hexadecyltrimethylammonium

bromide and ethanol treatments) containing about 1.0 mg PRP / ml, placed in 20 mM sodium phosphate buffer, pH 6.9, and mixed for 1 hour in an ice-cold water bath (1 to 4 ° C). The mixture is centrifuged in the Sorvall RC2-B for 30 minutes at 1600 g. The supernatant is then passed through a 0.65 µ millipore filter and subjected to the procedure described above two more times, each time treating with 2.5 g of hydroxyapatite. The solution obtained is by 0.65 Li and 0.45 u. Millo liporfilter filtered, dialyzed against pyrogen-free distilled water and lyophilized. The lyophilized product shows a strong immunogenic activity (see below, combination vaccine and animal experiments) and contains only very few impurities, such as nucleic acids, proteins 15 and endotoxins (see Table I). About 170 mg of lyophilized PRP are obtained, which corresponds to about 70% of the polysaccharide used. The PRP must be stored under suitable conditions, for example at 4 ° C, in a desiccator over phosphorus pentoxide and silica gel.

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Example 2

A partially purified PRP with a PRP content of 300 mg is dissolved in 100 ml of 20 mM sodium phosphate buffer, pH 5.8 (i.e. 3 mg PRP / ml). This solution is applied at room temperature to a column (5.0 × 45 cm) of hydroxyapatite (bed volume approximately 250 ml, equilibrated with 20 mM sodium phosphate buffer, pH 5.8) and with 20 mM, 50 mM and 100 mM sodium phosphate buffer , pH 5.8, eluted. The fractions obtained with 20 mM and 50 mM sodium phosphate buffer (pH 5.8) eluting 3o fractions (200 ml) which are pentose positive (pentose determination according to the orcin method) are dialyzed against pyrogen-free distilled water and lyophilized. About 206 mg of lyophilized product, PRP, are obtained.

35 The purity of the polysaccharide PRP is checked by determining the degree of contamination with nucleic acids, proteins and endotoxins. The protein concentration is determined by the method of Lowry et al. in J. Biol.

Chem. 193: 265 (1951) with bovine serum albumin as standard 40. Nucleic acid is determined by the absorption of the PRP solution at 260 nm. The absorption of 50 µg of nucleic acid in 1 ml of water in a cell with a light path of 1 cm is equated to 1.0. The molecular weight is determined by means of Sepharose 4B or 2B gel filtration on columns 45 of 1.5 × 90 cm (Pharmacia-Fine Chemicals, Piscataway, N.J.). The values of the distribution coefficient (Kd) are calculated from the elution image developed by the orcin reaction (Herbert et al., Methods in Microbiology, Vol. 5B, 285-291).

Table 1

Physico-chemical properties of PRP

Testing endotoxin

Mode of operation Mol. Size (Kd) Pentose (%) Nucleic acid (%) Protein (%) Rabbit Pyrogen Limulus Lysate Testd

Testc

Example 1 0a 36.0 0.8 0.7 10.0 laoo

Example 2 0.44b 33.8 0.3 0.7 10.0 '/ iooo a Using Sepharose 2B and 4B, molecular weight 2 x 107.

b Using Sepharose 4B.

c jj.g PRP / kg body weight (rabbits) that do not cause a fever.

d Reciprocal dilution of PRP (100 µg PRP / ml) compared to Bureau of Biologics El (endotoxin standard).

Example 1 relates to the characteristics of Haemophilus influenzae type b CK strain, ACC 31441.

Example 2 relates to the characteristics of Haemophilus influenzae type b Rab strain available from Babies Hosp. Columbia Univ. New York, N.Y., USA.

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Example 3

A combination vaccine containing PRP from H. influenzae type b and B. pertussis antigens is prepared as follows: 140 mg PRP (prepared as described in Example 2) are in 350 ml sterile 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) dissolved and filtered through 0.45 u Millipor filter. This concentrated PRP solution (400 µg / ml) is used to make a combination vaccine consisting of PRP and B. pertussis antigens.

150 ml of the concentrated PRP solution (400 μg / ml) are mixed with 150 ml of a fresh cell suspension of B. pertussis (strain 138) in PBS, pH 7.0 (containing 149 opacity (op) unit cells / ml), whereby the stock solution of the combination vaccine is obtained. This stock solution, 300 ml, is kept at 4 ° C until the degree of endotoxin of pertussis is reduced (at least 90 days). The sterility of the stock solution is checked with thioglycollate media at 32 to 33 ° C. The pH of the stock solution after an incubation of 90 days is checked and adjusted to 7.0 + 1. The finished product used for animal testing (vaccine)

is prepared by diluting the combined stock solution with PBS and contains 10 µg PRP and 3.5 op units per-tussis cells / 0.5 ml (dose).

The results of the antibody response to this combination vaccine in young rats are shown in FIGS. 1 and 2.

Example 4

The stock solution of PRP (400 jig / ml) is prepared by dissolving 30 mg of the PRP prepared as described in Example 1 in 75 ml of sterile phosphate-buffered saline (PBS) and filtering through 0.45-ji millipore filters. To 25 ml of this concentrated PRP solution, an equal volume, i.e. 25 ml fresh cell suspension of B. pertussis 5 (strain 138) in PBS, pH 7.0 (containing 149 op-units cells / ml), whereby the stock solution of the combination vaccine is obtained. This stock solution (50 ml) is left to stand at 4 ° C for at least 90 days. The sterility of the stock solution is checked with thioglycol-io latmedien as mentioned above. The pH of the stock solution is 7.0 + 1. The finished product (vaccine) used for animal experiments is prepared by diluting the combination stock solution with PBS and contains 10 jxg PRP and 3.7 op units of pertussis cells / 0.5 ml (dose).

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Example 5

The PRP stock solution (200 µg / ml in PBS, pH 7.0) is prepared as described in Example 4. The pertussis stock solution (containing 75 op units / ml) is prepared by diluting 20 ml fresh cell suspension of B. pertussis (149 op units / ml) with an equal volume of PBS. Both stock solutions are incubated separately at 90 ° C for 90 days or a little longer. The combination vaccine is prepared using 14 ml PRP stock solution (200 (ig / ml), 14 ml (detoxified) stock solution of pertussis antigens (75 op units / ml) and 112 ml PBS (final volume 140 ml) This vaccine used for animal experiments contains 10% PRP and 3.7 op units B. pertussis / 0.5 ml (dose).

3o The results of the antibody response to this combination vaccine in young rats appear in Fig. 3 and in Table II.

Table II

Immunogenicity of PRP-pertussis complex in young rats3

Anti-PRP antibody activity (cpm 3H-PRP bound / 50 | il serum)

Dose one or week after injection 3rd week after injection

Vaccine multiple 0 1 2 3 ... Times increase by

Pre-immunization

Phosphate buffered

(M) b

99c

116

107

158

1.6

Saline solution

Pertussis

(M)

113

108

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149

1.3

PRP (K) (example 1)

(M)

110

136

148

142

1.3

PRP (K) (3 mo) + pertussis

(S) d

108

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132

147

1.4

(3 mo) (example 5)

(M)

101

135

1002

1771

17.5

PRP (K) + pertussis (3 mo)

(S)

93

146

135

140

1.5

(Example 4)

(M)

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751

1410

11.8

a 10 (ig PRP or 3.5 op units pertussis / dose (0.5 ml) b best value 2nd and 3rd week (multiple injection) c mean value of 10 rats d single injection

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3 sheets of drawings

Claims (14)

649 781 1. Process for the isolation and purification of immunologically active polyribosyl ribitol phosphate, called PRP, by fermentation of strains of the organism Haemophilus influenzae type b, isolation of the PRP by ethanol precipitation, subsequent isolation of the PRP as hexadecyltrimethylammonium bromide complex, release of the PRP from the complex and Purification of the PRP obtained, characterized in that the partially purified PRP by adding hydroxyapatite, ie 3Ca3 (PO <i) 2 ■ Ca (OH) 2, in a 20 mM sodium phosphate buffer at a pH of 6.5 to 7.0, mixing at a temperature of 2 to 2. Process for the isolation and purification of immunologically active polyribosyl ribitol phosphate, called PRP, by fermentation of strains of the organism Haemophilus influenzae type b, isolation of the PRP by ethanol precipitation, subsequent isolation of the PRP as hexadexyltrimethylammonium bromide complex, release of the PRP from the complex and Purification of the obtained PRP, characterized in that the PRP by column chromatography in a 20 mM sodium buffer at pH 5.8 using hydroxylapatite, ie 3Ca3 (P04) 2-Ca (0H) 2, elution with a graded 20 mM to 50 mM sodium phosphate buffer at pH 5.8, isolation of fractions by analysis on PRP, dialysis of these fractions against pyrogen-free distilled Water and lyophilizing is cleaned. 2nd PATENT CLAIMS 3. The method according to claim 1 or 2, characterized in that the Rab strain of Haemophilus influenzae type b is used for the fermentation. 4. The method according to claim 1 or 2, characterized in that the CK strain from Haemophilus influenzae type b ATCC 31441 is used for the fermentation. 5. Isolated and purified by the method according to claim 1, immunologically active polyribosyl ribitol phosphate. 6. Isolated and purified, immunologically active polyribosyl ribitol phosphate by the process according to claim 2. 7. polyribosyl ribitol phosphate according to claim 5 or 6, isolated and purified by the method according to claim 3. 8. polyribosyl ribitol phosphate according to claim 5 or 6, isolated and purified by the method according to claim 4. 9. A method of making a combination vaccine for the formation of anti-PRP, i.e. Anti-polyribosylribitolphos phat and antipertussis antibodies in young warm-blooded animals, characterized in that polyribosylribitolphosphate obtained by the process according to claim 1 is mixed with Bordetella pertussis antigens. 10. A method of making a combination vaccine for the formation of anti-PRP, i.e. Anti-polyribosylribitolphos phat and antipertussis antibodies in young warm-blooded animals, characterized in that polyribosylribitolphosphate obtained by the process according to claim 2 is mixed with Bordetella pertussis antigens. 10 ° C, centrifuging, separating the supernatant liquid and repeating these measures at least twice, filtering the supernatant liquid and then dialyzing against pyrogen-free distilled water and lyophilizing. 11. The method according to claim 9 or 10, characterized in that the Bordetella pertussis antigens have been obtained from Bordetella pertussis ATCC 10380. 12. The method according to claim 9 or 10, characterized in that the polyribosyl ribitol phosphate has been obtained from the Rab strain of Haemophilus influenzae type b. 13. The method according to claim 9 or 10, characterized in that the Bordetella pertussis antigens from Bordetella pertussis ATCC 10380 and the polyribosyl ribitol phosphate from the Rab strain of Haemophilus influenzae type b have been obtained. 14. The method according to claim 9 or 10, characterized in that the Bordetella pertussis antigens from Bordetella pertussis ATCC 10380 and the polyribosyl ribitol phosphate from the CK strain of Haemophilus influenzae type b ATCC 31441 have been obtained.