DD140701A5 - PROCESS FOR THE ISOLATION AND PURIFICATION OF IMMUNOLOGICALLY ACTIVE POLYRIBOSYLRIBITOL PHOSPHATE - Google Patents

Description

The invention is in the field of vaccines for immunization against infections by Haemophilus influenzae type b, such as meningitis. More specifically, it relates to (1) a method of isolating the polysaccharide poly (polysaccharide) pyrilosyl-ribitol-phosphate (PRP) from Haemophilus influenzae type b cultures and (2) a method of producing a combination vaccine, PRP and B. pertussis Contains antigens. The PRP according to the invention is also effective in conjunction with other non-phosphate strains of bacteria such as E. coli, B.subtilis, S. aureus, B. punilus and plantarum for inducing a _t antibody response in warm-blooded animals.

Ch ^ akteristilc the known technical solutions

The purified polyribitol phosphate (PRP) from Haemophilus influenzae type b is tested as a protective immunogen. However, an effective antigenic response has not yet been achieved in young animals and children. Ethanol and hexadecyltrimethylammonium bromide (Cetavlon) are used in the known method of purification. The impurities,. Endotoxins and fumed substances are removed by use of cold phenol or chloroform and t-butanol, which may result in the loss of the antigenic nature of this polysaccharide.

Object of the invention '''·

A new method for the isolation and purification of immunologically active PRP from Haemophilus · influenzae type b has now been found.

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

More importantly, the PRP prepared by the new method is highly immunogenic, in contrast to the warm-blooded animal PRP prepared by known methods.

DISCLOSURE OF THE INVENTION 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 conditions indicated.

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

(X) Isolation and Purification of Polyribosylribitol Phosphate , the envelope polysaccharide of Haemophilus influenzae type b

Organisms, culture medium and culture

Two strains of Haemophilus influenzae type b are used. The Rab tribe has been obtained from Grace Leidy, Babies Hospital, Columbia University, New York City, New York.

The CK strain has been isolated from a patient at Waterbury Hospital, Waterbury, Conn. The organisms are subjected to multiple passages through mice to ensure their virulence. They are isolated from mouse brain tissue at autopsy, and their growth is achieved using one of the following media: 3.7% brain heart infusion (BHI) (Difco Lab., Detroit, Mich.) Medium or 5% BHI Agar supplemented with 0.01% nicotinamide adenine dinucleotide (NAD) (PL Biochemicals, Milwaukee, Wis.) And 1% (v / v) defibrinated horse blood (Animal Blood Center, Syracuse, NY). For further culturing aliquots of 1 ml are distributed into vials, lyophil.i- 'Siert and stored at -70 ⁰ C.

Base medium used for breeding the organisms is 3.7% BHI. The basal medium is supplemented with TO mg NAD and 20 mg hemin (Eastman Kodak, Rochester, N.Y.) per liter. Each 50 ml of seed culture is added to 1% (v / v) defibrinated horse blood. The supplements are made fresh and filtered through a Ο, 45μ Nalgene filter unit (Nalge Sybron Corp., Rochester, N.Y.) before use. For the cultivation of the organism in a 14 1 fermentation vessel, the medium is also supplemented with 0.5% 'glucose.

For the preparation of organisms for insemination, 1 ml of frozen stock culture is thawed and added to 50 ml of enriched BHI medium supplemented with defibrinated horse blood and NAD. The culture is grown for 8 hours at 37 ⁰ C on a Rundschüttelvorrichtung at 150 revolutions / Mxnute. The organisms as 1-owned inoculum to portions of 500 ml of the enriched BHI-medium in 2 1 flask, the (then at 37 ⁰ C with moderate shaking on a Rundschüttelvorrichtung 8 hours (for the isolation of PRP) or 14 hours a as seed cultures).

The fermentation of each portion in a 14 L fermentor is initiated by aseptic transfer of 700 mL of seed culture to 7 L of enriched BHI medium supplemented with hemin in place of defibrinated horse blood. The culture is kept at 37 + 1 ^0C. While stirring at 150 rpm, an air flow of 0.25 l air per liter of medium and minute is maintained. During growth, 0.001% of a silicone defoamer (FD-82, Hodag Chemical Corp.) is added as needed. Culturing of the cultures is continued until only exponential growth (8 to 10 × 10 living cells / ml) occurs, usually about 8 hours, and then the cultivation is increased to 4 by addition of 0.4% formaldehyde and overnight ' ⁰ C ended.

Isolation of polyribosylribitol phosphate (PRP)

The slurry obtained as described above is at 4 ⁰ C at 30 min 27 000. g centrifuged. The cell-free supernatant fluid is separated and treated as follows:

Stage 1, ethanol precipitation '·'.

The supernatant liquid of the culture is added to sodium acetate to a final concentration of 4%. After adjusting the pH of the solution to 6.0-6.2, 44 L of 3A ethanol is slowly added at 4 ^° C with vigorous stirring. The mixture is adjusted with glacial acetic acid to a pH value of 6.8 and then allowed to stand for 12 hours at 3 ⁰ C. The precipitate formed is recovered by decanting and centrifuging to give crude PRP.

Step 2, treatment with hexadecyltrimethylammonium bromide (Cetavlo

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 added slowly 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 L of 0.3 M sodium chloride. The cloudy solution is centrifuged to remove insoluble matter, such as the Nuclein-Hexadecyltrimethylammoniumbromid salts.

The supernatant is diluted with an equal volume of water to precipitate the PRP-hexadecyltrimethylammonium bromide salt. The mixture is stirred for 1 hour and the precipitate is recovered by centrifugation and then dissolved in 2 L of 0.3 M sodium chloride solution.

Stage 3, E thanolfael tion

Hexadecy 1 tr ime thy la mi orium bromide and the contaminating nucleic acids and proteins are further removed by at least two ethanol precipitations. The PRP is precipitated as described in Step 1 while hexadecyltrimethylammonium bromide is dissolved in the alcoholic solution. The PRP is centrifuged off, dissolved in 2 l of pyrogen-free distilled water and then precipitated again as described above. The final PRP precipitate is dissolved in 20 mM 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 a calcium phosphate-containing adsorbent, such as hydroxylapatite / 3Ca ₃ (PO 4 ₎ 2Ca (OH) ₂ - /. ,

The invention is based on the perception that the polysaccharide PRP is replaced by the calcium phosphate adsorbent, the phosphate buffer (20 mM) with a pH of 6.7 to 6.9 or a phosphate buffer (50 mM) with a pH of 5.8 contains, in contrast to the impurities, such as nucleic acids, proteins and endotoxins, is not adsorbed under these conditions. The process according to the invention can be carried out batchwise or in column mode. In batch work, the hydroxyapatite is added to the partially purified PRP preparation (in 20 mM phosphate, pH 6.9). After thorough mixing, centrifugation is performed to remove unwanted solids (adsorbent and the contaminants adsorbed thereon). The supernatant liquid is subjected to the procedure described above at least two more times. The resulting solution is filtered through Millipore filters, dialyzed against pyrogen-free distilled water and lyophilized. , ,

In column mode, the partially purified PRP in 20 mM phosphate buffer (pH at least 5.8) is loaded on a column containing the adsorbent hydroxyapatite equilibrated with 20 mM phosphate buffer, pH 5.8, and with a Stepwise modified sodium phosphate buffer (pH 5.8) eluted from 20 mM to 100 mM. The collected fractions are tested for pentose (polyribosylribitol phosphate). "Those fractions that are positive for pentose are dialyzed against pyrogen-free distilled water and lyophilized.

(II) A method for producing a combination vaccine from PRP from H. influenzae type b and B. pertussis antigens

To produce a PRP vaccine, lyophilized PRP prepared as described above is dissolved in phosphate buffered saline (PBS) at a concentration of 20 μ 9 / μl (0.113 g kaiium 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 μ Milliporfiltereinheiten, placed in glass vials and stored at 4 ⁰ C. , :

A concentrated solution of the PRP is prepared 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). manufactured. This concentrated solution of PRP is then mixed with fresh B. pertussis cell suspensions to give the stock solution. The characteristics of B. pertussis strain 138 are given in Bergey's Manual of Determinative Bacteriology, VIII, ed. Buchanan and Gibbons, WMS and Wilkins Co., Maryland, USA ./1974, at page 283. This strain is available from the American Type Culture Collection, Maryland, V.St.A. available under the accession number 10380. The combination vaccine in this stock solution contains 200 μl PRP and about 70 opacity units (op) cells / ml. The stock solution is allowed to de-toxinize pertussis antigens for 90 days at 4 ⁰ C before the final product. (Vaccine) containing 10 μ9 PRP and 3.5 op units pertussis cells / 0.5 ml dose.

embodiments

The following examples further illustrate the invention.

.Example 1

2.5 g of hydroxyapatite (eg, Bio-Gel HTP, Bio-Rad Laboratories, Richmond, Calif.) Are added to 250 ml of partially purified PRP preparations (after hexadecyltrimethylammonium bromide and ethanol treatments) containing about 1.0 mg PRP / ml. in 20 mM sodium phosphate buffer, pH 6.9, and 1 hour

in an ice-cold water bath (1 to 4 ⁰ C) mixed. The mixture is in Sorvall. RC2-B 30 minutes at 1600. g centrifuge. The supernatant liquid is then passed through a 0.65 μ millipore filter and subjected to two additional operations as described above, treating each time with 2.5 g of hydroxyapatite. The resulting solution is filtered through 0.65 μ and 0.45 μ Millipore filters, dialyzed against pyrogen-free distilled water and lyophilized. The lyophilized product shows strong immunogenic activity (see below, combination vaccine and animal experiment) and contains very little impurities, nucleic acids, proteins and endotoxins (see Table I). Approximately 170 mg of lyophilized PRP are obtained 70% of the polysaccharide used. The PRP must ..Bedingungen under suitable, for example, at 4 ⁰ C, stored in a desiccator over phosphorus pentoxide and silica gel.

Example 2

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

The purity of the polysaccharide PRP is tested by determining the level of contamination 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 determined by the absorbance of the PRP solution at 260 nm. The absorbance of 50 μg of nucleic acid in 1 ml of water in a cell with a light path of 1 cm is set equal to 1.0. The molecular weight is determined by Sepharose 4B or 2B gel filtration on 1.5 x 90 cm columns (Pharmacia-Fine Chemicals, Piscataway, N.J.). The values of the partition coefficient (Kd) are calculated from the elution pattern developed by the orcin reaction (Herbert, et al., Methods in Microbiology, Vol. 5B, 285-291).

T a-b e I

Physicochemical properties of PRP

exam

endotoxin

Mol. GröjSe pentose Procedure (Kd) (%)

Nucleic acid protein rabbit pyrogenic

test ° '

Limulus lysate test ^d

example 1

Example 2 0.44

36.0

33.8

0.7

0.7

10.0 10.0

1/400 1/1000

a) Using Sepharose 2B and 4B, molecular weight 2 x '10

b) Using Sepharose 4B

c) μg PRP / kg of body weight (rabbits) that do not cause fever

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

Example 1 relates to the features of Haemophilus influenzae type b CK strain, ATCC 31551

Example 2 refers to the features of Haemophilus influenzae type b Rab strain, available from Babies Hosp. Columbia Univ. New York, NY , USA

Example 3

A combination vaccine containing PRP from H. influenzae type b and B. pertussis antigens is prepared as follows: 140 mg of PRP (prepared as described in Example 2) are dissolved in 350 ml of 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) and filtered through 0.4 5 μ millipore filter. This concentrated PRP solution (400 μg / ml) is used to prepare a combination vaccine consisting of PRP and B. pertussis antigens.

To 150 ml of the concentrated PRP solution (400 μg / ml) is added 150 ml of a fresh cell suspension of B pertussis (strain 138) in PBS, pH 7.0 (containing 149 opacity (op) unit cells / ml) the stock solution of Kombinationsvaccine is obtained. This stock solution, 300 ml, is kept at 4 ^° C. until the endotoxic grade of pertussis is reduced (at least 90 days). The sterility of the stock solution is tested with thioglycollate media at 32 to 33 ^° C. The pH of the stock solution after incubation for 90 days is checked and adjusted to 7.0 + _ 1. The finished product (vaccine) used for animal experiments is prepared by diluting the pooled stock solution with PBS and contains 10 μL of PRP and 3.5 ounces of pertussis / O, 5 mL (dose).

The results of the antibody reaction to these combination vaccines in young rats are shown in FIGS. 1 and 2. '.

Example 4

The stock solution of PRP (400 μg / ml) is obtained by dissolving 30 mg of the as in Example. 1 described PRP in

20871

75 ml of sterile phosphate buffered saline (PBS) and filtered through 0.45 μ millipore filters. To 25 ml of the concentrated PRP solution is added an equal volume, i. h 25 ml fresh cell suspension of B. pertussis (strain 138) in PBS, pH 7.0 (containing 149 op-units cells / ml) to give the stock solution of the combination vaccines. This stock solution (50 ml) is kept at 4 for at least 90 days ⁰ C left. The sterility of the stock solution is tested as mentioned above with thioglycollate media. The pH of the stock solution is 7.0 + 1. The finished product used for animal experiments (vaccine) is prepared by diluting the combination stock solution with PBS and contains 10 ^ g PRP and 3.7 op units of pertussis cells / 0.5 ml (Dose).

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 25 ml fresh cell suspension of B. pertussis (149 op units / ml) with an equal volume of PBS. Both stock solutions are incubated separately for 90 days or a little longer at 4 ⁰ C. The combination vaccine is prepared using 14 ml of PRP stock solution (200 μg / ml), 14 ml (de-poisoned) stock solution of pertussis antigens (75 op units / ml) and 112 ml of PBS (final volume 140 ml). These for animal The vaccine used contains 10 μg PRP and 3.7 op units B. pertussis / 0.5 ml (dose). ,

The results of the antibody reaction to these combination vaccines in young rats appear in FIG. 3 and in Tabula II.

T '& be 1 1 e II

Immunogenicity of PRP-pertussis complex in young rats

Vaccine

Dose single or multiple

Anti-PRP antibody activity (cpm H-PRP bound / 50 μ! Serum)

3rd week after injection (fold increase by pre-immunization

Week after injection 0 '1 2

Phosphate buffered saline

pertussis

PRP (K) (Example 1) (M)

PRP (K) (3 mo). (S) '

Pertussis (3 mo) (M). (Example 5)

PRP (K) + Perussis (S)

(3 mo) (Example 4) (M)

99 116 107 158 113 108 117 149 110 136 148 142 108 136 132 147 101 135 1002 1771 93 146 135 140 119 154 751 · 1410

1.6 1.3

1.3

1.4

17.5

1.5 11.8

a) 10 μg PRP or 3.5 μl units of pertissus / dose (0.5 ml)

b) best value 2nd and 3rd week (multiple injection)

c) Mean of 10 rats

d) single injection

Claims (2)

Sponsor claims 1. A process for the isolation and purification of immunologically active polyribosylribitol phosphate (PRP), the envelope polysaccharide of Haemophilus influenzae type b by fermentation of strains of the organism Haemophilus influenzae type b under known conditions, isolation of the PRP by conventional ethanol precipitation and further known per se isolation of the PRP as Hexadecyltrimethylammoniumbromidkomplex, characterized in that PRP by addition of hydroxylapatite / 3Ca ₃ (PO ₄ ) ₂ Ca (OH) __ / in a 20 millimolar sodium phosphate buffer at a pH of 6.5 - 7.0, mixing at a temperature of 2-1O ° C, centrifuging , Separation of the supernatant liquid and at least two repetition of these measures, filtration of the supernatant liquid and subsequent dialysis against pyrogen-free distilled water and lyophilization is purified. 2. Method according to item 1, characterized in that the PRP is purified by column chromatography in a 0.02M sodium phosphate buffer at pH 5.8 using hydroxyapatite / 3Ca., (PO ₂ ) ₂ .Ca (OH) ₂ _ /, eluted with a graded 0.02 M to 0.05 M sodium phosphate buffer at pH 5.8, isolation of fractions by analysis for PRP, dialysis of these fractions against pyrogen-free distilled water and lyophilization becomes.