NO149611B - PROCEDURE FOR THE ISOLATION AND CLEANING OF IMMUNOLOGICALLY ACTIVE POLYRIBOSYL-RIBITOL PHOSPHATE (PRP), THE CAPSULAR POLYSACCARIDE OF HAEMOPHILUS INFLUENZAE - Google Patents

Description

This invention relates to a method for isolating and purifying the antigenic polysaccharide polyribosylribitol phosphate (PRP) from Haemophilus influenzae type b cultures.

The purified polyribitol phosphate (PRP) from Haemophilus influenzae type b is currently being investigated as a protective immunogen. However, an active antigenic response in young animals and children has not been achieved. According to the previously known method of purification, ethanol and "Cetavolon" (hexadecyltrimethylammonium bromide) are used. The contaminants, endotoxins and pyrogenic substances were removed using cold phenol or chloroform and t-butanol, which may result in the loss of the antigenic character of this polysaccharide.

A new method for isolating and purifying immunologically active PRP from Haemophilus influenzae type b has been developed.

The present method shows clear advantages compared to previously known methods, in that all contaminants (nucleic acids, proteins and endotoxins) are removed to the lowest possible level by treatment with hydroxylapatite. PRP produced by the method described here gives a higher molecular weight polysaccharide than previously reported.

It is also important that PRP produced according to this new method is highly immunogenic in warm-blooded animals in contrast to the PRP produced according to previously known methods.

A method for removing contaminants (proteins, nucleic acids and endotoxins) in the polysaccharide PRP is achieved by treating the partially purified polysaccharide with a phosphate-containing adsorbent which does not absorb the polysaccharide under the specified conditions.

(I) Isolation and purification of polyribosylribitol phosphate,

the capsular polysaccharide of Haemophilus influenzae type b.

Organisms, culture medium and culture

Two strains of Haemophilus influenzae type b are used.

The Rab strain was obtained from Grace Leidy, Babies Hospital, Columbia University, New York City, New York. The CK strain was isolated from a patient at Waterbury Hospital Waterbury, Conn. The organisms are passed through mice several times to ensure their virulence.

The organisms are isolated at autopsy from mouse brain tissue, sub-cultured on either 3.7% brain-heart infusion medium (BHI)

(Difco Lab., Detroit, Mich.) or on 5% BHI agar supplemented with 0.01% nicotinamide adenine dinucleotide (NAD) (P-L Biochemicals, Milwaukee, Wis.) and 1% (vol/vol ) defibrinated horse blood (Animal Blood Center, Syracuse, N.Y.), and then dispensed in 1-ml portions into vials, lyophilized, and stored at -70°C.

Basic medium used for cultivation of the organisms is

3.7% BHI. The basic medium is supplemented with 10 mg NAD and 20 mg hemin (Eastman Kodak, Rochester, N.Y.) per litres. 1% (volume/volume) defibrinated horse blood is added per 50 ml inoculum. The supplements are freshly prepared and filtered through a 0.45 µm Nalgene filter unit (Nalge Sybron Corp., Rochester, N.Y.) prior to use. For cultivation of the organism in a 14 liter fermenter, the medium is further supplemented with 0.5% glucose.'

To prepare a flask of inoculum organisms, optines

1 ml of frozen stock culture and transfer to 50 ml of the enriched BHI medium supplemented with defibrinated horse blood and NAD. The culture is grown for 8 hours at 37°C on a gyro device at a speed of 150 rpm. The organisms are added as a 1% inoculum to 500 ml portions of enriched BHI culture medium in 2 liter flasks, which are then incubated at 37°C with moderate shaking on a gyro shaker for 8 hours (for isolation of PRP) or 14 hours ( as graft cultures).

The fermentation of each batch in a 14 liter fermenter is initiated by aseptically transferring 700 ml of the inoculum to 7 liters of the enriched BHI culture fluid supplemented with hemin instead of defibrinated horse blood. The culture is maintained continuously at 37 - 1°C. The tank is stirred at a speed of 150 rpm, and an air flow of 0.25 liters of air per liter of culture liquid per minute is maintained. During cultivation, 0.001% of a silicone antifoam agent (FD-82, Hodag Chemical Corp.) is added as needed. Cultures are grown to only exponential growth (8-10 x 10 9 viable cells per ml), usually approx. 8 hours, after which the cultivation is terminated by adding 0.4% formaldehyde and storing the cultures overnight at 4°C.

Isolation of polyribosylribitol phosphate (PRP)

Culture fluid prepared according to the above is centrifuged at 27,000 x g for 30 minutes at 4°C. The cell-free liquid on top is stored and processed as follows:

Step 1, ethanol sweep

Sodium acetate is added to the liquid at the top of the culture (final concentration 4%). The solution is adjusted to pH 6.0-6.2, and 44 liters of methanol denatured ethanol are added slowly with vigorous stirring at 4°C. The mixture is adjusted to pH 6.8 with glacial acetic acid and then stored for 12 hours at 3°C. The resulting precipitate is collected by decantation and then centrifuged to form impure PRP.

Step 2, "Cetavlon" (hexadecyltrimethylammonium bromide) treatment

The precipitate from 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% water solution of "Cetavlon" while mixing (final concentration 0.5%). The mixture is stirred for 1 hour and then centrifuged. The precipitation of nucleic acids and PRP "Cetavlon" complex is mixed with 2 liters of 0.3M sodium chloride. The cloudy solution is centrifuged to remove insoluble material such as Imklein "Cetavlon" salt.

The liquid on top is diluted with an equal volume of water, whereby the PRP "Cetavlon" salt is precipitated. The mixture is stirred for 1 hour, the precipitate is collected by centrifugation and then dissolved in 2 liters of 0.3M sodium chloride solution.

Step 3, ethanol sweep

"Cetavlon" and the contaminants of nucleic acids and proteins are further removed by ethanol purification (at least twice). PRP is precipitated according to step 1, while "Cetavlon" is dissolved in the alcohol solution. The PRP is collected by centrifugation, dissolved in 2 liters of pyrogen-free, distilled water and then precipitated again as described above. The final PRP sweep is solubilized in 20 mM sodium phosphate, pH 6.8.

Step 4, hydroxylapatite treatment

Impurities (such as nucleic acids, proteins and endotoxins) in the partially purified PRP preparations are selectively removed by adsorption on hydroxylapatite [3-Ca^(PO4) 2Ca(OH)^\ .

The invention is based on the discovery that the polysaccharide PRP is not adsorbed by the hydroxylapatite-containing phosphate buffer (0.02M) with a pH of 6.7-6.9; or a phosphate buffer (0.02M) med

a pH of approx. 5.8; while, on the other hand, the contaminants (such as nucleic acids, proteins and endotoxins) are adsorbed under these conditions. The method according to the invention can be carried out in batches or in column operation. In batch execution, the hydroxyl apatite is added to the partially purified PRP preparation (in 0.02M sodium phosphate buffer; pH 6.7-6.9). The mixture is carefully mixed at 2-10°C, and centrifuged to remove unwanted solids (adsorbent and the contaminants adsorbed by the adsorbent). The liquid on top is subjected to the aforementioned procedure at least two more times. The resulting solution is filtered through a "Millipore" filter, dialyzed against pyrogen-free distilled water and lyophilized.

During column operation, partially purified PRP is applied in 0.02M sodium phosphate buffer (pH approx. 5.8) in a column containing the adsorbent hydroxylapatite, which has been brought to equilibrium with 0.02M sodium phosphate buffer, pH approx. 5.8, and eluted with a stepwise gradient of sodium phosphate buffer, pH approx. 5.8 from 0.02M to 0.05M. Fractions are collected and analyzed for pentose (and thus for polyribosylribitol phosphate). The fractions that are positive with respect to pentose are dialyzed against pyrogen-free, distilled water and lyophilized.

The invention shall be illustrated in more detail by means of the following examples where the indicated temperatures are in °C.

Example 1

2.5 g of hydroxylapatite (e.g. Bio. gel HTP, Bio-Rad Laboratories, Richmond, California) is added to 250 ml of partially purified PRP preparations (after "Cetavlon" and ethanol treatments)

(containing approx. 1.0 mg PRP/ml) in 20 mM sodium phosphate buffer,

pH 6.9, and mixed in an ice-cold water bath (1-4°C) for 1 hour. The mixture is centrifuged in a Sorvall RC2-B for 30 minutes at 16,000 x g. The supernatant is then passed through a 0.65 m millipore filter and subjected to the aforementioned procedure (each time treated with 2.5 g of hydroxylapatite) twice more. The solution obtained is filtered through 0.65 u and 0.45 m millipore filters,

dialyzed against pyrogen-free, distilled water and lyophilized.

The lyophilized product exhibits strong immunogenic activity

(see below regarding animal testing with combined vaccine)

and contains a very small amount of contaminants (such as nucleic acids, proteins and endotoxins) (see Table I below). About. 170 mg of lyophilized PRP is obtained. The recovery of PRP by this method is approx. 70%, calculated on the starting polysaccharide. PRP should be stored under suitable conditions, e.g. at 4° in a desiccator over phosphorus pentoxide and silica gel.

Example 2

Partially purified PRP (300 mg PRP) is dissolved in 100 ml 20 mM sodium phosphate buffer, pH 5.8 (ie 3 mg PRP/ml). This solution is applied to a column at room temperature (5.0 x 45 cm) of hydroxylapatite (approx. 250 ml layer volume), which has been brought to equilibrium with 20 mM sodium phosphate buffer, pH 5.8, and eluted with a stepwise gradient of 20 mM , 50 mM and 100 mM sodium phosphate buffer, pH 5.8.

The individual fractions (200 ml), eluted with 20 mM and 50 mM sodium phosphate buffer (pH 5.8), which are positive for pentose (pentose determination according to the original method), are stored, dialyzed against pyrogen-free distilled water and lyophilized. You get approx. 206 mg of the lyophilized product,

PRP.

The purity of the polysaccharide, PRP, is analyzed by assessing the extent to which they are contaminated with nucleic acids, proteins and endotoxins. The protein concentration is determined according to Lowry et al. in J. Biol. Chem. 193:265 (1951) with bovine serum albumin as standard. The nucleic acid is determined by absorption of the PRP solution at 2 60 nm. The absorbance of 50 ug nucleic acid

in 1 ml of water in a cell with a light path of 1 cm is considered to be equal to 1.0. Molecular size is assessed by "Sepharose" 4B or 2B gel filtration on 1.5 x 90 cm columns (Pharmacia-Fine Chemicals, Pissataway, N.J.). The partition coefficient (Kd) is determined from the elution pattern produced by the original reaction (Herbert et al, Methods in Microbiology, vol. 5B, 285-291).

To investigate the effect of a vaccine containing PRP from H. influenzae, a combination vaccine with B. pertussis antigens was prepared: A. A combination vaccine containing PRP from H. influenzae type b and B. pertussis antigens is prepared according to the following: 140 mg of PRP (prepared according to example 2) is dissolved in 350 ml of sterile, phosphate-buffered sodium chloride solution (PBS) (0.113 g of potassium phosphate, 0.83 g of disodium phosphate and 8.5 g of sodium chloride per liter, pH 7.0, containing 0.01% thimerosal) and filtered through a 0.45 µm millipore filter. This concentrated PRP solution (400 µg/ml) is used for the production of a combination vaccine, which consists of PRP and B. pertussis antigens.

150 ml of the concentrated PRP solution (400 µg/ml) is added to 150 ml of fresh B. pertussis (strain 138) cell suspension in PBS, pH 7.0 (with opacity 149; op, unit cells/ml) to prepare the stock solution of the combined vaccine. This stock solution (300 ml) is kept at 4° until the pertussis endotoxin level has decreased (at least 90 days). The sterility of the stock solution is checked with thioglycolate media (at 32-33°). The pH of the stock solution after 90 days of incubation is checked and adjusted to pH 7.0 in 1. The final product (vaccine) used for animal experiments is prepared by diluting the combined stock solution with PBS, and it contains 10 yg of PRP and 3.5-op -units of pertussis cells/0.5 ml (dose).

B. The stock solution of PRP (400 ug/ml) prepared by dissolving 30 mg of PRP (prepared according to Example 1) in 75 ml of sterile, phosphate buffered sodium chloride solution (PBS) and filtering through a 0.45 um millipore filter. To 25 ml of this concentrated PRP is added an equal volume (ie 25 ml) of fresh B. pertussis (strain 138) cell suspension in PBS, pH 7.0 (containing 149 op-units cells/ml) to prepare the stock solution of the combined vaccine. This stock solution (50 ml) is kept for at least 90 days at 4°. The sterility of the stock solution is tested as indicated above with thioglycolate media. The pH of the stock solution is 7.0 ± 1. The final product (vaccine) used for animal experiments is prepared by diluting the combined stock solution with PBS, and it contains 10 µg of PRP and 3.7 op units

of pertussis cells/O.5 ml (dose).

C. The PRP stock solution (200 ug/ml) in PBS (pH 7.0) is prepared as in B. 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 for 90 days or slightly longer at 4°. The combined vaccine is prepared by taking 14 ml of PRP stock solution (200 yq/ml) and 14 ml (detoxified) stock solution of pertussis antigens (75 op units/ml) and diluting it with 112 ml of PBS (final volume 140 ml). The finished vaccine for animal experiments contains 10 Mg PRP and 3.7 pertussis pertussis units/0.5 ml (dose).

The results regarding the antibody response to this combined vaccine in young rats are shown in Table II.

Comparison experiment

. In the following table it is shown that PRP is produced

according to the invention. Jean is combined with B. pertussis cells and that the resulting combined vaccine elicits a significantly greater antibody reaction in young individuals than is the case with vaccines containing PRP prepared by a phenol extraction method.

Comparison of antibody response elicited by vaccines containing PRP prepared by different methods

Antibody mirror - 0.15 ug/ml is considered protective

Claims (1)

Process for the isolation and purification of immunologically active polyribosyl-ribitol phosphate (PRP), the capsular polysaccharide of Haemophilus influenzae, after fermentation of strains of the organism Haemophilus influenzae type b, isolation of PRP by ethanol precipitation and further isolation of PRP as hexadecyltrimethylammonium bromide complex, characterized in that the PRP isolated after ethanol precipitation and treatment with hexadecyltrimethylammonium bromide is purified using hydroxylapatite either (a) by adding hydroxylapatite [3 Ca^(PO4)2Ca(OH)21 in a 0.02 M sodium phosphate buffer at a pH from 6 .7 to 6.9, mixing at a temperature of 2 to 10°C, centrifugation, removing the supernatant and repeating the preceding process at least two more times, filtering the supernatant through appropriate filters, dialyzing against pyrogen-free distilled water and then lyophilization, or (b ) by column chromatography in 0.02 M sodium phosphate buffer at a pH of approx. 5.8 through hydroxylapatite [3Ca.j (PO^) 2 • Ca (OH) , elution with a stepwise gradient of 0.02M and 0.05M sodium phosphate buffer at a pH of approx. 5.8, isolation of fractions by analysis after PRP, dialysis of said fractions against pyrogen-free, distilled water and then lyophilization.