SE430753B - COMBINED HAEMOPHILUS INFLUENZAE-BORDETELLA PERTUSSIS-VACCIN - Google Patents

Description

Elimination of impurities (proteins, nucleic acids and endotoxins) in the polysaccharide PRP is carried out 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 polyribosyl ribitol phosphate, the capsular polysaccharide of Haemophilus influenzae type b Organisms, culture medium and culture Two strains of Haemophilus influenzae type b were used. The rhubarb strain is 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 allowed to pass through mice several times to ensure their virulence. The organisms are isolated by autopsy from brain tissue in mice, subcultured 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) (PL Biochemicals, Milwaukee, Wis.) and 1% (v / v) defibrinated horse blood (Animal Blood Center, Syracuse, NY) and then aliquoted into 1 ml aliquots on ampoules, lyophilized and stored at -70 ° C.

The basic medium used for culturing the organisms consists of 3.7% BHI. The base medium is supplemented with 10 mg NAD and 20 mg hemin (Eastman Kodak, Rochester, N.Y.) per liter. 1% (v / v) defibrinated horse blood is added per 50 ml of seed culture.

The complements are freshly prepared and filtered through a 0.45u Nalgene filter unit (Nalge Sybron Corp., Rochester, N.Y.) prior to use. For culturing the organism in a 14-liter fermentor, the medium is further supplemented with 0.5% glucose.

To prepare a flask of inoculum, 1 ml of frozen stock culture is thawed and transferred 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 shaker at a speed of 150 rpm. The organisms are added in the form of a 1% inoculum to 500 ml portions of enriched BHI culture broth in 2-liter flasks, which are then incubated at 37 ° C with moderate shaking on a gyro shaker for 8 hours (to isolate PRP ) or 14 hours (such as inoculum cultures).

The fermentation of each batch in a 14-liter fermentor is initiated by aseptic transfer of 700 ml of the inoculum to 7 liters of the enriched BHI culture broth supplemented with hemin instead of defibrinated horse blood. The culture is maintained continuously at 37 ° 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 fluid per minute is maintained. During culture, 0.001% of a silicone antifoam (FD-82, Hodag Chemical Corp.) is added as needed. Cultures are grown to the late logarithmic growth (8-10 x 109 culture is terminated by adding 0.4% formaldehyde viable cells / ml), usually about 8 hours, after which the cultures are stored overnight at 4 ° C.

Isolation of polyribosylribitol phosphate (PRP) Culture fluid prepared as above is centrifuged at 27,000 x g for 30 minutes at 4 ° C. The cell-free supernatant is collected and treated as follows: Step 1, ethanol precipitation The supernatant in the culture is added with sodium acetate (final concentration 4%). The solution is adjusted to pH 6.0-6.2 and 44 liters of 3A-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 recovered by decantation and then centrifuged to form crude PRP.

Step 2, Cetavlon (hexadecyltrimethylammonium bromide) treatment The precipitate from step 1 is dissolved in pyrogen-free distilled water and centrifuged to eliminate the residue. The clear brown solution is then slowly added to 100 ml of 10% aqueous solution of Cetavlon with mixing (final concentration 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 eliminate insoluble materials, such as nucleic Cetavlon salt.

The supernatant is diluted with an equal amount of volume (water, whereby the PRP-Cetavlon salt precipitates. The mixture is stirred for one hour, the precipitate is collected by centrifugation and then dissolved in 2 liters of 0.3 M sodium chloride solution. H Step 3, ethanol precipitation Cetavlon and precipitation of nucleic acids and proteins are further removed by ethanol precipitation (at least twice).

PRP is precipitated according to step 1, while Cetavlon is dissolved in the alcohol solution. PRP is collected by centrifugation, dissolved in 2 liters of pyrogen-free distilled water and then precipitated again as above. The final PRP precipitate is solubilized in 20 mM sodium phosphate, pH 6.8.

Step 4, hydroxylapatite treatment Contaminants (such as nucleic acids, proteins and endotoxins) in the partially purified PRP preparations are selectively removed by adsorption on a calcium phosphate-containing adsorbent, such as hydroxyl apatite [3.Ca3 (PO4) 2.Ca (OH) 2].

The invention is based on the discovery that the polysaccharide PRP is not adsorbed by the calcium phosphate adsorbent-containing phosphate buffer (20 mM) with a pH of approximately 6.7-6.9; or a phosphate buffer (50 mM) having a pH of about 5.8; however, the impurities (such as nucleic acids, proteins and endotoxins) are adsorbed under these conditions. The process according to the invention can also be carried out batchwise or in column operation. In the batch process, the hydroxylapatite is added to the partially purified PRP preparation (in 20 mM phosphate; pH 6.9). The mixture is mixed thoroughly and centrifuged to eliminate unwanted solids (adsorbent and the impurities adsorbed by the adsorbent). The supernatant is subjected to the said procedure at least two more times. The resulting solution is filtered through a Millipor filter, dialyzed against pyrogen-free distilled water and lyophilized.

During column operation, partially purified PRP is applied in 20 mM phosphate buffer (pH at least 5.8) in a column containing adsorbent hydroxylapatite, which is equilibrated with 20 mM phosphate buffer, pH 5.8, and eluted with a step gradient of sodium phosphate buffer, pH 5.8 from 20mM to 100mM. Fractions are collected and analyzed for pentose (and thus for polyribosyl ribitol phosphate). The pentose-positive fractions are dialyzed against pyrogen-free distilled water and lyophilized.

(II) Process for the preparation of a combination vaccine, consisting of PRP from H. influenzae type b and B. pertussis antigens For the preparation of a PRP vaccine, lyophilized PRP (prepared as above) is dissolved in a concentration of 20 g / ml in phosphate buffered sodium chloride solution (PBS) (0.13 g of potassium diphosphate, 0.83 g of disodium phosphate and 8.5 g of sodium chloride per liter, pH 7.0, containing 0,01% thimerosal). The vaccine is sterile filtered through 0.45 millipore filter units, distributed in glass vials and stored at 4oC.

A concentrated solution of PRP is prepared by weighing predetermined amounts of the polysaccharide and dissolving it in phosphate buffered sodium chloride solution (0.13 g of potassium diphosphate, 0.83 g of disodium phosphate and 8.5 g of sodium chloride per liter, pH 7.0, containing 0 .0l% timerosal). This concentrated solution of PRP is then mixed with an appropriate volume of fresh B. pertussis cell suspensions to prepare the stock solution. 1a11192-9 6 Characteristics of B. pertussis, strain 138 are described in Bergeys Manual of Determinative Bacteriology, Eight Ed., Buchanan and Gibbons Editors, Williams & Wilkins Co., Maryland, USA. This strain is available from the American Type Culture Collection, -Maryland, USA, Deposit No. 10380. The combined vaccine in this stock solution contains 200 ng PRP and approximately 70 opacity units (op) cells per ml. The stock solution is maintained for 90 days at 400 to allow detoxification of pertussis antigens before the final product (vaccine) is prepared (containing 10 μg PRP and 3.5 op units of pertussis cells / 0.5 ml dose). .

The invention is further illustrated by the following examples, in which the temperatures indicated refer to degrees Celsius.

Example 1. 2.5 g of hydroxylapatite (such as Bio. Gel HTP, Bio-Rad Laboratories, Richmond, California) is added to 250 ml of partially purified PRP preparations (after Cetavlon and ethanol treatments) (containing approximately 1.0 mg PRP / ml ) in 20 mM sodium phosphate buffer, pH 6.9, and mix in an ice-cold water bath (1-400) for one 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 μ millipore filter and subjected to the said procedure (each time treated with 2.5 g of hydroxylapatite) twice more. The resulting solution is filtered through 0.65 μl and 0.45 μm millipore filters, dialyzed against pyrogen-free distilled water and lyophilized. The lyophilized product shows strong immunogenic activity (see the following for animal experiments with a combined vaccine) and contains a very small amount of impurities (such as nucleic acids, proteins and endotoxins) (see Table I below). Approximately 170 mg of lyophilized PRP is obtained. The recovery of PRP according to this procedure is about 70%, calculated on the starting polysaccharide. PRP should be stored under appropriate conditions, such as at 40 in a desiccator over phosphorus pentoxide and silica gel. 7811192-9 Example 2.

Partially purified PRP (300 mg PRP) is dissolved in 100 ml of 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 hydroxyl apatite (approximately 250 ml bed volume), equilibrated with 20 mM sodium phosphate buffer, pH 5.8, and eluted with a step gradient of 20 mM, 50 mM. 100mM 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 collected, dialyzed against pyrogen-free distilled water and lyophilized. Approximately 206 mg of the lyophilized product, PRP, is obtained.

The purity of the polysaccharide, PRP, is analyzed by estimating the extent to which they are contaminated with nucleic acids, proteins and endotoxins. The protein concentration is determined according to Lowry, et al. and J. Biol. Chem. l93¿265 (1951) with bovine serum albumin as standard. The nucleic acid is determined by the absorption of the PRP solution at 260 nm. The absorbance of 50 ng of nucleic acid in 1 ml of water in a cell with a light path of 1 cm is assumed to be equal to 1.0. Molecular size is estimated by Sepharose ÉD4B or 2B gel filtration on columns 1.5 x 90 cm (Pharmacia-Fine Chemicals, Piscataway, N.J.).

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A combination vaccine containing PRP from H. influenzae type b and B. pertussis antigens is prepared as follows: 140 mg PRP (prepared according to Example 2) is dissolved in 350 ml sterile phosphate buffered sodium chloride solution (PBS) (0.113 g potassium phosphate, 0, 83 g disodium phosphate and 8.5 g sodium chloride per liter, pH 7.0, containing 0.01% thimerosal) and filtered through a 0.45 μ millipore filter. This concentrated PRP solution (400 ug / ml) was used to prepare a combination vaccine consisting 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 (showing opacity 149; op, unit cells / ml) for preparation of the stock solution of the combined vaccine.

This stock solution (300 ml) is maintained at 40 until the endotoxin level of pertussis has dropped (minimum 90 days). The sterility of the stock solution is tested with thioglycolate media (at 32-330). The pH of the stock solution after 90 days of incubation is checked and adjusted to pH 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 PRP and 3.5 op units of pertussis cells / 0.5 ml (dose).

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

Example 4.

The stock solution of PRP (400 ng / ml) is 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 μ millipore filter. 25 ml of this concentrated PRP is added with an equal volume (ie 25 ml) of fresh B. pertussis (strain 138) cell suspension in PBS, pH 7.0 (containing 149 op units of cells / ml) to prepare the stock solution of the combined vaccine. This stock solution (50 ml) is maintained for at least 90 days at 40. The sterility of the stock solution is tested as 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 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 according to 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 storage solutions are incubated separately at 40 for 90 days or slightly longer. The combined vaccine is prepared by taking 14 ml of PRP stock solution (200 ng / 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 animal test vaccine contains 10 ng PRP and 3.7 op units of pertussis / 0.5 ml (dose).

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Claims (4)

7. 711112-9 H .. CLAIMS 1. Combined vaccine which produces effective levels of anti-PRP (polyribosyl-ribitol phosphate) and antipertussis antibodies in young warm-blooded animals, characterized in that it consists of polyribosyl ribitol phosphate, isolated and purified from the haemophilus influenzae type b capsular polysaccharide by adding hydroxylapatite in 2-20 molar phosphate buffer with a pH of 6.5-7.0, mixing at a temperature of 2-110 ° C , centrifugation, elimination of the supernatant and repetition of said procedure at least twice, filtration of the supernatant, dialysis against pyrogen-free distilled water and lyophilization, and Bordetella pertussis antigens; wherein the combined vaccine contains 10% polyribosyl ribitol phosphate and 3.5 units of opacity Bordetella pertussis cells per 0.5 ml of vaccine dissolved in the phosphate buffer. A vaccine according to claim 1, characterized in that the Bordetella pertussis antigens are obtained from the Bordetella pertussis strain 138. 3. A vaccine according to claim 1, characterized in that polyribosyl ribitol phosphate is obtained from the Haemophilus influenzae type b Rab strain. 4. A vaccine according to claim 1, characterized in that the Bordetella pertussis antigens are obtained from the Bordetella pertussis strain 138 and the polyribosyl ribitol phosphate is obtained from the Haemophilus influenzae type b Rab strain.