DK154327B - PROCEDURE FOR ISOLATING AND CLEANING IMMUNOLOGICALLY ACTIVE POLYRIBOSYLRIBOLPHOSPHATE - Google Patents

Description

DK 154327 B

The present invention relates to a method for isolating and purifying immunologically active polyribosylribitol phosphate (PRP), the capsule polysaccharide of Haemophilus influenzae type b, in which strains of the organism Haemophilus influenzae type b are grown under known conditions, PRP is isolated by ethanol precipitation, and PRP is further isolated as a hexadecyltrimethylammonium bromide complex.

The process of this invention is characterized in that PRP is further purified by hydroxylapatite either a) by addition of hydroxylapatite [3 * Ca 2 (PO 2) 2Ca (OH) 2J in a 20 mM sodium phosphate buffer at a pH of 6, 5 to 7.0,

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of the above liquid and repeating the procedure described at least twice, filtering the above liquid through suitable filters, dialyzing against pyrogen-free, distilled water and then lyophilizing or b) by column chromatography in a 0.02 M sodium phosphate buffer at a pH of ca. 5.8 through hydroxylapatite [3'Ca 2 (PO 2) 2 * Ca (OH) 21, eluting with a stepwise gradient of 0.02 M and 0.05 M sodium phosphate buffer at a pH of approx. 5.8, isolating the fractions by assay for PRP, dialyzing the fractions against pyrogen-free, distilled water and then lyophilizing.

Thus produced PRP is effective in a combined vaccine containing PRP and B. pertussis antigens as well as when used in conjunction with other non-pathogenic bacterial strains such as E. coli, B. subtilis, S. aureus, B. punilus and L. plantarum , to trigger an antibody reaction in warm-blooded animals.

The purified polyribosylribitol phosphate (PRP) from Haemophilus influenzae type b has been previously investigated as a protective immunogen. However, no active antigenic response has been achieved in young animals and young children. In the known purification method, ethanol and "Cetavlon" ® (hexadecyltrimethylammonium bromide) are used. The impurities, endotoxins and pyrogenic substances are removed using cold phenol or chloroform and tert-butanol, which may result in loss of the antigenic nature of this polysaccharide.

The present method for isolating and purifying immunologically active PRP from Haemophilus influenzae type b has clear advantages over the known methods in that all impurities (nucleic acids, proteins and endotoxins) are removed to the least possible amount by treatment with hydroxylapatite.

The PRP prepared by the process of the present invention gives a higher molecular weight polysaccharide than those previously known.

Of even greater importance is that the PRP produced by the present process is highly immunogenic in warm-blooded animals as opposed to the PRP produced by the known methods.

Hydroxylapatite has previously been used to remove endotoxin impurities from Meningococcus group A and group C - polysaccharides, cf. U.S. Patent No. 3,978,209. However, it is a disadvantage of this known method that it is also desired

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polysaccharide is adsorbed together with the impurities, so that a subsequent selective desorption step is necessary to separate polysaccharide and impurities. In contrast, by the process of the invention, PRP is not adsorbed on the hydroxyl apatite but remains in solution such that a desorption step is superfluous.

The method of the invention allows a combined PRP and pertussis vaccine to be highly immunogenic in young animals.

(I) Isolation of purification of polyribosylribitol phosphate, the capsule polysaccharide of Haemophilus influenzae type b.

Organisms, culture substrate and culture.

Two strains of Haemophilus influenzae type b are used. The Rab strain is available from Grace Leidy, Babies Hospital, Columbia University, New York City, New York. The CK strain is isolated from a patient at Waterbury Hospital, Waterbury, Connecticut. The organisms are repeatedly cultured on mice to ensure their virulence. The organisms are isolated by autopsy from mouse brain tissue, then cultured in subculture on either 3.7% brain-heart infusion substrate (BHI) (Difco Lab., Detroit, Michigan) or on 5% BHI agar supplemented with 0.01% nicotinamide adenine dinucleotide ( NAD) (PL Bio-chemicals, Milwaukee, Wisconsin) and 1% (r / r) defibrinated horse blood (Animal Blood Center, Syracuse, NY) and then distributed in 1 ml portions in ampoules, lyophilized and left at -70 ° C.

The basal substrate used for growing the organisms is 3.7% (BHI). The basal substrate is supplemented with 10 mg NAD and 20 mg hemin (Eastman Kodak, Rochester, N.Y.) per ml. liter. 1% (r / r) of defibrinated horse blood is added per day. 50 ml seed culture. The added materials are freshly prepared and filtered through a 0.45 µm "Nalgene" ® filter (Nalge Sybron Corp., Rochester, N.Y.) before use. To grow the organisms in a 14 liter fermentation vessel, the substrate is added an additional 0.5% glucose.

To prepare a flask of graft organisms, 1 ml of frozen stock culture is thawed and transferred to 50 ml of the enriched BHI substrate added to defibrinated horse blood and NAD. The culture is grown for 8 hours at 37 ° C on a shaking table at 150 rpm. The organisms are added as 1% inoculum to 500 ml portions of the enriched BHI substrate in 2 liter flasks, which are then incubated at 37 ° C under moderate shaking on a shaking table for 8 hours (for isolation of PRP) or for 14 hours ( as seed cultures).

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The culture of each batch in a 14 liter fermentation vessel is initiated by aseptically transferring 700 ml of the seed culture to 7 liters of the enriched BHI substrate added to hemin instead of defibrinated horse blood. The culture is maintained continuously at a temperature of 37 - 1 ° C. The vessel is stirred at a speed of 150 rpm and an air flow of 0.25 liters of air per minute is maintained. liter of mask per During cultivation, 0.001% of a silicone foam damper ("FD-82", Hodag Chemical Corp.) is added if necessary. The cultures are grown to the 9 late log phase (8-10 x 10 viable cells / ml), usually approx.

8 hours and then the culture is stopped by the addition of 0.4% formaldehyde and standing overnight at 4 ° C.

Polyribosylribitol phosphate (PRP) isolation

Culture substrate prepared as described above is centrifuged at 27,000 x g for 30 minutes at 4 ° C. The cell-free supernatant is isolated and treated as follows:

Step 1) Ethanol precipitation

Sodium acetate (final concentration 4%) is added to the fracentrifuged liquid. The solution is adjusted to pH 6.0-6.2 and 44 liters of ethanol are slowly added with vigorous stirring at 4 ° C. The mixture is adjusted to pH 6.8 with glacial acetic acid and then left for 12 hours at 3 ° C. The precipitate formed is isolated by decantation and then centrifuged to give crude PRP.

Step 2) "Cetavion" ® (hexadecyltrimethylammonium bromide) treatment

The precipitate formed in 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 "Cetavlon" ® with mixing (final concentration 0.5%). The mixture is stirred for 1 hour and then centrifuged. The precipitate of nucleic acids and the PRP "Cetavlon" ® complex are mixed with 2 liters of 0.3 M sodium chloride. The turbid solution is centrifuged to eliminate insoluble materials such as nuclein "Cetavlon" ® salt.

The above liquid is diluted with a similar volume of water, whereby the PRP "Cetavlon" ® salt precipitates. The mixture is stirred for 1 hour, the precipitate is isolated by centrifugation and then dissolved in 2 liters of 0.3 M sodium chloride.

Step 3) Ethanol precipitation "Cetavlon" ® and the impurities of nucleic acids and proteins are further removed by ethanol precipitation (at least 2 times). The PRP is precipitated as described in step 1), while "Cetavlon" ® is dissolved in the alcoholic solution.

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solution. PRP is isolated by centrifugation, dissolved in 2 liters of pyrogen-free, distilled water and then precipitated again as described above. The final PRP precipitate is solubilized in 20 mmol sodium phosphate, pH 6.8.

Step 4) Hydroxylapatite treatment

Impurities (e.g., nucleic acids, proteins, and endotoxins) in the partially purified PRP materials are selectively removed by adsorption on hydroxylapatite [3 · Ca 2 (PO 2) 2 * Ca (OH) 0].

The present invention is based on the recognition that the polysaccharide PRP is not adsorbed by the calcium phosphate-containing adsorbent containing 20 mmol phosphate buffer having a pH of 6.7-6.9 or 50 mmol of phosphate buffer having a pH of about

5.8. However, the impurities (such as nucleic acids, proteins and endotoxins) are adsorbed under these conditions. The present process can be carried out batchwise or by column operation. In the batch process, hydroxylapatite is added to the partially purified PRP material (in 20 mmol phosphate, pH 6.9). The mixture is thoroughly mixed and centrifuged to remove undesirable solids (adsorbent and impurities adsorbed by the adsorbent). The above liquid is treated with hydroxylapatite as described above, at least 2 times more. The resulting solution is filtered through millipore filters, dialyzed against pyrogen-free, distilled water and lyophilized.

In column operation, the partially purified PRP in 20 mmol phosphate buffer (pH at least 5.8) is added to a column containing the adsorbent hydroxylapatite equilibrated with 20 mmol phosphate buffer pH 5.8 and eluted with a stepwise gradient of sodium phosphate buffer (pH 5.8) from 20 mmol to 100 mmol. Fractions are isolated and analyzed for pentose (for polyribosylribitol phosphate). Fractions with a positive pentose reaction are dialyzed against pyrogen-free, distilled water and lyophilized.

Example 1 2.5 g of hydroxylapatite (e.g., "Biogel® HTP", Bio-Rad Laboratories, Richmond, California) is added to 250 ml of partially purified PRP material (after "Cetavlon" ® and ethanol treatment) (containing ca. 1.0 mg PRP / ml) in 20 mmol sodium phosphate buffer, pH 6.9, and mixed on an ice-cold water bath (1-4 ° C) for 1 hour. The mixture is centrifuged in a "Sorvall RC2-B" centrifuge for 30 minutes

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The above liquid is then passed through a 0.65 μιη millipore filter and subjected to the above-described treatment (treated each time with 2.5 g of hydroxylapatite) an additional 2 times. The resulting solution is filtered through 0.65 µm and 0.45 μπι millipore filters, dialyzed against pyrogen-free, distilled water and lyophilized. The lyophilized product exhibits strong immunogenic activity (cf. subsequent animal studies with combined vaccine) and contains very small amounts of impurities (such as nucleic acids, proteins and endotoxins), cf. the following table I. Approx. 170 mg lyophilized PRP. The yield of PRP in this method is approx. 70% of the starting polysaccharide.

PRP should be stored under suitable conditions, such as at 4 ° C in a phosphorus pentoxide and silica gel desiccator.

Example 2

Partially purified PRP (300 mg PRP) is dissolved in 100 ml 20 mmol sodium phosphate buffer, pH 5.8 (ie 3 mg PRP / ml). This solution is added to a 5.0 x 45 cm column of hydroxylapatite (about 250 ml mass volume ), equilibrated with 20 mmol sodium phosphate buffer, pH 5.8, and eluted with a stepwise gradient of 20 mmol, 50 mmol and 100 mmol sodium phosphate buffer, pH 5.8. The individual fractions (200 ml) eluted with 20 mmol and 50 mmol sodium phosphate buffer (pH 5.8) with positive pentose reaction (pentose assessment by the orcinol method) are isolated, dialyzed against pyrogen-free, distilled water and lyophilized. There are approx. 206 mg of the lyophilized product PRP.

The purity of the polysaccharide PRP is examined by calculating the extent to which it is contaminated with nucleic acids, proteins and endotoxins. Protein concentration is determined by the method of Lowry et al., Cf. "J. Biol. Chem." 193: 265 (1951) with bovine serum albumin as standard. The nucleic acid content is measured by absorption of the PRP solution at 260 nm. The absorption of 50 µg of nucleic acid in 1 ml of water in a cell where the light passes a layer thickness of 1 cm is assumed to correspond to 1.0. The molecular size is calculated using "Sepharose" ® 4B "or -2B" gel filtration on a 1.5 x 90 cm column (Pharmacia-Fine Chemicals, Piscataway, New Jersey). The partition coefficient values (Kd) (cf. Gel Filtration, Theory and Practice, Pharmacia Fine Chemicals, (1979) pages 31-33 are calculated from the elution pattern induced by the orcinol reaction (cf. Herbert et al., "Methods in Microbiology", Vol. 5B, 285 -291).

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To investigate the effect of a vaccine containing PRP from Haemophilus influenzae, a combination vaccine with B. pertussis antigens has been prepared.

A. A combination vaccine containing PRP from Haemophilus influenzae type b and B. pertussis antigens is produced 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 of calcium diphosphate, 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 mm millipore filter. This concentrated PRP solution t (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) add 150 ml of fresh B. pertussis (strain 138) cell suspension in PBS, pH 7.0 (containing 149 opacity cell units per ml) to prepare the stock solution of the combined vaccine. . This stock solution (300 ml) is maintained at 4 ° C until the amount of endotoxin in pertussis is reduced (at least 90 days). The sterility of the stock solution is tested using thioglycollate substrate at 32-33 ° C. The pH - = value of the stock solution after 90 days of incubation is measured and adjusted to 7.0 µl 1. The final product (vaccine) used for animal testing is prepared by diluting the combined stock solution with PBS and containing 10 PRP and 3.5 pertussis opacity cell units / Q, 5 ml (dose).

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

B. A stock solution of PRP (400 µg / ml) is prepared by dissolving 30 mg of PRP (prepared as described in Example 1) in 75 ml of sterile, phosphate-buffered saline (PBS) and filtered through a 0.45 Mm millipore filter. To 25 ml of this concentrated PRP is added a corresponding volume (i.e. 25 ml) of fresh B. pertussis (strain 138) cell suspension in PBS, pH 7.0 (containing 149 opacity cell units per ml) to prepare the stock solution of the combined vaccine. This stock solution (50 ml) is stored at 4 ° C for at least 90 days. The sterility of this stock solution is tested as described above with thioglycollate substrate. The pH of the stock solution is 7.0 - 1. The end product (vaccine) used for animal testing,

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is prepared by diluting the stock solution with IPBS and containing 10 µg of PRP and 3.7 pertussis opacity cell units / 0.5 ml (dose).

C- A PRP stock solution (200 µg # ml in PBS, pH 7.0) is prepared as described under B. The Pertussis stock solution ((containing 75 opacities / ml}) is prepared by diluting 25 ml of fresh ert pertussis cell suspension ( 149 units of opacity per ml) with a similar volume of PBS. Both tamper solutions are incubated separately at 4 ° C for 90 days or slightly longer.The combined vaccine is prepared by taking 14 ml of the PRP stock solution ((200 µg / ml) plus 14 ml (detoxified) stock solution of pertussis antigens (75 opacities / ml) and dilute this with 112 ml PBS (final volume 140 ml). 5 ml (dose).

The results of antibody response to this combined vaccine in young rats are shown in Figure 3 of the drawing sanfit of Table II.

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Comparison test.

Table III below shows that the PRP produced leather method of the invention can be combined with B. pertaiss ice cells, and that the resulting combination vaccine induces a significant cytochrome antibody response in many individuals than in vaccines containing ® RP prepared by a phenol extraction method.

Table III

Comparison of antibody response elicited with vaccines containing variously prepared PRP.

Vaccine Individual No. PRP Antibody µg / ml) PRP prepared by the 16 0 "46 method 17 ^ 0, 54 according to the invention 18 JD" 27 + B. pertussis-19 0,, 48-cell 20, 31, 31

Phenol extracted 1 <3), / 02 PRP + B. pertus 2 <0, / 02 sis cells 3 <0, / 02 4 <0, / 02 5 0, / 06 6 0, / 05 7 <0 "i02 8 0 ,, (04 9 <0, / 02 10 <0, / 02

An antibody level of> 0.15 Mg / ml Sor is considered protective.

Claims (1)

Method for isolating and purifying immunologically active polyribosylribitol phosphate (PRP), the capsule polysaccharide of Haemophilus influenzae type b, in which strains of the organism Haemophilus influenzae type b are cultured under known conditions, characterized in that the PRP is further purified by hydroxylapatite either a) by the addition of hydroxylapatite [3 'Ca 2 (PO 2) (OH) 2] in a 20 mM sodium phosphate buffer at a pH of 6.5 to 7 Mixing at a temperature of 2-10 ° C, centrifuging, removing the above liquid and repeating the procedure described at least twice, filtering the above liquid through suitable filters, dialyzing against pyrogen-free, distilled water and then lyophilizing or b) by column chromatography in a 0.02 M sodium phosphate buffer at a pH of ca. 5.8 through hydroxylapatite [S'Ca ^ (PO ^) 2'Ca (OH) / elution with a stepwise gradient of 0.02 M and 0.05 M sodium phosphate buffer at a pH of approx. 5.8, isolating the fractions by assay for PRP, dialyzing the fractions against pyrogen-free, distilled water and then lyophilizing.