IE47474B1 - Purifying polyribosyl ribitol phosphate (prp) and vaccines containing immunologically active prp - Google Patents

Abstract

The invention provides methods for the purification of polyribosyl ribitol phosphate (PRP) from Haemophilus influenzae type b cultures, as well as novel forms of immunologically active PRP obtainable by such methods and combined vaccines containing immunologically active PRP and Bordetella pertussis antigens. The purification methods involve removing contaminants to a low level by treating the PRP in the form of a hexadecyltrimethyl ammonium bromide complex with a calcium phosphate adsorbent (hydroxylapatite) in a phosphate buffer, and may be practiced either as a batch process or as a column process. The purified PRP thus obtainable is immunologically active and may be used with B. pertussis antigens to form a combined vaccine which elicits anti-PRP antibody and antipercussis antibody formations in young animals. This sera with anti-PRP antibody exhibits a strong bactericidal activity.

Description

This invention is in the field of vaccines for immunization against Haemophilus influenzae type b infections, such as meningitis. More specifically, this invention relates to: (1) methods for the preparation of an immunologically active polysacchari de polyribosyl ribitol phosphate (PRP) from Haemophilus influenzae type b cultures: (2) a novel immunologically active PRP; and (3) combined vaccines containing immunologically active PRP and B. pertussis antigens. The PRP of this invention should also be effective when used in conjunction with other nonpathogenic strains of bacteria, such as E. coli, B.. subtilis, .S. aureus, pumilus and L_. plantarum, to trigger an antibody response in warm-blooded animals.

The purified polyribitol phosphate (PRP) from Haemophilus influenzae type b has been the subject of investigation as a protective immunogen. However, the active antigenic response in young animals and infants has not hitherto been achieved. The prior art method for the purification employed ethanol and hexadecyltrimethyT ammonium bromide (available under the Trade Mark Cetavlon).

The contaminants, endotoxins, and pyrogenic substances, were removed by the use of cold phenol or chloroform and jt-butanol which may have results in the loss of the antigenic nature of this polysaccharide.

A new method for the preparation of immunologically active PRP from Haemophilus influenzae type b has now been established.

The methods to be described have distinct advantages over the prior art procedures in that all contaminants (nucleic acids, proteins and endotoxins) are removed by a low level. By means of the present invention we have obtained PRP which has a higher molecular weight than those reported previously.

More importantly, the PRP obtainable herein is highly immunogenic in warm-blooded animals as compared with the PRP produced by prior art procedures. This PRP can be used to prepare combined PRP and pertussis vaccine which is highly immunogenic in young animals.

The present invention is predicated upon our discovery that partially purified polysaccharide PRP can be further treated, under conditions hereinafter explained, for the removal of contaminants (proteins, nucleic acids and endotoxins) with a phosphate-containing adsorbent which does not absorb the polysaccharide and which does not deleteriously affect its antigenic nature.

Thus, in accordance with one aspect, the present invention provides a method of purifying polyribosyl ribitol phosphate (PRP), the capsular polysaccharide of Haerophilus influenzae type b, obtained by fermenting a strain of the organism Haemophilus influenzae type b, isolating the PRP produced by ethanol precipitation and further isolating the PRP as a hexadecyltrimethyl ammonium bromide complex, comprising the steps of: adding hydroxyl apatite [j.Cag^Q^CaiOH^j to said PRP hexadecyl trimethyl ammonium bromide complex in a from 2 to 20 millimolar phosphate buffer at a pH from 6.5 to 7.0; mixing the resulting mixture at a temperature of 2°tolO°C., centrifuging; removing the supernatant and repeating the foregoing procedure at least two more times; filtering the supernatant; dialyzing against pyrogen-free distilled water; and then lyophilizing, whereby there is obtained an immunologically active PRP.

Preferred embodiments of this method of purifying the PRP have the following characteristics: (1) the phosphate buffer, preferably sodium phosphate, has a pH from 6.7 to 6.9, and preferably a strength of about 20 millimoles; and (2) the buffer and hydroxyl apatite are mixed at about 4°C.

As will be appreciated, the above method is a batch process, and is currently preferred. The same principles can be applied to column operation, and in accordance with this aspect of the present invention there is provided a method of purifying polyribosyl ribitol phosphate (PRP), the capsular polysaccharide of Haemophilus influenzae type b, obtained by fermenting a strain of the organism Haemophilus influenzae type b, isolating the PRP produced by ethanol precipitation, and further isolating the PRP as a hexadecyltrimethyl ammonium bromide complex, comprising the steps of: purifying the PRP by column chromatography in a from 2 to 20 millimolar phosphate buffer through hydroxylapatitejjCa^PO^.CafOH^]; eluting with a phosphate buffer of a concentration which increases upwards from 0.2M, isolating fractions which analyse positive from PRP; dialyzing said fractions against pyrogen-free distilled water; and then lyophilizing, whereby there is obtained an immunologically active PRP.

Preferred embodiments of this column method have the following characteristics: (1) the buffer, preferably sodium phosphate, in which the PRP is applied to the column has a strength of 20 minimoles, and preferably a pH of about 5.8; and (2) the column is eluted with a step-wise gradient of phosphate buffer, preferably sodium phosphate, and preferably having a pH of about 5.8, v/hich increases in strength from 0.02 M to 0.05 M.

A novel form of purified PRP is obtainable by means of the present purification methods. This novel PRP is characterized by being immunologically active and having a molecular v/eight greater than 20 millions.

As hereinbefore indicated, PRP obtainable by this invention can be used to prepare a combined PRP and pertussis vaccine.

DESCRIPTION OF PREFERRED EMBODIMENTS (I) Isolation and purification of polyribosyl ribitol phosphate, the capsular polysaccharide of Haemophilus influenzae type b Organisms, Growth Medium and Culture In our work, two strains of Haemophilus influenzae type b have been used. The Rab strain is obtainable from the Rabies Hospital, Columbia University, New York City, New York, U.S.A.

The CK strain was isolated from a patient at Waterbury Hospital, Waterbury, Conn., and has been deposited at the American Type Culture Collection, Maryland, U.S.A. under number ATCC 31441 on 11 October 1978.

The organisms are passed through mice several times to insure their virulence. The organisms are isolated at autopsy from the brain tissue of mice, subcultured on either 3.7% Brain Heart Infusion (BHI) (Difco Lab., Detroit,Mich.) medium or on 5% BHI agar supplemented with 0.01% nicotinamide adenine dinucleotide (NAD) (P-L Biochemicals, Wilwaukee, Wis.) and 1% (v/v) defibrinated horse blood (animal Blood Center, Syracuse, N.Y.) and then distributed in one ml portions in ampules, and stored at -ZO°C., or lyophilized and stored at 4°C.

The microorganisms may be fermented under art-recognized conditions, and the following is a description of a suitable procedure.

Basal medium used for the growth of the organisms is 3.7% (BHI). The basal medium is supplemented with 10 mg of NAD and 20 mg of hemin (Eastman Kodak, Rochester, Ν. Y.) per liter. One percent (v/v) defibrinated horse blood is added per 50 ml of seed culture. The supplements are freshlyprepared and filtered through a 0.45^Nalgene filter unit Nalge Sybron Corp., Rochester, Ν. Y.) before use. For growth of the organism in a 14 liter fermentor, the medium is further supplemented with 0.5% glucose.

To prepare a flask of seed organisms, one 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 gyrotory shaker at 150 rpm. The organisms are added as a 1% inoculum to 500 ml portions of the enriched BHI broth in 2 liter flasks which are then incubated at 37 °C with moderate shaking on a gyrotory shaker for 8 hours (for isolation of PRP) or 14 hours (as seed cultures).

The fermentation of each batch in a 14 liter fermentor is initiated by aseptically transferring 700 ml of the seed culture to 7 liters of the enriched BHI broth supplemented with hemin instead of defibrinated horse blood. The culture is continuously maintained at 37‘J+1°C. The tank is stirred at a rate of 150 rpm and an air flow of 0.25 liter of air per liter of mash per minute is maintained. During the growth 0.001% of a silicone antiform (FD-82, Hodag Chemical Corp.) is added as needed. Cultures are grown to the late Q logarithmic of growth (8 to 10 x 10 viable cells/ml), usually about 8 hours and then growth is terminated by adding 0.4% formaldehyde and standing overnight at 4°C.

Isolation of Polyribosyl Ribitol Phosphate (PRP) Culture broth prepared as described above is centri5 fuged at 27,000 x g for 30 minutes at 4°C. The cell free supernatant is collected and partially purified by conventional procedures preferably as follows: Step 1, Ethanol Precipitation To the culture supernatant is added sodium acetate (final concentration 4%). The solution is adjusted to pH 6.Οβ. 2 and 44 liters of 3A ethanol are added slowly with vigorous stirring at 4°C. The mixture is adjusted to pH 6.8 vzith glacial acetic acid and then allowed to stand for 12 hours at 3°C. The resulting precipitate is collected by decantation and then cen15 trifugation to afford crude PRP.

Step 2, Cetavlon(hexadecyltrimethy1 ammonium 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% aqueous solution of Cetavlon with mixing (final cone. 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 egual volume of water causing the PRP-Cetavlon salt to precipitate. The mixture is stirred for one hour, the precipitate is collected by centrifugation and is then dissolved in 2 liters of 0.3M sodium chloride.

Step 3, Ethanol Precipitation Cetavlon and the contaminants 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. The PRP is recovered by centrifugation, dissolved in 2 liters of pyrogen-free distilled water and then reprecipitated as described above. The final PRP precipitate is solubilized in 20mM sodium phosphate, pH 6.8.

Step 4, Hydroxylapatite Treatment Contaminants (e.g. nucleic acids, proteins and endotoxins) in the partial purified PRP preparations are selectively removed by adsorption on a calcium phosphate containing adsorbent such as hydroxylapatite [3.Ca^(PO^)2.Ca(OH)2].

This invention is based on the discovery that the polysaccharide PRP is not adsorbed by the calcium phosphate adsorbent containing nhosphate buffer, under certain conditions; however, the contaminants (such as nucleic acids, proteins and endotoxins) are adsorbed under these conditions. The process of the present invention may be carried out in batch-wise or column operation. In the batch process, the hydroxylapatite is added to the partially purified PRP preparation (in 2-20mM phosphate; pH about 6.5 - 7.0, preferably about 6.9). This mixture is mixed well and centrifuged to remove non-desired solids (adsorbent and the contaminants adsorbed by the adsorbent). The supernatant fluid is subjected to the foregoing procedure at least 2 more times. The resulting solution is filtered through *MiTli pore filters, dialyzed against pyrogen-free distilled water, and lyophilized.

In column operation, the partially purified PRP in about 2-20mM phosphate buffer (preferably pH of at least 5.8) is applied to a column containing the adsorbent hydroxylapatite which had been equilibrated with the phosphate buffer, preferably pH 5.8 and eluted with a preferably stepwise, gradient of sodium phosphate buffer (pH b.8; from 20mM to lOOmM. Fractions are collected and assayed for pentose (for polyribosyl ribitol phosphate). Those fractions which are positive for pentose are dialyzed against pyrogen-free distilled water and lyophilized.

(II) Preparation of a combination vaccine consisting of PRP from H. influenzae type b and B. pertussis antigens - Preferred Procedure To prepare a PRP vaccine, lyophilized PRP (prepared as aforementioned) is dissolved at a concentration of 20 ug/ml in ohosphate 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 0.45ji millipore filter units, dispensed into glass vials, and stored at 4°C.

A concentrated solution of the PRP is prepared by weighing predetermined amounts of the polysaccharide and dissolving it in phosphate buffered saline (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). This concentrated solution of PRP is then mixed with an appropriate volume of fresh B. pertussis cell suspensions to prepare the *Miliipore is a trade mark 7 474 stock solution. The combined vaccine in this stock solution contains 200 pg of PRP and approximately 70 opacity units (op) of cells per ml.

The stock solution is kept at 4°C for 90 days to allow for detoxification of pertussis antigens before the final product (vaccine) is prepared (which contains 10 pg of PRP and 3.5 op units of pertussis cells/0.5ml dose).

Suitably, the Bordetella pertussis antigens are obtained from Bordetella pertussis strain 138 (ATCC 10380). The characteristics of IB. pertussis strain 138 are described in Bergey's Manual of Determinative Bacteriology, 8th Edition 1974, edited by Buchanan and Gibbons, published by The Williams and Wilkins Company, Baltimore, Maryland, United States of America, on page 282 et seq. This strain is available from the American Type Culture Collection, Maryland, United States of America under deposit number 10380.

The invention is illustrated by the Examples which follow.

EXAMPLE 1 Hydroxylapatite (e.g. Bio. gel HTP, Bio-Rad Laboratories, Richmond, Calif.), 2.5 g is added to 250 ml partially purified PRP preparations (after Cetavlon and ethanol treatments) (containing approximately 1.0 mg PRP/ml) in 20mM sodium phosphate buffer, pH 6.9 and mixed at ice-cold water bath (1-4°C) for one hour. The mixture is centrifuged in the Sorvall RC2-B for 30 minutes at 16,000xg. The supernatant fluid is then passed through 0.65 μ millipore filter and subjected to the foregoing procedure (each time treated with 2.5g hydroxyl apatite) 2 more times. The resulting solution is filtered through 0.65μ and 0.45μ millipore filters, dialyzed against progen-free distilled water and lyophilized. The lyophilized product exhibits strong immunogenic activity (see below combined vaccine and animal experiment) and contains very low contaminants (such as nucleic acids, proteins and endotoxins) see Table 1 below). Approximately 170 mg of the lyophilized PRP is obtained. The recovery of PRP by this process is approximately 70% of the starting polysaccharide. The PRP should be stored under suitable conditions, such as at 4°C in a desiccator over phosphorus pentoxide and silica gel. 7 4 7 4 Example 2 A partially purified PRP (300 mg PRP) is dissolved in 100 ml of 20mM sodium phosphate buffer, pH 5.8 (i.e. 3 mg PRP/ml). This solution is applied to a column at ambient temperature (5.0 x 45 cm) of hydroxylapatite (approximately 250 ml bed volume which has been equilibrated with 20mM sodium phosphate buffer, pH 5.8 and eluted with a stepwise gradient of 20mM, 50mM, lOOmM sodium phosphate buffer, pH 5.8. The individual fractions (200 ml) eluted with 20mM and 1° 50mM sodium phosphate buffer (pH 5.8), positive for pentose (pentose determination by the orcinal 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 assayed by estimating to what extent they are contaiminated 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 boving serum albumin as standard. Nucleic 2θ acid is measured by the absorption of the PRP solution at 260nm. The absorbance of 50ug of nucleic acid in one ml of water in a cell of 1-cm light path is assumed to be equal to 1.0. Molecular size is estimated by means of Sepharose 4B or 2B gel filtration on columns 1.5 x 90 cm (Pharmacia-Fine Chemicals, Piscataway, N. J.). Partition coefficient (Kd) values are calculated from the elution pattern developed by the orcinal reaction (Herbert, et al., Methods in Microbiology, Vol. 5B, 285-291. <0 υ •ri Φ Χί υ ι r-f Hf Ο •rl in >4 Λ Endotoxin Φ Ρ nf ω >1 (JO «Ρ ω ω β φ Η Εη •Η 1/400 1/1000 β φ tn Ο ρ >ιϋ CM -Ρ W Ρ Φ •Η Εη XI 43 Φ ί£ 10.0 lo.o 1 Assay 1 Protein (%) 0.7 i 0.7 1 j Nucleic Acid (%) CO o 0.3 Pentose (%) 1 36.0 1 J 33.8 jMol. Size (Kd) nJ o •O* o Procedure Example 1 j 1 Example 2 h- O j— XJ X P Φ CM *0 A c nJ P «Ρ XJ P β w tn φ •P > XS c Φ Φ ? •H Ji um X r-r 0 U Ji O’r-f 4J id j; c e o 3^Ό ft C O 0)rS K r-4 i >1 CPH ε^«Μ - o O W M « C Ο O Η -rf 4-) — Ul xi= xs o β V tPft -1 Hf g-rf PS 0 ° Φ ft --1 ιΟΜ4 ini’ o a Ο—.Ω 0 3 M -rl nf n)“ +J +) ¢1 XS^-h 3 M Q. φΧ3 H 3 0) in .Ω -rf CO in 9«fl b M 0 XS jz -^+1 p Q. (Γ nJ ο φ 44 υ Ό £ω\ ο α) cl ρ ρ O' tpre ο· nJ β β CM ·ρ α •Η ·Η ϋ β ω ω tp Φ ο •-ι η « 05 η «3 Λ υ·ϋ υ P tn p in 2 o c 2C Φ XJ P O Φ TS 3 -c •r- XJ X O Φ P f“ C T3 O E C <0 β u- Φ •σ to φ •r— XJ xj <0 Φ P in ε P •r— 10 ω P Φ c X +4 P <0 υ u QC Φ •r- £ X x XJ P XJ u p C fti Φ Φ c W X φ ·Γ“ 3 P r— ε P •r- P 3 P Φ CM u- o cs X Φ Φ E= C X o O. c tn £ c •r* (0 (ti in X o r— P LU t— to £ Φ X -σ 10 U P c P T3 (ti P in Φ Q. (ti 2 x: X o in o in P υ in Φ Ό 10 X O 4- co o f— P O > P> H- P XJ c • (0 >> >7 P X c *φ (ti •r— > 1—· •r— r— in >> P +4 •ι-· p i— p υ O C Φ (Λ cu (0 Φ > X. X Ε τ- IZ w o Φ P o Φ υ P o P o •r- Φ o 3 Ω. X o cr in P in φ Φ £ Φ P P Ό •Γ- > Φ ΙΟ P in P P o*-> 3 P (ti P r— in cn φ U (ti Φ r- Φ in £ u- ε Φ Φ X x ft r—* X P P c a. P 2 2 φ ε O o o <ϋ μ- P ρ σ X Ld α σ> cn*-* Example 3 A combination vaccine containing PRP from H. influenzae type b and B. pertussis antigens is prepared as follows: 140 mg PRP (as prepared in Example 2) is dissolved 5 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), and filter through 0.45tJ millipore filter. This concentrated PRP solution (400Ug/ml) is used to prepare a combination vaccine which consists of PRP and B. pertussis antigens.

To 150 ml of the concentrated PRP solution (400 flg/ml) add 150 ml of fresh B. pertussis (strain 138) cell suspension in PBS, pH 7.0 (containing 149 opacity; op, unit cells/ml) to prepare the stock solution of the combined vaccine. This stock solution (300 ml) is kept at 4°C until the endotoxin level of the pertussis is decreased (at least 90 days). The sterility of the stock solution is tested with thioglycollate media (at 32-33°C). The pH of the stock solution after 90 days incubation is checked and adjusted to pH 7.0 -1. The final product (vac20 cine) used for animal experiments is prepared by diluting the stock combined solution with PBS and it contains 10 pg of 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 appears in Figures 1 and 2 as shown below.

Example 4 The stock solution of PRP (400 ug/ml) is prepared by dissolving 30 mg PRP (as prepared in Example 1) in 75 ml sterile phosphate buffered saline (PBS) and filtered through 0.45ΰ millipore filter. To 25 ml of this concentrated PRP is added an equal volume (i.e. 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 at 4°C for (at least) 90 days. The sterility of the stock solution is tested as aforementioned with thioglycollate media. The pH of the stock solution is pH 7.0+1. The final product (vaccine) used for animal experiments is prepared by diluting the stock combined solution with PBS, and it contains 10 Hg of PRP and 3.7 op units of pertussis cells/0.5 ml (dose).

Example 5 The PRP stock solution (200 ug/ml in PBS (pH 7.0) is prepared as in 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 stock solutions are incubated separately at 4°C for 90 days or slightly longer. The combined vaccine is made by taking 14 ml of PRP stock solution (200 ag/ml), plus 14 ml (detoxified) stock solution of per25 tussis antigens (75 op units/ml) and diluting it with 112 ml PBS (final val. 140 ml). The final vaccine used for animal experiments contains 10 ug PRP and 3.7 op units pertussis/0.5 ml (dose).

The results of the antibody response to this com30 bined vaccine in young rats appears in Figure 3 and in Table II.

H H W HI Eh nJ ra P ra tt Cn C fi O fi •ri X Φ rt tn •H (0 in fi rt p Φ nj I a Pi Inununogenicity of P Φ fi cn 0 •ri ε rt rt o V» 0 P o Φ y-i in •ΓΊ ΰ X fi Φ 0 Ό tn *H C ra P fi P (1) ra 0 w P N ID CO tt 0 o rt • • Pj fi fi rt rt Pj •ri fi Pi λ: fS Pj φ ό fc- I Φ rt rt tt & 0 Φ ro IW P Tl ·*-* Oj ε P Oj (O >1 P 03 cn ro in •o* > fi rt rt •rl 0 rt •ri 0 -P rt 0 Φ r- Γ- Po •m n o rt τί fi rt rt 0 H Λ •H «Ρ -P ω fi 0 ID CO rt Pi rt rt O rt rt A δ Q) Pj (I) 1 s ϋ •P o σι CO fi σι rt rt rt fi 0 Φ Φ Φ rt •Q ω rt Qj n Oirt s a Q fi rt ♦ri rt CQ fi ε TJ Φ P Φ rt rt 0 Φ tt c rt Φ ω o rt •rl n ra φ Ul ra jS C in p> Girt 2 W rt rt o ra p λ cn Φ Pi Pi ro

Claims (24)

1. A method of purifying polyribosyl ribitol phosphate (PRP), the capsular polysaccharide of Haemophilus influenzae type b. obtained by fermenting a strain of the organism Haemophilus influenzae type b, isolating the PRP produced by ethanol precipitation and further isolating the PRP as a hexadecyltrimethyl ammonium bromide complex, comprising the steps of: adding hydroxylapatite rS.Ca^PO^CaiOH^ in a from 2 to 20 millimolar phosphate buffer at a pH from 6.5 to 7.0; mixing the resulting mixture at a temperature of 2° to 10°C., centrifuging; removing the supernatant and repeating the foregoing procedure at least two more times; filtering the supernatant; dialyzing against pyrogen-free distilled water; and then lyophilizing, whereby there is obtained an immunologically active PRP.

2. A method according to Claim 1, wherein said pH is from

3. A method according to Claim 1 or Claim 2, wherein said buffer is sodium phosphate.

4. A method according to any preceding claim, wherein said buffer has a strength of approximately 20 millimolar. 5. Isolating the PRP as a hexadecyltrimethyl ammonium bromide complex, comprising the steps of: purifying the PRP by column chromatography in a from 2 to 20 millimolar phosphate buffer through hydroxylapatite L3Ca 3 (P0 4 ) 2 .Ca(0H)2 7 ; eluting with a phosphate buffer of a

5. A method according to any preceding claim, wherein said mixing is carried out at about 4°C.

6. A method according to any preceding claim, wherein said PRP is obtained by fermenting Haemophilus influenzae type b Rab strain. 6.7 to 6.9.

7. A method according to any one of Claims 1-5, wherein said PRP is obtained by fermenting Haemophilus influenzae type b CK strain (ATCC 31551).

8. A method of purifying PRP, according to Claim 1 and substantially as described in Example 1 herein.

9. A method of purifying polyribosyl ribitol phosphate (PRP), the capsular polysaccharide of Haemophilus influenzae type b, obtained by fermenting a strain of the organism Haemophilus influenzae type b, isolating the PRP produced by ethanol precipitation, and further 10. 20. An immunologically active PRP, substantially as described in Table 1 herein.

10. A method according to Claim 9, wherein said buffer is 10 concentration which increases upwards from 0.2 M, isolating fractions which analyse positive for PRP; dialyzing said fractions against pyrogen-free distilled water; and then lyophilizing, whereby there is obtained an immunologically active PRP.

11. A method according to Claim 9 or Claim 10, wherein said buffer in which said PRP is applied to the column has a strength of approximately 20 millimoles.

12. A method according to any one of Claims 9-11, wherein the

13. A method according to any one of Claims 9 - 12, wherein the column is eluted with a stepwise gradient of phosphate buffer of a strength increasing from 20 to 50 millimoles.

14. A method accoording to any one of Claims 9 - 13, wherein 25 the eluting buffer has a pH of about 5.8.

15. A method according to any one of Claims 9 - 14, wherein the eluting buffer is sodium phosphate. 15 sodium phosphate.

16. A method according to any one of Claims 9-15, wherein said PRP is obtained by fermenting Haemophilus influenzae type b Rab strain.

17. A method according to any one of Claims 9-15, wherein said PRP is obtained by fermenting Haemophilus influenzae type b CK strain (ATCC 31551).

18. A method of purifying PRP, according to Claim 9 and 5 substantially as described in Example 2 herein.

19. Substantially pure polyribosyl phosphate (PRP), the capsular polysaccharide of Haemophilus influenzae type b, characterized in that said PRP is immunologically active and has a molecular weight greater than 20 millions.

20. k ATCC 10380). 20 pH of the buffer applied to the column is about 5.8.

21. An immunologically active PRP, obtained by a method according to any one of Claims 1-18.

22. A combined vaccine that elicits anti-PRP (poly-ribosyl ribitol 15 phosphate) and antipertussis antibody formations in young warm-blooded animals which consists of polyribosyl ribitol phosphate according to any one of Claims 19-21 and Bordetella pertussis antigens.

23. A combined vaccine according to Claim 22, wherein said Bordetella pertussis antigens are obtained from Bordetella pertussis strain 138

24. A combined vaccine, according to Claim 22 and substantially as described in any one of Examples 3-5 herein.