MXPA00009378A - Vaccine composition - Google Patents

Abstract

This invention relates to a general method by which either extemporaneously prepared or liqui Hib/DTPa combination vaccines can be made in order to avoid Hib interference whilst being able to maintain the maximum, stable adsorption of each antigen onto the aluminium-based adjuvant on which it is most immunogenic. In so doing, pertussis antigens in combination vaccines of the present invention are stably retained in their most potent form.

Description

VACCINE COMPOSITION The present invention relates to a method for reducing the interference of the capsular polysaccharide component of a Haemophilus influenzae B (Hib) conjugate vaccine in a combined vaccine comprising DTPa, a well known "trivalent" combined vaccine comprising diphtheria toxoid (DT), tetanus toxoid (TT) and acellular Pertussis components [typically Pertussis toxoid (PT) detoxified, and filamentous hemagglutinin (FHA) with optional pertactin (PRN), and / or agglutinin 1 + 2]; for example, the commercial vaccine INFANRIX-DTPa ™ (SmithKine Beecham Biologicals), which contains DT, TT, PT.FHA and PRN antigens. The invention also relates to combined vaccines that are prepared by this method. Vaccines using polysaccharides are known in the art. For example, a Hib vaccine for the prevention of Haemophilus influenzae B infections is based on the capsular polysaccharide (PRP) of H. influenzae B, conjugated to a carrier protein. The polysaccharide is a polymer of ribarosa, ribitol and phosphate. Examples of carrier protein include diphtheria or tetanus toxoid, or an outer membrane protein of N. meningitidis. See, for example, US 4,365,170, US 4,673,574, EP 208375, EP 477508 and EP 161188. It is convenient to administer said conjugate vaccines with other antigens or vaccines at the same time, and this may mean multiple injections. The problems associated with multiple injections include a more complicated administration procedure and a large total volume of injection. This is a particularly acute problem when the vaccine is intended for infants. Both for the infant and the practitioner it is convenient to inject all the necessary antigens in a single injection of normal volume, thus making the vaccination procedure less traumatic and painful for the infant, and more efficient and easier to handle for the practitioner . Therefore, it has been proposed to combine said conjugate vaccines with polysaccharide, with other vaccines, such as DTPa to produce more elaborate combined vaccines. Additionally, the inclusion of other antigens in this combined vaccine has also been proposed for the prevention of diseases such as hepatitis B or poliomyelitis (combined vaccines comprising an antigen against hepatitis B and antigens against diphtheria, tetanus and whooping cough). (HepB, DTPa) have been described in WO 93/24148 and WO 97/46255). However, it has been found that simple mixing of the components of a combined vaccine is complicated, in that not all antigens can be mixed together effectively. The reduction in the immunogenicity of an antigen, when combined with other components (as compared to the particular antigen administered alone) is known as "interference". It is known, for example, that extemporaneous mixing of a combined DTPa vaccine with Hib without adjuvant results in the reduction of antibody titers for the polysaccharide component of Hib (WO 97/00697). Additionally, WO 97/00697 showed that if Hib is adsorbed on aluminum hydroxide there is a significant reduction of the antibody titers for the polysaccharide component. These results indicated that there was interference between the aluminum hydroxide of the DTPa vaccine and Hib. In order to treat and minimize this interference in that combined vaccine, prepared extemporaneously, Hib was preadsorbed in aluminum phosphate. WO 96/37222 also describes that problem. In this case the antigenicity of Hib is stabilized by adsorbing it and also the other components of DTPa, in an aluminum-based adjuvant with a zero point charge of less than 7.2, for example, aluminum phosphate or aluminum hydroxide, to which I had added anions. However, when aluminum phosphate is used as an approximation, an increase in the desorption of other antigens from the combined vaccine can be obtained, which work best when adsorbed on the aluminum hydroxide adjuvant.; which can also be a problem when using aluminum hydroxide plus anions (the surface of the aluminum hydroxide has been essentially coated by the anion). Another problem encountered in the formulation of monovalent vaccines and combined vaccines is the inherent stability of their mixed antigens over time. Vaccines in solution may suffer from processes, over time, that diminish the immunogenicity of their antigenic components; for example, the degradation of the antigen or the desorption of the antigen from the adjuvant in which it had been adsorbed. Although in general it is convenient to try to reach the maximum possible degree of antigen adsorption in the adjuvant, for a reliable vaccine, the main goal is to obtain the maximum level of adsorption that can be maintained throughout the vaccine's storage life. Even when lyophilized vaccines can be solubilized extemporaneously, it is advantageous that the solubilized vaccine is stable, so that it is effective for a prolonged time frame, in order to give the practitioner maximum flexibility with the minimum waste of vaccine. Ideally, a stable vaccine in solution (a "liquid" vaccine) could be provided. A stable vaccine in solution would not only have a lower manufacturing cost than a lyophilized equivalent, but would also eliminate the additional manipulation step by the practitioner, who may run the risk of being carried out incorrectly.

BRIEF DESCRIPTION OF THE DRAWINGS • Figure 1 is a graph showing the elimination activity in the mouse lung with pertussis, of formulations in "light aluminum hydroxide" (lot 21750 and 21751) and "total aluminum phosphate" (lot 21752) (with and without Hib), compared to a standard DTPa vaccine, commercially available (indicated by square symbols).

DESCRIPTION OF THE INVENTION This invention relates to a general method by which combined Hib / DTPa vaccines prepared extemporaneously or liquid can be prepared in order to better avoid the problem of Hib interference, with respect to what has been described in the prior art; at the same time that it is able to maintain the maximum, stable adsorption of each antigen on the aluminum-based adjuvant, in which it is highly immunogenic. In doing so, the vaccines combined with pertussis antigens of the present invention are stably retained in their most potent form. Additionally, the invention relates to the manner in which the method is compatible with a further method for improving the adsorption of the components of a liquid combined vaccine, on aluminum-based adjuvants, in a stable manner. In that way, the immunogenicity of the polysaccharide component of Hib in the combined vaccines can be consistently maintained; and can remain stable in a liquid state for a longer period of time. Consequently, the vaccines thus produced are of greater use when administered to subjects, in particular to children. Specifically, this invention teaches a reliable method by which aluminum hydroxide can be added to an adjuvant in a combined DTPa-Hib vaccine, without causing interference to the Hib component over time, in formulations prepared extemporaneously or liquid, and without adding in a manner free anions to the formulation, as does the method described in WO 96/37222. As no anions are added to the formulation, the stability of all the adsorbed antigens is maximized and, in addition, the method is compatible with another method described to optimally elevate the antigen adsorption in aluminum-based adjuvants, a combined vaccine, stably; specifically, to raise its adsorption (and minimize subsequent desorption) to the optimum point in the adjuvant, over time, in a liquid formulation; using "extra-washed" aluminum phosphate. Accordingly, the present invention provides a method for reducing the interference of a capsular polysaccharide component of a conjugate vaccine of Haemophilus influenzae B (Hib) in a combined vaccine comprising DTPa; wherein said method comprises: i) selecting one or more antigens to be adsorbed in an aluminum hydroxide adjuvant; ii) presaturating the adjuvant aluminum hydroxide with the selected antigens; iii) select Hib plus one or more additional antigens, to be adsorbed on aluminum phosphate; iv) adsorb Hib and the additional antigens in aluminum phosphate; v) combine all the antigens in the vaccine. By "presaturate" is meant that the antigen or antigens requiring the adjuvant aluminum hydroxide are preadsorbed in the aluminum hydroxide, so that there is no excess of aluminum hydroxide present in sufficient quantity to cause significant interference to Hib ( either not adsorbed or adsorbed on aluminum phosphate) in the final combined vaccine; and that time is given for the adsorption to take place before the antigens adsorbed on the aluminum hydroxide are combined with other antigens of the vaccine. Preferably the adsorption on the aluminum hydroxide should be allowed to complete (at least a period of approximately 10 minutes at room temperature). The presaturation of the aluminum hydroxide, in such a manner, results in a minimization of any interference to the Hib antigen, caused by the aluminum hydroxide adjuvant. Frequently the state of flocculation (or aggregation) of the adjuvant aluminum hydroxide in the combined vaccine may be an indicator of Hib interference.; (the greater the flocculation, the greater the probability of Hib interference.) In addition, the use of antigens to presaturate aluminum hydroxide has the additional advantage of incorporating less aluminum hydroxide per fixed dose of antigen, which can potentially reduce the local reactogenicity, associated with aluminum It should be noted that, although the above method is the most preferred, the method can also be carried out when the Hib component has no adjuvant when added to the formulation. invention is a method by which Hib without adjuvant or Hib adsorbed on aluminum phosphate can be combined extemporaneously and effectively with the combined vaccine, with a minimum decrease in its immunogenicity, which comprises the following steps: i) selecting one or more antigens to be adsorbed to the adjuvant aluminum hydroxide; ii) presaturating the adjuvant aluminum hydroxide with the selected antigens; iii) selecting one or more additional antigens to be adsorbed on aluminum phosphate; iv) adsorbing the additional antigens in aluminum phosphate; v) combine all the antigens in the vaccine; vi) extemporaneously add Hib without adjuvant or Hib adsorbed on aluminum phosphate. By "DTPa" we mean the definition given above. DT, TT, PT, FHA and PRN are well known in the art. The PT component can also be formed to a toxoid, either chemically or genetically, for example, as described in EP 515415. See also EP 427462 and WO 91/12020, for the preparation of pertussis antigens. Optionally, the PT component can be recombinant (for example, as described in European patent applications EP 306318, EP 322533, EP 396964, EP 322115 and EP 275689). Optionally, the DT and TT components can also be recombinants. Typically the components PT, FHA, PRN, HBsAg (surface antigen for hepatitis B) and Hib will be in the range of 8-25 μg per dose of 0.5 ml of total vaccine. DT, TT and IPV components (trivalent, inactivated polio virus vaccine) should typically be present as approximately 15-25 Lf (flocculating units), 10 Lf and 40/8/32 (types l / ll / III) DU, respectively, per dose of 0.5 ml of total vaccine. It is preferred that the carrier protein used in the Hib conjugate be any diphtheria toxoid, tetanus toxoid, Diphtheria Crm197 protein or an outer membrane protein of a bacterium, such as N. meningitidis. The synthesis of capsular polysaccharide conjugate (PRP) of Haemophilus influenzae type B, and tetanus toxoid (T) is described in WO 97/00697. The polysaccharide can be prepared by any known coupling technique. For example, the polysaccharide can be coupled by means of a thioether ligation. This method of conjugation is based on the activation of the polysaccharide with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) to form a cyanate ester. In this way, the activated polysaccharide can be coupled, or by means of a spacer group, to an amino group in the carrier protein. It is preferred that the cyanate ester be coupled with hexanediamine and that the amino derivative polysaccharide be conjugated to the carrier protein, using heteroligation chemistry, which comprises the formation of the thioether linkage. Said conjugates are described in the published application of TCP WO 93/15760 (Uniformed Services University). Conjugates can also be prepared by reductive amination methods such as those described in US 4365170 (Jennings) and US 467574 (Anderson). Other methods are described in EP 161188, EP 208375 and EP477508. Yet another method comprises coupling an activated polysaccharide with cyanogen bromide, derivative with adipic acid hydrazide (ADH) to the protein carrier, by means of carbodiimide condensation. Said conjugation is described in Chu C. and co-authors, Infec. Immunity, 1983, 245, 256. In addition to the DTPa and Hib antigens, the combined vaccine may also comprise other antigens, preferably one or more of the Hepatitis B surface antigen (HBsAg) group, inactivated Hepatitis A virus, antigens of poliovirus (for example, inactivated trivalent poliovirus (IPV)), capsular polysaccharide of. meningitidis A (and its protein conjugates), capsular polysaccharide of N. meningitidis A (and its protein conjugates), capsular polysaccharide of Streptococcus pneumoniae (and its protein conjugates, proteins of Streptococcus pneumoniae, outer membrane proteins of Moraxella catarrhalis; non-determinable outer membrane proteins in type, from Haemophilus influenzae, outer membrane proteins from N. Meningitidis B. It is preferred that HBsAg is adsorbed to aluminum phosphate, as described in WO 93/24148. In addition, methods for adsorbing DTPa and Hib antigens in aluminum adjuvants are known in the art. See, for example, WO 93/24148 and WO 97/00697. The components suitable for use in these vaccines are now available commercially and details of them can be obtained from the World Health Organization. For example, the IPV component can be the Salk vaccine against inactivated polio. The hepatitis B surface antigen may consist of the "S" antigen in EngerixB ™ (SmithKine Beecham Biologicals). The inactivated hepatitis A virus may preferably comprise the commercial vaccine known as Havrix ™ SmithKine Beecham Biologicals), which is a killed attenuated vaccine derived from the HM-175 strain of the virus. The addition of freeze-dried or liquid Hib (without adjuvant or adsorbed on aluminum phosphate) to a solution of the other components of the combined vaccine can be carried out extemporaneously, or before the vaccine leaves the manufacturer. In any of these cases, the immunogenicity of the Hib component must remain stable and effective, with minimal interference. Híb can be added either by itself or in admixture with other components without aluminum hydroxide adjuvant. Vaccine preparation is generally described in Vaccine Design - The Subunit and adjuvant approach, Ed Powel and Newnan; Pellum Press. Advantageously, the combined vaccine according to the invention is a pediatric vaccine. The amount of conjugated antigen in each vaccine dose is selected as an amount that induces an immunoprotective response without the significant adverse side effects of typical vaccines. Said amount will vary depending on the specific immunogens used. In general, it is expected that each dose comprises from 1 to 1000 μg of conjugated polysaccharide (expressed in amount of polysaccharide), preferably from 2 to 100 μg and, most preferably, from 4 to 40 μg. An optimal amount for a particular vaccine can be determined by normal studies involving the observation of antibody titers and other responses in subjects. After an initial vaccination, subjects may receive one or two booster injections at approximately 4 week or more intervals. In a preferred embodiment of the invention the ratio of aluminum phosphate to aluminum hydroxide adjuvant (expressed as the relative amounts of Al3") present in the combined vaccine, preferably ranges from 1: 1 to 20: 1, more preferably, 2 : 1 to 14: 1 and, most preferably, 1.5: 1 to 14: 1 It is preferred to adsorb the PRN component in aluminum hydroxide in the formulation, since the inventors have discovered that, while the antigen is completely adsorbed on Aluminum hydroxide, on a wide range of pH values, when adsorbed on aluminum phosphate, is rapidly adsorbed poorly (and is less potent) at pH values above 5.1 After 24 hours of adsorption at pH 6.1, there is approximately less than 10% unadsorbed PRN, using aluminum hydroxide, against approximately more than 50%, using aluminum phosphate.As the final pH specified by WHO for DTP vaccines is between 6 and 7, therefore, it is important that The PRN is adsorbed on aluminum hydroxide for maximum efficiency. PRN has been shown to be one of the most important components in the pertussis vaccine, and it has now been discovered that minimization of adsorption reduces the T cell response and the potency of the pertussis vaccine, in its whole. In another preferred embodiment of the invention, all antigens of the combined vaccine are adsorbed onto aluminum phosphate, provided that the pertactin is adsorbed on aluminum hydroxide. The combined vaccine uses what is called the "totally aluminum phosphate" approach. It is preferred that the pertactin adsorbed on the aluminum hydroxide be combined with inactivated, non-adsorbed polio virus antigens, before combining them with the antigens adsorbed on aluminum phosphate (thus ensuring, furthermore, that aluminum hydroxide is preset ). An example of this is described in example 1). The "fully aluminum phosphate" approach can also be used for the alternative method detailed above, which adds Hib extemporaneously, either in an unadjuvanted form or adsorbed to aluminum phosphate. In another preferred embodiment of the invention all antigens of the combined vaccine are adsorbed to aluminum hydroxide, provided that HBsAg and Hib are adsorbed to the aluminum phosphate. This combined vaccine uses the approach called "light aluminum hydroxide." It is preferable that the antigens adsorbed on aluminum hydroxide be combined before adding HBsAg adsorbed on aluminum phosphate, it is preferred to combine the HBsAg with the other antigens before of adding the adsorbed Hib to aluminum phosphate. An example of this is described in Example 2. However, if the total amount of adjuvant (aluminum salts) used is not sufficient to generate an adjuvant effect, phosphate can be added. Free aluminum at this time, without denying the advantages of the invention The "light aluminum hydroxide" approach can also be used for the alternative method detailed above, which adds Hib extemporaneously in un-adjuvanted or adsorbed form on aluminum phosphate. In this case, any additional free aluminum phosphate that is added to the combined vaccine should preferably be added before adding Hib. The "full aluminum phosphate / light aluminum hydroxide hybrid" approach is another more preferred embodiment of the invention. In that hybrid formulation, the antigens selected in addition to pertactin are adsorbed to the aluminum hydroxide, but not as many as in the "light aluminum hydroxide" formulations. Once again, all the aluminum hydroxide used is presaturated with antigen, which minimizes the risk of interference by Hib. It is preferred that all antigens of the combined vaccine be adsorbed on aluminum phosphate, conditional upon the pertactin, diphtheria toxoid, pertussis toxoid, and filamentous haemagglutinin being adsorbed on the aluminum hydroxide. Again the Hib can be without adjuvant or it can be adsorbed on aluminum phosphate when it is added to the liquid or to formulations prepared extemporaneously. Preferably, pertactin, diphtheria toxoid, pertussis toxoid, filamentous haemagglutinin, adsorbed on aluminum hydroxide, are combined with inactivated, non-adsorbed poliovirus antigens, before being combined with antigens adsorbed on aluminum phosphate. An example of this is described in example 3. It should be further appreciated that one or more of the antigens selected to presaturate the aluminum hydroxide adjuvant may already be adsorbed to another adjuvant, preferably aluminum phosphate. These formulations are called "fully aluminum phosphate / lightweight aluminum hydroxide formulations"; and provide a further preferred embodiment of the invention. Again all the aluminum hydroxide used is presaturated with the antigen, so that the risk of interference with Hib is minimized. In such formulations, Hib must be added to the formulation extemporaneously, in an adjuvanted state, or adsorbed on aluminum phosphate. It is preferred that all antigens of the combined vaccine be adsorbed to aluminum phosphate, provided that pertactin, pertussis toxoid, filamentous haemagglutinin, are adsorbed to aluminum hydroxide, and diphtheria toxoid (which had previously been adsorbed) in aluminum phosphate) is also combined with free aluminum hydroxide, before combining it with the other antigens. An example of this is described in Example 4. It is preferred that the aluminum phosphate used to carry out the method of the invention be "extra-washed", before the adsorption of the antigen, so that the concentration of free phosphate ion is reduce to less than 10 mM and preferably be 3 mM or less, more preferably, 2.5 mM or less. Extra-lamellar aluminum phosphate should be sterilized (preferably by autoclaving) before adsorption of the antigen. The inventors of the present have shown that using extralayered aluminum phosphate improves the level of adsorption of Hib and HBsAg in aluminum phosphate, and helps to prevent the desorption of all antigens in the combined DTPa-Hib vaccine (and, therefore, , improves the stability of the antigens). A washing method of the aluminum phosphate adjuvant is described in the examples. Due to the above disadvantageous effect of the phosphate anions in solution, shown by the inventors, any regulator used in the combination vaccine to stabilize the pH should preferably be cationic in nature. L-histidine monohydrate / monochloride should be used, most preferably. In a further aspect according to the invention, a combined vaccine obtained by the method of the invention described above is provided. Additionally, a combined vaccine comprising a conjugated capsular polysaccharide of Haemophilus influenzae B (Hib) and DTPa is provided, characterized in that the Hib is adsorbed onto the aluminum phosphate and any free aluminum hydroxide does not cause significant interference with Hib. The extra-washed aluminum phosphate method can also be used independently to increase the adsorption of the antigens on the aluminum phosphate and to decrease its subsequent desorption in a combined vaccine comprising DTPa, where the method comprises the following steps: i) washing the aluminum phosphate adjuvant, so as to reduce the concentration of the free phosphate ion to 3 mM or less (preferably less than 1 mM); I) sterilizing the washed aluminum phosphate from (i); iii) select the antigens to be adsorbed in the aluminum phosphate adjuvant; iv) adsorbing said antigens on the washed aluminum phosphate; v) if necessary, combine the antigens of the combined vaccine. It is preferable that the phosphate ions be removed by repeated centrifugation and dilution steps (see example 8) or by subsequent diafiltration steps, to those commonly carried out by the aluminum phosphate manufacturers, using an ultrafiltration device. It is preferred to carry out the sterilization of the extra-washed adjuvant from step (ii) by autoclaving. Additionally, a combined vaccine of this invention comprising DTPa and aluminum phosphate adjuvant is provided; characterized in that the aluminum phosphate adjuvant has been washed so that the concentration of free phosphate ion is reduced to 3 mM or less. Also provided is an immunogenic composition comprising aluminum phosphate adjuvant and an antigen, characterized in that the aluminum phosphate adjuvant has been washed so that the concentration of free phosphate ion is reduced to 3 mM or less. • The following examples illustrate, but do not limit, the scope of the invention.

EXAMPLES EXAMPLE 1 PREPARATION OF A VACCINE FORMULATION USING THE APPROXIMATION "TOTALLY ALUMINUM PHOSPHATE" The method used to prepare the combined liquid vaccine DTPa-Hib-HBsAg-IPV was as follows (the quantities are to scale to represent a dose of vaccine): (1) 8 μg of preadsorbed PRN was mixed in 0.05 mg of Superfos aluminum hydroxide, with 40/8/32 DU of IPV without adjuvant (type 1/2/3, respectively), and stirred for one hour at room temperature. The IPV antigen coating kept the adjuvant aluminum hydroxide stable in its antigen-saturated state in the final combined vaccine. (2) 17 Lf of DT, 10 Lf of TT, 25 μg of PT, 25 μg of FHA and 10 μg of HBsAg were mixed, all preadsorbed in 0.17, 0.1, 0.05, 0.05 and 0.2 mg of extra-washed Superfos aluminum phosphate, respectively (expressed as to the amount of Al3 +), together with the PRN of step 1), for one hour, at room temperature. (3) 10 μg of Hib (PRP conjugated to tetanus toxoid) was added, preadsorbed on 0.12 mg of extra-washed Superfos aluminum phosphate, to the result of step (2) and the mixture was stirred for 15 minutes at room temperature. (4) The mixture for the cationic regulator monohydrate / L-histidine monochloride was made 10 mM and the pH was adjusted to 6.1 ± 0.1. Histidine has a pKa close to the desired final pH of 6.1 and, due to its cationic nature, does not cause desorption of the antigen. (5) 5 mg / ml 2-phenoxyethanol was added to the mixture, which was stirred for sixty minutes at room temperature. (6) It was found that the pH of the mixture was 6.1 ± 0.1 (between 6 and 7, as specified by WHO for DT and DTP vaccines). The final mix volume was 0.5 ml and contained 140 mM NaCl. (7) The combined vaccine was stored for at least two weeks at room temperature before its use in tests of immunogenic potency. The ratio of aluminum phosphate to aluminum hydroxide adjuvant (expressed as the relative amounts of Al3 +) DT, TT, PT, FHA, PRN, HBsAg and Hib were adsorbed to their respective adjuvants at pH 5.1 ± 0.1, 5.7 ± 0.1, 5.1 ± 0.1, 5.1 ± 0.1, 5.5 ± 0.1 and 5.1 ± 0.1, respectively.

EXAMPLE 2 PREPARATION OF A VACCINE FORMULATION USING THE APPROXIMATION "LIGHT ALUMINUM HYDROXIDE" The method used to prepare the combined DTPa-Hib-HBsAg-IPV liquid vaccine was as follows (the amounts are scaled to represent a dose of vaccine): (1) 25 Lf of DT, 10 Lf of TT, 25 were mixed μg of PT, 25 μg of FHA, 8 μg of PRN and 40/8/32 DU of IPV (types 1/2/3, respectively), all preadsorbed in 0.1, 0.025, 0.005, 0.005, 0.05 and 0.01 mg of aluminum hydroxide Superfos, respectively (expressed in the amount of Al3 +), for one hour, at room temperature. With these amounts of aluminum hydroxide the adjuvant was stably saturated with the antigen. (2) 10 μg of HBsAg, preadsorbed in 0.2 mg unwashed Superfos aluminum phosphate, was mixed with the result of step 1) and stirred for one hour at room temperature. (3) 10 μg of Hib (PRP conjugated to tetanus toxoid), preadsorbed in 0.12 mg of extra-washed Superfos aluminum phosphate was added to the result of step (2) and the mixture was stirred for 15 minutes at room temperature. (4) 10mM mixture was made for the cationic regulator monohydrate / L-histidine monochloride and the pH was adjusted to 6.1 ± 0.1. Histidine has a pKa close to the desired final pH of 6.1 and, due to its cationic nature, does not cause desorption of the antigen. (5) 5 mg / ml 2-phenoxyethanol was added to the mixture, which was stirred for 60 minutes at room temperature. (6) It was found that the pH of the mixture was 6.1 ± 0.1 (between 6 and 7, as specified by WHO for DT and DTP vaccines). The final mixed volume was 0.5 ml and contained 140 mM NaCl. (7) The combined vaccine was stored for at least two weeks at 4 ° C before its use in immunogenic potency tests. The ratio of aluminum phosphate to aluminum hydroxide adjuvant (expressed as the relative amounts of Al3 +) present in the combined vaccine was 1.7: 1. DT, TT, PT, FHA, PRN, HBsAg, IPV and Hib were adsorbed in their respective adjuvants at pH 6.1 ± 0.1, 6.1 ± 0.1, 6.1 ± 0.1, 6.1 ± 0.1, 6.1 ± 0.1, 5.5 ± 0.1, 6.1 ± 0.1 and 5.1 ± 0.1, respectively.

EXAMPLE 3 PREPARATION OF A VACCINE FORMULATION USING ONE APPROXIMATION "HYBRID OF TOTALLY ALUMINUM PHOSPHATE / LIGHTWATER HYDROXIDE" The method used to prepare the combined DTPa-Hib-HBsAg-IPV vaccine was as follows (the amounts are to scale to represent a dose of vaccine): (1) 25 Lf of DT, 25 μg of PT, 25 μg of FHA and 8 μg of PRN, all preadsorbed in 0.1, 0.05, 0.05 and 0.05 mg of Superfos aluminum hydroxide, respectively, with 40/8/32 DU of IPV without adjuvant (type 1/2/3, respectively). (2) 10 Lf of TT and 10 μg of HBsAg, preadsorbed in 0.25 and 0.2 mg of unwashed Superfos aluminum phosphate, respectively (expressed as the amount of Al3 +), were mixed together with the mixture from step 1) and it was stirred for 15 minutes at room temperature. (3) The mixture for the cationic regulator monohydrate / L-histidine monochloride was made 10 mM. The mixture was stirred at room temperature for fifteen minutes and then the pH was adjusted to 6.1 ± 0.1. Histidine has a pKa close to the desired final pH of 6.1 and, due to its cationic nature, does not cause antigen desorption. (4) 5 mg / ml 2-phenoxyethanol was added to the mixture, which was stirred for 60 minutes at room temperature. (5) The pH of the mixture was checked to be 6.1 ± 0.1 (between 6 and 7, as specified by WHO for DT and DTP vaccines). The final mixed volume was 0.5 ml and contained 140 mM NaCl. (6) The combined vaccine was stored for at least three weeks at 4 ° C before being used in immunogenic potency tests. (7) 10 μg of Hib (PRP conjugated to tetanus toxoid) was added extemporaneously, to the result of step (6). DT, TT, PT, FHA, PRN and HBsAg were adsorbed in their respective adjuvants at pH 6.1 ± 0.1, 5.7 ± 0.1, 6.1 ± 0.1, 6.1 ± 0.1, 6.1 ± 0.1 and 5.3 ± 0.1, respectively.

EXAMPLE 4 PREPARATION OF A VACCINE FORMULATION USING AN APPROACH "COMPLETELY ALUMINUM PHOSPHATE / LIGHT, ADAPTED HYDROXIDE" The method used to prepare the combined vaccine DTPa-Hib-HBsAg-lPV was as follows (the amounts are to scale to represent a dose of vaccine): (1) 25 Lf of DT was adsorbed on 0.15 mg of unwashed (normal) Superphos aluminum phosphate (expressed as quantity of Al3 +). This was added to 0.1 mg of Superfos aluminum hydroxide and the mixture was stirred for more than 30 minutes at room temperature. The pH was adjusted to 6.1 ± 0.1. (2) 25 μg of PT, 25 μg of FHA and 8 μ of PRN were mixed, all preadsorbed at 0.05, 0.05 and 0.05 mg of Superfos aluminum hydroxide, respectively, with the DT of step (1), and 40/8/32 DU of IPV without adjuvant (type 1/2/3, respectively). (3) 10 Lf of TT and 10 ± of HBsAg, preadsorbed in 0.1 and 0.2 mg of unwashed Superfos aluminum phosphate (normal), respectively (expressed in the amount of Al3 +), were mixed with the mixture of step (2) ) and stirred for 15 minutes at room temperature. (4) The mixture for the cationic regulator monohydrate / L-histidine monochloride was made 10mM and the pH was adjusted to 6.1 ± 0.1. Histidine has a pKa close to the desired final pH of 6.1 and, due to its cationic nature, does not cause desorption of the antigen. (5) 5 mg / ml 2-phenoxyethanol was added to the mixture, which was stirred for 60 minutes at room temperature. (6) It was found that the pH of the mixture was 6.1 ± 0.1 (between 6 and 7, as specified by WHO for DT and DTP vaccines). The final volume was 0.5 ml and contained 140 mM NaCl. (7) The combined vaccine was stored for at least 3 weeks at 4 ° C before its use in immunogenic potency tests. (8) 10 μg of Hib (PRP conjugated to tetanus toxoid) was added extemporaneously to the result of step (7). DT, TT, PT, FHA, PRN and HBsAg were adsorbed in their respective adjuvants at pH 5.4 ± 0.1, 5.7 ± 0.1, 6.1 ± 0.1, 6.1 ± 0.1, 6.1 ± 0.1 and 5.3 ± 0.1, respectively.

EXAMPLE 5 MEASURES OF INTERFERENCE IN CLINICAL TRIALS OF THE COMBINED VACCINES The following clinical tests show the interference problem that was found in the anti-PRP antibody titers in combination with vaccines comprising DTPa (conventionally adsorbed on an excess of aluminum hydroxide) and Hib.

Healthy, initially seronegative, volunteers were immunized with three doses of the combined vaccine, administered according to the programs given below. The responses were defined as subjects with antibody titers significantly above the background. Titers were expressed in mlU / ml. The following results are expressed as geometric mean titers (GMT) in mlU / ml. For anti-PRP, the number in parentheses represents the percentage of individuals who had GMT values greater than 1.0. The results show that the problem of interference with Hib could be alleviated using the "totally aluminum phosphate" approach, without affecting the efficacy of the other antigens in the combined vaccine.

TABLE 1 Reference for antibody titer PRP (Hib) - DTPa in one arm and Hiberix ™ (PRP without adjuvant, conjugated to tetanus toxoid) (SmithKine Beecham Biologicals) in the other arm. DTPa was adsorbed in 500 μg / ml of aluminum hydroxide. ** DTPaHepB in combination with Hiberix ™. Ixophilized Hib I was reconstituted extemporaneously with DTPa-HepB vaccine. DTPa was adsorbed in 100 μg / ml of aluminum hydroxide. HepB, HBsAg, was adsorbed to aluminum phosphate and then combined with DTPa as described in WO 93/24148. *** The "totally aluminum phosphate" approach with Hib without adjuvant. DTPa-HepB was adsorbed to aluminum phosphate (except that pertactin was adsorbed onto aluminum hydroxide) using the method of Example 1, except that no Hib was added. Hib lyophilized, without adjuvant (Hiberix ™) was reconstituted extemporaneously with the resulting DTPa-HepB vaccine. The previous group of three experiments was carried out in a group of 18 individuals. The individuals were vaccinated at the age of 3, 4 and 5 months, with doses of 0.5 ml. 4 DTPaHepB in combination with Hiberix ™. Hib lyophilized was reconstituted extemporaneously with DTPa-HepB vaccine. DTPa was adsorbed in 500 μg / ml of aluminum hydroxide. HepB, HBsAg, was adsorbed to aluminum phosphate and then combined with DTPa, as described in WO 93/24148. The "totally aluminum phosphate" approach with Hib without adjuvant. DTPa-HepB was adsorbed to aluminum phosphate (except that the pertactin was adsorbed on aluminum hydroxide) using the method of Example 1, except that no Hib was added. Freeze-dried Hib, without adjuvants (Hiberix ™), was reconstituted extemporaneously with the resulting DTPa-HepB vaccine. The previous group of two experiments was carried out in Turkey, in a group of 30 individuals. The individuals were vaccinated at the age of 3, 4 and 5 months, with doses of 0.5 ml.

EXAMPLE 6 CLEANING ACTIVITY OF TOSFERIN LUNG IN MOUSE, OF THE FORMULATIONS "LIGHT ALUMINUM HYDROXIDE" AND "TOTALLY ALUMINUM PHOSPHATE" The "light aluminum hydroxide" and "fully aluminum phosphate" formulations without Hib, shortly after being formulated, were tested to examine their mouse lung clearing activity with pertussis, compared to the commercial DTPa vaccine formulation (INFANRIX) ™, SmithKine Beecham Biologicals). The addition of Hib without adjuvant to the formulations was determined by its effect on the effectiveness. The analyzes were performed with a two-dose immunization schedule (using a quarter of a human dose). Mice immunized with Bordetella pertussis were infected and the CFU / bacteria lung was measured against the time after the challenge.

The results can be seen in figure 1. The challenge data in animals with pertussis show that the activity of lung clearing with pertussis is equivalent, using the formulations "light aluminum hydroxide" and "fully aluminum phosphate" (with or without Hib adjuvant), compared to the classical DTPa formulations.

EXAMPLE 7 IMMUNOGENICITY STUDIES OF Hib IN INFANT RAT, WITH THE FORMULATIONS "LIGHT ALUMINUM HYDROXIDE" AND "TOTALLY ALUMINUM PHOSPHATE" A infant rat model was used to determine the immunogenicity of the Hib component in vaccine formulations "light aluminum hydroxide" and "totally aluminum phosphate". The experiments were carried out administering 1/10 of the human dose (injection of 50 μl), subcutaneously, with a program of 0-14-28 days (starting 7 days after birth), taking blood samples on day 42. 24-30 animals were determined per group. Hib adsorbed on aluminum phosphate was added well to the formulation (the "liquid" formulation of examples 1 and 2), two weeks before the experiments, either extemporaneously. The mean geometric antibody (GMT) titers against PRP were measured. The results indicated that the PRP titers for both "light aluminum hydroxide" and "fully aluminum phosphate" formulations (either in a "liquid" formulation or in an extemporaneously prepared formulation) were equivalent, or at most minimally interfered with in comparison with the titers measured after the administration of monovalent Hib adsorbed on aluminum phosphate.

EXAMPLE 8 PREPARATION OF "ADJUVANT EXTRALAVED ALUMINUM PHOSPHATE" To wash the aluminum phosphate, the following steps were carried out: The aluminum phosphate solution was centrifuged at 3500 r.p.m. for 10 minutes, and the supernatant was collected. The pellet was resuspended with 150 mM NaCl. These steps were repeated at least 3-4 times. The result of this was filtered through a 100 μm filter (to remove the potential aggregates) and the aluminum phosphate was sterilized by autoclaving. The sterile adjuvant was stored at 4CC until it was used. The autoclaved, extra-washed aluminum phosphate is stable for at least 45 days at room temperature. Superfos aluminum phosphate was used for this process. The product provided by the manufacturer contains up to 5 mM of free phosphate ion. When washed thoroughly, the concentration of free phosphate can be reduced to less than 0.5 mM, which rises to 1 mM if the washed solution is incubated at 37 ° C for 7 days. If the washed aluminum phosphate is subsequently autoclaved, the concentration of free phosphate can rise to 2.0-3.0 mM.

RESULTS The effect of washing the aluminum phosphate on the adsorption of the antigens (H = hours, TA = room temperature) is shown in Table II.

TABLE II As can be seen from the results, the use of extra-washed aluminum phosphate improves the adsorption of all antigens, especially for DT and TT. It can also be seen that adding free phosphate anions to the adsorption process results in a significant decrease in HBsAg adsorption.

EXAMPLE 9 THE EFFECT OF NON-ADSORBID ANTIGENS AND FREE PHOSPHATE ON THE DESADSORTION OF PRN PRN should be adsorbed on aluminum hydroxide in order to stably adsorb the antigen at pH 6.1 (and retain the potency) as described above. When it comes to small amounts of adjuvant (in order to be presaged with the antigen) it is important to examine the stability of the antigen in a combined vaccine to determine if the aluminum hydroxide is supersaturated in the context of the combination. The following table shows the effect of mixing PRN preadsorbed in aluminum hydroxide (8 μg per 10 μg or 50 μg of Al3 +), with a dose of a non-adsorbed antigen (or aluminum phosphate) for seven days at 4 ° C or 37 ° C, pH 6.1. The final concentrations of antigen were equivalent to their concentrations in a typical combined vaccine. The percentage of PRN in the supernatant was measured using ELISA; The PRN was measured in the supernatant and in the pellet (500 mM of phosphate anions was used to dessorb the PRN and the PRN released was measured). Then a percentage was calculated.

TABLE III As the table shows, in the context of the combined vaccine the aluminum hydroxide is supersaturated with 8 μg of PRN, using 10 μg of aluminum hydroxide, but it is stable using 50 μg of the adjuvant. The effect of free aluminum phosphate to cause some desorption of PRN is also shown. The use of extra-washed aluminum phosphate would limit this effect.

Claims (27)

1. - A method for reducing the interference of a capsular polysaccharide component of a conjugated Haemophilus influenzae B (Hib) vaccine in a combined vaccine comprising DTPa, characterized in that said method comprises: i) selecting one or more antigens to be adsorbed in adjuvant aluminum hydroxide; ii) presaturating the aluminum hydroxide adjuvant with the selected antigens, so that there is no excess of aluminum hydroxide; iii) selecting Hib plus one or more additional antigens, to be adsorbed on aluminum phosphate; iv) Adsorb Híb and the additional antigens in aluminum phosphate; v) combine all the antigens in that vaccine.

2. The method according to claim 1, further characterized in that the Hib adsorbed on the aluminum phosphate is mixed extemporaneously with the other antigens of the combined vaccine.

3. The method according to claim 1 to 2, further characterized in that the combined vaccine additionally comprises one or more antigens selected from the group of: hepatitis B surface antigen (HBsAg), inactivated hepatitis A virus, inactivated poliovirus, capsular polysaccharide of N. meningitidis A, capsular polysaccharide of N. meningitidis C, capsular polysaccharide of Streptococcus pneumoniae, proteins of Streptococcus pneumoniae, outer membrane proteins of Moraxella catarrhalis, outer membrane proteins of Haemophilus influenzae, without determination of type; outer membrane proteins of N. meningitidis B.

4. The method according to claim 3, further characterized in that the combined vaccine comprises hepatitis B surface antigen, adsorbed to aluminum phosphate.

5. The method according to any of claims 1 to 4, further characterized in that the ratio of aluminum phosphate to aluminum hydroxide adjuvant, present in the combined vaccine, varies from 1: 1 to 20: 1.

6. The method according to any of claims 1 to 5, further characterized in that all the antigens of the combined vaccine are adsorbed on aluminum phosphate; as long as pertactin is adsorbed on aluminum hydroxide.

7. The method according to claim 6, further characterized in that the pertactin adsorbed on aluminum hydroxide is combined with inactivated, non-adsorbed poliovirus antigens, before combining it with the antigens adsorbed on aluminum phosphate.

8. The method according to any of claims 1 to 5, further characterized in that all the antigens of the combined vaccine are adsorbed on aluminum hydroxide; as long as HBsAg and Hib are adsorbed to aluminum phosphate.

9. The method according to claim 8, further characterized in that the antigens adsorbed on aluminum hydroxide are combined before the HBsAg antigen, adsorbed on aluminum phosphate, is added and Hib is combined, adsorbed on aluminum phosphate, then that the HBsAg antigen has been added.

10. The method according to claims 8 to 9, further characterized in that additional free aluminum phosphate is added to the combined vaccine.

11. A method for reducing the interference of a capsular polysaccharide component of a conjugated Haemophilus influenzae B (Hib) vaccine in a combined vaccine comprising DTPa, characterized in that said method comprises: i) selecting one or more antigens to be adsorbed in adjuvant aluminum hydroxide; ii) presaturating the aluminum hydroxide adjuvant with the selected antigens, so that there is no excess of aluminum hydroxide; iii) selecting one or more additional antigens, to be adsorbed on aluminum phosphate; iv) adsorbing the additional antigens in aluminum phosphate; v) combine all the antigens in that vaccine.

12. A method for reducing the interference of a capsular polysaccharide component of a conjugated Haemophilus influenzae B (Hib) vaccine in a combined vaccine comprising DTPa, characterized in that said method comprises: i) selecting one or more antigens to be adsorbed in adjuvant aluminum hydroxide; ii) presaturating the aluminum hydroxide adjuvant with the selected antigens, so that there is no excess of aluminum hydroxide; iii) selecting one or more additional antigens, to be adsorbed on aluminum phosphate; iv) adsorbing the additional antigens in aluminum phosphate; v) combine all the antigens in that vaccine; vi) extemporaneously add Hib without adjuvant or Hib adsorbed on aluminum phosphate.

13. The method according to claim 12, further characterized in that the combined vaccine additionally comprises one or more antigens selected from the group: hepatitis B surface antigen (HBsAg), inactivated hepatitis A virus, inactivated poliovirus, capsular polysaccharide of N. meningitidis A, capsular polysaccharide of N. meningitidis C, capsular polysaccharide of Streptococcus pneumoniae, proteins of Streptococcus pneumoniae, outer membrane proteins of Moraxella catarrhalis, outer membrane proteins of Haemophilus influenzae, without determination of type; outer membrane proteins of N. meningitidis B.

14. - The method according to claim 13, further characterized in that the combined vaccine comprises hepatitis B surface antigen, adsorbed on aluminum phosphate.

15. The method according to any of claims 12 to 14, further characterized in that the ratio of aluminum phosphate or aluminum hydroxide adjuvant, present in the combined vaccine, varies from 1: 1 to 20: 1.

16. The method according to any of claims 12 to 15, further characterized in that all the antigens of the combined vaccine are adsorbed on aluminum phosphate, provided that pertactin is adsorbed on aluminum hydroxide and that Hib is added without adjuvant or adsorbed on aluminum phosphate.

17. The method according to claim 16, further characterized in that the pertactin adsorbed on aluminum hydroxide is combined with inactivated, non-adsorbed poliovirus antigens, before combining it with the antigens adsorbed on aluminum phosphate.

18. The method according to any of claims 12 to 15, further characterized in that all the antigens of the combined vaccine are adsorbed on aluminum phosphate, as long as pertactin, diphtheria toxoid, pertussis toxoid, filamentous hemagglutinin is adsorbed. in aluminum hydroxide; and that Hib is added without adjuvant or adsorbed on aluminum phosphate.

19. - The method according to claim 18, further characterized by combining pertactin, diphtheria toxoid, pertussis toxoid, filamentous haemagglutinin, adsorbed on aluminum hydroxide, with inactivated, non-adsorbed poliomyelitis virus antigens, before combining them with the antigens adsorbed on aluminum phosphate.

20. The method according to any of claims 12 to 15, further characterized in that all the antigens of the combined vaccine are adsorbed on aluminum hydroxide, as long as HBsAg is adsorbed on aluminum phosphate and that Hib is added without adjuvant or adsorbed on aluminum phosphate.

21. The method according to claim 20, further characterized in that the antigens adsorbed on aluminum hydroxide are combined before adding the HBsAg antigen, adsorbed on aluminum phosphate.

22. The method according to claim 20 or 21, further characterized in that additional free aluminum phosphate is added to the combined vaccine, before the extemporaneous addition of the Hib without adjuvant.

23. The method according to any of claims 12 to 22, further characterized in that one or more of the antigens selected to presaturate the aluminum hydroxide, has been previously adsorbed on aluminum phosphate.

24. The method according to claim 23, further characterized in that the diphtheria toxoid, adsorbed on aluminum phosphate, is one of the antigens selected to presaturate the aluminum hydroxide.

25. The method according to any of claims 1 to 24, further characterized in that the combined vaccine is regulated with L-histidine. 26.- A combined vaccine, characterized in that it is obtainable by the method according to any of claims 1 to 25 ..}. 27. A method for vaccinating against diphtheria, tetanus, pertussis and H. influenzae type B, characterized in that it comprises administering a pharmaceutically effective amount of the combined vaccine of claim 26.