The subject of the present invention is a stabilizer, a stabilized aqueous vaccine composition and a method for preparing a dry vaccine composition enabling the IPV polio antigen to be dried without a large loss of titer during the drying process and with a loss of titer, during storage of the resulting dry composition for at least 7 days at 37°C, that is less than what has been disclosed to date. Another subject of the present invention is a dry vaccine composition comprising an inactivated poliovirus (IPV) of at least one serotype and a stabilizer, characterized by an extraordinary thermostability.

Two types of poliomyelitis vaccine exist. They are made up of three virus serotypes (1 , 2 and 3). One, the oral vaccine (OPV - Oral Polio Vaccine), uses three live attenuated strains, the other, the inactivated vaccine (IPV - Inactivated Polio Vaccine), uses three inactivated strains. The attenuated vaccine (OPV) has been widely used to combat this terrible disease, despite its instability. An initiative was launched by the WHO between 1990 and 1995 (Children's vaccine initiative product development group on thermostable Oral Poliovaccine) in order to encourage the development of a liquid or freeze-dried, thermostable OPV vaccine. This action was carried out in the context of the poliomyelitis eradication program, the instability of the OPV vaccine limiting the success of vaccination with this vaccine.

The risks of poliovirus dissemination, due to the use of an oral vaccine containing a live attenuated virus having the possibility of reversion during intestinal transit of the latter, drive the use of the inactivated polio vaccine. The use of this inactivated vaccine, as a replacement for the unstable attenuated oral vaccine, leads to its stability being increased as much as possible so as not to limit the successful use thereof. The re-emergence of cases of poliomyelitis in certain African countries in 2009, where the disease had been eradicated, shows the topicality of the need for a stable vaccine.

The stabilization of fragile, heat-sensitive products is mainly obtained using a drying technique known as freeze-drying. Freeze-drying is a technique which uses successively freezing and then sublimation in order to dry and stabilize fragile products, despite a reasonably large loss of titer or of activity, observed during the process.

It is not obvious to use freeze-drying for stabilizing the poliovirus since a large loss of virus is observed after freeze-drying both for OPV (loss of infectious titer) and for IPV (loss of D-antigen titer). In addition, former publications (Tex Rep Biol Med. 1951 ; 9(4): 749-54) have shown inactivation of the poliovirus by freeze-drying.

This technical problem has been addressed in various ways in the prior art. Application WO 89/06542, for example, teaches that the decrease in efficacy which results in a loss of infectious titer of a type 3 poliovirus, dried at 37°C and stored for 7 days at this temperature, can be brought back from 6 logs to 3.7 logs by simple drying of the virus stock solution at 37°C in the presence of a stabilizing solution made up of 10% trehalose as sole protective agent. However, the drop in infectious titer remains large and greater than that observed for the nondried vaccine. This shows the difficulty in drying this attenuated poliovirus.

For the IPV vaccine, application US 2006/0127414 (WO 2004/039399 Al) provides a method for stabilizing the poliovirus (IPV vaccine) by drying, and also a stabilizing solution containing saccharides, which make it possible to obtain a highly viscous vaccine, the residual water content of which is less than or equal to 15%, and showing, for IPV, alone, a loss during the method of greater than or equal to 50%, irrespective of the serotype. It should be noted that, in this case, the degree of drying does not make it possible to obtain a solid (either crystalline or amorphous) product, the total solidification temperature being below the storage temperature of the vaccine (5°C).

In the literature, other authors have claimed a method for stabilizing the poliovirus, in this case OPV, by freeze-drying in the presence of a stabilizing solution based on sorbitol, glutamine and BSA, followed by rehydration with a solution of peptone and sucrose (Jpn. J. Infect. Dis., 56, 70-72, 2003). The better stability (1.29 log during the method for serotype 3) in this case was also obtained for drying conditions that were for several days and retained a high residual water content in the product after drying, in comparison with the standard residual water contents for pharmaceutical products, namely < 3%.

Consequently, the technical problem on which the present invention is based is that of providing a stabilizer, a bulk aqueous vaccine composition and a method for preparing a dry vaccine composition comprising at least one of the three inactivated poliovirus (IPV) serotypes which enable the IPV polio antigen to be dried, for example by freeze-drying, without a large loss of titer during the drying process and which enable the resulting composition to be stored for at least 7 days at 37°C with a loss of titer which is less than anything which has been disclosed to date.

In the context of the present invention, the term "dry" denotes a product which is characterized by a residual water content of less than 3% (measured by the method according to Karl Fischer) and which is solid, either crystalline or amorphous, both at 5°C and at ambient temperature (21 to at least 37°C).

The abovementioned technical problem is solved by a stabilizer for the preparation of a dry vaccine composition made up of at least one inactivated poliovirus (IPV) serotype, comprising:

(i) urea or derivatives thereof,

(ii) a non-reducing disaccharide,

(iii) a gelling polymer,

(iv) amino acids, at least one of which is an antioxidant amino acid,

(v) a buffer, and

(vi) a surfactant.

Among the urea derivatives, mention may, for example, be made of thiourea, allylurea, acetamide, methylcarbamate or butylcarbamate.

A non-reducing disaccharide is a saccharide formed by two monosaccharides, the reducing groups of which are blocked by the acetal linkage between the two monosaccharides. Non- reducing disaccharides are, for example, trehalose and sucrose. According to one embodiment of the present invention, the stabilizer comprises sucrose as non-reducing disaccharide.

Gelling polymers are substances which make it possible to give in particular food products the consistency of a gel. Examples for gelling polymers are gelatin, pectins, alginates, carrageenans and xanthan. According to one embodiment of the present invention, the stabilizer comprises xanthan as gelling polymer.

According to one embodiment of the present invention, the antioxidant amino acid in the stabilizer is methionine and, according to yet another embodiment, the amino acids included in the stabilizer comprise glutamine and glycine.

All the buffers normally used for vaccine compositions can be used for the stabilizer according to the present invention, for example TRIS, PBS or HEPES. According to one embodiment of the present invention, the buffer included in the stabilizer is HEPES.

Examples for surfactants which can be included in the stabilizer according to the present invention are poloxamers (Pluronic®), CTAB (hexadecyltrimethylammonium bromide), SDS (sodium dodecyl sulfate or sodium lauryl sulfate) or a polysorbate (Tween® 20, Tween® 80). According to one embodiment of the present invention, the surfactant included in the stabilizer is a polysorbate (Tween® 20, Tween® 80). The stabilizer according to the present invention can therefore comprise, in 1000 ml of aqueous solution:

(i) 10 to 30 g of urea,

(ii) 100 to 300 g of sucrose,

(iii) 0.5 to 3 g of xanthan,

(iv) 5 to 14 g of methionine,

1 to 30 g of glutamine,

1 to 30 g of glycine,

(v) HEPES (10 to 50 mM), and

(vi) 0.01 to 0.1 % Tween® 80.

As mentioned above, another aspect of the present invention is a stabilized aqueous vaccine composition, which is for example a bulk composition, comprising 1 volume of at least one of the three known inactivated poliovirus (1PV) serotypes and 2 volumes of the stabilizer described above. This stabilized aqueous composition may comprise other protein or polysaccharide antigens optionally conjugated to carrier proteins. For example, according to one embodiment of the present invention, the composition comprises at least one bacterial polysaccharide or one bacterial oligosaccharide. These bacterial polysaccharides or oligosaccharides comprise capsular polysaccharides from any bacterium, for example one or more from Neisseria meningitidis (for example, capsular polysaccharides derived from one or more serogroups A, C, W-135 and Y), from Haemophilus influenzae b. from Streptococcus pneumoniae (preferably serotypes 1 , 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F), group A streptococci, group B streptococci, Staphylococcus aureus or Staphylococcus epidermis. According to one preferred embodiment of the present invention, the stabilized aqueous composition comprises a capsular polysaccharide or oligosaccharide antigen originating from Haemophilus influenzae b (Hib), preferably conjugated to a carrier protein.

According to another embodiment of the present invention, the stabilized aqueous composition comprises, in addition, a capsular polysaccharide or oligosaccharide originating from N. meningitidis A, C, Y or W or any combination thereof, preferably conjugated to a carrier protein.

Examples for carrier proteins conjugated to bacterial polysaccharides or oligosaccharides are tetanus toxoid, tetanus toxoid fragment C, diphtheria toxoid, the nontoxic mutant form CRM 197 of diphtheria toxin, pneumolysin and protein D. When several conjugated bacterial oligosaccharides are present in the aqueous stabilized composition according to the present invention, the oligosaccharides can be conjugated to the same carrier protein or to different carrier proteins, preferably while adhering to the teaching of application WO 98/51339 (AU 748716B) regarding the maximum load (amount) of carrier proteins in one dose.

As mentioned above, another aspect of the present invention is a method for preparing a dry vaccine composition comprising at least one inactivated poliovirus (IPV) serotype, which comprises drying the bulk aqueous vaccine composition described previously. According to one embodiment of the present invention, the drying is carried out by freeze- drying which is a method comprising a freezing step, a sublimation step and a desorption step. The drying is preferably for a period of less than 40 hours.

Another aspect of the present invention is a dry vaccine composition comprising an inactivated poliovirus (IPV) of at least one serotype and a stabilizer, wherein its residual water content is less than 3% and the maximum loss of D-antigen of each IPV serotype remains less than 15% during storage for 0 to at least 7 days at 37°C following drying (compared with its content directly after drying).

This dry vaccine composition can, for example, be obtained by drying the stabilized bulk aqueous vaccine composition described previously. According to one embodiment of the present invention, the drying is carried out by freeze-drying. Another aspect of the present invention is a vaccine comprising the abovementioned vaccine composition. This vaccine can be reconstituted in an aqueous solution before its use.

Figures

Figure 1 shows the synopsis of the formulation of this intermediate bulk

Figure 2 shows the formulation of the stabilizing composition

Materials and Methods Vaccine: The same production scheme is applied to the three serotypes, which are obtained separately (B.J. Montagnon, B. Fanget and J. Nicolas: Develop, biol. Standard. 1980-47, 55-64). A culture of Vero cells, on microcarriers, is infected with a viral seed batch (type 1 : Mahoney; type 2: MEF 1 ; type 3 : Saukett). The culturing of virus is carried out in M 199 medium and ends at cell lysis. The virus is harvested by drawing off the viral culture supernatant. The virus purification is carried out in several steps:

1 - ultrafiltration-concentration on membranes;

2 - ion exchange chromatography (DEAE);

3 - ultrafiltration-concentration on membranes;

4 - gel filtration chromatography;

5 - ion exchange chromatography (DEAE).

Virus inactivation is obtained with formol. The IPV is then stored in a Hanks M 199 medium with 0.02% Tween® 80.

D-antigen titration

The D-antigen titration is an ELISA titration, determined for each viral serotype. The method used is an indirect method which follows the following steps:

1 - sensitizing the wells of the ELISA plates with heifer anti-polio antibodies for 16 h at

5°C;

2 - saturation of the wells of the plates using a solution of semi-skimmed milk and Tween® 20;

3 - bringing the various dilutions of the sample to be titrated into contact with the heifer antibodies in the wells of the sensitized plate, covering the plate with an adhesive film before incubation at 37°C for 2 h;

4 - adding the rabbit anti-polio antibody, after rinsing of the wells, and covering the plate with an adhesive film before incubation at 37°C for 1 h;

5 - adding the anti-rabbit IgG antibody, after rinsing of the wells, and covering the plate with an adhesive film before incubation at 37°C for 1 h;

6 - adding the substrate (ABTS) solution to the wells after rinsing, and covering the plate with an adhesive film before incubation at 37°C for 10 to 15 min; and then stopping the reaction with the blocking solution (SDS); and reading the plates at 405 nm;

7 - calculating the titers according to the parallel line method. Method for stabilizing IPV a) Preparation of the intermediate bulk solution containing the IPV

An intermediate bulk solution containing at least one of the 3 IPV serotypes and adjusted to the desired titer is obtained by mixing the bulks of the desired serotypes with a diluting solution, which is Hanks M 199 medium containing 0.02% Tween® 80. For all the tests described below, the volumes of the bulks of each of the IPV serotypes and the volumes of diluting solution were calculated so as to obtain an intermediate bulk with the following titers:

- serotype 1 : 450 DU/ml

- serotype 2: 105 DU/ml

- serotype 3: 375 DU/ml.

Figure 1 shows the synopsis of the formulation of this intermediate bulk.

b) Formulation of the stabilizing composition

The stabilizing composition is obtained by mixing one volume of the intermediate bulk as described in the paragraph above with two volumes of the stabilizer (Figure 2).

The composition of the IPV stabilizer according to the present invention was determined by very wide screening of numerous molecules and by optimizing the amount thereof. An example for a composition of this stabilizer is described below:

- Urea: 25.5 g/1

Sucrose: 225 g/1

- L-methionine: 13.4289 g/1

- Glutamine: 24 g/1

- Glycine: 22.5 g/1

Concentrated solution of Tween® 80 at 5%: 3 ml per liter of stabilizer

- HEPES: 30 mM Xanthan: 1 g/1

pH adjusted to 7.0 +/- 0.2 with sodium hydroxide or hydrochloric acid. c) Freeze-drying of the stabilizing composition

The stabilizing composition obtained is then distributed in a proportion of 0.5 ml per 5 ml flask, the flasks are placed on the shelves of the freeze-dryer pre-cooled to 5°C, and then the following freeze-drying cycle is applied:

- freezing: ramp of 5°C to -50°C in 90 min, followed by a hold of 60 min at -50°C cooling of the trap and placing the freeze-dryer under vacuum

sublimation: ramp of -50°C to -35°C in 30 min, followed by a hold of 1920 min at - 35°C; pressure equal to 50 microbar

- desorption: ramp of -35°C to 30°C in 120 min, followed by a hold of 180 min at 30°C; pressure equal to 50 microbar

stoppering of the freeze-dried flasks under 800 mbar of nitrogen.

All the products freeze-dried and tested by the method according to Karl Fischer had a residual water content of less than 3%. The freeze-dried materials were in the solid state both at 5°C and at ambient temperature. d) Measuring of the D-antigen titer after freeze-drying

The freeze-dried product is rehydrated with 0.5 ml of injectable water or of isotonic PBS solution.

Example 1 : Comparison of the stability obtained with the stabilizer and the stability of the prior art compositions

The stability obtained after freeze-drying and the thermostability for 7 days at 37°C were compared, firstly, with that obtained with another stabilizer proposed for the stabilization of live viruses according to application WO 2010/003670 and, secondly, with a composition containing Hib polysaccharide and sucrose as described in application US 2006/0127414.

The formulating of the stabilizing composition according to application WO 2010/003670 was carried out as described above, replacing the IPV stabilizer according to the present invention with the stabilizer according to application WO 2010/003670, namely:

The stabilizing composition described in application US 2006/0127414 was prepared as follows: the bulk concentrates of each serotype were diluted in a buffer containing sucrose and Hib polysaccharide so as to obtain, in the stabilizing composition to be freeze-dried, 3.15% of sucrose and 24 μg/ml of conjugated Hib polysaccharide.

The results obtained are shown in the table below, expressed for each serotype as percentage loss of D-antigen titer. The term "loss freeze-drying To" corresponds to the loss of D-antigen titer between the product before and after freeze-drying; the term "loss thermostability 7d at 37°C" corresponds to the loss of titer between the product after freeze-drying and the freeze-dried product incubated for 7 days at 37°C.

The IPV stabilizer clearly gives greater stability and provides, per se, something new for IPV stabilization.

Example 2: Demonstration of the importance of the excipients contained in the IPV stabilizer according to the present invention

In order to demonstrate the importance of the excipients contained in the IPV stabilizer according to the present invention for obtaining optimum stability, the following formulations were tested:

formulation 1 : IPV stabilizer for which the sucrose has been replaced with lactose, which is another disaccharide, but one which is a reducing disaccharide;

formulation 2: IPV stabilizer without xanthan (gelling polymer):

formulation 3: stabilizer without urea;

formulation 4: IPV stabilizer without glutamine and without glycine.

The results obtained are shown in the table below, expressed for each serotype as percentage loss of D-antigen titer. The term "loss freeze-drying To" corresponds to the loss of D-antigen titer between the product before and after freeze-drying: the term "loss thermostability 7d at 37°C" corresponds to the loss of titer between the product after freeze-drying and the freeze-dried product incubated for 7 days at 37°C. The freeze-dried products were rehydrated with injectable water.

Example 3: Study of the impact of the concentration of the excipients of the IPV stabilizer according to the present invention

A factorial experiment design was implemented in order to study the impact of the concentration of urea and of sucrose and to determine the optimum concentration. The concentration ranges studied are the following:

sucrose: 150 g/1 for the level - and 225 g/1 for the level +

- urea: 15 g/1 for the level - and 25.5 g/1 for the level +

the amount of the other excipients of the IPV stabilizer remained unchanged.

The results obtained are shown in the table below, expressed for each serotype as percentage loss of D-antigen titer. The term "loss freeze-drying To" corresponds to the loss of D-antigen titer between the product before and after freeze-drying; the term "loss thermostability 7d at 37°C" corresponds to the loss of titer between the product after freeze-drying and the freeze-dried product incubated for 7 days at 37°C. Only serotypes 1 and 3 were analyzed. The freeze-dried products were rehydrated with an isotonic PBS solution.