AU2013203067A1 - Process for stabilizing an adjuvant containing vaccine composition - Google Patents

Abstract

H:\anr\Intenvoven\NRonbl@CC\AAR\5057264_1.de-9/12013 - 37h The present invention relates to a process for stabilizing an adjuvant containing vaccine composition, an adjuvanted vaccine composition in dry form and in particular a process for stabilizing an influenza vaccine composition, particularly an adjuvanted influenza vaccine composition in dry form.

Description

Process for stabilizing an adjuvant containing vaccine composition This application is a divisional of Australian Application No. 2009221180, the entire contents of which are incorporated herein by reference. The present invention relates to a process for stabilizing an adjuvant containing vaccine composition, an adjuvanted vaccine composition in dry form and in particular a process for stabilizing an influenza vaccine composition, particularly an adjuvanted 5 influenza vaccine composition In dry form. US 3,655,838 discloses a method of pelletizing analytical or immunological reagents by freezing droplets of solutions in a freezing medium, such as liquid nitrogen, and subsequent freeze drying in order to obtain freeze-dried, reagent containing micro particles, spherical beads or lyospheres. EP 0 081 913 BI describes a method for 10 producing spherical frozen particles by freezing droplets in an inert liquid freezing medium with a higher density than the droplets and removing the frozen particles from the surface of the liquid freezing medium. WO 94/25005 discloses the stabilization of gonadotropin in lyospheres, a method for making such lyospheres and pharmaceutical preparations comprising the same. EP 0 799 613 (US 5,897,852) 15 discloses a vaccine pack that comprises a vaccine container containing one ore more freeze-dried bodies containing the vaccine components, at least one of which being a lyosphere or micro-particle having a diameter of at least 0.2 mm. WO 2006/008006 (US 2008/0060213) discloses a process fbr producing containers filled with a freeze dried product wherein droplets of the product are frozen to form pellets, the pellets .0 are freeze dried, assayed and loaded into the containers. Other techniques for obtaining frozen particles or pellets are known for application in the food industry (e.g. US 5,036,673 or US 2007/0281067 Al). The freeze drying technology allows improving the stability of a lot of products which can be a vaccine with or without adjuvant. For example EP 0 475 409 disposes a 5 method for preserving a buffer and optionally a cryoprotectant containing suspension of microscopic biological material by nebulizing the suspension to microdroplets, freezing the droplets on a rotating cryogenic surface and drying the microdroplets. Preferably the droplets have a diameter of about less than 200 pm. The freeze-drying of flu antigens has been studied in the literature and a detailed ) review is available (Amorij et al. 2008: Development of stable Influenza vaccine WO 2009/109550 PCTr2009/052460 2 powder formulations: challenges and possibilities. Pharmaceutical Research, Vol 25, 1256-1273). US 3 674 864 discloses a process for stabilizing influenza virus vaccines essentially by suspending the virus in an aqueous sucrose containing solution and freeze-drying the suspension. Also the stabilization of tetanus and diphtheria toxoids 5 has been discussed in the literature (see e.g. S.P. Schwendeman et at, Proc. Nati. Acad. Sci. USA Vol. 92, pp. 11234-11238, 1995). Very recently, optimized formulations for lyophilizing tetanus toxoid have been proposed (P. Matejtschuk et al. Biologicals 37 (2009) 1-7). Usually the freeze drying is a final step in the pharmaceutical industry, coming after 10 the filling step in vials, syringes or larger containers. In this case the dried product has to be rehydrated (synonyms in this document: reconstituted or dissolved) before its use. Freeze drying in the form of micropellets allows the same stabilization of the dried vaccine product as for mere freeze-drying alone or it improves stability for storage. 15 Furthermore, the micropellets technology offers several advantages in comparison to the current freeze drying, since it allows e.g. - blending of the dried products before filling (or by sequential filling) - titer adjustment before filling (which can be used in case of stock piling) - minimizing the interaction between products (there is only product interaction after !0 rehydration ), and - improvement of the stability in some cases. For these reasons the advantage of the micropellets technology allows several approaches for the drying of adjuvanted vaccine: -,The drying of the antigens together with the adjuvant (being in the same phase): to 5 stabilize the two (antigens and adjuvant) and to stabilize the interaction between them by trapping them in a glassy matrix in which all molecular motions and chemical reactions are greatly hindered. This solid state allows to maintain throughout storage (even at higher temperature) potency of the antigen, physical and immunological properties of the adjuvant and nature and force of the interaction between the two.

WO 2009/109550 PCT/EP2009/052460 3 - The drying of the antigens and the separate drying of the adjuvant (antigen and adjuvant being in different phases), followed by blending of the two before the filling or by a sequential filling. In some cases, stability of the adjuvant alone can be a problem (chemical stability of emulsions, physical stability of aluminum gels, 5 liposomes and others...) at the liquid state for long-term storage at +5 0 C or higher or at lower temperatures. The micropellet technology allows improving stability of the adjuvanted vaccine by generating separate micropellets of antigen and adjuvant. The stabilizing formulation can be optimized independently for each antigens and the adjuvant. The micropellets of antigens and adjuvants can be then subsequently filled 10 into the vials or blended before filling into the vials. The separated solid state allows to avoid throughout storage (even at higher temperature) interactions between antigen and adjuvant, to maintain the potency of the antigen and physical and the immunological properties of the adjuvant. In such a configuration, the content of the vial can be more stable than any other configurations with either one of the antigens 15 or the adjuvants in the liquid state or when antigens and adjuvant are dried within the same pellets. Interactions between.antigens and adjuvants are this way standardized as they occur only after rehydration of the dry combination with a selected diluent which may comprise water for injection, buffers and stabilizing excipients. Interactions are therefore to be controlled only during the short period of time 0 between rehydration and injection of the vaccine. It is therefore possible to improve the overall stability of the two products can be improved (optimization of the formulation for each product and not a compromise for the two together) and the vaccine itself, adjust the titer of one out of the two after the storage and before filling, to facilitate the manufacturing process by separation of the 5 two products drying. The present invention provides a process for stabilizing an adjuvant containing vaccine composition comprising the steps of diluting a liquid bulk composition comprising an antigen or an antigenic preparation by an aqueous solution comprising a stabilizer, subjecting the diluted composition to a process in order to form regular 0 droplets having a diameter of approximately fmorn about 200pm to about 1500 pm, -subjecting the regular droplets to freezing in a freezing medium to form frozen regular spherical micropellets or particles and drying the frozen regular spherical micropellets WO 20091109550- PCT/EP2009/052460 4 or particles to form dry regular spherical micropeliets or particles having a diameter from about 200pm to about 1500 pm. Suitable vaccine compositions are for example compositions comprising whole viruses or antigens, such as Influenza, Rotavirus , cytomegalo virus, Hepatitis A and 5 B , whole bacteria or bacterial protein or polysaccharide antigens ( conjugated or non-conjugated ), such as Haemophilus influenzae , meningococcal polysaccharides, Tetanus, Diphteria, cellular and acellular pertussis, Botulism, Anthrax or C. Difficile. The freezing of the droplets can e.g. be achieved in that the diluted composition, i.e. the formulated liquid product, is prilled in order to generate calibrated droplets which 10 diameter ranges from 200pm to 1500 pm, with a very narrow size distribution. These droplets fall in a cryogenic chamber in which the temperature is maintained below I 10*C by a freezing medium, either by direct injection/nebulization of liquid nitrogen or by flowing counter currently a very cold (T<-1 10*C) gas such as nitrogen, C02 or air. The droplets freeze during their fall in order to form calibrated frozen particles. 15 Other techniques are known in the art for obtaining calibrated droplets, such as ultrasonic atomization (Sindayihebura D., Dobre M., Bolle L., 1997 - Experimental study of thin liquid film ultrasonic atomization. ExHFT'97, Bruxelles.), and, subsequently, calibrated frozen particles or, as mentioned before being known from the food industry, by means of a particular freezing drum as disclosed in !0 US 5,036,673. The process may further comprise the addition of an aqueous solution comprising one or more adjuvants and, optionally, a stabilizer to the liquid bulk antigen composition. Alternatively, the process according to the present invention further comprises 25 diluting a separate liquid bulk solution or emulsion of one or more adjuvants by an aqueous solution comprising a stabilizer, subjecting the diluted adjuvant solution or emulsion to a process in order to form regular droplets having a diameter of approximately from about 200pm to about 1500 pm, subjecting the regular droplets to freezing in a freezing medium to form frozen regular spherical micropellets or 0 particles, drying the frozen regular spherical micropeliets or particles to form dry WO 2009/109550 PCTEP2009/052460 5 regular spherical micropellets or particles having a diameter from about 200pm to about 1500 pm and blending and filling into a vial or other appropriate recipient together with the antigen containing dry regular spherical micropellets. The liquid bulk composition may further comprise one or more additional antigens or 5 antigenic preparations, each from a different pathogen or serotype of a pathogen, in order to obtain dry regular spherical micropellets or particles, each comprising two or more antigens or antigenic preparations from two or more different pathogens or serotypes of a pathogen. Alternatively, the liquid bulk composition comprises an antigen or antigenic 10 preparation from one single pathogen or serotype of a pathogen, in order to obtain dry regular spherical micropellets or particles, each comprising antigens or antigenic preparations from the same pathogen or serotype of a pathogen. In this case the process may optionally further comprise the dosing, blending and filling into a vial or other appropriate recipient two or more types of dry regular spherical micropellets or 15 particles, characterized in that each type of micropellets comprises antigens or antigenic preparations from two or more different pathogens or serotypes of a pathogen. The process according to the present invention may be further characterized in that the drying occurs by the method of lyophilization (i.e. sublimation of ice and !0 dissorption of bound water). Suitable drying methods are also atmospheric freeze drying, fluidized bed drying, vacuum rotary drum drying, stirred freeze-drying, vibrated freeze-drying and micmwave freeze-drying. Advantageously, the stabilizer comprises a monosaccharide, such as mannose, an oligosaccharide, such as sucrose, lactose, trehalose, maltose or a sugar alcohol, !5 such as sorbitol, mannitol or inositol, or a mixture of two or more different of these before mentioned stabilizers, such as mxtures of sucrose and trehalose. Advantageously, the concentration of carbohydrates, sugar alcohols and stabilizing excipients ranges from 2% (w/v) to limit of solubility in the formulated liquid product. In general, the concentration of carbohydrates, sugar alcohols and stabilizing 0 excipients ranges between 5% (w/v) and 40% (w/v), 5% (w/v) and 20% (w/v) or 20% WO 20091109550 PCT/EF2009/052460 6 (w/v) and 40% (w/v). Examples for the concentration of a 1:1 mixture of sucrose and ttehalose in the formulated liquid product comprising e.g. tetanus or diphtheria toxoids and aluminum gel (AIOOH) are 18,1% (w/v) and 17,5% (w/v), respectively. The present invention particularly relates to a process for stabilizing an influenza 5 vaccine composition comprising the steps of diluting a liquid bulk composition comprising an influenza antigen or an antigenic preparation from a seasonal, pre pandemic or pandemic influenza virus strain by an aqueous solution comprising a carbohydrate and/or a sugar alcohol or a mixture of two or more different carbohydrates and/or sugar alcohols in order to obtain a 2% (wlv) to limit of solubility 10 of carbohydrate and/or sugar alcohol content of the resulting diluted composition, subjecting the diluted composition to a process in order to form regular droplets having a diameter of approximately from about 200pm to about 1500 pm, subjecting the regular droplets to freezing in a freezing medium to form frozen regular spherical micropellets or particles and drying the frozen regular spherical micropellets or 15 particles to form dry regular spherical micropellets or particles having a diameter from about 200pm to about 1500 pm. The liquid bulk composition comprising an influenza antigen or an antigenic preparation from a seasonal, pre-pandemic or pandemic influenza virus strain can be obtained by the method as disclosed in US 6,048,537 (WO 96/05294). !0 The drying occurs advantageously by the method of lyophilization (i.e. sublimation of ice and dissorption of bound water). Further suitable drying methods for the frozen micropellets are atmospheric freeze-drying, fluidized bed drying, vacuum rotary drum drying, stirred freeze-drying, vibrated freeze-drying, and microwave freeze-drying. Advantageously, the carbohydrate is a disaccharide, particularly sucrose. Also !5 suitable disaccharides are for example lactose, maltose or trehalose. Also suitable for stabilizing the influenza vaccine composition are monosaccharides, such as mannose, or sugar alcohols, such as sorbitol, inositol or mannitol. Advantageously, the concentration of the carbohydrates or sugar alcohols ranges between 5% and 40% (w/v), alternatively between 5% and 20% (w/v) or between WO 2009/109550 PCT/EP2009/052460 7 20% and 40% in the formulated liquid product. Examples for the concentrations of e.g. sucrose in the formulated liquid product are 2% (w/v), 10% (w/v) or 15,4% (wlv). The liquid bulk composition may further comprise one or more additional influenza antigens or antigenic preparations, each from a different seasonal, pre-pandemic or 5 pandemic influenza virus strain, in order to obtain dry regular spherical micropellets or particles, each comprising two or more influenza antigens or antigenic preparations from two or more different seasonal, pre-pandemic or pandemic influenza virus strains. In this case the process optionally further comprises dosing and filling the dry regular spherical micropellets or particles into a vial or other 10 appropriate recipient. Alternatively, the liquid bulk composition comprises one or more influenza antigens or antigenic preparations from one seasonal, pre-pandemic or pandemic influenza virus strain, in order to obtain dry regular spherical micropellets or particles, each comprising influenza antigens or antigenic preparations from the same seasonal, pre 15 pandemic or pandemic influenza virus strain. In this case the process may optionally further comprise dosing, blending and filling into a vial or other appropriate recipient two or more types of dry regular spherical micropellets or particles, characterized in that each type of micropeilets comprises influenza antigens or antigenic preparations from a different seasonal, pre-pandemic or pandemic influenza virus strain than the .0 other type. The process optionally further comprises the addition of an aqueous solution comprising one or more adjuvants and, optionally, a stabilizer to the liquid bulk antigen composition. Alternatively, the process according to the present invention further comprises !5 diluting a separate liquid bulk solution or emulsion of one or more adjuvants by an aqueous solution comprising a stabilizer, subjecting the diluted adjuvant solution or emulsion to a process in order to form regular droplets having a diameter of approximately from about 200pm to about 1500 pm, subjecting the regular droplets to freezing in a freezing medium to form frozen regular spherical micropellets or 0 particles, drying the frozen regular spherical micropellets or particles to form dry regular spherical micropellets or particles having a diameter from about 200pm to WO 20O9/109550 PCT/EP2009/052460 8 about 1500 pm and blending and filling into a vial or other appropriate recipient together with the antigen containing dry regular spherical micropellets. Particularly suitable adjuvants are for example liposomes, lipid or detergent micellIes or other lipidic particles, polymer nanoparticles or microparticles, soluble polymers, 5 -protein particles, mineral gels, mineral micro- or nanoparticles, polymer/aluminum nanohybrids, oil in water or water in oil emulsions, saponin extracts, bacterial cell wall extracts, stimulators of innate immunity receptors, in particular natural or synthetic TLR agonists, natural or synthetic NOD agonists and natural or synthetic RIG agonists. A suitable adjuvant for the process according to the present invention for JO example is that disclosed in WO 20071006939. The influenza virus strains are for example H5NI, H9N2, H7N7, H2N2, H7N1 and HI N1 (Emergence of multiple genotypes of H5N1 avian influenza viruses in Hong Kong SAR: Y Guan et al, 8950 - 8955, PNAS - June 25, 2002 - vol 99 n" 13; H9N2 Influenza A Viruses from Poultry in Asia have Human Virus-like Receptor Specificity: 15 MN Matrosovich , S Krauss and R Webster, Virology 281, 156 - 162 ( 2001 ) Evolution and Ecology of Influenza A Viruses: R Webster et al., Microbiological ReviewsMar 1992, p. 152 - 179). Alternatively, it could be an influenza strain selected from the group of the seasonal influenza virus strains. The influenza antigen can be in a form selected from the group consisting of purified t0 whole influenza virus, inactivated influenza virus or sub-unit components of influenza virus, or a split influenza virus. The influenza antigen may be derived from cell culture. Alternatively, the influenza antigen is produced in embryonic eggs. The present invention also concerns a stabilized dry vaccine composition, particularly !5 a stabilized dry influenza vaccine composition or stabilized dry vaccine compositions containing other antigens, such as inactivated whole viruses or antigenic components of viruses, Influenza, Rotavirus, cytomegalo virus and Hepatitis A and B, and whole bacteria or bacterial protein or polysaccharide antigens, conjugated or- non conjugated, such as Haemophilus influenzae , meningococcal polysaccharides, 30 tetanus, diphtheria, cellular and acellular pertussis, Botulism, Anthrax, C. Difficile in WO 20091109550 PCT/EP2009/052460 9 the form of dry regular spherical micropellets or particles having a diameter from about 200pm to about 1500 pm obtainable by the process according to the present invention. Advantageously, each regular bead or particle comprises only one type of antigen, 5 for example one or more influenza antigens from only one influenza virus strain or for example only Tetanus or only Diphtheria antigens. Alternatively, each regular bead or particle comprises one or more types of antigens, for example influenza antigens from one or more different influenza virus strains or for example Tetanus and Diphtheria antigens. 10 The composition may further comprise an adjuvant which is optionally contained in separate dry regular spherical micropellets or particles. The present invention further relates to a process for the preparation a vaccine comprising the step of reconstitution of the stabilized dry vaccine composition, for example the before mentioned stabilized dry influenza vaccine composition in the 15 form of dry regular spherical micropellets or particles in an aqueous solution. The aqueous solution may optionally comprise an adjuvant. The present invention further relates to a vaccine kit, comprising a first recipient containing a stabilized dry vaccine composition, for example a stabilized dry influenza vaccine composition, in the form of dry regular spherical micropellets or 0 particles and a second recipient containing an aqueous solution for the reconstitution of the vaccine. The kit may further comprise a recipient containing dry regular spherical micropellets or particles comprising an adjuvant. Alternatively, the aqueous solution comprises an adjuvant. The present invention also relates to a method of stockpiling a stable dry bulk of 5 antigens wherein the antigen or the antigens is/are stabilized by the method described before and the resulting stabilized dry vaccine composition (e.g. an influenza, diphtheria or tetanus vaccine composition) is reconstituted with an adequate solvent and optionally fiormulated prior to liquid filling into vials or syringes after storage in the form of dry regular spherical micropellets or particles having a 0 diameter from about 200pm to about 1500 pm.

WO 20091109550 PCT/EP20091052460 10 The process for thermo-stabilization of dry vaccine in micro-pellet form according to the present invention Is explained in more detail in the following. In order to be processed by the micro-pellet technology, biological substances such as antigens require to be formulated in order to protect it against the physical and 5 chemical stresses undergone throughout the process. Formulated liquid products to be dried are obtained by mixing the concentrated vaccine bulk (containing the antigen) and a stabilizing formulation comprising at least one carbohydrate and/or sugar alcohol, so that the formulated liquid product obtained contains the targeted amounts per ml of stabilizing excipients and antigens. The 10 concentration of carbohydrates, sugar alcohols and stabilizing excipients ranges between 2% (w/v) to limit of solubility in the formulated liquid product. To give an example, the concentration at the limit of solubility of sucrose in water at 20*C is at about 66,7% (w/v). Figure 1 shows a flow chart of the general formulation and drying procedure. 15 The process as shown in Figure 2 is used to generate the dry micropellets. Drilling, also known as laminar jet break-up technique, is a well known technique to generate calibrated droplets of liquid commonly used in the field of biocatalysts and living cells immobilization (Hulst et al., 1985. A new technique for the production of immobilized biocatalyst and large quantities. Biotechnol. Bioeng. 27, 870-876; Gotoh !0 et al., 1993. Mass production of biocatalyst-entrapping alginate gel particles by a forced oscillation method. Chem. Eng. Commun. 120, 73-84.; Seifert and Philips, 1997. Production of small, monodispersed alginate beads for cell immobilization. Biotechnol. Prog. 13, 562-568). Lord Rayleigh was the first to analyze instability of capillary jets coming out of a nozzle and to propose a model to describe it (Rayleigh !5 L, 1978. On the stability of jets. Proc. London Math. Soc. 10, 4-13) for Newtonian fluids. Weber (Weber C, 1931. Zurn Zerfall eines Flssigkeitsstrahles. Z. Angew. Math. Mech. 11, 136-154) extended the analysis including the effect of the viscosity. The optimal wavelength for the fastest growing disturbance and jet beak-up is given WO 20091109550 PCTEP2009/052460 by: V 3 Where X is the optimal wave-length for jet break-up, d is the diameter of the jet, i1 is the viscosity of the fluid, p is the density of the fluid and a is the surface tension of 5 the fluid. The diameter d of the droplets formed can be calculated by: d= 4/L 5-djO4 t The frequency f to apply to the fluid to achieve the desired results is related to the jet velocity (and therefore the flow rate of the fluid) u and the wavelength by: 0 Therefore; optimal conditions can be calculated knowing process parameters and fluid characteristics. However, a range of frequencies and jet velocities exist to form uniform droplets depending on the nozzle diameter, rheology of the fluid and surface tension (Meesters G., 1992. Mechanisms of droplet formation. Delft University Press, Delft, NL). Suitable working frequencies can be also be determined experimentally by 5 visual assessment of the stability of the droplet formation. Standard prilling equipment are equipped with light stroboscope to observe the droplet formation: for a given product and given working conditions, one can adjust manually the frequency until observing a stable and still droplets chain with this stroboscope light; Moreover, multinozzle systems have been developed for aseptic prilling applications 0 (Brandenberger H. et al.1998. A new multinozzle encapsulation/immobilization system to produce uniform beads of alginates. J. Biotechnol. 63, 73-80) In conclusion, working frequencies can be determined both theoretically and experimentally depending on the available tools and knowledge of the product.

WO 20091109550 PCT/EP20091052460 12 The prilling process is adapted to viscous liquids and saturated sugar solutions. The maximum acceptable viscosity according to current suppliers is approximately 300 mPa.s. Therefore, formulation content can range from very low concentration up to the limit of solubility of the selected excipients at room or processing temperature. 5 Moreover, the temperatures in the process tubing and in the nozzle have to be controlled in order to avoid sugar or solvent crystallization before droplet formation. The person skilled in the art of formulation will adjust different excipient concentrations in the product in order to avoid non-controlled crystallization and viscosities above the given limit, taking into account eventual interactions between 10 excipients. The formulated liquid product is prilled in order to generate calibrated droplets the diameter of which ranges from 200pm to 1500 pm, with a very narrow size distribution. These droplets fall in a cryogenic chamber in which temperature is maintained below -11 0*C either by direct injection/nebulization of liquid nitrogen or by 15 flowing counter currently a very cold (T*<-1 10"C) gas such as nitrogen, C02 or air. The droplets freeze during their fall in order to form calibrated frozen particle. The minimum failing height to freeze the droplets (i.e. ice crystals formation that solidifies the pellets) can be minimized by limiting super-cooling. Super-cooling can be reduced by seeding ice nucleation in the droplets either by contact with liquid 20 nitrogen fog or droplets (direct injection of liquid nitrogen in the chamber) or with microscopic ice crystals (direct nebulization of water or superheated steam in the cold chamber). These frozen particles are then collected and transferred on pre-cooled trays at -50 0 C and loading on the pre-cooled shelves of the freeze-drier (-50*C) in order to !5 always keep the frozen pellets below the glass transition Tg' of their cryo concentrated phase (Tg' value can range from -10 0 C to -45 0 C) and to avoid any melting or aggregation of the particles. Once the freeze-drier is loaded, vacuum is pulled in the freeze-drying chamber to initiate conventional freeze-drying (sublimation of the ice) of the pellets as know by the state of the art. The following freeze-drying 0 parameters are an example of what can be used for a formulation which Tg' ranges from -30 0 C to -450C: WO 2009/109550 PCT/EP2009/052460 13 Primary drying: shelf temperature equal to -35 0 C, pressure equal to 50pbars during I Oh. Secondary drying: shelf temperature equal to 20 0 C, pressure equal to 50pbars during 3h. 5 Freeze-drying cycle has to be designed in order to get residual moistures preferentially lower than 3%. However, the moisture content can be optimized at higher value, on a case by case basis, if the stability of the dry antigen requires it. Other drying technologies such as atmospheric freeze-drying, fluidized bed drying, vacuum rotary drum drying, stirred freeze-drying, vibrated freeze-drying, microwave 10 freeze-drying can be used to dry the pellets. Dry pellets are then collected in bulk to be analyzed and stored. Storage conditions are suitable fbr dry, friable and hygroscopic particles. Dry samples.are rehydrated prior to analysis using a diluent which may contain water for injection, buffers, stabilizing excipients and adjuvants. Dissolution is instantaneous. 15 Bulk dry pellets are then filled into vials using dry powder filling technologies that are currently on the market. Also, in a perspective of stockpiling a stable dry bulk of antigen, the pellets can be reconstituted with adequate solvent and formulated (if needed) prior to liquid filling into vials or into syringes. Thanks to the stability of such pellets, the storage of the 0 stockpile can be performed at +5 0 C or higher temperatures, which is more convenient than for a frozen liquid material (-20*C or lower). Finally, stockpiling dry bulk material is much less space consuming than stockpiling filled dry products in their final container. In the case of adjuvanted vaccine, the stability during storage at +5 0 C or higher and 5 lower (freezing) temperature is often a problem when antigens and adjuvants are in the same liquid state. As an example, the table below describes the impact of a freeze-thawing cycle at -20 0 C on oxyhydroxyl aluminum gel measured by dynamic WO 2009/109550 PCT/EP2009052460 14 light scattering: Particles size (pm) E mean d10 d50 d90 Initial + 5*C 8,9 2,6 4,9 10,3 1 freeze thawing II cycle- 20*C 49,6 15,3 43,7 92,4 The micropellet technology allows overcoming stability problems with 3 different 5 strategies to be selected on a case by case basis: 1. Drying of the antigens alone under micropellet form and dissolution with the adjuvant (aluminum gel, emulsion, etc.) in the diluent. This strategy is suitable when the antigen stability is increased under micropellet form and when the adjuvant alone is highly thermostable 10 -> No interaction during shelf life -> Increased stability of each phase when stored independently Standardization of adsorption behavior by extemporaneous dissolution of the dry antigen with the liquid adjuvant 2. Drying of the antigens with the adjuvant to stabilize both within the same pellet and 15 to stabilize the interaction between them by trapping them in a glassy matrix in Which all molecular motions and chemical reactions are greatly hindered. This solid state allows to maintain throughout storage (even at higher temperature) potency of the antigen, physical and immunological properties of the adjuvant and nature and force of the interaction between the two. )0 3. Drying of the antigens and the separate drying of the adjuvant, followed by blending of the two before the filling or by a sequential filling. In some cases, stability WO 2009/109550 PCT/EP2009/052460 15 of the adjuvant alone can be a problem (chemical stability of emulsions, physical stability of aluminum gels, liposomes and others...) at the liquid state for long term storage at +5"C or higher temperatures. The micropellet technology allows improving stability of the adjuvanted vaccine by generating separate micropellets of antigen and 5 adjuvant. Stabilizing formulation can be optimized independently for each antigens and the adjuvant. micropellets of antigens and adjuvants can be then subsequently filled into the vials. The separated solid state allows to avoid throughout storage (even at higher temperature) interactions between antigen and adjuvant, to maintain potency of the antigen and physical and immunological properties of the adjuvant. In 10 such a configuration, the content of the vial is more stable than any other configurations with either one of the antigens or in the adjuvant in the liquid state or - when antigens and adjuvant are dried within the same pellets. Interactions between antigens and adjuvants are this way standardized as they occur only after rehydration of the dry combination with a selected diluent which may comprise water 15 for injection, buffers and stabilizing excipients. Interactions therefore exist only during the short period of time between rehydration and injection of the vaccine (fast interactions and very short aging time). It is therefore possible to improve the overall stability of the two products (optimization of the formulation for each product and not a compromise for the two together) and the vaccine itself, adjust the titer of one out t0 of the two after the storage, to facilitate the manufacturing process by separation of the two products drying. In order to be processed by the micropellet technology, biological substances such as antigens and adjuvants require to be formulated in order to protect them against the physical and chemical stresses undergone throughout the process. !5 In case of adjuvants, the stabilizing fbrmulation has to maintain their quality during processing (formulation, pelletizing, drying, storage, filling and rehydration) If the strategy is to dry both antigens and adjuvants in the same micro-pellet, formulated liquid product to be dried is obtained by mixing the concentrated vaccine bulk (containing the antigens), the concentrated adjuvant bulk and a stabilizing 0 formulation comprising at least one carbohydrate and or sugar alcohol, so that the formulated liquid product obtained contains the targeted amounts per ml of stabilizing WO 2009/109550 PCT/EP2009/052460 16 excipients, adjuvants and antigens. The concentration of the stabilizing excipients ranges between 2% (w/v) and limit of solubility in the formulated liquid product. Figure 3 is a flow chart showing the formulation and drying procedure comprising an adjuvant. 5 If the strategy is to dry antigens and adjuvants in separate micropellets, the formulated liquid product to be dried (comprising the antigens) is obtained by mixing the concentrate vaccine bulk (comprising the antigens) and a stabilizing formulation, so that the formulated liquid product obtained contains the targeted amounts per ml of stabilizing excipients and antigens. The concentration of the carbohydrates and 10 the stabilizing excipients ranges between 2% (wlv) and limit of solubility in the formulated liquid product. The same way for the adjuvant, the formulated liquid product to be dried is obtained by mixing the concentrated adjuvant bulk and a stabilizing formulation, so that the formulated liquid product obtained contains the targeted amounts per ml of stabilizing 15 excipients and adjuvants. The carbohydrates and stabilizing excipients concentration ranges are between 2% (w/v) and limit of solubility in the formulated liquid product. Figure 4 is a flow chart showing the formulation and drying procedure in order to produce the antigen containing micropellets, Figure 5 is a flow chart showing the formulation and drying procedure in order to produce the adjuvant containing !0 micropellets. Figure 6 shows the combination of the different micropeliets in order to complete the dry vaccine. The general process for the generation of the dry micropellets is shown in Figure 2. It is applicable independently from the chosen strategy for the adjuvanted vaccine. Fast freezing kinetics of the micro-pellet technology is also adapted for drying !5 adjuvants. As an example, the graph in Figure 9 shows size distribution results after drying of an aluminum phosphate gel in presence of a carbohydrate and using the micro-pellet technology. It can be seen that drying and rehydration has no significant negative effect on the size distribution of the adjuvant particles. Suitable adjuvants that may be considered are exemplified in the following: WO 200919550 PCTEP2009/052460 17 1) The particulate adjuvants such as: Jiposomes and in particular cationic liposomes (e.g. DC-Chol, see e.g. US 2006/0165717, DOTAP, DDAB and 1,2-Dialkanoyl-sn glycero-3-ethylphosphocholin (EthyiPC) liposomes, see US 7,344,720), lipid or detergent micelles or other lipid particles (e.g. Iscomatrix from CSL or from fsconova, 5 virosomes and proteocochleates), polymer nanoparticles or microparticles (e.g. PLGA and PLA nano- or microparticles, PCPP particles, Alginate/chitosan particles) or soluble polymers (e.g. PCPP, chitosan), protein particles such as the Neisseria meningitidis proteosomes, mineral gels (standard aluminum adjuvants: AIOOH,

AIPO

4 ), microparticles or nanoparticles (e.g. Cas(P04)2), polymer/aluminum 10 nanohybrids (e.g. PMAA-PEG/AJOOH and PMAA-PEG/AJPO 4 nanoparticles) O/W emulsions (e.g. MF59 from Novartis, AS03 from GlaxoSmithKline Biologicals) and W/O emulsion (e.g. ISA51 and ISA720 from Seppic, or as disclosed in WO 2008/009309). For example, a suitable adjuvant emulsion for the process according to the present invention is that disclosed in WO 2007/006939. 15 2) The -natural extracts such as: the saponin extract QS21 and its semi-synthetic derivatives such as those developed by Avantogen, bacterial cell wall extracts (e.g. micobacterium cell wall skeleton developed by Corixa/GSK and micobaterium cord factor and its synthetic derivative, trehalose dimycholate). 3) The stimulators of innate immunity receptors such as: natural or synthetic TLR W agonists (e.g. synthetic lipopeptides that stimulate TLR2/1 or TLR2/6 heterodimers, double stranded RNA that stimulates TLR3, LPS and its derivative MPL that stiuult 1LR4,E6-2OanaR r-t529iht-stimolate--T-LR4-fageltin-that-stimr ates TLR5, single stranded RNA and 3M's synthetic imidazoquinolines that stimulate TLR7 and/or TLR8, CpG DNA that stimulates TLR9, natural or synthetic NOD !5 agonists (e.g. Muramyl dipeptides), natural or synthetic RIG agonists (e.g. viral nucleic acids and in particular 3' phosphate RNA). These adjuvants may also be used in combination. Preferred combinations are those between the particulate adjuvants and natural extracts and those between particulate adjuvants and stimulators of innate immunity receptors. 0 For the following examples, the prilling apparatus IE-50R Encapsulator Research, from Inotech (CH) and a 300pm nozzle head were used to generate the micropellets.

WO 20091109550 PCT/EP2009/052460 18 Example 1: Manufacturing of thermo-stable dry vaccine under micropellet form containing Flu antigens This study compared the thermo-stability of the current liquid trivalent flu vaccine to dry formulations of this same vaccine processed with the micro pellet technology. 5 Trivalent flu vaccine contains 30 pg/ml of each A/Salomon, B/Malaysia and A/Wisconsin strains in the vaccinal buffer. Formulated liquid products to be dried were obtained by mixing one volume of trivalent flu vaccine with one volume of a stabilizing formulation comprising at least one carbohydrate which concentration ranges between 4% (wlv) and limit of solubility. This corresponds to a concentration 10 range from 2% to 32% (weight by volume) in the formulated liquid product to be dried. Two formulations were evaluated: SGI and SG2 which compositions are given in tables I and 2: Table 1 SG1 COMPONENTS quantity for 1000 Mi Sucrose 200 G Adjustment pH @ 7,0+/- 0,2 (NaOH. HCI) Water PPI 1000 MI 15 Table 2 SG2 COMPONENTS quantity for 1000 ml Sucrose 200 g Glutamine 2 g Urea 10 9 Dextrane 70 10 9 Adjustment pH @ 7,0+/- 0,2 (NaOH, HCl) Water PPI 1000 ml WO 2009/109550 PCTFEP2009/052460 19 Figure 7 shows a flowchart of the formulation and drying procedure. Figure 2 shows the process that was used to generate the dry micropellets of formulations SG1 and SG2. 5 Formulated liquid product SG1 and SG2 were prilled in order to generate calibrated droplets. Prilling parameters for this formulation and a 300pm nozzle head were: - Product flow rate: 8ml/min - nozzle frequency: 954 Hz These droplets fell in a cryogenic chamber in which the temperature was maintained [0 below -110*C by direct injection of liquid nitrogen or by flowing countercurrent very cold gas (t*<-110*C). The droplets froze during their fall and formed calibrated frozen particles. These frozen particles were then transferred on pm-cooled trays at -50 0 C and loaded on the pre-coded shelves of the freeze-drier (-50 0 C) in order to always keep the 5 frozen pellets below their glass transition (which was evaluated between -30 0 C and 40*C) and to avoid any melting or aggregation of the particles. Once the freeze-dryer was loaded, vacuum was pulled in the freeze-drying chamber to initiate conventional freeze-drying of the pellets as know by the state of the art. For these formulations, the following freeze-drying parameters were used: Primary drying: shelf temperature 0 equal to -35 0 C, pressure equal to 50pbars during 10h. Secondary drying: shelf temperature equal to 20 0 C, pressure equal to 50pbars during 3h. Residual moisture of the micropellets was below 2% for both SG1 and SG2 formulations. Figure 8 gives an idea of the very narrow size distribution obtained with this pelletizing and drying technology. 5 Therefore, 3 products were available for a thermo-stability study: The marketed trivalent flu vaccine (VAXIGRIP*, Sanofi Pasteur), the dry micropellets SG1 formulation (trivalent flu vaccine) and the dry micropellets SG2 formulation (trivalent flu vaccine).

WO.2009/109550 PCT/EP2009052460 20 Samples of each of these 3 formulations were exposed to different time at 37'C and 45"C. Potency (pg of antigen/mi) was then measured for each sample by SRD (SRID) method (M. S. Williams, Veterinary Microbiology, 37 (1993) 253-262). Dry samples were re-hydrated using water for injection prior to analysis. Dissolution was 5 instantaneous. The tables 3, 4 and 5 summarize the obtained results. Results are expressed in mean value pglml of antigen. Table 3 A/Salomon Stability at 37C (days) 0 7 30 90 Liquid formulation 29,8 24,3 12.1 micropellets SGI 23,2 22,5 24,9 27 micropellets SG2 26,5 24,7 28,2 29.4 Stability at 45

"

C (days) 0 7 Liquid formulation 29,8 16,4 micropellets SG1 23,2 24,3 micropellets SG2 26,5 25,8 Table 4 B/Malaysia Stability at 37*C (days) 0 7 30 90 Liquid formulaion 30 21,4 14.4 micropellets SG1 25,4 25,7 24,9 28.2 micropellets SG2 26,6 25,4 27,2 30.4 Stailitat 45"Cays 0 7. Liquid formulation 30 16,3 micropellets SG1 25,4 24 micropellets SG2 26,6 26,71 WO 2009/109550 PCT/EP2009/052460 21 Table 5 A/Wisconsin Stability at 37*C (days) 0 7 30 90 Liquid formulation 30 24,7 13.2 micropellets SG1 25,4 24,6 25,5 26.8 micropellets SG2 28,7 26 28,4 30.7 _Stability at45"OC dags .0 7 Liquid formulation 30 16 micropellets SG1 25,4 25 micropellets SG2 28,7 27,8 These results show that the dried formulations SG1 and SG2 in the micro-pellet form are much more stable than the current liquid formulation. No significant antigenicity 5 loss was measured after up to 3 months at 370C and I week at 45*C. Example 2: Manufacturing of thermo-stable dry vaccine under micropellet form containing Flu H5N1 (Indonesia) antigens adjuvanted with Aluminum oxyhydroxide gel This study compared the thermo-stability of the liquid H5N1 Indonesia flu vaccine to .0 dry formulations of this same vaccine processed with the micropellet technology according to the present invention. The vaccine contains 65.4 pg/mI of H5N1 indonesia strain in the vaccinal buffer, adjuvanted by aluminum oxyhydroxide gel, Formulated liquid product to be dried was obtained by mixing one volume of H5N1 5 vaccine with one volume of the stabilizing formulation SGI. (cf. Example I for SGI composition) Figure 10 shows a flowchart of the formulation and drying procedure.

WO 2009/109550 PCT/EP2009052460 22 Formulated liquid product SG1 was prilled In order to generate calibrated droplets. Prilling parameters for this formulation and a 300pm nozzle head were: - Product flow rate: 8mi/min - nozzle frequency: 916 Hz 5 These droplets fell in a cryogenic chamber in which temperature was maintained below -11 0C by direct injection of liquid nitrogen or by flowing countercurrerit very cold gas (t <-1 10 C). The droplets froze during their fall and formed calibrated frozen particles. These frozen particles were then transferred on pre-cooled trays at -50"C and 10 loading on the pre-cooled shelves of the freeze-drier (-50 0 C) in order to always keep the frozen pellets below their glass transition (which was evaluated between -30*C and -40*C) and to avoid any melting or aggregation of the particles. Once the freeze drier was loaded, vacuum was pulled in the freeze-drying chamber to initiate conventional freeze-drying of the pellets as know by the state of the art. For these 15 formulations, the following freeze-drying parameters were used: Primary drying: shelf temperature equal to -35 0 C, pressure equal to 50pbars during 10h. Secondary drying: shelf temperature equal to 20*C, pressure equal to 50pbars during 3h. Residual moisture of the micropellets was below 2%. Micropellets samples were exposed 7 days at 37 0 C and 450C. Potency (pg of 0 antigen/ml) was then measured for each sample by SRD method. Dry samples were rehydrated using water for injection prior to analysis. Dissolution was instantaneous. The-table 6 summarizes the obtained results. Results are expressed in mean value pg/ml of antigen; N.D. stands for "Non Detectable Antigenicity". Table 6 SRD Titers pg/ml Liquid formulation Micropellet SGI TO 65,4 53,6 7d@37*C 52,5 61,4 745*C <7,0 47,9 7d55C N.D. 44,9 !5 WO 2009/109550 PCTfxP2009/052460 23 These results show that the dried formulation SGI in the micro-pellet form is much more stable than the current liquid formulation. No significant antigenicity loss was measured after I week at 37*C and at 45 0 C. A loss of approximately 15% was observed after 1 week at 55'C, which is close to the precision of the assay itself. 5 The impact of this drying process and the rehydration of the pellets on the adjuvant properties were evaluated by measuring the aluminum oxyhydroxide particle size distribution In the formulated bulk before drying, and after dissolution of the pellets using a particle size analyzer Malvern MasterSizer 2000. The results are given figure 11. We observed that this process did not induced aggregation of the gel. 10 Figure 12 also shows that the gel's size is maintained after thermostability incubation for at least 7 days at 37*C, 45*C and 55*C, and demonstrates the stability of alum gel adjuvant in a micropellet form. This example confirms the applicability of this technology to alum gel adjuvanted Flu antigens, and most generally, the feasibility to dry adjuvanted antigens with alum gels 5 using the micropeilet technology. Example 3: Manufacturing of thermo-stable dry vaccine under micropellet form containing non-adjuvanted Flu H5NI (Indonesia) and rehydration with an adjuvant emulsion 0 This study compared the thermo-stability of the liquid H5N1 Indonesia flu vaccine to dry formulations of this same vaccine processed with the micropeliet technology. The vaccine contains 176 pg/mI of H5N1 Indonesia strain in the vaccinal buffer. Formulated liquid product to be dried was obtained by mixing the H5N1 vaccine with the stabilizing formulation SG1, in order to target the desired antigen concentration 5 and stabilizer contents. The formulation SG1 was evaluated (cf. Example 1 for SG1 composition) Figure 13 shows a flowchart of the formulation and drying procedure.

WO 20091109550 PCT/EP2009/052460 24 Formulated liquid product was prilled in. order to generate calibrated droplets. Prilling parameters for this formulation and a 300pm nozzle head were: - Product flow rate: 8ml/min - nozzle frequency: 997 Hz 5 These droplets fell in a cryogenic chamber in which the temperature was maintained below -11 0"C by direct injection of liquid nitrogen or by flowing countercurrent very cold gas (t*<-1 I 0"C). The droplets froze during their fall and formed calibrated frozen particles. These frozen particles were then transferred on pre-cooled trays at -50 0 C and 10 loading on the pre-cooled shelves of the freeze-drier (-50*C) in order to always keep the frozen pellets below their glass transition (which was evaluated between -30*C and -40"C) and to avid any melting or aggregation of the particles. Once the freeze drier was loaded, vacuum was pulled in the freeze-drying chamber to initiate conventional freeze-drying of the pellets as know by the state of the art. For these 15 formulations, the following freeze-drying parameters were used: Primary drying: shelf temperature equal to -35*C, pressure equal to 50pbars during .10h. Secondary drying: shelf temperature equal to 20*C, pressure equal to 50pbars during 3h. Residual moisture of the micropellets was below 2%. In parallel, the same formulated product was generated, filled into vials and freeze 0. dried normally in a standard freeze-dryer. Filled vials were loaded on pre-cooled shelves at 5 0 C; the product was then frozen down to -50*C at 1"C/min and vacuum was pulled to initiate sublimation. Primary drying parameters were: shelf temperature equal to -18 0 C, pressure equal to 50pbars during 16h. Secondary drying parameters were: shelf temperature equal to 37 0 C, pressure equal to 50pbars during 2h. !5 Residual moisture of the freeze-dried product was also below 2%. Micropellet and liquid samples were exposed to different time at 37 0 C, 45 0 C and 55*C. Freeze-dried samples were exposed at 37 0 C and 55*C. Potency (pg of antigen/ml) was then measured for each sample by SRD method. Dry samples were rehydrated using water for injection (WFI) prior to analysis. Dissolution was WO 2009/109550 PCT/EP2009/052460 25 instantaneous. The tables 7, 8 and 9 summarize the obtained results respectively for a standard liquid formulation, dried micropeliets and the freeze-dried product. Results are expressed in mean value pg/ml of antigen. Initial SRD Titer at To corresponds to the measured titer after reconstitution of the micropellets after processing. 5 Table 7 InItial SRD Titer: To=14, lpg/mi Liquid H5N41 Stability study Time SRD Titers pginl days I month 3 months Thermostabilty at 37*C 11,6 5 <5 Thermostability at 45*C 3,4 <5 <5 Thermostability at 55"C 5<5 <5 _ Table 8 -nitial SRD Titer: To= 47,2 pg/lml Dry micropellets HSN1 Time Stability study SRD Triters pg/m, Rehydration WFI 7days I month 3 months Thermostability at 37*C 53 - 47,3 50,1 Thermostability at 45*C 51,6 47 41,1 Thermostability at 55 0 C 49,5 45,4 47,3 Table 9 Initial SRD Titer: To= 39.4 pg/ml Freeze-dried H5N1 Stability Time study SRD Titers pg/ml, Rehydration WFI 14days I month Thermostability at 37 0 C 36.9 38.1 Thermostability at 55"C 35.6 35,1 0 These results show that the dried formulation SGI in the micro-pellet form is much more stable than the current liquid formulation. No significant antigenicity loss was WO 2009/109550 PCT/EP2009/052460 26 measured after 3 months at 37*C, 45*C and 55*C. Results given table 9 confirm that standard freeze-drying also provides good thermostabilty. Moreover, the data given table 10 show that no antigenicity loss was measured after 9 months at +5*C. These results are very promising for long term stability at +5 0 C 5 and room temperature over a several year time period. The feasibility of rehydrating the H5NI micropellets with an emulsion has been studied. The emulsion used for this study is the one described in the patent application WO 2007/006939. The same experimental plan as table 8 was performed but samples were rehydrated with the emulsion rather than water for injection. The J0 results are given table 10 and table 11: Table 10 H5N1 micropellet H5N1 micropellet formulation formulation (pg/in# To+9months at (pg/mlja t To +" After Drying. Rehydration WFI 47,2 54,8 After Drying. Rehydration Emulsion 49,2 61 Table 11 Initial SRD Titer: To= 49,2 pg/ml Dry H5NI Stability Time study SRD Titers pg/ml, Rehydration Emulsion days I month 3 months Thermostability at 465 42,6 37 0 C 46,6 42,6 47,8 Thermostability at 4361 46,1 45*C . 46,5 Thermostability at 41,8 42,7 55 0 C 41,7 WO 2009/109550 PCT/EP2009/052460 27 Table'10 proves that the dissolution of the micropellets with an emulsion as adjuvant does not impact the stability of the antigen and therefore its recovery. Table 11 confirms that this statement is also verified after thermostability incubation of the micropellets as all measured titers are comparable with titers measured after 5 dissolution with water for injection. Figure 14 shows a comparison of the size of the emulsion before rehydration of these micropellets and after dissolution. The superposition of the two size distributions confirms that the size of the emulsion, and therefore its integrity, is not significantly altered by the rehydration of a freeze-dried matrix. The size distribution remains 10 monomodal and centered on 1 00nm. Moreover, Figure 15 confirms the stability of the size of the emulsion after dissolution of the micropellets and over a one hour storage period at room temperature. This study confirms that, thanks to the micropellet technology, it is possible to use the adjuvant as a dissolution buffer and to avoid all interaction between the adjuvant and 15 the antigen during the shelf life of the product. Example 4a: Manufacturing of a thermo-stable dry vaccine under micropellet form containing non-adjuvanted Flu H5N1 (Indonesia) with different sugars used as stabilizers :0 This study shows the preparation of 3 stabilized dry influenza vaccine compositions each comprising a different disaccharide (trehalose, lactose and maltose respectively) and the thermo-stability of such dry compositions processed with the micropellet technology. The vaccine contains 131 pg/mi of H5N1 Indonesia strain in the vaccinal buffer. 5 The formulated liquid products to be dried were obtained by mixing the H5N1 vaccine with the stabilizing formulations SG5, SG6 and SG7, respectively (see tables 12a, 12b and 12c), in order to target the desired antigen concentrations and stabilizer contents.

WO 2009/109550 PCT/EP20091052460 28 Table. 12a SG5 COMPONENTS quantity for 1000 ml Trehalose 200 g Adjustment pH @ 7,0+/- 0,2 (NaOH, HCI) Water PPI 1000 ml Table 12b SG6 COMPONENTS quantity for 1000 ml Lactose 200 Adjustment pH @ 7,0+/- 0,2 (NaOH, HCI) Water PPI 1000 ml 5 Table 12c SG7 COMPONENTS quantity for 1000 ml Maltose 200 g Adjustment pH @ 70+/- 0.2 (NaOH, HCI) Water PPI 1000 ml Figure 13 shows a flowchart of the principle of the formulation and drying procedure with SG1 stabilizer as an example. Identical flowchart was used with SG5, SG6 and SG7 to obtain a disaccharide concentration of 15,4%(w/v) in the formulated product 10 prior to prilling.

WO 2009/109550 PCT/EP2009 /052460 29 Each of the 3 formulated liquid products was prilled in order to generate calibrated droplets. Prlliing parameters for these formulation and a 300pm nozzle head were: - Product flow rate: 8ml/min - nozzle frequency: ranging from 994Hz and 1001 Hz depending on the formulation 5 These droplets fell in a cryogenic chamber in which the temperature was maintained below -110*C by direct injection of liquid nitrogen or by flowing countercurrent very cold gas (t*<-1 I 0C). The droplets froze during their fall and formed calibrated frozen particles. These frozen particles were then transferred on pre-cooled trays at -50*C and 10 loading on the pre-cooled shelves of the freeze-drier (-50*G) in order to always keep the frozen pellets below their glass transition (which was evaluated between -30*C and -40*C) and to avoid any melting or aggregation of the particles. Once the freeze drier was loaded, vacuum was pulled in the freeze-drying chamber to initiate conventional freeze-drying of the pellets as know by the state of the art. For these 15 formulations, the following freeze-drying parameters were used: Primary drying: shelf temperature equal to -35"C, pressure equal to 50pbars during 10h. Secondary drying: shelf temperature equal to 200C, pressure equal to 50pbars during 3h. Residual moisture of the micropellets was below 2%. Micropellets samples were exposed to different time at 37 0 C and 550C. Potency (pg !0 of antigen/ml) was then measured for each sample by SRD method. Dry samples were rehydrated using water for injection (WFI) prior to analysis. Dissolution was instantaneous. The tables 12d, 12e and 12f summarize the obtained thermostability results respectively for each of the 3 compositions in the form of dried micropellets. Results are expressed in mean value pg/ml of antigen. Initial SRD Titer at To !5 corresponds to the measured titer after reconstitution of the micropellets after processing.

WO 2009/109550 PCT/EP2009/052460 30 Table 12d Initial SRD Titer: To= 61,5 pg/mi Dry micropeflets Time H5N1+SG5 Stability study SRD Titers pg/ml, Rehydration WFl 14 days I month Thermostability at 57,7 58,7 370C Thermostability at 57,2 58,9 550C5725, Table 12e Initial SRD Titer: To= 60,2 pg/ml Dry micropellets - Time H5NI+SG6 Stability study SRD Titers pg/mi, Rehydration WFi 14 days I month Thermostablity at 53,0 50,5 37C Thermostability at 52,4 53,1 550C 5 Table 12f initial SRD Titer: To= 52,9 pg/mi Dry micropellets H5NI+SG7 Stability Time study SRD Titers pgfml, Rehydration WFI 14 days 1 month Thermostability at 52,8 49,6 370C Thermostability at 50,4 51,2 550C 5 These results confirm a similar stability profile for a wide range of saccharide excipients used as stabilizer.

WO 20091109550 PCTEP2009/052460 31 Example 4b: Manufacturing of a thermo-stable dry vaccine under micropellet form containing non-adjuvanted Flu H5N1 (Indonesia) with 2% (w/v) sucrose in the formulated bulk ready to be processed. The vaccine contains 176 pg/ml of H5N1 Indonesia strain in the vaccinal buffer. 5 The formulated liquid product to be dried was obtained by mixing the H5N1 vaccine with the stabilizing formulation SG8, in order to target the desired antigen concentration and a stabilizer content of 2 % (w/v), Table 12g shows the composition of the stabilizing formulation SG8. Table 12g SG8 COMPONENTS quantity for 1000 mI Sucrose 1 26 g Adjustment pH @ 7,0+1- 0,2 (NaOH, HCI) Water PPI 1000 m1 0 The micropellets made from the formulated liquid bulk containing 2% w/v sucrose (liquid bulk formulated with SG8) were obtained as described in example 4a. Figure 16 shows a flowchart of the formulation and drying procedure. The micropellet samples were exposed to different time at 370C and 55 0 C._Potency 5 (pg of antigen/ml) was then measured for each sample by SRD method. Dry samples - were rehydrated using water for injection (WFI) prior to analysis. Dissolution was instantaneous. Table 12h shows the obtained results for the dried micropellets. The results are expressed in mean value pg/mi of antigen. Initial SRD Titer at To corresponds to the measured titer after reconstitution of the micropellets right after 0 processing.

WO 2009/109550 PCTEP2009/052460 32 Table 12h Initial SRD Titer: To= 39,6 pg/m Dry micropellets rime H5N1+SG8 Stablifty study SRD Titers pg/ml, Rehydration WFI 14days 1 month Thermostablility at 37*C 32,9 33,6 Thermostability at 55*C. 34,71 34,0 -Example 5: Study of the impact of micropellet processing on adjuvanted Diphtheria 5 toxoid (Dt) and Tetanus toxoid (Tt) vaccines The first part of this study evaluated the stability of TetanusTt and Dt antigens freeze dried in a micropellet form either with or without pre-adsorption on aluminum gel ALOOH. Five formulations were prepared as described below. Formulated liquid products to be dried containing DiphtheriaDt or TetanusTt in 10 presence of aluminum gel were obtained by mixing a given volume of Diphtheria or Tetanus toxoid concentrated bulk with aluminum gel and a stabilizer in order to obtain the following composition: - Dt (Diphteria toxoid) formulated product: 200 LfIml of antigen and 0.8mg/ml of aluminum gel 15 - Tt (Tetanus toxoid) formulated product: 40 Lf/ml of antigen and 0.8mg/ml of aluminum gel Figure 17 shows a flowchart of the formulation and drying procedure of these two formulations Formulated liquid products to be dried containing DiphtheriaDt or TetanusTt without !0 aluminum gel were obtained by mixing a given volume of Diphtheria or Tetanus WO 2009/109550 PCTZP20091052460 33 toxoid concentrated bulk with a stabilizer in order to obtain the following composition: - Dt (Diphteria toxoid) formulated product: 500 Lf/ml of antigen - Tt (Tetanus toxoid) formulated product: 100 Lf/ml of antigen Figure 18 shows a flowchart of the formulation and drying procedure of these two 5 formulations. Antigen contents for Diphtheria toxoid and Tetanus toxoid were measured using the rocket immuno-electrophoresis test, as performed by the state of the art. Formulated liquid product to be dried containing only aluminum gel is obtained by mixing a given volume aluminum gel with a stabilizer in order to obtain the following 0 composition: - Aluminum gel ALOOH formulated product: 2.4mg/ml Figure 19 shows a flowchart of the formulation and drying procedure of this formulation. The composition of the stabilizer used for this experiment, called SDT-1, is given in i the table below: Table 13 SDT-1 Quantity for I000ml Sucrose 100 G Trehalose 100 G Tris 50mM 6,055 G pH adjustment 7.0+1- 0.2 (NaOH. HCI) Water for Injection 1000 mL WO 2009/109550 PCT/EP2009/052460 34 Formulated liquid products were prilled in order to generate calibrated droplets. Prilling*parameters for these formulations and a 300pm nozzle head are summarized in the table below: Table 14 Diphtheria Tetanus Diphtheria Tetanus toxoid toxoid taxoid toxoid +AI gel + Al gel Al gel Flow rate 8mllmin 8mImin 8m|/min 8mVmin 8mil/min Frequency - (Hz) 1487 1543 1223 1282 1479 5 These droplets fell in a cryogenic chamber in which temperature was maintained below -110*C by direct injection of liquid nitrogen or by flowing countercurrent very cold gas (t<1 10C). The droplets froze during their fall and formed calibrated frozen particles. 10 These frozen particles were then transferred on pre-cooled trays at -50"C and loading on the pre-cooled shelves of the freeze-drier (-50 0 C) in order to always keep the frozen pellets below their glass transition (which was evaluated between -30 0 C and -40*C) and to avoid any melting or aggregation of the particles. Once the freeze drier was loaded, vacuum was pulled in the freeze-drying chamber to initiate 15 conventional freeze-drying of the pellets as know by the state of the art. For these formulations, the following freeze-drying parameters were used: Primary drying: shelf temperature equal to -35"C, pressure equal to 50pbars during 10h. Secondary drying: shelf temperature equal to 20 0 C, pressure equal to 50pbars during 3h. Residual moisture of the micropelets was below 2%. !O Micropellets samples were exposed at 37*C and 55*C. Potency (Lf/ml) was Measured for each sample by rocket immuno-electrophoresis test. Dry samples were re-hydrated using water for injection prior to analysis. Dissolution was instantaneous.

WO 2009/109550 PCT/EP2009/052460 35 Diphtheria toxoid stability results: Table 15 Diphtheria Toxoid Time (days Target=10L f/mi 0 14 30 90 Stability at 37*C(Lf/ml) 98,5 100,3 100 86,7 Stability at 55*C(Lf/mi) 98,5 102,2 104 87,6 These results confirm no significant loss after up to 1 month at 37*C and 55 0 C and 5 only about 13% loss after 3 months, which is at the limit of significance. Moreover, incubation of micropellets containing Diphtheria toxoid and alum gel showed that at all time points and all tested temperatures, 100% of the antigen remained adsorbed to the gel after dissolution. The observed stability of the gel size distribution after dissolution and thermo-stability study showed similar results as for flu H5N1 10 adjuvanted with alum gel (see example 2). Tetanus toxoid stability results: Table 16 Tetanus Taxoid Time ys) Target=20Lf/mi 0 14 30 90 Stability at 37"C(Lf/mI) 18,1 21,8 20,1 17,1 Stabillt at 55 0 C(Lf/ml 18,1 21,8 20,9 18 These results confirm no significant loss after up to 3 months at 37*C and 55*C. 5 Moreover, incubation of micropeliets containing Tetanus toxoid and alum gel showed that at all time points and all tested temperatures, 100% of the antigen remained adsorbed to the gel after dissolution. The observed stability of the gel size distribution after dissolution and .thermo-stabiity study showed similar results as for flu H5N1 adjuvanted with alum gel (see example 2). D These data confirm that the micropeliet process allows obtaining thermostable dry adjuvanted or non-adjuvanted Diphtheria and Tetanus toxoid vaccines, when properly formulated, in terms of potency, adsorption state and adjuvant quality, up to WO.2009/1f9550 PCTEP2009/052460 36 3 months at 55*C without significant degradation. Moreover, aluminum gel could successfully be freeze-dried using the micropellet technology maintaining a comparable size distribution and avoiding massive aggregation. Example 6: Study of the impact of micropellet processing on aluminum gel 5 characteristics: interactions with T (Tetanus Toxoid) in this example, micropellets generated in example 5 were used. The goal of this study was to evaluate the impact of the micropellet processing on aluminum gel AFOOH adsorption capacity. In 3ml vials, a constant quantity of aluminum gel AJOOH of 0.3mg, initially in a liquid 10 or micropellet form, was mixed with different quantity of Tetanus toxoid (0.05, 0.1, 0.2, 0.3, 0.4, 0.5 et 0.75 mg total), initially in a liquid or micropellet form as well. 5 experiments were performed: -Series 1: liquid mix of liquid aluminum gel AIOOH and bulk Tetanus toxoid antigen, in the absence of stabilizer 15 - Series 2: liquid mix of dissolved Aluminum gel AIOOH micropellets and bulk Tetanus toxoid antigen -Series 3: liquid mix of liquid aluminum gel AIOOH , bulk Tetanus toxoid antigen and stabilizer -Series 4: liquid mix of liquid aluminum gel AIOOH and dissolved Tetanus !0 toxoid micropellets - Series 5: Liquid mix of dissolved aluminum gel AIOOH micropellets and dissolved Tetanus toxoid micropellets Water and stabilizer contents were adjusted before dissolution of the micropellets in order to obtain a rehydrated solution strictly identical in all stabilizer containing !5 formulations (Series 2 to 5). After mixing the liquid products, the vials were incubated at room temperature in a wheel agitator during 2 hours and then centrifuged at 3000 rpm during 5 minutes.

WO 2009/109550 PCTIEP2009/052460 37 The non adsorbed Tetanus toxoid was quantified in the supernatant by Micro Bradford technique (Bio Rad protein assay). A Tetanus toxoid reference was tested for each series of samples in order to have a quantitative assay. The obtained results are summarized in the table below: 5 Table 17 Presence Tetanus toxoid of adsorption capacity mg toxoid/mg gel stabilizer mg toxoid/ mg gel m Series 1 No 0,49 0,61 ± 0,12 0,72 0,67 0,60 ± 0,07 Series 2 yes 54 0,82 Series 3 yes 0,54 0,76 ± 0,22 0,92 Series 4 yes 0,7 0,64 0,08 ____ ____ ___ ____ ___0,72 0,59 Series 5 yes 1,06 0,86 ± 0,26 '0,91 These results confirm that the presence of a stabilizer does not have any significant negative impact on the adsorption capacity of the gel. Moreover, micropellet 10 processing does not impact significantly, taking into account the variability of the method, adsorption capacity of alum gel when applied on Tetanus toxoid and/or alum gel.

C:\NRPoctbIWCC\AARW320953_LDDC-1 8f)2/20122 -37a Throughout this specification, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or method step or group of elements or integers or method steps but not the exclusion of any element or integer or method step or group of elements or integers or method steps. Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country.

HMiniiierwov eI\NRPonbhDCC\AAR15057799 IAQc-W04/2013 - 37b 1. A process for stabilizing an influenza vaccine composition comprising the steps of diluting a liquid bulk composition comprising an influenza antigen or an antigenic preparation from a seasonal, pre-pandemic or pandemic influenza virus strain by an aqueous solution comprising a carbohydrate and/or a sugar alcohol or a mixture thereof in order to obtain a 2% (w/v) to limit of solubility of carbohydrate and/or sugar alcohol content of the resulting diluted composition, subjecting the diluted composition to a process in order to form regular droplets having a diameter of approximately from about 200 jim to about 1500 jim, subjecting the regular droplets to freezing in a freezing medium to form frozen regular spherical micropellets or particles and drying the frozen regular spherical micropellets or particles to form dry regular spherical micropellets or particles having a diameter from about 200 jim to about 1500 pim. 2. The process as in 1, characterized in that the drying occurs by the method of lyophilization. 3. The process as in 1, characterized in that the drying occurs by the method selected from the group consisting of atmospheric freeze-drying, fluidized bed drying, vacuum rotary drum drying, stirred freeze-drying, vibrated freeze-drying, microwave freeze-drying. 4. The process as in any of 1 to 3, characterized in that the carbohydrate is a disaccharide. 5. The process as in 4, characterized in that the carbohydrate is sucrose. 6. The process as in any of 1 to 5, wherein the liquid bulk composition further comprises one or more additional influenza antigens or antigenic preparations, each from a different seasonal, pre-pandemic or pandemic influenza virus strain, in order to obtain dry regular spherical micropellets or particles, each comprising two or more influenza antigens or antigenic preparations from two or more different seasonal, pre-pandemic or pandemic influenza virus strains. 7. The process as in 6, further comprising dosing and filling the dry regular spherical micropellets or particles into a vial or other appropriate recipient.

H:aar\]nenvovenINRPortbhDCC\AAR5057799_ doc-9/04/2013 -37c 8. The process as in any of I to 5, wherein the liquid bulk composition comprises one or more influenza antigens or antigenic preparations from one seasonal, pre-pandemic or pandemic influenza virus strain, in order to obtain dry regular spherical micropellets or particles, each comprising influenza antigens or antigenic preparations from the same seasonal, pre-pandemic or pandemic influenza virus strain. 9. The process as in 8, further comprising dosing, blending and filling into a vial or other appropriate recipient two or more types of dry regular spherical micropellets or particles, characterized in that each type of micropellets comprises influenza antigens or antigenic preparations from a different seasonal, pre-pandemic or pandemic influenza virus strain than the other type. 10. The process as in 1 to 9, further comprising the addition of an aqueous solution comprising one or more adjuvants and, optionally, a stabilizer to the liquid bulk antigen composition. 11. The process as in any of 1 to 9, further comprising diluting a separate liquid bulk solution or emulsion of one or more adjuvants by an aqueous solution comprising a stabilizer, subjecting the diluted adjuvant solution or emulsion to a process in order to form regular droplets having a diameter of approximately from about 200gm to about 1500 pm, subjecting the regular droplets to freezing in a freezing medium to form frozen regular spherical micropellets or particles, drying the frozen regular spherical micropellets or particles to form dry regular spherical micropellets or particles having a diameter from about 200gm to about 1500 pim and blending and filling into a vial or other appropriate recipient together with the antigen containing dry regular spherical micropellets, 12. The process as in 10 or 11, wherein the adjuvant is selected from the group consisting of liposomes, lipid or detergent miceilles or other lipidic particles, polymer nanoparticles or microparticles, soluble polymers, protein particles, mineral gels, mineral micro- or nanoparticles, polymer/aluminum nanohybrids, oil in water or water in oil emulsions, saponin extracts, bacterial cell wall extracts, stimulators of innate immunity receptors, in particular natural or synthetic TLR agonists, natural or synthetic NOD agonists and natural or synthetic RIG agonists.

R:\arnhtenvoven\NRPoatbI\DCC\AAR\5O57799_ Idec-9/04/2*I3 -37d 13. The process as in any of 1 to 12, wherein the influenza virus strains are selected from the group consisting of H5Nl, H9N2, H7N7, H2N2, 147N1 and H1N1. 14. The process as in any of 1 to 12, wherein the influenza virus strains are selected from the group of the seasonal influenza virus strains. 15. The process as in any of I to 14, wherein the influenza antigen is in a form selected from the group consisting of purified whole influenza virus, inactivated influenza virus or sub-unit components of influenza virus. 16. The process as in 15, wherein the inactivated influenza virus is a split influenza virus. 17. The process as in any of 1 to 16, wherein the influenza antigen is derived from cell culture. 18. The process as in any of 1 to 16, wherein the influenza antigen is produced in embryonic eggs. 19. A stabilized dry influenza vaccine composition in the form of dry regular spherical micropellets or particles having a diameter from about 200pm to about 1500 pm obtainable by the process according to one or more of I to 18. 20. The composition as in 19 wherein each regular bead or particle comprises one or more influenza antigens from only one influenza virus strain. 21. The composition as in 19 wherein each regular bead or particle comprises one or more influenza antigens from one or more different influenza virus strains. 22. The composition as in any of 19 to 21 further comprising an adjuvant. 23. The composition of 22, wherein the adjuvant is contained in separate dry regular spherical micropellets or particles. 24. A process for stabilizing an adjuvant containing vaccine composition comprising the steps of diluting a liquid bulk composition comprising an antigen or an antigenic H:Mjrkln wooven\NRPcnbl\DCC\AAR\5057799 .doc-9M4/2013 - 37e preparation by an aqueous solution comprising a stabilizer, subjecting the diluted composition to a process in order to form regular droplets having a diameter of approximately from about 200 pim to about 1500 ptm, subjecting the regular droplets to freezing in a freezing medium to form frozen regular spherical micropellets or particles and drying the frozen regular spherical micropellets or particles to form dry regular spherical micropellets or particles having a diameter from about 200 pim to about 1500 prm. 25. The process as in 24, further comprising the addition of an aqueous solution comprising one or more adjuvants and, optionally, a stabilizer to the liquid bulk antigen composition. 26. The process as in 24, further comprising diluting a separate liquid bulk solution or emulsion of one or more adjuvants by an aqueous solution comprising a stabilizer, subjecting the diluted adjuvant solution or emulsion to a process in order to form regular droplets having a diameter of approximately from about 200 p m to about 1500 pm, subjecting the regular droplets to freezing in a freezing medium to form frozen regular spherical micropellets or particles, drying the frozen regular spherical micropellets or particles to form dry regular spherical micropellets or particles having a diameter from about 200 pm to about 1500 pm and blending and filling into a vial or other appropriate recipient together with the antigen containing dry regular spherical micropellets. 27. The process as in any of 24 to 26, wherein the liquid bulk composition further comprises one or more additional antigens or antigenic preparations, each from a different pathogen or serotype of a pathogen, in order to obtain dry regular spherical micropellets or particles, each comprising two or more antigens or antigenic preparations from two or more different pathogens or serotypes of a pathogen. 28. The process as in any of 24 to 26, wherein the liquid bulk composition comprises an antigen or antigenic preparation from one single pathogen or serotype of a pathogen, in order to obtain dry regular spherical micropellets or particles, each comprising antigens or antigenic preparations from the same pathogen or serotype of a pathogen.

H:ar\eIrwovcIn\NRPorbl\DCC\AAR\557799_i doc-9/O42L3 - 37f 29. The process as in 28, further comprising dosing, blending and filling into a vial or other appropriate recipient two or more types of dry regular spherical micropellets or particles, characterized in that each type of micropellets comprises antigens or antigenic preparations from two or more different pathogens or serotypes of a pathogen. 30, The process as in any of 24 to 29, characterized in that the drying occurs by the method of lyophilization. 31. The process as in any of 24 to 29, characterized in that the drying occurs by the method selected from the group consisting of atmospheric freeze-drying, fluidized bed drying, vacuum rotary drum drying, stirred freeze-drying, vibrated freeze-drying, microwave freeze-drying. 32. The process as in any of 24 to 31, characterized in that the stabilizer comprises a monosaccharide, an oligosaccharide or a sugar alcohol or a mixture thereof. 33. A stabilized dry vaccine composition comprising one or more antigens which composition is in the form of dry regular spherical micropellets or particles having a diameter from about 200 ptm to about 1500 pim obtainable by the process according to one or more of 24 to 32. 34. The composition as in 33 wherein each regular bead or particle comprises only one type of antigen. 35. The composition as in 33 wherein each regular bead or particle comprises one or more different types of antigen. 36. The composition as in any of 33 to 35 further comprising an adjuvant. 37. The composition of 36, wherein the adjuvant is contained in separate dry regular spherical micropellets or particles. 38. The composition as in any of 33 to 37 wherein the antigen or the antigens are selected from the group consisting of live attenuated and inactivated whole viruses or antigenic components of viruses, such as Polio, Influenza Rotavirus , cytomegalo virus H:\a~lenvovon\NR~onbl\DCC\AAR\50571799_1.dc-WO42}I3 - 37g and Hepatitis A and B , and whole bacteria or bacterial protein or polysaccharide antigens, conjugated or non-conjugated, such as meningococcal polysaccharides , Tetanus Diphteria, cellular and acellular pertussis, Botulism and Anthrax. 39. A process for the preparation a vaccine comprising the step of reconstitution of the composition according to any of 19 to 23 or according to any of 33 to 38 in an aqueous solution. 40. The process as in 39, wherein the aqueous solution comprises an adjuvant. 41. A vaccine kit, comprising a first recipient containing a stabilized dry vaccine composition in the form of dry regular spherical micropellets or particles according to any of 19 to 25 or according to any of 33 to 38 and a second recipient containing an aqueous solution for the reconstitution of the vaccine. 42. The kit as in 41 characterized in that aqueous solution comprises an adjuvant. 43. The kit as in 41 characterized in that it comprises a third recipient containing a stabilized dry adjuvant composition in the form of dry regular spherical micropellets or particles. 44. A method of stockpiling a stable dry bulk of antigens wherein the antigen or the antigens is/are stabilized by a method according to any of 1 to 18 or 24 to 32 and the resulting stabilized vaccine composition is reconstituted with an adequate solvent and optionally formulated prior to liquid filling into vials or syringes after storage in the form of dry regular spherical micropellets or particles having a diameter from about 200 pm to about 1500 pim.

Claims (79)

1. A process for stabilizing a vaccine composition comprising the steps of diluting a liquid bulk composition comprising an antigen or an antigenic preparation by an aqueous solution comprising a stabilizer, prilling the diluted composition to form regular droplets having a diameter from about 200 im to about 1500 itm, subjecting the regular droplets to freezing to form frozen regular spherical micropellets or particles and drying the frozen regular spherical micropellets or particles to form dry regular spherical micropellets or particles having a diameter from about 200 pm to about 1500 im.

2. The process as claimed in Claim 1, wherein the liquid bulk composition further comprises one or more additional antigens or antigenic preparations, each from a different pathogen or serotype of a pathogen, in order to obtain dry regular spherical micropellets or particles, each comprising two or more antigens or antigenic preparations from two or more different pathogens or serotypes of a pathogen.

3. The process as claimed in Claim 1, wherein the liquid bulk composition comprises an antigen or antigenic preparation from one single pathogen or serotype of a pathogen, in order to obtain dry regular spherical micropellets or particles, each comprising antigens or antigenic preparations from the same pathogen or serotype of a pathogen.

4. The process as claimed in Claim 2, further comprising dosing, blending and filling into a vial or other appropriate recipient two or more types of dry regular spherical micropellets or particles, characterized in that each type of micropellets comprises antigens or antigenic preparations from two or more different pathogens or serotypes of a pathogen,

5. The process as claimed in any of Claims 1 to 4, characterized in that the drying occurs by the method of lyophilization.

6. The process as claimed in any of Claims 1 to 4, characterized in that the drying occurs by the method selected from the group consisting of atmospheric freeze-drying, H AarMimerwoven\NRPonbIUJCC\AAR5057180_L.DOC-9M/4203 -39 fluidized bed drying, vacuum rotary drum drying, stirred freeze-drying, vibrated freeze drying, microwave freeze-drying.

7. The process as claimed in any of Claims 1 to 6, characterized in that the stabilizer comprises a monosaccharide, an oligosaccharide, a sugar alcohol or a mixture thereof.

8. The process as claimed in any of Claims 1 to 7 characterized in that the stabilizer comprises a carbohydrate within a concentration range from 2% (w/v) to the limit of solubility in the formulated liquid product.

9. The process as claimed in any of Claims 1 to 8, wherein the antigen or the antigens are selected from the group consisting of live attenuated and inactivated whole viruses, antigenic components of viruses, whole bacteria, bacterial protein or polysaccharide antigens, conjugated or non-conjugated.

10. The process as claimed in Claim 9, wherein the viruses are selected from the group of poliovirus, Influenza virus, Rotavirus, cytomegalovirus, Hepatitis A and Hepatitis B virus.

11. The process as claimed in Claim 9, wherein the antigen or the antigens are whole bacteria, bacterial protein or polysaccharide antigens, conjugated or non-conjugated and selected from Haemophilus influenzae, meningococcal polysaccharides, tetanus, Diphtheria, cellular and acellular pertussis, Botulism, Anthrax and Clostridium difficile.

12. A stabilized dry vaccine composition comprising one or more antigens which composition is in the form of dry regular spherical micropellets or particles having a diameter from about 200 jtm to about 1500 pm obtainable by the process according to one or more of Claims I to 11.

13. The composition as claimed in Claim 12, wherein each regular spherical micropellet or particle comprises only one type of antigen. H:m\Intenvcven\NRPorbl\CCAAR\5057]SO1.DOC-924/3 -40

14. The composition as claimed in Claim 12, wherein each regular spherical micropellet or particle comprises one or more different types of antigens.

15. A process for the preparation a vaccine comprising the step of reconstitution of the composition according to any of Claims 12 to 14 in an aqueous solution.

16. A vaccine kit, comprising a first recipient containing a stabilized dry vaccine composition in the form of dry regular spherical micropellets or particles according to any of Claims 12 to 14 and a second recipient containing an aqueous solution for the reconstitution of the vaccine.

17. A method of stockpiling a stable dry bulk of antigens wherein the antigen or the antigens is/are stabilized by the process according to any of Claims 1 to 11 and the resulting stabilized vaccine composition is reconstituted with an adequate solvent and optionally formulated prior to liquid filling into vials or syringes after storage in the form of dry regular spherical micropellets or particles having a diameter from about 200 Pm to about 1500 pvm.

18. A process for producing dry regular spherical micropellets or particles adjuvant comprising: a. mixing a liquid bulk adjuvant solution or emulsion comprising one or more adjuvants with an aqueous solution comprising a stabilizer; b. prilling the mixed adjuvant solution or emulsion to form regular droplets of the adjuvant solution or emulsion having a diameter from about 200 pm to about 1500 ptm; c. subjecting the regular droplets of the adjuvant to freezing to form frozen regular spherical micropellets or particles of the adjuvant; and d. drying the frozen regular spherical micropellets or particles of the adjuvant to form dry regular droplets of the adjuvant having a diameter from about 200 xm to about 1500 pm. H:\irjNitcmnvoven\NRoilDCC\AAR\505718 I1.DOC-90412013 -41

19. The process as claimed in Claim 18, wherein the adjuvant is selected from the group consisting of liposomes, lipid or detergent micellles or other lipidic particles, polymer nanoparticles or microparticles, soluble polymers, protein particles, mineral gels, mineral micro- or nanoparticles, polymer/aluminum nanohybrids, oil in water or water in oil emulsions, saponin extracts, bacterial cell wall extracts, stimulators of innate immunity receptors, in particular natural or synthetic TLR agonists, natural or synthetic NOD agonists and natural or synthetic RIG agonists.

20. The process as claimed in Claim 18 or 19, wherein the adjuvant is an aluminium salt such as aluminium hydroxide or aluminium phosphate.

21. The process as claimed in any of Claims 18 to 20, wherein the stabilizer is an aqueous solution comprising a carbohydrate within a concentration range from 2% (w/v) to the limit of solubility of carbohydrate in the mixed adjuvant solution.

22. The process as claimed in any of Claims 18 to 21, wherein the stabilizer comprises a monosaccharide, an oligosaccharide, a sugar alcohol or a mixture thereof.

23. The process as claimed in any of Claims 18 to 22, further comprising filling the dry regular spherical micropellets or particles of adjuvant into a vial or other appropriate recipient.

24. A stabilized dry adjuvant composition comprising one or more adjuvants which composition is in the form of dry regular spherical micropellets or particles of adjuvant having a diameter from about 200 pim to about 1500 jim obtainable by the process according to one or more of Claims 18 to 23.

25. A kit comprising a container containing a stabilized dry adjuvant composition as claimed in Claim 24. [K arIiterwo, cI\NRPonbl\DCC\AARhIl7Klg0_ I DDC-9At/20 ]3 - 42

26, A process for stabilizing an adjuvant containing vaccine composition comprising the steps of diluting a liquid bulk composition comprising an antigen or an antigenic preparation by an aqueous solution comprising a stabilizer, prilling the diluted composition to form regular droplets having a diameter from about 200 Pm to about 1500 pm, subjecting the regular droplets to freezing to form frozen regular spherical micropellets or particles and drying the frozen regular spherical micropellets or particles to form dry regular spherical micropellets or particles having a diameter from about 200 pim to about 1500 gim.

27. The process as claimed in Claim 26, further comprising the addition of an aqueous solution comprising one or more adjuvants and, optionally, a stabilizer to the liquid bulk antigen composition.

28. The process as claimed in Claim 26, further comprising diluting a separate liquid bulk solution or emulsion of one or more adjuvants by an aqueous solution comprising a stabilizer, prilling the diluted adjuvant solution or emulsion to form regular droplets having a diameter from about 200 p m to about 1500 pim, subjecting the regular droplets to freezing to form frozen regular spherical micropellets or particles, drying the frozen regular spherical micropellets or particles to form dry regular spherical micropellets or particles having a diameter from about 200 pm to about 1500 Pm and blending and filling into a vial or other appropriate recipient together with the antigen containing dry regular spherical micropellets.

29. The process as claimed in Claim 26 further comprising a step of reconstituting the dry regular spherical micropellets or particles having a diameter from about 200 pm to about 1500 pim with an aqueous solution comprising an adjuvant.

30. The process as claimed in any of Claims 26 to 29, wherein the liquid bulk composition further comprises one or more additional antigens or antigenic preparations, each from a different pathogen or serotype of a pathogen, in order to obtain dry regular Hr \At~enovenNRPNthlDCCAAR150571X30DOC-9A4213 - 43 spherical micropellets or particles, each comprising two or more antigens or antigenic preparations from two or more different pathogens or serotypes of a pathogen.

31. The process as claimed in any of Claims 26 to 29, wherein the liquid bulk composition comprises an antigen or antigenic preparation from one single pathogen or serotype of a pathogen, in order to obtain dry regular spherical micropellets or particles, each comprising antigens or antigenic preparations from the same pathogen or serotype of a pathogen.

32. The process as claimed in Claim 30, further comprising dosing, blending and filling into a vial or other appropriate recipient two or more types of dry regular spherical micropellets or particles, characterized in that each type of micropellets comprises antigens or antigenic preparations from two or more different pathogens or serotypes of a pathogen.

33. The process as claimed in any of Claims 26 to 32, characterized in that the drying occurs by the method of lyophilization.

34. The process as claimed in any of Claims 26 to 32, characterized in that the drying occurs by the method selected from the group consisting of atmospheric freeze-drying, fluidized bed drying, vacuum rotary drum drying, stirred freeze-drying, vibrated freeze drying, microwave freeze-drying.

35. The process as claimed in any of Claims 26 to 34, characterized in that the stabilizer comprises a monosaccharide, an oligosaccharide, a sugar alcohol or a mixture thereof.

36. The process as claimed in any of Claims 26 to 35 wherein the antigen or the antigens are selected from the group consisting of live attenuated and inactivated whole viruses, antigenic components of viruses, whole bacteria, bacterial protein or polysaccharide antigens, conjugated or non-conjugated. H:NaarlIewote,\NRPoribI\DCC AAR50578BI_ DOC-9M/21;±3 - 44

37. The process as claimed in Claim 36 wherein the viruses are selected from the group of poliovirus, Influenza virus, Rotavirus, cytomegalovirus, Hepatitis A and Hepatitis B virus.

38. The process as claimed in Claim 36 wherein the antigen or the antigens are whole bacteria, bacterial protein or polysaccharide antigens, conjugated or non-conjugated and selected from Haemophilus influenzae, meningococcal polysaccharides, tetanus, Diphtheria, cellular and acellular pertussis, Botulism, Anthrax and Clostridium difficile.

39. A stabilized dry vaccine composition comprising one or more antigens which composition is in the form of dry regular spherical micropellets or particles having a diameter from about 200 gm to about 1500 gim obtainable by the process according to one or more of Claims 26 to 38.

40. The composition as claimed in Claim 39 wherein each regular spherical micropellet or particle comprises only one type of antigen.

41. The composition as claimed in Claim 39 wherein each regular spherical micropellet or particle comprises one or more different types of antigens.

42. The composition as claimed in any of Claims 39 to 41 further comprising an adjuvant.

43. The composition of Claim 42, wherein the adjuvant is contained in separate dry regular spherical micropellets or particles.

44. A process for the preparation of a vaccine comprising the step of reconstitution of the composition according to any of Claims 39 to 43 in an aqueous solution.

45. The process as claimed in Claim 44, wherein the aqueous solution comprises an adjuvant. Ha\ArrterwoiIe\NRPortbDCC\AAR\5157180l.DOC-)O4/20j 3 - 45

46. A vaccine kit, comprising a first recipient containing a stabilized dry vaccine composition in the form of dry regular spherical micropellets or particles according to any of Claims 39 to 43 and a second recipient containing an aqueous solution for the reconstitution of the vaccine.

47. The kit as claimed in Claim 46 characterized in that aqueous solution comprises an adj uvant.

48. The kit as claimed in Claim 46 characterized in that it comprises a third recipient containing a stabilized dry adjuvant composition in the form of dry regular spherical micropellets or particles.

49. A method of stockpiling a stable dry bulk of antigens wherein the antigen or the antigens is/are stabilized by the process according to any of Claims 26 to 38 and the resulting stabilized vaccine composition is reconstituted with an adequate solvent and optionally formulated prior to liquid filling into vials or syringes after storage in the form of dry regular spherical micropellets or particles having a diameter from about 200 pm to about 1500 pim.

50. A process for stabilizing an influenza vaccine composition comprising the steps of diluting a liquid bulk composition comprising an influenza antigen or an antigenic preparation from a seasonal, pre-pandemic or pandemic influenza virus strain by an aqueous solution comprising a carbohydrate, a sugar alcohol, or a mixture thereof in order to obtain a 2% (w/v) to limit of solubility of carbohydrate and/or sugar alcohol content of the resulting diluted composition, prilling the diluted composition to form regular droplets having a diameter from about 200 pm to about 1500 pim, subjecting the regular droplets to freezing to form frozen regular spherical micropellets or particles and drying the frozen regular spherical micropellets or particles to form dry regular spherical micropellets or particles having a diameter from 200 pm to about 1500 pm. R II r lnLenGverINRPoribl\DCC\AARf505712O..l.D IC--9M4/2013 - 46

51. The process as claimed in Claim 50, characterized in that the drying occurs by the method of lyophilization.

52. The process as claimed in Claim 51, characterized in that the drying occurs by the method selected from the group consisting of atmospheric freeze-drying, fluidized bed drying, vacuum rotary drum drying, stirred freeze-drying, vibrated freeze-drying, microwave freeze-drying.

53. The process as claimed in any of Claims 50 to 52, characterized in that the carbohydrate is a disaccharide.

54. The process as claimed in Claim 53, characterized in that the carbohydrate is sucrose.

55. The process as claimed in any of Claims 50 to 54, wherein the liquid bulk composition further comprises one or more additional influenza antigens or antigenic preparations, each from a different seasonal, pre-pandemic or pandemic influenza virus strain, in order to obtain dry regular spherical micropellets or particles, each comprising two or more influenza antigens or antigenic preparations from two or more different seasonal, pre-pandemic or pandemic influenza virus strains.

56. The process as claimed in Claim 55, further comprising dosing and/or filling the dry regular spherical micropellets or particles into a vial or other appropriate recipient.

57. The process as claimed in any of Claims 50 to 54, wherein the liquid bulk composition comprises one or more influenza antigens or antigenic preparations from one seasonal, pre-pandemic or pandemic influenza virus strain, in order to obtain dry regular spherical micropellets or particles, each comprising influenza antigens or antigenic preparations from the same seasonal, pre-pandemic or pandemic influenza virus strain. H !i'arntrwcoven\NRPortbl\DCCAAR\50578ql IDOC-9.'0412013 -47

58. The process as claimed in Claim 57, further comprising dosing, blending and filling into a vial or other appropriate recipient two or more types of dry regular spherical micropellets or particles, characterized in that each type of micropellets comprises influenza antigens or antigenic preparations from a different seasonal, pre-pandemic or pandemic influenza virus strain than the other type.

59. The process as claimed in any of Claims 50 to 58, further comprising the addition of an aqueous solution comprising one or more adjuvants and, optionally, a stabilizer to the liquid bulk antigen composition.

60. The process as claimed in any of Claims 50 to 58, further comprising diluting a separate liquid bulk solution or emulsion of one or more adjuvants by an aqueous solution comprising a stabilizer, prilling the diluted adjuvant solution or emulsion to form regular droplets having a diameter from about 200 gm to about 1500 gm, subjecting the regular droplets to freezing to form frozen regular spherical micropellets or particles, drying the frozen regular spherical micropellets or particles to form dry regular spherical micropellets or particles having a diameter from about 200 gm to about 1500 gm and blending and filling into a vial or other appropriate recipient together with the antigen containing dry regular spherical micropellets.

61. The process as claimed in Claims 59 or 60, wherein the adjuvant is selected from the group consisting of liposomes, lipid or detergent micelles or other lipidic particles, polymer nanoparticles or microparticles, soluble polymers, protein particles, mineral gels, mineral micro- or nanoparticles, polymer/aluminum nanohybrids, oil in water or water in oil emulsions, saponin extracts, bacterial cell wall extracts, stimulators of innate immunity receptors, in particular natural or synthetic TLR agonists, natural or synthetic NOD agonists and natural or synthetic RIG agonists.

62. The process as claimed in any of Claims 50 to 61, wherein the influenza virus strains are selected from the group consisting of H5Nl, H9N2, H7N7, H2N2, H7N1 and H1Nl. K a;.r\1numeiovno bi~ rtDfCCiAAR\ 818! DOC.9/U4/20 D - 48

63. The process as claimed in any of Claims 50 to 61, wherein the influenza virus strains are selected from the group of the seasonal influenza virus strains.

64. The process as claimed in any of Claims 50 to 63, wherein the influenza antigen is in a form selected from the group consisting of purified whole influenza virus, inactivated influenza virus or sub-unit components of influenza virus.

65. The process as claimed in Claim 64, wherein the inactivated influenza virus is a split influenza virus.

66. The process as claimed in any of Claims 50 to 65, wherein the influenza antigen is derived from cell culture.

67. The process as claimed in any of Claims 50 to 65, wherein the influenza antigen is produced in embryonic eggs.

68. A stabilized dry influenza vaccine composition in the form of dry regular spherical micropellets or particles having a diameter from about 200 pm to about 1500 ptm obtainable by the process according to one or more of Claims 50 to 67.

69. The composition as claimed in Claim 68 wherein each regular spherical micropellet or particle comprises one or more influenza antigens from only one influenza virus strain.

70. The composition as claimed in Claim 68 wherein each regular spherical micropellet or particle comprises one or more influenza antigens from one or more different influenza virus strains.

71. The composition as claimed in any of Claims 68 to 70 further comprising an adjuvant. HAnartecnvoven\NRPNenbl\DCC\AA\505718 . 0DOC-904/20l3 - 49

72. The composition of Claim 71, wherein the adjuvant is contained in separate dry regular spherical micropellets or particles.

73. A process according to any one of Claims 1 to 11, Claim 15, Claims 18 to 23, Claims 26 to 38, 44, 45 or 50 to 67 substantially as herein described with reference to the Figures and/or Examples.

74. A stabilized dry vaccine composition according to any one of Claims 12 to 14 substantially as herein described with reference to the Figures and/or Examples.

75. A vaccine kit according to any one of Claims 16 or 46 substantially as herein described with reference to the Figures and/or Examples.

76. A method according to any one of Claims 17 or 49 substantially as herein described with reference to the Figures and/or Examples.

77. A stabilized dry adjuvant composition according to any one of Claim 24, 39 or 68 substantially as herein described with reference to the Figures and/or Examples.

78. A kit according to any one of Claims 25, 47 or 48 substantially as herein described with reference to the Figures and/or Examples.

79. A composition according to any one of Claims 40 to 43 or 69 to 72 substantially as herein described with reference to the Figures and/or Examples.