Title of Invention	A PROCESS FOR THE PREPARATION OF A POWDER SUITABLE FOR VACCINE AND THE POWDER PREPARED BY THEREOF
Abstract	A gel-forming free-flowing powder suitable for use as a vaccine is prepared by spray-drying or spray freeze-drying an aqueous suspension that contains an antigen adsorbed to an aluminum salt or calcium salt adjuvant, a saccharide, an amino acid or a salt thereof, and a colloidal substance. Powder for vaccine purposes are also prepared by spray freeze-drying an aqueous suspension of such an adjuvant having an antigen adsorbed therein. Processes for forming these powder compositions are also described, as well as methods of using the compositions in a vaccination procedure.

Title of Invention

A PROCESS FOR THE PREPARATION OF A POWDER SUITABLE FOR VACCINE AND THE POWDER PREPARED BY THEREOF

Abstract

A gel-forming free-flowing powder suitable for use as a vaccine is prepared by spray-drying or spray freeze-drying an aqueous suspension that contains an antigen adsorbed to an aluminum salt or calcium salt adjuvant, a saccharide, an amino acid or a salt thereof, and a colloidal substance. Powder for vaccine purposes are also prepared by spray freeze-drying an aqueous suspension of such an adjuvant having an antigen adsorbed therein. Processes for forming these powder compositions are also described, as well as methods of using the compositions in a vaccination procedure.

Full Text	The invention relates to a process for the preparation of a powder suitable for vaccine and the powder prepared by thereof More specifically, the invention relates to vaccine compositions suitable for transdermal particle delivery from a needleless syringe system. Background to the Invention The ability to deliver pharmaceutical agents into and through skin surfaces (transdermal delivery) provides many advantages over oral or parenteral delivery techniques. In particular, transdermal delivery provides a safe, convenient and uoninvasive alternative to traditional administration systems, conveniently avoiding the major problems associated with oral delivery (e.g. variable rates of absorption and metabolism, gastroibtestinal irritation and/or bitter or unpleasant drug tastes) or parenteral delivery (e.g. needle pain, the risk of introducing infection to treated individuals, the risk of contamination or infection of health care workers caused by accidental needle-sticks and the disposal of used needles). However, despite its clear advantages, transdermal delivery presents a number of its own inherent logistical problems. Passive delivery through intact skin necessarily entails the transport of molecules through a number of structurally different tissues, including the stratum comeum, the viable epidemiis, the papillary dermis and the capillary walls in order for the drug to gain entry into the blood or lymph system. Transdermal delivery systems must therefore be able to overcome the various resistances presented by each type of tissue. In light of the above, a number of alternatives to passive transdermal delivery have been developed. These altematives include the use of skin penetration enhancing agents, or "permeation enhancers," to increase skin penneability, as well as non-chemical modes such as the use of iontophoresis, elcctroporation or ultrasound However, these alternative techniques often give rise to their own unique side effects such as skin irritation or sensitization. Thus, the spectrum of agents that can be safely and effectively administered using traditional transdermal delivery methods has remained limited. More recently, a novel transdermal drug delivery system that entails the use of a needleless syringe to fire powders (i.e., solid drug-containing particles) in controlled doses into and through intact skin has been described. In particular, commonly owned U.S. Patent No. 5,630,796 to Bellhouse et al describes a needleless syringe that delivers pharmaceutical particles entrained in a supersonic gas flow. The needleless syringe is used for transdennal delivery of powdered drug compounds and compositions, for delivery of genetic material into living cells (e.g., gene therapy) and for the delivery of biophannaceuticals to skin, muscle, blood or lymph. The needleless syringe can also be used in conjunction with surgery to deliver drugs and biologies to organ surfaces, solid tumors and/or to surgical cavities (e.g., tumor beds or cavities after tumor resection). In theory, practically any pharmaceutical agent that can be prepared in a substantially solid, particulate form can be safely and easily delivered using such devices. One area of the pharmaceuticals field which is of particular interest for delivery via this new system is that of vaccine compositions. Suitable vaccines include those comprising an antigen adsorbed into a salt adjuvant. Such compositions are known in the art (see for example U.S.Patent No, 5,902,565) and are advantageous since the adjuvant branches the immunogenicity of the vaccine. However, the storage and transportation of adjuvant vaccines is problematic. Commercial vaccine compositions containing salt adjuvants cannot be fixizen without causing damage to the vaccine. Further, one of the common storage techniques currently used for vaccines, fi-eeze-drying, is also unavailable for salt adjuvant containing compositions. Previous research has demonstrated that t freeze-drying causes the collapse of the gel structure of the vaccine composition, resulting in aggregation and precipitation of the adjuvant salt on resuspension in water (Warren et al, 1986, Annu. Rev. Immunol. 4: pages 369-388; Alving et al, Ann, N. Yi Acad. Sci. 690: pages 265-275). This is believed to be due to crystallisation of the water contained in the composition into large crystals on freezing and hence the concentration of the solute into specific regions, known as freeze concentrate regions. In the freeze concentrate regions, adjuvant salt particles are brought into close proximity and repulsive forces are overcome, thereby resulting in coagulation. Once the salt has coagulated, the original suspension cannot be reproduced. This effect has been found to significantly reduce the immunogenicity of the vaccine, one report demonstrating a complete loss in immunogenicity of a freeze-dried alum-adsorbed hepatitis B surface antigen (HBsAg) after storage at 4'C for two years (Diminsky et al. Vaccine, 18: pages 3-17)- An altemative method for storing adjuvant vaccine compositions is therefore required, which addresses the problems of aggregation associated with freeze-drying and which provides maximum retention of immunogenicity. Prolonged storage of vaccines is essential, both for use with the novel transdermal drug delivery systems mentioned above and also for use with conventional vaccination techniques. The provision of an effective altemative to freeze-drying is therefore of considerable commercial importance. It is also desired that the vaccine be produced in a form suitable for needleless injection. Needleless injection requires the vaccine composition to be in powder form, each particle having a suitable size and strength for transdermal delivery and being capable of forming a gel on resuspension. Alternatives to conventional freeze-drying techniques that have previously been reported include the incorporation of additives in the vaccine composition to improve the stability of an alum adjuvant, U.S. Patent No. 4,578-70 describes the addition of large amounts of both dextran and protein in order to achieve partial retention of the aluminum gel structure. This large addition of protein could however act to displace vaccine antigens from the aluminum gel and in addition would, in most cases, be immunogenic and as a result tend to swamp the immune response to the vaccine antigen. EP-B-0130619 is also concerned with the addition of stabilisers to lyophilised, or freeze-dried, vaccine preparations. Lyophilised preparations of a hepatitis B vaccine .comprising an inactivated purified hepatitis B virus surface antigen absorbed an aluminum gel and stabiliser are described The stabiliser is composed of at least one amino acid or salt thereof, at least one saccharide and at least one colloidal substance. Very low concentrations of aluminum salt adjuvant are used, typically less than 0.1% by weight. However, this document relates only to the hepatitis B vaccine and does not disclose a generic process, which is non-immunogen-specific. Spray-dried vaccine preparations comprising an immunogen adsorbed into an aluminum salt are disclosed in U,S, Patent No. 5,902,565, Immediate-release preparations are described which are prepared by spray-drying an aqueous suspension of aluminum salt-adsorbed immunogen. In the only Example, Example 1, in which such information is given, the resultant microspheres had a size range around 3 µ.m in diameter. According to U.S. Patent No. 5,902,565 the gel-forming nature of aluminum gels is completely retained during spray-drying even in the absence of any other materials which could exert a stabilising effect (apart from minimal quantities of vaccine antigen, typically 1 to 10 \|ig/ml). Addition of water to the spray-dried powder was said to result in the instant formation of a typical gel, with sedimentation properties similar to the startmg material. Summary of the Invention We investigated whether a gel-forming spray-dried powder of an aluminum salt could indeed be formed as described in U.S. Patent No, 5,902,565, We found that spray drying a suspension of aluminum hydroxide or aluminum phosphate in water caused submicron particles of the aluminum salt to aggregate to larger particles in the resulting spray-dried powder. Upon reconstitution of this powder in water, these larger particles did not disintegrate into small particles. A gel suspension did not form. Rather, the aggregated particles of aluminum hydroxide or aluminum phosphate sedimented and precipitated out of the suspension. Further experiments were carried out. We found that a suitable powder could be formed by spray-drying when an aluminum salt was utilised with a specific combination of other agents. Additionally, the aluminum salt and other agents needed to be used in specific proportions. We found too that the particular drying method used has a significant effect on the degree of coagulation of the adjuvant salt These investigations led to the finding that a powder suitable for needleless injection, and which substantially retained its gel structure on reconstitution in water, was obtainable by spray freeze-diying an alum adjuvant vaccine composition. The spray freeze-drying method involves atomizing the suspended vaccine composition into liquid nitrogen. This process has two important effects: firstly, the liquid nitrogen acts as a heat transfer agent and provides rapid freezing of the suspension; and secondly, the atomisation reduces the volume of each droplet to be frozen, further increasing the freezing rate. This combined effect causes extremely rapid freezing of very small droplets of suspension and leads to the formation of smaller ice crystals in the solid. The freeze concentrate regions which form during a standard freeze-drying technique are therefore significantly reduced in size. The rapid freezing of the particles, and their small size leads to powders having little or no aggregated adjuvant. The present invention therefore provides simple, yet effective techniques that generate salt adjuvant-containing vaccine compositions in a powder form which is suitable for long-term storage. The vaccine compositions of the invention show substantially no aggregation on reconstitution and therefore immunogenicity is substantially retained. The compositions also have well-defined particle size, density and mechanical properties which collectively are suitable for powders for transdermal delivery from a needleless syringe. The invention has the further, significant advantage that is suitable for use with a wide range of vaccine compositions and may well also be applicable to other pharmaceutical compositions, in particular where similar aggregation problems are encountered. As yet, the spray freeze-drying technique has been found to be entirely formulation independent within the field of adjuvant vaccine compositions. Accordingly, the present invention provides a process for the preparation of a powder suitable for use as a vaccine, which process comprises spray freeze-drying an aqueous suspension comprising an aluminum salt or calcium salt adjuvant having an antigen adsorbed therein. Freeze-flowing powder compositions suitable for vaccine use can thus be produced. The compositions have well-defined particle size, density and mechanical properties which collectively are suitable for powders for transdermal delivery from a needleless syringe. The invention further provides: a powder suitable for use as a vaccine, said powder being obtained by a process of the invention; a dosage receptacle for a needleless syringe, said receptacle containing an effective amount of powder of the invention; a needleless syringe which is loaded with a powder of the invention; a vaccine composition comprising a pharmaceutically acceptable carrier or diluent and a powder of the invention; and a gel-fonning free-flowing powder suitable for use as a vaccine, which powder comprises: (i) from 5 to 60% by weight of an aluminium salt or calcium salt adjuvant having an antigen adsorbed thereon; (ii) from 25 to 90% by weight of a saccharride; (iii) from 4.5 to 40% by weight of an amino salt or salt thereof; and (iv) from 0.5 to 10% by weight of a colloidal substance. IJ Figure 1 shows the particle size distribution of an HBsAg adsorbed alum gel (i) before drying and (ii) after drying using a spray freeze-drying technique followed by reconstitution in water. Figure 2 shows the particle size distribution of a second HBsAg adsorbed alum gel before drying and after drying via a conventional freeze drying method. Figure 3 illustrates the results of an immunogenicity study using mice injected with HBsAg absorbed alum vaccine which had been dried by either spray freeze-drying (SFD) according to present invention, or using freeze-drying (FD). The FD powders were sieved into different size fractions and tested for immxmogenicity. Two SFD formulations, varying in alum contact, were tested. Figure 4 illustrates the immunogenicity of three different spray freeze-dried powders in mice immunized by either intramuscular injection using a needle or epidermal powder immunization using a powder delivery device. Figure 5 illustrates the immunogenicity of spray freeze-dried diphtheria-tetanus toxoid vaccine in guinea pigs. Spray freeze-dried powders of 20-38 µm and 38-53 µm in diameter were administered as a powder to the abdominal skin using a powder delivery device. Detailed Description of the Preferred Embodiments Before describing the present invention in detail, it is to be understood that this invention is not limited to particularly exemplified compositions or process parameters as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting. All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety. It must be noted that, as used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a particle" includes a mixture of two or more such particles, reference to "an excipient" includes mixtures of two or more such excipients, and the like. A. Definitions Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commornly understood by one of ordinary skill in the art to which the invention pertains. Although a number of methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the preferred materials and methods are described herein, In describing the present invention, the following terms will be employed, and are intended to be defined as indicated below. By "antigen" is meant a molecule which contains one or more epitopes that will stimulate a host's immune system to make a cellular antigen-specific immune response or a humoral antibody response. Thus, antigens include polypeptides including antigenic protein fragments, oligosaccharides, polysaccharides and the tike. Furthermore, the antigen can be derived from any known virus, bacterium, parasite, plant, protozoan or fungus, and can be a whole organism. The term also includes-tumor antigens. Similarly, an oligonucleotide or polynucleotide which expresses an antigen, such as in DNA immunization appHcations, is also included in the definition of an antigen. Synthetic antigens are also included, for example polyepitopes, flanking epitopes and other recombinant or synthetically derived antigens (Bergmann et al (1993) Eur. 1 Immunol 23:277T-2781; Bergmann et al (1996) J., Immunol 157:3242-3249; Suhrbier, A, (1997) Immunol and Cell Biol 75:402-408; Gardner et aL (1998) 12^ World AIDS Conference, Geneva, Switzerland, June 28-July 3,1998). The aduvants having antigen adsorbed thereon of the present invention, alone or in combination, are typically combined with one or more added materials such as carriers, vehicles, and/or excipients. "Carriers," "vehicles" and "excipients" generally refer to substantially iaert materials which are nontoxic and do not interact with other components of the composition in a deleterious manner. These materials can be used to increase the amount of solids in particulate pharmaceutical compositions. Examples of suitable carriers include water, silicone, gelatin, waxes, and like materials. Examples of normally employed "excipients," include pharmaceutical grades of carbohydrates including monosaccharides, disaccharides, cyclodextrans, and polysaccharides (e.g., dextrose, sucrose, lactose, trehalose, rafSnose, mannitol, sorbitol, inositol, dextrans, and maltodextrans); starch; cellulose; salts (e,g. sodium or calcium phosphates, calcium sulfate, magnesimn sulfate); citric acid; tartaric acid; glycine; high molecular weight polyethylene glycols (PEG); Pluronics; surfactants; and combinations thereof Generally, when carriers and/or excipients are used, they are used in amounts ranging from about 0.1 to 99 wt% of the pharmaceutical composition. The term "powder' as used herein refers to a composition that consists of substantially solid particles that can be delivered transdermally using a needleless syringe device. The particles that make up the powder can be characterized on the basis of a number of parameters including, but not limited to, average particle size, average particle density, particle morphology (e.g. particle aerodynamic shape and particle surface characteristics) and particle penetration energy (P.E,). The average particle size of the powders according to the present invention can vary widely and is generally from 0.1 to 250 µm, for example from 10 to 100 µmand more typically from 20 to 70 µm. The average'particle size of the powder can be measured as a mass mean aerodynamic diameter (MMAD) using conventional techniques such as microscopic techniques (where particles are sized directly and individually rather than grouped statistically), absorption of gases, permeability or time of flight If desired, automatic particle-size counters can be used (e.g. Aerosizer Counter, Coulter Counter, KAC Counter, or Gelman Automatic Particle Counter) to ascertain the average particle size. Actual particle density or "absolute density" can be readily ascertained using known quantification techniques such as helium pycnometry and the like. Alternatively, envelope ("tap") density measurements can be used to assess the density of a powder according to the invention. The envelope density of a powder of the invention is generally from 0.1 to 25 g/cm3, preferably from 0.8 to 1,5 g/cm3. Envelope density information is particularly useful in characterizing the density of objects of irregular size and shape. Envelope density is the mass of an object divided by its volume, where the volume includes that of its pores and small cavities but excludes interstitial space. A number of methods of determining envelope density are known in the art, including wax immersion, mercury displacement, water absorption and apparent specific gravity techniques, A number of suitable devices are also available for determining envelope density, for example, the GeoPycTM Model 1360, available from the Micromeritics Instrument Corp, The difference between the absolute density and envelope density of a sample pharmaceutical composition provides information about the sample's percentage total porosity and specific pore volume. Particle morphology, particularly the aerodynamic shape of a particle, can be readily assessed using standard light microscopy. It is preferred that the particles which make up the instant powders have a substantially spherical or at least substantially ellipti cal aerodynamic shape. It is also preferred that the particles have an axis ratio of 3 or less to avoid the presence of rod- or needle-shaped particles. These same microscopic techniques can also be used to assess the particle surface characteristics, e,g. the amount and extent of surface voids or degree of porosity. Particle penetration energies can be ascertained using a number of conventional techniques, for example a metallized film P.E, test A metallized film material (e.g. a 125 µm polyester film having a 350 A layer of aluminum deposited on a single side) is used as a substrate into which the powder is fired from a needleless syringe (e.g, the needleless syringe described in U.S. Patent No. 5,630,796 to Bellhouse et al) at an initial velocity of about 100 to 3000 m/sec. The metallized film is placed, with the metal-coated side facing upwards, on a suitable surface, A needleless syringe loaded with a powder is placed with its spacer contacting the film, and then fired. Residual powder is removed from the metallized film surface using a suitable solvent. Penetration energy is then assessed using a BioRad Model GS-700 imaging densitometer to scan the metallised film, and a personal computer with a SCSI interface and loaded with MultiAnalyst software (BioRad) and Matlab software (Release 5.1, The MathWorks, Inc.) is used to assess the densitometer reading- A program is used to process the densitometer scans made using either the transmittance or reflectance method of the densitometer. The penetration energy of the spray-coated powders should be equivalent to, or better than that of reprocessed mannitol particles of the same size (mannitol particles that are freeze-dried, compressed, groimd and sieved according to the methods of commonly owned International Publication No. WO 97/48485, incorporated herein by reference). The term "subject" refers to any member of the subphylum cordata including, without limitation, humans and other primates including non-hmnan primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs; birds, including domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like. The term does not denote a particular age. Thus, both adult and newbom individuals are intended to be covered. The methods described herein are intended for use in any of the above vertebrate species, since the immune systems of all of these vertebrates operate similarly. The term "transdermal dehvery" includes both transdermal ("percutaneous") and transmucosal routes of administration, i.e, delivery by passage through the skin or mucosal tissue. SEE G,g., Transdermal Drug Delivery: Developmental Issues and Research Initiatives, Hadgraft and Guy (eds,). Marcel Dekker, Inc., (1989); Controlled Drug Delivery: Fundamentals and Applications, Robinson and Lee (eds.). Marcel Dekker Retroviradae (e.g. HTLV-I, HTLV-II; HIV-1 and HIV-2); and simian immunodeficiency vims (SIV) among others. Peptides: Analysis, Synthesis, Biology, supra, Vol. 1, for classical solution synthesis. One or more saccharrides may be present in the aqueous suspension as component (b). The saccharride content is typically 1.5 to 5% by weight, preferably 2 to 4% by weight. The saccharride may be monosaccharide such as glucose, xylose, galactose, fructose, D-mannose or sorbose; a disaccharide such as lactose, maltose, saccharose, trehalose or sucrose; or a sugar alcohol such as mannitol, sorbitol, xylitol, glycerol, erythritol or arabitol. One or more amino acids or amino acid salts is present in the aqueous suspension as component (c). Any physiologically acceptable amino acid salt may be employed. The salt may be an alkali or alkaline earth metal salt such as sodium, potassium or magnesium salt. The amino acid may be an acidic, neutral or basic amino acid. Suitable amino acids are glycine, alanine, glutamine, arginine, lysine and histidine. Monosodium glutamate is a suitable amino acid salt. The aqueous suspension generally contains from 0.5 to 1.5% by weight, more preferably from 0.75 to L25 by weight, of the amino acid and/or amino acid salt. The colloidal substance (d) is a divided substance incapable of passing through a semi-permeable membrane, comprised of fine particles which, in suspension or solution, fail to settle out. Suitable colloidal substances axe disclosed in EP-B-0130619, Component (d) may be selected from polysaccharides such as dextran or maltodextran; hydrogels such as gelatine or agarose; or proteins such as human serum albumin. The substance may have a molecular weight of 500 to 80,000 or higher, for example from 1000 or 2000 to 30,000 or from 5,000 to 25,000, Component (d) is generally present in the aqueous suspension in an amount of from 0.05 to 0.5% by weight, preferably from 0.07 to 0.3% by weight. The adjuvant having antigen adsorbed thereon and the saccharide, amino acid or salt thereof and colloidal substance are suspended in water. The aqueous suspension is spray freeze-dried. The spray freeze-drying conditions are selected to enable the desired particles to be produced. The air inlet temperature, air outlet temperature, feed rate of the aqueous suspension, air flow rate, etc, can thus be varied as desired. Any suitable spray-drier may be used. The nozzle size may vary as necessary. Particular spray freeze-drying conditions are described in more detail below. A gel-forming free-flowing powder can thus be provided which is suitable for use as a vaccine. The proportions of the various components of the powder can be adjusting by adjusting the composition of the suspension that is spray freeze-dried. However, the powder typically comprises or, in some embodiments, consists essentially of: (i) from 5 to 60%, for example from 7 to 50% such as from 10 to 30%, by weight of an aluminium salt or calcium salt adjuvant having an antigen adsorbed thereon; (ii) from 25 to 90%, for example from 30 to 80% such as from 40 to 70% by weight of a saccharide; (iii) from 4.5 to 40%, for example from 7 to 30% such as from 10 to 20% by weight of an amino acid or salt thereof; and (iv) from 0.5 to 10%, for example from 0.8 to 6% such as from 1 to 3%, by weight of a colloidal substance. The invention is concerned generally with powders suitable for use as vaccines that are formed by spray freeze-drying an aqueous suspension comprising an aluminum salt or calcium salt adjuvant having an antigen adsorbed therein. Such powders are suitable for transdermal administration from a needleless syringe deUvery system. As such, the particles which make up the powdered composition must have sufficient physical strength to withstand sudden acceleration of up to several times the speed of sound and the impact with, and passage through, the skin and tissue. Preferably, the aqueous suspension, prior to spray freeze-drying, contains less than 10% by weight, for instance less than 5% weight and preferably less than 3% by weight, of the salt adjuvant having antigen adsorbed thereon. The aqueous suspension typically contains at least 0.05% by weight, for instance at least 0.1 % by weight or at least 0.6% by weight, of the adjuvant having antigen adsorbed thereon. More preferably, the suspension contains from 0,2 or 0.3 to 0.6%, 0.75% or 1% by weight, preferably from 0.2 to 0.4% by weight, of adjuvant onto which antigen is adsorbed. At concentrations above about 10%, by weight of adjuvant salt, the aqueous suspension becomes highly viscous. This limits the ability to atomize the suspension. It should be understood that the preferred upper limit of adjuvant concentration applies to the aqueous suspension prior to spray freeze-drying. The content of adjuvant salt having antigen adsorbed thereon may be as high as 50% by weight or more in the spray freeze-dried powders of the invention. The adjuvant is generally an aluminum salt, for example aluminum hydroxide or aluminum phosphate. Alternatively, the adjuvant salt may be aluminum sulfate or calcium phosphate. Again, any suitable antigen as defined herein may be employed. The antigen may be a viral antigen. The antigen may therefore be derived from members of the families Picomaviridae (e.g. poliovinises, etc.); Caliciviridae; Togaviridae (e.g. rubella virus, dengue virus, etc.); Flaviviridae; Coronaviridae; Reoviridae; Bimaviridae; Rhabodoviridae (e.g. rabies virus, etc.); Filovuidae; Paramyxoviridae (e.g, mumps virus, measles virus, respiratory syncytial virus, etc.); Orthomyxoviridae (e.g. influenza virus types A, B and C, etc); Bunyaviridae; Arenaviridae; Retroviradae (e.g. HTLV-I; HTLV-II; HIV-l and HIV-2); and simian immunodeficiency vims (SIV) among others. Alternatively, viral antigens may be derived from papillomavirus (e.g. HPV); a herpesvirus; a hepatitis virus, e.g. hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C (HCV), the delta hepatitis virus (HDV), hepatitis E virus (HEV) or hepatitis G. virus (HGV); and the tick-bome encephalitis viruses. See, e.g. Virology, 3rd Edition (WX. Joklik ed. 1988); Fundamental Virology, 2nd Edition (B,N, Fields andD.M. Knipe, eds. 1991) for a description of these viruses. Bacterial antigens for use in the invention can be derived from organisms that cause diphtheria, cholera, tuberculosis, tetanus, pertussis, meningitis and other pathogenic states, including, e.g,. Meningococcus A, B and C, Hemophilus influenza type B (HIB), Helicobacter pylori. Vibrio cholerae, Escherichia coli, Campylobacter, Shigella, Salmonella, Streptococcus sp, and Staphylococcus sp. A combination of bacterial antigens may be provided, for example diphtheria, pertussis and tetanus antigens. Suitable pertussis antigens are pertussis toxin and/or filamentous haemagglutinin and/or pertactin, alternatively teraied P69. An anti-parasitic antigen may be derived from organisms causing malaria and Lyme disease. disaccharides such as lactose, maltose, saccharose, trehalose or sucrose, sugar alcohols such as mannitol, sorbitol, xylitol, glycerol, erythritol or arabitol, polymers such as dextran, starch, cellulose or high molecular weight polyethylene glycols (PEG), amino acids or their salts, such as glycine, alanine, glutamine, arginine, lysine or histidine or their salts with alkali or alkaline earth metals such as a sodium, potassium or magnesium salts, or sodium or calcium phosphates, calcium carbonate, calcium sulfate, sodium citrate, citric acid, tartaric acid, and combinations thereof. Suitable solvents include, but are not limited to, methylene chloride, acetone, methanol, ethanol, isopropanol and water. Typically, water is used as the solvent. Generally pharmaceutically acceptable salts having molarities ranging from about 1 mM to 2M can be used. Pharmaceutically acceptable salts include, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like, A thorough discussion of pharmaceutically acceptable excipients, vehicles and auxiliary substances is available in REMINGTON'S PHAEMACEUTICAL SCIENCES (Mack Pub. Co., NJ. 1991), incorporated herein by reference. Preferred excipients for use in the aqueous suspension include saccharides, amino acids or salts thereof and polymers. Typically, the suspension contains one or more saccharides, such as a combination of mannitol and trehalose. Saccharides are typically present in an amount of from 0,5 to 30% by weight. An amino salt, such as arginine glutamate or aspartate in an amount of from 0.1 to 30% by weight, and/or a polymer, such as dextran, in an amount of from 0 to 30% may also be included, typically in an amount of from 0 to 30 % by weight. Typical excipient combinations include one or more saccharides and a polymer and include substantially no amino salt The total amount of excipients present in the aqueous suspension is typically from 0 to 50%, more preferably from 10 to 30%. The particles of the invention are formed by first suspending the adjuvant having an antigen adsorbed therein, and any required additives, in water. The aqueous suspension is then spray freeze-dried. Any known technique in the art (for example the methods described by Mumenthaler ef al, Int J, Pharmaceutics (1991) 72, pages 97-110 and Maa et al, Phar. Res. (1999) VoL 16, page 249) may be used to carry out the spray freeze- . panicles to mnimally surgical sites containing a suitable dosage of the powder of the invention. The loaded syiringe can be packaged in a hermetically sealed container, which may further be labeled as described above. test is similar to that used in the metallized film test. The depth of penetration is measured using precision calipers. For each experiment a processed mannitol standard is run as comparison and all other parameters such as device pressure, particle size range, etc., are held constant. Data also show this method to be sensitive to differences in particle size and pressure. Processed mannitol standard as an excipient for drugs has been proven to deliver systemic concentrations in preclinical experiments, so the relative performance measure in the foam penetration test has a practical in vivo foundation. Promising powders can be expected to show equivalent or better penetration to mannitol for anticipation of adequate performance in preclinical or clinical studies. This simple, rapid -1. test has value as a relative method of evaluation of powders and is not intended to be considered in isolation. Particle Attrition Test A further indicator of particle performance is to test the ability of various candidate compositions to withstand the forces associated with high-velocity particle injection techniques, that is, the forces from contacting particles at rest with a sudden, high velocity gas flow, the forces resulting from particle-to-particle impact as the powder travels through the needleless syringe, and the forces resulting from pardcle-to-device collisions also as the powder travels through the device. Accordingly, a simple particle attrition test has been devised which measures the change in particle size distribution between the initial composition, and the composition after having been delivered from a needleless syringe device. The test is conducted by loading a particle composition into a needleless syringe as described above, and then discharging the device into a flask containing a carrier fluid in .which the particular composition is not soluble (e.g,, mineral oil, silicone oil, etc.). The carrier fluid is then collected, and particlq size distribution in both the initial compositior] and the discharged composition is calculated using a suitable particle sizing apparatus, e,g an AccuSizer® model 780 Optical Particle Sizex. Compositions that demonstrate less than about 50%, more preferably less than about 20% reduction in mass mean diameter (as determined by one of skill in the art. Dosage treatment may be a single dose schedule or a multiple dose schedule. A multiple dose schedule is one in which a primary course of vaccination may be with 1-10 separate doses, followed by other doses given at subsequent time intervals, chosen to maintain and/or reinforce the immune response, for example at 1-4 months for second dose and, if needed, a subsequent dose(s) after several months. The dosage regimen will also, at least in part, be determined by the need of the subject and be dependent on the judgement of the practitioner. Vaccination will of course generally be effected prior to primary infection with the pathogen against which protection is desired. C. Experimental Below are examples of specific embodiments for carrying out the present invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc), but some experimental error and deviation should, of course, be allowed for. Example 1 Two vaccine formulations were prepared as follows: formulation having an alum concentration of 0.6%. The two formulations were, dried using the techniques set out in Table 1 below: Freeze Drying: A Dura-Stop freeze dryer (FTS Systezn, Stone Ridge, NY) was used to freeze dry the alum-adsorbed HBsAg fomrulation based on the freeze-drying cycle in Table 2, Secondary Drying ramp at 1.0°Dmin to ST == 10°C, hold for 4 hours ramp at 1.0°C/niin to ST = 20'C, hold for 1-1 hours A vacuum of 100 mT (13-3 Pa) was maintained throughout primary and secondary drying. Spray-freeze-drying: Each suspension solution was sprayed into liquid nitrogen stirred in a stainless steel pain using an ultrasonic atomizer (Sono Tek Corporation, Milton, NY) with a nozzle frequency of 60 kHz. Sonic energy for atomization was set at 5.0 watts. Liquid feed was delivered hy a MasterFlex C/L peristaltic pump at 1.5 mL/mia, The pan containing frozen particles in liquid nitrogen was loaded into the Dura-lyophilizer pre-cooled to -50 °C and freeze-dried based on the condition of Table 3, Table 3: Freeze-drviag cycle Stage/Cycle Conditions Freezing pre-cool shelf temperature (ST) = -50°Cramp at 1.0°C/min to ST = -55°C, hold for 15 min wait for product temp (PT) = -48°C, hold for 120 min Primary Drying condenser/vacuum (CAO switched "on"when condenser temp, reaches -40 °C, vacuum pump tumeconwait for chamber vacuum to reach 150 mT (20.0 Pa)wait for foreline vacuum to reach 100 mT (13.3 Pa)ramp at l,0°C/min to ST = -25°C, hold for 18 hours Secondary Drying ramp at 1.0'°C/min to ST = 20°C, hold for 9 hours A vacuum of 200 mT (16.6 Pa) was maintained throughout primary and secondary drying. Compress/Grind/Sieve: The lyophilized material was rendered into particulate form using a compress, grind and sieve ("C/G/S") technique. More particularly, the lyophilized material was A study was carried out to assess the effect of alnm coagulation on the immunogenicity of alum-absorbed hepatitis B vaccine. As stated earlier, severe coagulation occurred when hepatitis B vaccine (containing alum) was dried by the "freeze-drying process, whereas spray-freeze-drying of hepatitis B vaccine did not cause coagulation, In this mouse experiment, the immunogemcity of freeze-dried and spray-freeze-dried hepatitis B vaccines were compared. Further, the immunogemcity of unsieved freee-dried vaccine and various sieved fractions ( Table 4: Experimental design of the mouse immunogenicitu study The larger particle fractions were less immunogenic than the smaller particle size fraction.. This clearly indicated that large size particles associated with coagulation had lost its Experiment 3: Effect of excipient and drying processes on the stability of spray-freeze-dried hepatitis B vaccine 42 in an ELISA- The antibody titers were determined by comparing to reference a serum. -,. Table 6: Experimental design of the mouse immunogenicity study compress/grind/sieve to generate particles with mean size of 20-38µm and 38-53 µm in diameter. The formulation information is summarised in Table 7. These particles do not have coagulation problems when reconstitated in water and examined under optical microscopy (data not shown). The immnnogenicity of spray-freeze-dried diphtheria-tetanus-toxoid vaccine was determined in guinea pigs (Charles River). Guinea pigs (4/group) were vaccinated on days 0 and 28 by administering powders to the abdominal skin using a powder delivery device. Each animal received 0,5 mg powders containing 1.5 Lf diphtheria toxoid and 1.5 Lf tetanus toxoid absorbed on 250µg of aluminum phosphate. Control animals were vaccinated with untreated vaccine by intramuscular injection using a.23 1/2 needle. Serum antibody responses to diphtheria toxoid and tetanus toxoid were measured in an ElIS A using sera collected on days 42. The results of the immunogenicity study are shown in Figure 5. Epidermal powder immunisation with spray-freeze-dried diphtheria toxoid absorbed on alum elicited antibody responses to each of the vaccine components and the tiers are comparable to that elicited by intramuscular injection of untreated vaccine. The size of the spray-freeze-dried powders did not appear to affect the immunogemcity significantly since these powders did not have coagulation problem in vivo. The smaller particle fraction of the spray-freeze dried formulation appears to have elicited slightly lower antibody titers to the diphtheria toxoid than the larger size fraction. This may reflect the relatively lower delivery efficiency for the smaller size fraction. This study again demonstrated that spray-freeze-drying process preserves the potency of alum-containing vaccine the dry solid dosage form. Accordingly, novel freeze spray-dried powder compositions and methods for producing these compositions have been described. Although preferred embodiments of the subject invention have be, described in some detail, it is understood that obvious variations can be made without departing from the spirit and the scope of the invention as defined by the appended claims. WE CLAIM 1. A gel-forming free-flowing powder suitable for use as a vaccine, said powder being obtainable by spray-drying or spray freeze-drying an aqueous suspension comprising: (a) from 0.1 to 0.95% by weight of an aluminum salt or calcium salt adjuvant having an antigen adsorbed therein; (b) from 0.5 to 6% by weight of a saccharide; (c) from 0.1 to 2% by weight of an amino acid or salt thereof; and (d) from 0.02 to 1% by weight of a colloidal substance. 2. A powder according to claim 1, wherein the adjuvant is aluminum hydroxide or aluminum phosphate. 3. A powder according to claim 1, wherem the adjuvant is aluminum sulfate or calcium phosphate. 4. A powder according to any one of the preceding claims, wherein the antigen is a bacterial or viral antigen. ' 5, A powder according to any one of the preceding claims, wherein the saccharide is a monosaccharide, disaccharide or sugar alcohol. 6, A powder according to any one of claims 1 to 4, wherein the saccharide is ; selected from the group consisting of glucose, xylose, galactose, fructose, D-mannose, sorbose, lactose, maltose, saccharose, trehalose, sucrose, mannitol, sorbitol, xyhtol, glycerin, glycerol, erythritol and arabitol. 7, A powder according to any one of the preceding claims, wherein the amino acid is an acidic, neutral or basic amino acid. 8, A powder according to any one of the preceding claims, wherein the amino acid or salt thereof is selected from the group consisting of glycine, alanine, glutamine, arginine, lysine, histidine and monosodium glutamate. 9, A powder according to any one of the preceding claims, wherein the colloidal substance is selected from the group consisting of polysaccharides, hydrogels and proteins. 10, A powder according to claim 9, wherein the said substance.is selected from the group consisting of dextran, maltodextran, gelatin, agarose and human serum albumin. 11, A powder according to any one of the preceding claims, wherein the aqueous suspension comprises from 0.2 to 0.4% by weight of the adjuvant having antigen adsorbed thereon, from 2 to 4% by weight of the saccharide, from 0.75 to 1.25% by weight of the amino acid or salt thereof and from 0.07 to 0.3% by weight of the colloidal substance. 12, A powder according to any one of the preceding claims, which comprises: (i) from 7 to 50% by weight of the adjuvant having an antigen* adsorbed therein, (ii) from 30 to 80% by weight of the saccharide, (iii) from 7 to 30% by weight of the amino acid or salt thereof, and (iv) from 0.8 to 6% by weigiht of the colloidal substance, 13, 'A powder according to any one of the preceding claims, having a mass mean aerodynamic diameter of from 10 to 100 µm and an envelope density of from 0,8 to 1.5 g/cm3 14, A powder according to any one of the preceding claims, which forms a gel-like suspension without any precipitate after having been added to distilled water (1:500 by weight) and shaken for 3 minutes. 15, A process for the preparation of a gel-forming free-flowing powder suitable for use as a vaccine, which process comprises spray-diying or spray freeze-drying an aqueous suspension comprising: (a) from 0.1 to 0.95% by weight of an aluminum salt or calcium salt adjuvant having an antigen adsorbed therein; . (b) from 0,5 to 6% by weight of a saccharide; (c) from 0.1 to 2% by weight of an amino acid or salt thereof; and (d) from 0.02 to 1% by weight of a colloidal substance. 16, A process according to claim 15- wherein the aqueous suspension comprises from 02 to 0.4% by weight of the adjuvant having antigen adsorbed thereon, from 2 to 4% by weight of the saccharide, from 0.75 to 1 ,l5% by weight of the amino acid or salt thereof and from 0.07 to 03% by weight of the colloidal substance. 17. A process according to claim 15 or 16, wherein the resultant powder forms a gel-like suspension without any precipitate after having been added to distilled water (1:500 by weight) and shaken for 3 minutes. 18. A dosage receptacle for a needleless syringe, said receptacle containing an effective amount of a gel-forming free-flowing powder as defined in any one of claims 1 to 14. 19. A receptacle according to claim 18, wherein the receptacle is selected from the group consisting of capsules, foil pouches, sachets and cassettes. 20. A needleless syringe which is loaded with a gel-forming free-flowing powder as defined in any one of claims 1 to 14, 21. A vaccine composition comprising a pharmaceutically acceptable carrier or diluent and a gel-forming free-flowing powder as defined in any one of claims 1 to 14- 22* A method of vaccinating a subject, which method comprises administering to the said subject an effective amount of a gel-forming free-flowing powder as defined in any one of claims 1 to 14, 23, A method according to claim 22, wherein the powder is administered by a needleless syringe. • 24, A method according to claim 22, wherein the powder is formulated with a pharmaceutically acceptable carrier or diluent. 25. A method according to claim 24, wherein the formulation is administered subcutaneously or intramuscularly. 26- A gel-forming free-flowing powder suitable for use as a vaccine, which powder comprises: .(i) from 5 to 60% by weight of an aluminum salt or calcium salt adjuvant having an antigen adsorbed thereon; (ii) from 25 to 90% by weight of a saccharide; (iii) from 4-5 to 40% by weight of an amino acid or salt thereof; and (iv) from 0.5 to 10% by weight of a colloidal substance, 27- A powder according to claim 26, which comprises: (i) from 7 to 50% by weight of the adjuvant having an antigen adsorbed therein, (ii) from 30 to 80% by weight of the saccharide, (iii) from 7 to 30% by weight of the amino acid or salt thereof and (iv) from 0.8 to 6% by weight of the colloidal substance, 28, A powder according to claim 26 or 27, which forms a gel-like suspension without any precipitate after having been added to distilled water (1:500 by weight) and shaken for 3 minutes. 29, A powder suitable for use as a vaccine, said powder being obtainable by spray freeze-drying an aqueous suspension comprising an aluminum salt or calcium salt adjuvant having an antigen adsorbed therein. 30, A powder according to claim 29, wherein the adjuvant is aluminum hydroxide, aluminum phosphate, aluminum sulfate or calcium phosphate, 3 L A powder according to claim 29 or 30, wherein the antigen is a bacterial or viral antigen, 32. A powder according to any one of claims 29 to 31, wherein the aqueous suspension comprises less than 10% by weight of the adjuvant having antigen adsorbed thereon. • 33. A powder according to any one of claims 29 to 32, having a mass mean aerodynamic diameter of from 1 to 100 nm and an envelope density of from 0.8 to 1.5 g/cm3. 34. A powder according to any one of claims 29 to 33, wherein the suspension further comprises an amorphous sugar, a crystalline sugar and optionally a polymer and/or an amino acid or a salt thereof. 35. A powder according to any one of claims 29 to 34, which forms a gel-like suspension without any precipitate after having been added to distilled water (1:500 by weight) and shaken for 3 minutes. 36. A process for the preparation of a powder suitable for use as a vaccine, which process comprises spray freeze-drying an aqueous suspension comprising an aluminum salt or calcium salt adjuvant having an antigen adsorbed therein. 37. A process according to claim 36, wherein the adjuvant is aluminium hydroxide, aluminum phosphate, aluminum sulfate or calcium phosphate. 38. A process according to claim 36 or 37, wherein the antigen is a bacterial or viral antigen. 39. A process according to anyone of claims 36 to 38, wherein the aqueous suspension comprises less than 10% by weight of the adjuvant having antigen adsorbed thereon 40. A process according to any one of claims 36 to 39, wherein the suspension further comprises an amorphous sugar, a crystalline sugar and optionally a polymer and/or an amino acid or a salt thereof. 41. A process according to any one of claims 36 to 40, wherein the resultant . spray freeze-dried powder forms a gel-like suspension without any precipitate after having been added to distilled water (1:500 by weight) and shaken for 3 minutes. 43. A dosage receptacle for a needleless syringe, said receptacle containing an effective amount of a powder as defined in any one of claims 29 to 35, 44. A receptacle according to claim 43, wherein the receptacle is selected from the group consisting of capsules, foil pouches, sachets and cassettes. 45. A needleless syringe which is loaded with a powder as defined in any one of claims 29 to 35. 46. A vaccine composition comprising a pharmaceutically acceptable carrier or diluent and a powder as defined in any one of claims 29 to 35. 47. A method of vaccinating a subject, which method comprises administering to the said subject an effective amount of a powder as defined in any one of claims 29 to 35, 48. A method according to claim 47, wherein the powder is administered by a needleless syringe, ' 49. A method according to claim 47, wherein the powder is formulated with a pharmaceutically acceptable carrier or diluent V ^ 50. A method according to claim 49, wherein the formulation is administered subcutaneously or intramuscularly. 51. A gel-forming free-flowing powder, substantially as hereinabove described and illustrated with reference to the accompanying drawings. 52. A process for the preparation of a powder, substantially as hereinabove described and illustrated with reference to the accompanying drawings.

Full Text

The invention relates to a process for the preparation of a powder suitable for vaccine and the powder prepared by thereof More specifically, the invention relates to vaccine compositions suitable for transdermal particle delivery from a needleless syringe system.
Background to the Invention
The ability to deliver pharmaceutical agents into and through skin surfaces (transdermal delivery) provides many advantages over oral or parenteral delivery techniques. In particular, transdermal delivery provides a safe, convenient and uoninvasive alternative to traditional administration systems, conveniently avoiding the major problems associated with oral delivery (e.g. variable rates of absorption and metabolism, gastroibtestinal irritation and/or bitter or unpleasant drug tastes) or parenteral delivery (e.g. needle pain, the risk of introducing infection to treated individuals, the risk of contamination or infection of health care workers caused by accidental needle-sticks and the disposal of used needles).
However, despite its clear advantages, transdermal delivery presents a number of its own inherent logistical problems. Passive delivery through intact skin necessarily entails the transport of molecules through a number of structurally different tissues, including the stratum comeum, the viable epidemiis, the papillary dermis and the capillary walls in order for the drug to gain entry into the blood or lymph system. Transdermal delivery systems must therefore be able to overcome the various resistances presented by each type of tissue.
In light of the above, a number of alternatives to passive transdermal delivery have been developed. These altematives include the use of skin penetration enhancing agents, or "permeation enhancers," to increase skin penneability, as well as non-chemical modes such as the use of iontophoresis, elcctroporation or ultrasound However, these alternative techniques often give rise to their own unique side effects such as skin irritation or sensitization. Thus, the spectrum of agents that can be safely and effectively administered

using traditional transdermal delivery methods has remained limited.
More recently, a novel transdermal drug delivery system that entails the use of a needleless syringe to fire powders (i.e., solid drug-containing particles) in controlled doses into and through intact skin has been described. In particular, commonly owned U.S. Patent No. 5,630,796 to Bellhouse et al describes a needleless syringe that delivers pharmaceutical particles entrained in a supersonic gas flow. The needleless syringe is used for transdennal delivery of powdered drug compounds and compositions, for delivery of genetic material into living cells (e.g., gene therapy) and for the delivery of biophannaceuticals to skin, muscle, blood or lymph. The needleless syringe can also be used in conjunction with surgery to deliver drugs and biologies to organ surfaces, solid tumors and/or to surgical cavities (e.g., tumor beds or cavities after tumor resection). In theory, practically any pharmaceutical agent that can be prepared in a substantially solid, particulate form can be safely and easily delivered using such devices.
One area of the pharmaceuticals field which is of particular interest for delivery via this new system is that of vaccine compositions. Suitable vaccines include those comprising an antigen adsorbed into a salt adjuvant. Such compositions are known in the art (see for example U.S.Patent No, 5,902,565) and are advantageous since the adjuvant branches the immunogenicity of the vaccine.
However, the storage and transportation of adjuvant vaccines is problematic. Commercial vaccine compositions containing salt adjuvants cannot be fixizen without causing damage to the vaccine. Further, one of the common storage techniques currently used for vaccines, fi-eeze-drying, is also unavailable for salt adjuvant containing compositions. Previous research has demonstrated that t freeze-drying causes the collapse of the gel structure of the vaccine composition, resulting in aggregation and precipitation of the adjuvant salt on resuspension in water (Warren et al, 1986, Annu. Rev. Immunol. 4: pages 369-388; Alving et al, Ann, N. Yi Acad. Sci. 690: pages 265-275). This is believed to be due to crystallisation of the water contained in the composition into large crystals on freezing and hence the concentration of the solute into specific regions, known as freeze concentrate regions. In the freeze concentrate regions, adjuvant salt particles are brought into close proximity and repulsive forces are overcome, thereby resulting in

coagulation. Once the salt has coagulated, the original suspension cannot be reproduced. This effect has been found to significantly reduce the immunogenicity of the vaccine, one report demonstrating a complete loss in immunogenicity of a freeze-dried alum-adsorbed hepatitis B surface antigen (HBsAg) after storage at 4'C for two years (Diminsky et al. Vaccine, 18: pages 3-17)-
An altemative method for storing adjuvant vaccine compositions is therefore required, which addresses the problems of aggregation associated with freeze-drying and which provides maximum retention of immunogenicity. Prolonged storage of vaccines is essential, both for use with the novel transdermal drug delivery systems mentioned above and also for use with conventional vaccination techniques. The provision of an effective altemative to freeze-drying is therefore of considerable commercial importance. It is also desired that the vaccine be produced in a form suitable for needleless injection. Needleless injection requires the vaccine composition to be in powder form, each particle having a suitable size and strength for transdermal delivery and being capable of forming a gel on
resuspension.
Alternatives to conventional freeze-drying techniques that have previously been reported include the incorporation of additives in the vaccine composition to improve the stability of an alum adjuvant, U.S. Patent No. 4,578-70 describes the addition of large amounts of both dextran and protein in order to achieve partial retention of the aluminum gel structure. This large addition of protein could however act to displace vaccine antigens from the aluminum gel and in addition would, in most cases, be immunogenic and as a result tend to swamp the immune response to the vaccine antigen.
EP-B-0130619 is also concerned with the addition of stabilisers to lyophilised, or freeze-dried, vaccine preparations. Lyophilised preparations of a hepatitis B vaccine .comprising an inactivated purified hepatitis B virus surface antigen absorbed an aluminum gel and stabiliser are described The stabiliser is composed of at least one amino acid or salt thereof, at least one saccharide and at least one colloidal substance. Very low concentrations of aluminum salt adjuvant are used, typically less than 0.1% by weight. However, this document relates only to the hepatitis B vaccine and does not disclose a generic process, which is non-immunogen-specific.

Spray-dried vaccine preparations comprising an immunogen adsorbed into an aluminum salt are disclosed in U,S, Patent No. 5,902,565, Immediate-release preparations are described which are prepared by spray-drying an aqueous suspension of aluminum salt-adsorbed immunogen. In the only Example, Example 1, in which such information is given, the resultant microspheres had a size range around 3 µ.m in diameter. According to U.S. Patent No. 5,902,565 the gel-forming nature of aluminum gels is completely retained during spray-drying even in the absence of any other materials which could exert a stabilising effect (apart from minimal quantities of vaccine antigen, typically 1 to 10 |ig/ml). Addition of water to the spray-dried powder was said to result in the instant formation of a typical gel, with sedimentation properties similar to the startmg material.
Summary of the Invention
We investigated whether a gel-forming spray-dried powder of an aluminum salt could indeed be formed as described in U.S. Patent No, 5,902,565, We found that spray drying a suspension of aluminum hydroxide or aluminum phosphate in water caused submicron particles of the aluminum salt to aggregate to larger particles in the resulting spray-dried powder. Upon reconstitution of this powder in water, these larger particles did not disintegrate into small particles. A gel suspension did not form. Rather, the aggregated particles of aluminum hydroxide or aluminum phosphate sedimented and precipitated out of the suspension.
Further experiments were carried out. We found that a suitable powder could be formed by spray-drying when an aluminum salt was utilised with a specific combination of other agents. Additionally, the aluminum salt and other agents needed to be used in specific proportions. We found too that the particular drying method used has a significant effect on the degree of coagulation of the adjuvant salt These investigations led to the finding that a powder suitable for needleless injection, and which substantially retained its gel structure on reconstitution in water, was obtainable by spray freeze-diying an alum adjuvant vaccine composition.
The spray freeze-drying method involves atomizing the suspended vaccine composition into liquid nitrogen. This process has two important effects: firstly, the liquid

nitrogen acts as a heat transfer agent and provides rapid freezing of the suspension; and secondly, the atomisation reduces the volume of each droplet to be frozen, further increasing the freezing rate. This combined effect causes extremely rapid freezing of very small droplets of suspension and leads to the formation of smaller ice crystals in the solid. The freeze concentrate regions which form during a standard freeze-drying technique are therefore significantly reduced in size. The rapid freezing of the particles, and their small size leads to powders having little or no aggregated adjuvant.
The present invention therefore provides simple, yet effective techniques that generate salt adjuvant-containing vaccine compositions in a powder form which is suitable for long-term storage. The vaccine compositions of the invention show substantially no aggregation on reconstitution and therefore immunogenicity is substantially retained. The compositions also have well-defined particle size, density and mechanical properties which collectively are suitable for powders for transdermal delivery from a needleless syringe.
The invention has the further, significant advantage that is suitable for use with a wide range of vaccine compositions and may well also be applicable to other pharmaceutical compositions, in particular where similar aggregation problems are encountered. As yet, the spray freeze-drying technique has been found to be entirely formulation independent within the field of adjuvant vaccine compositions.
Accordingly, the present invention provides a process for the preparation of a powder suitable for use as a vaccine, which process comprises spray freeze-drying an aqueous suspension comprising an aluminum salt or calcium salt adjuvant having an antigen adsorbed therein.
Freeze-flowing powder compositions suitable for vaccine use can thus be produced. The compositions have well-defined particle size, density and mechanical properties which collectively are suitable for powders for transdermal delivery from a needleless syringe. The invention further provides:

a powder suitable for use as a vaccine, said powder being obtained by a
process of the invention;
a dosage receptacle for a needleless syringe, said receptacle containing an
effective amount of powder of the invention;
a needleless syringe which is loaded with a powder of the invention;
a vaccine composition comprising a pharmaceutically acceptable carrier or
diluent and a powder of the invention; and
a gel-fonning free-flowing powder suitable for use as a vaccine, which
powder comprises:
(i) from 5 to 60% by weight of an aluminium salt or calcium salt
adjuvant having an antigen adsorbed thereon; (ii) from 25 to 90% by weight of a saccharride; (iii) from 4.5 to 40% by weight of an amino salt or salt thereof; and (iv) from 0.5 to 10% by weight of a colloidal substance.

IJ
Figure 1 shows the particle size distribution of an HBsAg adsorbed alum gel (i) before drying and (ii) after drying using a spray freeze-drying technique followed by reconstitution in water.
Figure 2 shows the particle size distribution of a second HBsAg adsorbed alum gel before drying and after drying via a conventional freeze drying method.
Figure 3 illustrates the results of an immunogenicity study using mice injected with HBsAg absorbed alum vaccine which had been dried by either spray freeze-drying (SFD) according to present invention, or using freeze-drying (FD). The FD powders were sieved into different size fractions and tested for immxmogenicity. Two SFD formulations, varying in alum contact, were tested.
Figure 4 illustrates the immunogenicity of three different spray freeze-dried powders in mice immunized by either intramuscular injection using a needle or epidermal powder immunization using a powder delivery device.
Figure 5 illustrates the immunogenicity of spray freeze-dried diphtheria-tetanus toxoid vaccine in guinea pigs. Spray freeze-dried powders of 20-38 µm and 38-53 µm in diameter were administered as a powder to the abdominal skin using a powder delivery device.
Detailed Description of the Preferred Embodiments
Before describing the present invention in detail, it is to be understood that this

invention is not limited to particularly exemplified compositions or process parameters as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting.
All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.
It must be noted that, as used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a particle" includes a mixture of two or more such particles, reference to "an excipient" includes mixtures of two or more such excipients, and the like.
A. Definitions
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commornly understood by one of ordinary skill in the art to which the invention pertains. Although a number of methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the preferred materials and methods are described herein,
In describing the present invention, the following terms will be employed, and are intended to be defined as indicated below. By "antigen" is meant a molecule which contains one or more epitopes that will stimulate a host's immune system to make a cellular antigen-specific immune response or a humoral antibody response. Thus, antigens include polypeptides including antigenic protein fragments, oligosaccharides, polysaccharides and the tike. Furthermore, the antigen can be derived from any known virus, bacterium, parasite, plant, protozoan or fungus, and can be a whole organism. The term also includes-tumor antigens. Similarly, an oligonucleotide or polynucleotide which expresses an antigen, such as in DNA immunization appHcations, is also included in the definition of an antigen. Synthetic antigens are also included, for example polyepitopes, flanking epitopes and other recombinant or synthetically derived antigens (Bergmann et al (1993) Eur. 1 Immunol 23:277T-2781; Bergmann et al (1996) J., Immunol 157:3242-3249; Suhrbier, A,

(1997) Immunol and Cell Biol 75:402-408; Gardner et aL (1998) 12^ World AIDS Conference, Geneva, Switzerland, June 28-July 3,1998).
The aduvants having antigen adsorbed thereon of the present invention, alone or in combination, are typically combined with one or more added materials such as carriers, vehicles, and/or excipients. "Carriers," "vehicles" and "excipients" generally refer to substantially iaert materials which are nontoxic and do not interact with other components of the composition in a deleterious manner. These materials can be used to increase the amount of solids in particulate pharmaceutical compositions. Examples of suitable carriers include water, silicone, gelatin, waxes, and like materials. Examples of normally employed "excipients," include pharmaceutical grades of carbohydrates including monosaccharides, disaccharides, cyclodextrans, and polysaccharides (e.g., dextrose, sucrose, lactose, trehalose, rafSnose, mannitol, sorbitol, inositol, dextrans, and maltodextrans); starch; cellulose; salts (e,g. sodium or calcium phosphates, calcium sulfate, magnesimn sulfate); citric acid; tartaric acid; glycine; high molecular weight polyethylene glycols (PEG); Pluronics; surfactants; and combinations thereof Generally, when carriers and/or excipients are used, they are used in amounts ranging from about 0.1 to 99 wt% of the pharmaceutical composition.
The term "powder' as used herein refers to a composition that consists of substantially solid particles that can be delivered transdermally using a needleless syringe device. The particles that make up the powder can be characterized on the basis of a number of parameters including, but not limited to, average particle size, average particle density, particle morphology (e.g. particle aerodynamic shape and particle surface characteristics) and particle penetration energy (P.E,).
The average particle size of the powders according to the present invention can vary widely and is generally from 0.1 to 250 µm, for example from 10 to 100 µmand more typically from 20 to 70 µm. The average'particle size of the powder can be measured as a mass mean aerodynamic diameter (MMAD) using conventional techniques such as microscopic techniques (where particles are sized directly and individually rather than grouped statistically), absorption of gases, permeability or time of flight If desired, automatic particle-size counters can be used (e.g. Aerosizer Counter, Coulter Counter,

KAC Counter, or Gelman Automatic Particle Counter) to ascertain the average particle size.
Actual particle density or "absolute density" can be readily ascertained using known quantification techniques such as helium pycnometry and the like. Alternatively, envelope ("tap") density measurements can be used to assess the density of a powder according to the invention. The envelope density of a powder of the invention is generally from 0.1 to 25 g/cm3, preferably from 0.8 to 1,5 g/cm3.
Envelope density information is particularly useful in characterizing the density of objects of irregular size and shape. Envelope density is the mass of an object divided by its volume, where the volume includes that of its pores and small cavities but excludes interstitial space. A number of methods of determining envelope density are known in the art, including wax immersion, mercury displacement, water absorption and apparent specific gravity techniques, A number of suitable devices are also available for determining envelope density, for example, the GeoPycTM Model 1360, available from the Micromeritics Instrument Corp, The difference between the absolute density and envelope density of a sample pharmaceutical composition provides information about the sample's percentage total porosity and specific pore volume.
Particle morphology, particularly the aerodynamic shape of a particle, can be readily assessed using standard light microscopy. It is preferred that the particles which make up the instant powders have a substantially spherical or at least substantially ellipti cal aerodynamic shape. It is also preferred that the particles have an axis ratio of 3 or less to avoid the presence of rod- or needle-shaped particles. These same microscopic techniques can also be used to assess the particle surface characteristics, e,g. the amount and extent of surface voids or degree of porosity.
Particle penetration energies can be ascertained using a number of conventional techniques, for example a metallized film P.E, test A metallized film material (e.g. a 125 µm polyester film having a 350 A layer of aluminum deposited on a single side) is used as a substrate into which the powder is fired from a needleless syringe (e.g, the needleless syringe described in U.S. Patent No. 5,630,796 to Bellhouse et al) at an initial velocity of

about 100 to 3000 m/sec. The metallized film is placed, with the metal-coated side facing upwards, on a suitable surface,
A needleless syringe loaded with a powder is placed with its spacer contacting the film, and then fired. Residual powder is removed from the metallized film surface using a suitable solvent. Penetration energy is then assessed using a BioRad Model GS-700 imaging densitometer to scan the metallised film, and a personal computer with a SCSI interface and loaded with MultiAnalyst software (BioRad) and Matlab software (Release 5.1, The MathWorks, Inc.) is used to assess the densitometer reading- A program is used to process the densitometer scans made using either the transmittance or reflectance method of the densitometer. The penetration energy of the spray-coated powders should be equivalent to, or better than that of reprocessed mannitol particles of the same size (mannitol particles that are freeze-dried, compressed, groimd and sieved according to the methods of commonly owned International Publication No. WO 97/48485, incorporated herein by reference).
The term "subject" refers to any member of the subphylum cordata including, without limitation, humans and other primates including non-hmnan primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs; birds, including domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like. The term does not denote a particular age. Thus, both adult and newbom individuals are intended to be covered. The methods described herein are intended for use in any of the above vertebrate species, since the immune systems of all of these vertebrates operate similarly.
The term "transdermal dehvery" includes both transdermal ("percutaneous") and transmucosal routes of administration, i.e, delivery by passage through the skin or mucosal tissue. SEE G,g., Transdermal Drug Delivery: Developmental Issues and Research Initiatives, Hadgraft and Guy (eds,). Marcel Dekker, Inc., (1989); Controlled Drug Delivery: Fundamentals and Applications, Robinson and Lee (eds.). Marcel Dekker

Retroviradae (e.g. HTLV-I, HTLV-II; HIV-1 and HIV-2); and simian immunodeficiency vims (SIV) among others.

Peptides: Analysis, Synthesis, Biology, supra, Vol. 1, for classical solution synthesis.
One or more saccharrides may be present in the aqueous suspension as component (b). The saccharride content is typically 1.5 to 5% by weight, preferably 2 to 4% by weight. The saccharride may be monosaccharide such as glucose, xylose, galactose, fructose, D-mannose or sorbose; a disaccharide such as lactose, maltose, saccharose, trehalose or sucrose; or a sugar alcohol such as mannitol, sorbitol, xylitol, glycerol, erythritol or arabitol.
One or more amino acids or amino acid salts is present in the aqueous suspension as component (c). Any physiologically acceptable amino acid salt may be employed. The salt may be an alkali or alkaline earth metal salt such as sodium, potassium or magnesium salt. The amino acid may be an acidic, neutral or basic amino acid. Suitable amino acids are glycine, alanine, glutamine, arginine, lysine and histidine. Monosodium glutamate is a suitable amino acid salt. The aqueous suspension generally contains from 0.5 to 1.5% by weight, more preferably from 0.75 to L25 by weight, of the amino acid and/or amino acid salt.
The colloidal substance (d) is a divided substance incapable of passing through a semi-permeable membrane, comprised of fine particles which, in suspension or solution, fail to settle out. Suitable colloidal substances axe disclosed in EP-B-0130619, Component (d) may be selected from polysaccharides such as dextran or maltodextran; hydrogels such as gelatine or agarose; or proteins such as human serum albumin. The substance may have a molecular weight of 500 to 80,000 or higher, for example from 1000 or 2000 to 30,000 or from 5,000 to 25,000, Component (d) is generally present in the aqueous suspension in an amount of from 0.05 to 0.5% by weight, preferably from 0.07 to 0.3% by weight.
The adjuvant having antigen adsorbed thereon and the saccharide, amino acid or salt thereof and colloidal substance are suspended in water. The aqueous suspension is spray freeze-dried. The spray freeze-drying conditions are selected to enable the desired particles to be produced. The air inlet temperature, air outlet temperature, feed rate of the aqueous suspension, air flow rate, etc, can thus be varied as desired. Any suitable spray-drier may be used. The nozzle size may vary as necessary. Particular spray freeze-drying conditions are described in more detail below.

A gel-forming free-flowing powder can thus be provided which is suitable for use as a vaccine. The proportions of the various components of the powder can be adjusting by adjusting the composition of the suspension that is spray freeze-dried. However, the powder typically comprises or, in some embodiments, consists essentially of:
(i) from 5 to 60%, for example from 7 to 50% such as from 10 to 30%, by weight of an aluminium salt or calcium salt adjuvant having an antigen adsorbed thereon; (ii) from 25 to 90%, for example from 30 to 80% such as from 40 to 70% by
weight of a saccharide; (iii) from 4.5 to 40%, for example from 7 to 30% such as from 10 to 20% by
weight of an amino acid or salt thereof; and (iv) from 0.5 to 10%, for example from 0.8 to 6% such as from 1 to 3%, by weight of a colloidal substance. The invention is concerned generally with powders suitable for use as vaccines that are formed by spray freeze-drying an aqueous suspension comprising an aluminum salt or calcium salt adjuvant having an antigen adsorbed therein. Such powders are suitable for transdermal administration from a needleless syringe deUvery system. As such, the particles which make up the powdered composition must have sufficient physical strength to withstand sudden acceleration of up to several times the speed of sound and the impact with, and passage through, the skin and tissue.
Preferably, the aqueous suspension, prior to spray freeze-drying, contains less than 10% by weight, for instance less than 5% weight and preferably less than 3% by weight, of the salt adjuvant having antigen adsorbed thereon. The aqueous suspension typically contains at least 0.05% by weight, for instance at least 0.1 % by weight or at least 0.6% by weight, of the adjuvant having antigen adsorbed thereon. More preferably, the suspension contains from 0,2 or 0.3 to 0.6%, 0.75% or 1% by weight, preferably from 0.2 to 0.4% by weight, of adjuvant onto which antigen is adsorbed. At concentrations above about 10%, by weight of adjuvant salt, the aqueous suspension becomes highly viscous. This limits the ability to atomize the suspension.

It should be understood that the preferred upper limit of adjuvant concentration applies to the aqueous suspension prior to spray freeze-drying. The content of adjuvant salt having antigen adsorbed thereon may be as high as 50% by weight or more in the spray freeze-dried powders of the invention.
The adjuvant is generally an aluminum salt, for example aluminum hydroxide or aluminum phosphate. Alternatively, the adjuvant salt may be aluminum sulfate or calcium phosphate.
Again, any suitable antigen as defined herein may be employed. The antigen may be a viral antigen. The antigen may therefore be derived from members of the families Picomaviridae (e.g. poliovinises, etc.); Caliciviridae; Togaviridae (e.g. rubella virus, dengue virus, etc.); Flaviviridae; Coronaviridae; Reoviridae; Bimaviridae; Rhabodoviridae (e.g. rabies virus, etc.); Filovuidae; Paramyxoviridae (e.g, mumps virus, measles virus, respiratory syncytial virus, etc.); Orthomyxoviridae (e.g. influenza virus types A, B and C, etc); Bunyaviridae; Arenaviridae; Retroviradae (e.g. HTLV-I; HTLV-II; HIV-l and HIV-2); and simian immunodeficiency vims (SIV) among others.
Alternatively, viral antigens may be derived from papillomavirus (e.g. HPV); a herpesvirus; a hepatitis virus, e.g. hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C (HCV), the delta hepatitis virus (HDV), hepatitis E virus (HEV) or hepatitis G. virus (HGV); and the tick-bome encephalitis viruses. See, e.g. Virology, 3rd Edition (WX. Joklik ed. 1988); Fundamental Virology, 2nd Edition (B,N, Fields andD.M. Knipe, eds. 1991) for a description of these viruses.
Bacterial antigens for use in the invention can be derived from organisms that cause diphtheria, cholera, tuberculosis, tetanus, pertussis, meningitis and other pathogenic states, including, e.g,. Meningococcus A, B and C, Hemophilus influenza type B (HIB),
Helicobacter pylori. Vibrio cholerae, Escherichia coli, Campylobacter, Shigella, Salmonella, Streptococcus sp, and Staphylococcus sp. A combination of bacterial antigens may be provided, for example diphtheria, pertussis and tetanus antigens. Suitable pertussis antigens are pertussis toxin and/or filamentous haemagglutinin and/or pertactin, alternatively teraied P69. An anti-parasitic antigen may be derived from organisms causing malaria and Lyme disease.

disaccharides such as lactose, maltose, saccharose, trehalose or sucrose, sugar alcohols such as mannitol, sorbitol, xylitol, glycerol, erythritol or arabitol, polymers such as dextran, starch, cellulose or high molecular weight polyethylene glycols (PEG), amino acids or their salts, such as glycine, alanine, glutamine, arginine, lysine or histidine or their salts with alkali or alkaline earth metals such as a sodium, potassium or magnesium salts, or sodium or calcium phosphates, calcium carbonate, calcium sulfate, sodium citrate, citric acid, tartaric acid, and combinations thereof. Suitable solvents include, but are not limited to, methylene chloride, acetone, methanol, ethanol, isopropanol and water. Typically, water is used as the solvent. Generally pharmaceutically acceptable salts having molarities ranging from about 1 mM to 2M can be used. Pharmaceutically acceptable salts include, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like, A thorough discussion of pharmaceutically acceptable excipients, vehicles and auxiliary substances is available in REMINGTON'S PHAEMACEUTICAL SCIENCES (Mack Pub. Co., NJ. 1991), incorporated herein by reference.
Preferred excipients for use in the aqueous suspension include saccharides, amino acids or salts thereof and polymers. Typically, the suspension contains one or more saccharides, such as a combination of mannitol and trehalose. Saccharides are typically present in an amount of from 0,5 to 30% by weight. An amino salt, such as arginine glutamate or aspartate in an amount of from 0.1 to 30% by weight, and/or a polymer, such as dextran, in an amount of from 0 to 30% may also be included, typically in an amount of from 0 to 30 % by weight. Typical excipient combinations include one or more saccharides and a polymer and include substantially no amino salt The total amount of excipients present in the aqueous suspension is typically from 0 to 50%, more preferably from 10 to 30%.
The particles of the invention are formed by first suspending the adjuvant having an antigen adsorbed therein, and any required additives, in water. The aqueous suspension is then spray freeze-dried. Any known technique in the art (for example the methods described by Mumenthaler ef al, Int J, Pharmaceutics (1991) 72, pages 97-110 and Maa et al, Phar. Res. (1999) VoL 16, page 249) may be used to carry out the spray freeze-

. panicles to mnimally surgical sites

containing a suitable dosage of the powder of the invention. The loaded syiringe can be packaged in a hermetically sealed container, which may further be labeled as described above.

test is similar to that used in the metallized film test. The depth of penetration is measured
using precision calipers. For each experiment a processed mannitol standard is run as
comparison and all other parameters such as device pressure, particle size range, etc., are
held constant. Data also show this method to be sensitive to differences in particle size and
pressure. Processed mannitol standard as an excipient for drugs has been proven to
deliver systemic concentrations in preclinical experiments, so the relative performance
measure in the foam penetration test has a practical in vivo foundation. Promising
powders can be expected to show equivalent or better penetration to mannitol for
anticipation of adequate performance in preclinical or clinical studies. This simple, rapid
-1.
test has value as a relative method of evaluation of powders and is not intended to be considered in isolation.
Particle Attrition Test
A further indicator of particle performance is to test the ability of various candidate compositions to withstand the forces associated with high-velocity particle injection techniques, that is, the forces from contacting particles at rest with a sudden, high velocity gas flow, the forces resulting from particle-to-particle impact as the powder travels through the needleless syringe, and the forces resulting from pardcle-to-device collisions also as the powder travels through the device. Accordingly, a simple particle attrition test has been devised which measures the change in particle size distribution between the initial composition, and the composition after having been delivered from a needleless syringe device.
The test is conducted by loading a particle composition into a needleless syringe as described above, and then discharging the device into a flask containing a carrier fluid in .which the particular composition is not soluble (e.g,, mineral oil, silicone oil, etc.). The carrier fluid is then collected, and particlq size distribution in both the initial compositior] and the discharged composition is calculated using a suitable particle sizing apparatus, e,g an AccuSizer® model 780 Optical Particle Sizex. Compositions that demonstrate less than about 50%, more preferably less than about 20% reduction in mass mean diameter (as

determined by one of skill in the art.

Dosage treatment may be a single dose schedule or a multiple dose schedule. A multiple dose schedule is one in which a primary course of vaccination may be with 1-10 separate doses, followed by other doses given at subsequent time intervals, chosen to maintain and/or reinforce the immune response, for example at 1-4 months for second dose and, if needed, a subsequent dose(s) after several months. The dosage regimen will also, at least in part, be determined by the need of the subject and be dependent on the judgement of the practitioner. Vaccination will of course generally be effected prior to primary infection with the pathogen against which protection is desired.
C. Experimental
Below are examples of specific embodiments for carrying out the present invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.
Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc), but some experimental error and deviation should, of course, be allowed for.

Example 1
Two vaccine formulations were prepared as follows:

formulation having an alum concentration of 0.6%.

The two formulations were, dried using the techniques set out in Table 1 below:

Freeze Drying:
A Dura-Stop freeze dryer (FTS Systezn, Stone Ridge, NY) was used to freeze dry the alum-adsorbed HBsAg fomrulation based on the freeze-drying cycle in Table 2,

Secondary Drying

ramp at 1.0°Dmin to ST == 10°C, hold for 4 hours ramp at 1.0°C/niin to ST = 20'C, hold for 1-1 hours

A vacuum of 100 mT (13-3 Pa) was maintained throughout primary and secondary
drying.
Spray-freeze-drying:
Each suspension solution was sprayed into liquid nitrogen stirred in a stainless steel pain using an ultrasonic atomizer (Sono Tek Corporation, Milton, NY) with a nozzle frequency of 60 kHz. Sonic energy for atomization was set at 5.0 watts. Liquid feed was delivered hy a MasterFlex C/L peristaltic pump at 1.5 mL/mia, The pan containing frozen particles in liquid nitrogen was loaded into the Dura-lyophilizer pre-cooled to -50 °C and freeze-dried based on the condition of Table 3,
Table 3: Freeze-drviag cycle

Stage/Cycle Conditions
Freezing pre-cool shelf temperature (ST) = -50°Cramp at 1.0°C/min to ST = -55°C, hold for 15 min
wait for product temp (PT) = -48°C, hold for 120 min
Primary Drying condenser/vacuum (CAO switched "on"when condenser temp, reaches -40 °C, vacuum pump tumeconwait for chamber vacuum to reach 150 mT (20.0 Pa)wait for foreline vacuum to reach 100 mT (13.3 Pa)ramp at l,0°C/min to ST = -25°C, hold for 18 hours
Secondary Drying ramp at 1.0'°C/min to ST = 20°C, hold for 9 hours
A vacuum of 200 mT (16.6 Pa) was maintained throughout primary and secondary
drying.

Compress/Grind/Sieve:
The lyophilized material was rendered into particulate form using a compress, grind and sieve ("C/G/S") technique. More particularly, the lyophilized material was

A study was carried out to assess the effect of alnm coagulation on the immunogenicity of alum-absorbed hepatitis B vaccine. As stated earlier, severe coagulation occurred when hepatitis B vaccine (containing alum) was dried by the "freeze-drying process, whereas spray-freeze-drying of hepatitis B vaccine did not cause coagulation, In this mouse experiment, the immunogemcity of freeze-dried and spray-freeze-dried hepatitis B vaccines were compared. Further, the immunogemcity of unsieved freee-dried vaccine and various sieved fractions (
Table 4: Experimental design of the mouse immunogenicitu study

The larger particle fractions were less immunogenic than the smaller particle size fraction..
This clearly indicated that large size particles associated with coagulation had lost its

Experiment 3: Effect of excipient and drying processes on the stability of spray-freeze-dried hepatitis B vaccine

42 in an ELISA- The antibody titers were determined by comparing to reference a serum. -,.

Table 6: Experimental design of the mouse immunogenicity study

compress/grind/sieve to generate particles with mean size of 20-38µm and 38-53 µm in diameter. The formulation information is summarised in Table 7. These particles do not have coagulation problems when reconstitated in water and examined under optical microscopy (data not shown).

The immnnogenicity of spray-freeze-dried diphtheria-tetanus-toxoid vaccine was determined in guinea pigs (Charles River). Guinea pigs (4/group) were vaccinated on days 0 and 28 by administering powders to the abdominal skin using a powder delivery device. Each animal received 0,5 mg powders containing 1.5 Lf diphtheria toxoid and 1.5 Lf tetanus toxoid absorbed on 250µg of aluminum phosphate. Control animals were vaccinated with untreated vaccine by intramuscular injection using a.23 1/2 needle. Serum antibody responses to diphtheria toxoid and tetanus toxoid were measured in an ElIS A using sera collected on days 42.
The results of the immunogenicity study are shown in Figure 5. Epidermal powder immunisation with spray-freeze-dried diphtheria toxoid absorbed on alum elicited antibody responses to each of the vaccine components and the tiers are comparable to that elicited by intramuscular injection of untreated vaccine. The size of the spray-freeze-dried powders did not appear to affect the immunogemcity significantly since these powders did not have coagulation problem in vivo. The smaller particle fraction of the spray-freeze dried formulation appears to have elicited slightly lower antibody titers to the diphtheria toxoid than the larger size fraction. This may reflect the relatively lower delivery efficiency for the smaller size fraction. This study again demonstrated that spray-freeze-drying process preserves the potency of alum-containing vaccine the dry solid dosage form.

Accordingly, novel freeze spray-dried powder compositions and methods for producing these compositions have been described. Although preferred embodiments of the subject invention have be, described in some detail, it is understood that obvious variations can be made without departing from the spirit and the scope of the invention as defined by the appended claims.

WE CLAIM
1. A gel-forming free-flowing powder suitable for use as a vaccine, said
powder being obtainable by spray-drying or spray freeze-drying an aqueous suspension
comprising:
(a) from 0.1 to 0.95% by weight of an aluminum salt or calcium salt adjuvant having an antigen adsorbed therein;
(b) from 0.5 to 6% by weight of a saccharide;
(c) from 0.1 to 2% by weight of an amino acid or salt thereof; and
(d) from 0.02 to 1% by weight of a colloidal substance.

2. A powder according to claim 1, wherein the adjuvant is aluminum hydroxide or aluminum phosphate.
3. A powder according to claim 1, wherem the adjuvant is aluminum sulfate or calcium phosphate.
4. A powder according to any one of the preceding claims, wherein the
antigen is a bacterial or viral antigen.
' 5, A powder according to any one of the preceding claims, wherein the saccharide is a monosaccharide, disaccharide or sugar alcohol.
6, A powder according to any one of claims 1 to 4, wherein the saccharide is ; selected from the group consisting of glucose, xylose, galactose, fructose, D-mannose, sorbose, lactose, maltose, saccharose, trehalose, sucrose, mannitol, sorbitol, xyhtol, glycerin, glycerol, erythritol and arabitol.
7, A powder according to any one of the preceding claims, wherein the amino acid is an acidic, neutral or basic amino acid.
8, A powder according to any one of the preceding claims, wherein the amino acid or salt thereof is selected from the group consisting of glycine, alanine, glutamine, arginine, lysine, histidine and monosodium glutamate.
9, A powder according to any one of the preceding claims, wherein the colloidal substance is selected from the group consisting of polysaccharides, hydrogels and proteins.
10, A powder according to claim 9, wherein the said substance.is selected

from the group consisting of dextran, maltodextran, gelatin, agarose and human serum albumin.
11, A powder according to any one of the preceding claims, wherein the aqueous suspension comprises from 0.2 to 0.4% by weight of the adjuvant having antigen adsorbed thereon, from 2 to 4% by weight of the saccharide, from 0.75 to 1.25% by weight of the amino acid or salt thereof and from 0.07 to 0.3% by weight of the colloidal substance.
12, A powder according to any one of the preceding claims, which comprises: (i) from 7 to 50% by weight of the adjuvant having an antigen* adsorbed
therein, (ii) from 30 to 80% by weight of the saccharide, (iii) from 7 to 30% by weight of the amino acid or salt thereof, and (iv) from 0.8 to 6% by weigiht of the colloidal substance,
13, 'A powder according to any one of the preceding claims, having a mass
mean aerodynamic diameter of from 10 to 100 µm and an envelope density of from 0,8 to
1.5 g/cm3
14, A powder according to any one of the preceding claims, which forms a gel-like suspension without any precipitate after having been added to distilled water (1:500 by weight) and shaken for 3 minutes.
15, A process for the preparation of a gel-forming free-flowing powder suitable for use as a vaccine, which process comprises spray-diying or spray freeze-drying an aqueous suspension comprising:
(a) from 0.1 to 0.95% by weight of an aluminum salt or calcium salt adjuvant
having an antigen adsorbed therein; . (b) from 0,5 to 6% by weight of a saccharide;
(c) from 0.1 to 2% by weight of an amino acid or salt thereof; and
(d) from 0.02 to 1% by weight of a colloidal substance.
16, A process according to claim 15- wherein the aqueous suspension
comprises from 02 to 0.4% by weight of the adjuvant having antigen adsorbed thereon,
from 2 to 4% by weight of the saccharide, from 0.75 to 1 ,l5% by weight of the amino

acid or salt thereof and from 0.07 to 03% by weight of the colloidal substance.
17. A process according to claim 15 or 16, wherein the resultant powder forms a gel-like suspension without any precipitate after having been added to distilled water (1:500 by weight) and shaken for 3 minutes.
18. A dosage receptacle for a needleless syringe, said receptacle containing an effective amount of a gel-forming free-flowing powder as defined in any one of claims 1 to 14.
19. A receptacle according to claim 18, wherein the receptacle is selected
from the group consisting of capsules, foil pouches, sachets and cassettes.
20. A needleless syringe which is loaded with a gel-forming free-flowing
powder as defined in any one of claims 1 to 14,
21. A vaccine composition comprising a pharmaceutically acceptable carrier or diluent and a gel-forming free-flowing powder as defined in any one of claims 1 to 14-
22* A method of vaccinating a subject, which method comprises administering to the said subject an effective amount of a gel-forming free-flowing powder as defined in any one of claims 1 to 14,
23, A method according to claim 22, wherein the powder is administered by a needleless syringe.
• 24, A method according to claim 22, wherein the powder is formulated with a pharmaceutically acceptable carrier or diluent.
25. A method according to claim 24, wherein the formulation is administered subcutaneously or intramuscularly.
26- A gel-forming free-flowing powder suitable for use as a vaccine, which
powder comprises:
.(i) from 5 to 60% by weight of an aluminum salt or calcium salt adjuvant
having an antigen adsorbed thereon; (ii) from 25 to 90% by weight of a saccharide; (iii) from 4-5 to 40% by weight of an amino acid or salt thereof; and (iv) from 0.5 to 10% by weight of a colloidal substance,
27- A powder according to claim 26, which comprises:

(i) from 7 to 50% by weight of the adjuvant having an antigen adsorbed
therein, (ii) from 30 to 80% by weight of the saccharide, (iii) from 7 to 30% by weight of the amino acid or salt thereof and (iv) from 0.8 to 6% by weight of the colloidal substance,
28, A powder according to claim 26 or 27, which forms a gel-like suspension without any precipitate after having been added to distilled water (1:500 by weight) and shaken for 3 minutes.
29, A powder suitable for use as a vaccine, said powder being obtainable by spray freeze-drying an aqueous suspension comprising an aluminum salt or calcium salt
adjuvant having an antigen adsorbed therein.
30, A powder according to claim 29, wherein the adjuvant is aluminum
hydroxide, aluminum phosphate, aluminum sulfate or calcium phosphate,
3 L A powder according to claim 29 or 30, wherein the antigen is a bacterial or viral antigen,
32. A powder according to any one of claims 29 to 31, wherein the aqueous suspension comprises less than 10% by weight of the adjuvant having antigen adsorbed thereon.
• 33. A powder according to any one of claims 29 to 32, having a mass mean aerodynamic diameter of from 1 to 100 nm and an envelope density of from 0.8 to 1.5 g/cm3.
34. A powder according to any one of claims 29 to 33, wherein the suspension further comprises an amorphous sugar, a crystalline sugar and optionally a polymer and/or an amino acid or a salt thereof.
35. A powder according to any one of claims 29 to 34, which forms a gel-like suspension without any precipitate after having been added to distilled water (1:500 by weight) and shaken for 3 minutes.
36. A process for the preparation of a powder suitable for use as a vaccine, which process comprises spray freeze-drying an aqueous suspension comprising an aluminum salt or calcium salt adjuvant having an antigen adsorbed therein.

37. A process according to claim 36, wherein the adjuvant is aluminium hydroxide, aluminum phosphate, aluminum sulfate or calcium phosphate.
38. A process according to claim 36 or 37, wherein the antigen is a bacterial or viral antigen.
39. A process according to anyone of claims 36 to 38, wherein the aqueous suspension comprises less than 10% by weight of the adjuvant having antigen adsorbed thereon
40. A process according to any one of claims 36 to 39, wherein the suspension further comprises an amorphous sugar, a crystalline sugar and optionally a polymer and/or an amino acid or a salt thereof.
41. A process according to any one of claims 36 to 40, wherein the resultant . spray freeze-dried powder forms a gel-like suspension without any precipitate after having been added to distilled water (1:500 by weight) and shaken for 3 minutes.

43. A dosage receptacle for a needleless syringe, said receptacle containing an effective amount of a powder as defined in any one of claims 29 to 35,
44. A receptacle according to claim 43, wherein the receptacle is selected from the group consisting of capsules, foil pouches, sachets and cassettes.
45. A needleless syringe which is loaded with a powder as defined in any one of claims 29 to 35.
46. A vaccine composition comprising a pharmaceutically acceptable carrier or diluent and a powder as defined in any one of claims 29 to 35.
47. A method of vaccinating a subject, which method comprises administering to the said subject an effective amount of a powder as defined in any one of claims 29 to 35,
48. A method according to claim 47, wherein the powder is administered by a
needleless syringe, '
49. A method according to claim 47, wherein the powder is formulated with a pharmaceutically acceptable carrier or diluent

V ^

50. A method according to claim 49, wherein the formulation is administered subcutaneously or intramuscularly.

51. A gel-forming free-flowing powder, substantially as hereinabove described
and illustrated with reference to the accompanying drawings.
52. A process for the preparation of a powder, substantially as hereinabove
described and illustrated with reference to the accompanying drawings.

Documents:

in-pct-2002-2012-che-abstract.pdf

in-pct-2002-2012-che-assignement.pdf

in-pct-2002-2012-che-claims filed.pdf

in-pct-2002-2012-che-claims granted.pdf

in-pct-2002-2012-che-correspondnece-others.pdf

in-pct-2002-2012-che-correspondnece-po.pdf

in-pct-2002-2012-che-description(complete)filed.pdf

in-pct-2002-2012-che-description(complete)granted.pdf

in-pct-2002-2012-che-drawings.pdf

in-pct-2002-2012-che-form 1.pdf

in-pct-2002-2012-che-form 26.pdf

in-pct-2002-2012-che-form 3.pdf

in-pct-2002-2012-che-form 5.pdf

in-pct-2002-2012-che-other documents.pdf

in-pct-2002-2012-che-pct.pdf

« Previous Patent

Next Patent »

Patent Number

212232

Indian Patent Application Number

IN/PCT/2002/2012/CHE

PG Journal Number

07/2008

Publication Date

15-Feb-2008

Grant Date

26-Nov-2007

Date of Filing

05-Dec-2002

Name of Patentee

M/S. POWDERJECT VACCINES, INC

Applicant Address

585 Science Drive, Madison, WISCONSIN 53711

Inventors:

#	Inventor's Name	Inventor's Address
1	MAA, Yuh-Fun	6511 Dumbarton Circle Fremont, CA 94555

PCT International Classification Number

A61K 9/00

PCT International Application Number

PCT/US2001/018494

PCT International Filing date

2001-06-08

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	60/210,581	2000-06-08	U.S.A.