CN1438874A - Powder compositions - Google Patents

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.

Description

powder composition

technical field

The present invention relates to vaccine compositions. More specifically, the present invention relates to vaccine compositions suitable for transdermal particle administration by needle-free injectors.

Background technique

Administration of an agent into and across the skin surface (transdermal administration) has several advantages over oral or parenteral administration. Specifically, transdermal drug delivery is a safe, convenient, and infection-free alternative to traditional drug delivery, which conveniently avoids major problems associated with oral drug delivery (e.g., uncertain rates of adsorption and metabolism). , irritation to the gastrointestinal tract, and/or bitter or unpleasant taste of the drug) or major problems associated with parenteral administration (e.g., needle sting, risk of infection to the patient, when health care workers do not Be aware of the risk of infection or infection from needle sticks and disposal of discarded needles).

However, despite its obvious advantages, transdermal delivery has a number of inherent associated problems. Passive drug delivery without damaging the skin must, of course, transport molecules across many structurally diverse tissues, such as the stratum corneum, living epidermis, dermal papilla, and capillary walls, allowing the drug to enter the blood or lymphatic system. Therefore, the way of transdermal drug delivery must overcome the barriers formed by various tissues.

For the above reasons, various alternative methods of passive transdermal drug delivery have been developed. These alternatives include the use of agents that enhance skin penetration, "penetration enhancers," used to increase skin penetration, and non-chemical methods such as iontophoresis, electroporation, or ultrasound. However, these alternatives often come with their own unique side effects, such as skin irritation or allergies. Therefore, the range of agents that can be safely and effectively administered by conventional transdermal delivery methods remains limited.

Recently, a new transdermal drug delivery device has been reported, which can inject a dose of powder (ie, drug-containing solid particles) through the skin using a needle-free syringe without damaging the skin. Specifically, US Patent No. 5,630,796 to co-owner Bellhouse et al. describes a needle-free injector that delivers drug particles by means of an ultrasonic stream of air. Uses of needle-free injectors include: transdermal delivery of powdered pharmaceutical compounds and compositions; delivery of genetic material into living cells (eg, gene therapy); and delivery of biological agents into the skin, muscle, blood or lymphatic system. Needle-free injectors can also be used in surgery to deliver drugs and biologics to organ surfaces, hardened tumors, and/or surgical cavities (eg, tumor beds or tumor cavities left after tumor resection). In theory, virtually any medicament that can be formulated as solid solid particles can be safely and conveniently administered using such devices.

Vaccine compositions that can be administered using this novel device are of particular interest in the field of medicine. Suitable vaccines include those containing antigen adsorbed by a saline adjuvant. Such compositions are known in the art (eg, see US Patent No. 5,902,565) and are advantageous because adjuvants enhance the immunogenicity of vaccines.

However, storage and transportation of adjuvanted vaccines presents problems. Commercial vaccine compositions containing salt adjuvants should not be frozen to avoid damage to the vaccine. Moreover, freeze-drying, one of the currently commonly used vaccine storage techniques, cannot be used for compositions containing salt adjuvants. Previous studies have shown that lyophilization causes the gel structure of vaccine compositions to collapse, leading to aggregation and precipitation of adjuvant salts upon resuspension in water (Warren et al., 1986, Annu. Rev. Immunol. 4: pp. 369-388; Alving et al., Ann. N.Y. Acad. Sci. 690: pp. 265-275). It is believed that this is due to the fact that the water contained in the composition freezes and crystallizes into large crystals, whereby the solutes are concentrated in certain specific regions, the so-called freeze-agglutination regions. In the freeze-agglutination zone, the adjuvant salt particles are brought into such close proximity that the repulsive forces between them are overcome and agglomerates are formed. Once the salt has coagulated, it cannot be restored to the original suspension. Agglomeration has been found to significantly reduce the immunogenicity of the vaccine. It has been reported that lyophilized alum-adsorbed hepatitis B surface antigen (HBsAg) completely lost its immunogenicity when stored at 4°C for 2 years (Diminsky et al., Vaccine, 18: 3-17) .

There is therefore a need for alternative methods of storing adjuvanted vaccine compositions that address the aggregation problems associated with lyophilization and that maximize immunogenicity. Whether for the above-mentioned new transdermal drug delivery device or for the application of traditional immunization techniques, prolonging the storage time of vaccines is of great significance. Therefore, it is of great commercial value to provide effective methods that can replace lyophilization. In addition, it would be desirable to prepare vaccines in a form suitable for needle-free injection. Needle-free injection technology requires that the vaccine composition be in powder form, each particle has a particle size and strength suitable for transdermal administration, and that the vaccine composition can form a gel when resuspended.

According to previous reports, the replacement technology of the traditional freeze-drying method includes adding adjuvants to the vaccine composition to increase the stability of the alum adjuvant. US Patent No. 4,578,270 describes adding large amounts of dextran and protein to partially maintain the structure of the aluminum gel. However, the large amount of added protein displaces the vaccine antigen from the aluminum colloid and is immunogenic in most cases, thus potentially masking the immune response to the vaccine antigen.

EP-B-0130619 also relates to the addition of stabilizers to lyophilized ie freeze-dried vaccine formulations. This patent describes a lyophilized hepatitis B vaccine formulation containing purified, inactivated HBV surface antigen adsorbed to an aluminum gel and a stabilizer. The stabilizer consists of at least one amino acid or a salt thereof, at least one sugar and at least one colloidal substance. This patent uses very low concentrations of aluminum salt adjuvants, typically less than 0.1% by weight. However, this document only relates to hepatitis B vaccine, and does not disclose a general non-specific immunization method.

US Patent No. 5,902,565 discloses spray-dried vaccine formulations containing immunogens adsorbed by aluminum salts. This patent describes an immediate release formulation prepared by spray drying an aqueous suspension of the immunogen adsorbed by an aluminum salt. This information is given in its sole Example 1, where the obtained microspheres have a particle size of approximately 3 μm. According to U.S. Patent No. 5,902,565, during the spray-drying process, even in the absence of any other stabilizing substances (except for a minimal amount of vaccine antigen, which is typically 1-10 μg/ml), Aluminum colloids completely retain their colloid-forming properties. Addition of water to the spray-dried powder is said to immediately form a typical colloid with precipitation characteristics similar to the starting material.

Contents of the invention

We investigated whether it was indeed possible to form a gel-forming aluminum salt spray-dried powder as described in US Patent No. 5,902,565. We have found that spray drying an aqueous suspension of aluminum hydroxide or aluminum phosphate yields a powder in which submicron particles of the aluminum salt agglomerate into larger particles. By rehydrating the powder, these larger particles cannot dissociate into smaller particles and form a gel suspension. Instead, aggregated particles of aluminum hydroxide or aluminum phosphate precipitate out of suspension.

Through further experiments, we found that when using aluminum salts in combination with other specific agents, suitable powders could be formed by spray drying. In addition, aluminum salts need to be used in certain proportions with other agents. We have also found that the specific drying method employed has a significant effect on the degree of coagulation of the adjuvant salts. Through these studies, we found that the powder suitable for needle-free injection can be obtained by spray-lyophilizing the alum-adjuvanted vaccine composition, and the powder can basically maintain the colloidal structure when rehydrated.

The spray freeze-drying method includes the step of spraying the suspended vaccine composition into liquid nitrogen. This method has two important effects: first, liquid nitrogen acts as a heat-conducting agent to rapidly freeze the suspension; second, atomization reduces the volume of each frozen droplet, thereby increasing the freezing rate. The action of these two aspects causes the tiny droplets of the suspension to freeze rapidly and form small ice crystals in the solid state. Thus, the size of the freeze-agglutination zone is significantly reduced compared to conventional freeze-drying techniques. Due to the very rapid freezing of the particles and the small size of the particles, there is little or no agglomeration of the adjuvant in the formed powder.

Thus, the present invention provides a simple and effective technique for the preparation of powdered vaccine compositions containing a salt adjuvant and suitable for long-term storage. The vaccine composition of the present invention does not substantially aggregate when rehydrated, thereby substantially maintaining its immunogenicity. The composition also has defined particle size, density and mechanical properties which in combination make the powder suitable for transdermal administration by needle-free injection.

Another important advantage of the present invention is that it is applicable to a wide variety of vaccine compositions and can be well adapted to other pharmaceutical compositions, especially when problems of agglomeration are also encountered. We have now found that in the field of adjuvanted vaccine compositions, the spray-lyophilization technique is completely independent of the formulation.

Accordingly, the present invention provides a powder suitable for use as a vaccine, which powder is capable of forming a gel and is free-flowing, said powder being formed by spray drying or spray lyophilization of an aqueous suspension comprising :

(a) 0.1-0.95% by weight of an aluminum or calcium salt adjuvant that has adsorbed the antigen;

(b) 0.5-6% by weight of sugar;

(c) 0.1-2% by weight of an amino acid or a salt thereof; and

(d) 0.02-1% by weight of colloidal substances.

This produces a free-flowing powder composition suitable for use in vaccines. The composition has defined particle size, density and mechanical properties which in combination make the powder suitable for transdermal administration by needle-free injection.

The present invention further provides:

- A process for the preparation of a powder suitable for use as a vaccine, which powder is capable of forming a gel and is free-flowing, said process comprising the steps of spray-drying or spray-lyophilizing an aqueous suspension comprising:

(a) 0.1-0.95% by weight of an aluminum or calcium salt adjuvant that has adsorbed the antigen;

(b) 0.5-6% by weight of sugar;

(c) 0.1-2% by weight of an amino acid or a salt thereof; and

(d) 0.02-1% by weight of colloidal substances;

- a dosage container for a needle-free injector containing an effective amount of a powder according to the invention;

- a needle-free injector filled with a powder according to the invention;

- a vaccine composition comprising a pharmaceutically acceptable carrier or diluent and a powder of the invention;

- A method of immunizing a subject comprising administering to said subject an effective amount of a powder of the invention; and

- a gel-forming free-flowing powder suitable for use as a vaccine, said powder comprising:

(i) 5-60% by weight of an aluminum or calcium adjuvant that has absorbed the antigen;

(ii) 25-90% by weight sugar;

(iii) 4.5-40% by weight of amino acids or salts thereof; and

(iv) 0.5-10% by weight of colloidal substances.

Furthermore, the present invention provides a powder suitable for use as a vaccine, which powder is obtained by spray-lyophilizing an aqueous suspension containing an adjuvant of aluminum or calcium salt to which antigen is adsorbed.

The present invention further provides:

- a method for the preparation of a powder suitable for use as a vaccine, said method comprising the step of spray-lyophilizing an aqueous suspension containing an adjuvant of aluminum or calcium salts to which antigens have been adsorbed;

- a dosage container for a needle-free injector containing an effective amount of the spray-lyophilized powder of the invention;

- a needle-free injector containing the spray-lyophilized powder of the present invention;

- a vaccine composition containing a pharmaceutically acceptable carrier or diluent and a spray-lyophilized powder of the present invention; and

- A method of immunizing a subject, said method comprising administering to said subject an effective amount of a spray-lyophilized powder of the invention.

Description of drawings

Figure 1 shows the particle size distribution of the alum colloid with adsorbed HBsAg before (i) and after (ii) drying, which was dried by spray freeze-drying technique and rehydrated.

Figure 2 shows the particle size distribution of another alum colloid with adsorbed HBsAg before and after drying, and the drying adopts the traditional freeze-drying method.

Figure 3 shows the results of an immunogenicity study using mice injected with HBsAg-adsorbed alum vaccine either dried using the spray freeze-drying (SFD) technique according to the present invention, or freeze-dried (FD ) method for drying. The lyophilized powder was sieved into fractions of different particle sizes and then tested for immunogenicity. Two formulations of spray-lyophilized powders with different alum contents were tested.

Figure 4 shows the immunogenicity of three different spray-lyophilized powders to mice, either by intramuscular injection with a needle or epidermal powder immunization with a powder delivery device.

Figure 5 shows the immunogenicity of spray-lyophilized diphtheria-tetanus toxoid vaccine in guinea pigs. Spray-lyophilized powders with a diameter of 20-38 μm and 38-53 μm were administered through the abdominal skin using a powder delivery device.

Detailed ways

Before the present invention is described in detail, it is to be understood that this invention is not limited to compositions or method parameters exemplified, as such may, of course, vary. It should also be understood that the terminology used herein is only used to describe specific embodiments of the present invention, rather than to limit the present invention.

All publications, patents, and patent applications cited above and below the text are hereby incorporated by reference in their entirety.

It must be noted that the singular nouns used in this specification and the appended claims include plural unless expressly defined otherwise. Thus, for example, reference to "a particle" includes two or more of that particle, reference to "an excipient" includes two or more of such excipients, etc.

A. Definition

Unless otherwise agreed, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although various 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 words and expressions will be used with the meanings defined below. "Antigen" refers to a molecule containing one or more antigenic determinants that elicit a host's immune system to mount a specific cellular antigen immune response or humoral antibody response. Thus, antigens include polypeptides, oligosaccharides/oligosaccharides, polysaccharides, etc., said polypeptides including antigenic protein fragments. Furthermore, antigens can be obtained from any known virus, bacterium, parasite, plant, protozoa or fungus and possibly all organisms. The term also includes tumor antigens. Similarly, the definition of antigen may also include, for example, oligonucleotides or polynucleotides expressing the antigen for DNA immunization applications. The term also includes synthetic antigens, such as multiple epitopes, side epitopes, and other recombinant or synthetically derived antigens (Bergmann et al. (1993), Eur. J. Immunol. 23 : 2777-2781; Bergmann (1996), Journal of Immunology (J.Immunol.) 157 :3242-3249; Suhrbier, A (1997), Immunology and Cell Biology (Immunol.and CellBiol.) 75 :402-408; Gardner et al. (1998), The 12th World AIDS Conference, Geneva, Switzerland, June 28-July 3, 1998).

The combination of one or more adjuvants with adsorbed antigens of the present invention is typically combined with one or more additional substances such as carriers, media and/or excipients. "Carrier", "vehicle" and "excipient" generally refer to substantially inert materials which are nontoxic and which do not react deleteriously with the other components of a composition. These materials can be used to increase the amount of solids in granular pharmaceutical compositions. Examples of suitable carriers include water, silicones, gelatin, waxes and the like materials. Examples of commonly used "excipients" include pharmaceutical grade carbohydrates, such carbohydrates include monosaccharides, disaccharides, cyclodextran and polysaccharides (such as glucose, sucrose, lactose, trehalose, raffinose, mannose, alcohol, sorbitol, inositol, dextran and maltose-dextran complex); starch; cellulose; salt (such as sodium or calcium phosphate, calcium sulfate, magnesium sulfate); citric acid; tartaric acid; glycine; High molecular weight polyethylene glycol (PEG); Pluronics; Surfactants; and combinations of the foregoing. When carriers and/or excipients are used, they are usually used in an amount of about 0.1-99% by weight of the pharmaceutical composition.

The term "powder" as used herein refers to a composition consisting essentially of solid particles which can be administered transdermally by a needle-free syringe. The particles making up the powder can be characterized by a range of parameters including, but not limited to, average particle size, average particle density, particle morphology (e.g., aerodynamic shape of the particles and surface characteristics of the particles), and osmotic energy of the particles (P.E. ).

The average particle size of the powders of the present invention can vary widely, typically from 0.1 to 250 μm, for example from 10 μm to 100 μm, more typically from 20 μm to 70 μm. The mass median aerodynamic diameter (MMAD) can be measured using conventional techniques such as microscopy (which directly measures the size of individual particles rather than determining the statistics of large numbers of particles), gas adsorption, osmotic force, or time-of-flight, Take this as the average particle size of the powder. The average particle size can be determined, if desired, using an automated particle size counter (eg, Aerosizer counter, Coulter counter, HIAC counter, or Gelman automatic particle counter).

The true or "absolute density" of the particles can be readily determined using known quantitative techniques such as helium pycnometric methods and the like. Alternatively, bulk densitometry can be used to determine the density of the powders of the invention. The bulk density of the powder of the present invention is generally 0.1-2.5 g/cm ³ , preferably 0.8-1.5 g/cm ³ .

Bulk density information is useful for characterizing the density of objects of irregular size and shape. Bulk density is the mass of an object divided by its volume, where volume includes the volume of pores and small cavities but excludes the volume of intergranular spaces. There are a number of methods known in the art for determining bulk density, such methods include wax soaking, mercury displacement, water adsorption and apparent specific gravity. There are many suitable instruments for measuring bulk density, such as Micromeritics Instrument Corp. Model 1360 GeoPyc. The difference between the bulk density and the absolute density of a sample of the pharmaceutical composition provides information on the overall percent porosity and specific pore volume of the sample.

The morphology of the particles, especially the aerodynamic shape of the particles, can be easily detected by conventional light microscopy methods. Preferably the particles making up the powder are substantially spherical, or at least substantially elliptical in aerodynamic shape. It is also preferred that the particles have an axial ratio of 3 or less in order to avoid rod-shaped or needle-shaped particles. These same microscopy techniques can also be used to examine particle surface characteristics, such as the number and proportion of surface pits or porosity.

The osmotic energy of particles can be determined using a variety of conventional techniques, such as metallized membrane osmotic energy testing. Using a metallized film material (for example, a 125 μm polyester film coated with 350 aluminum on one side) as a substrate, a needle-free injector (for example, the needle-free injector described in U.S. Patent No. 5,630,796 to Bellhouse et al.) The powder is launched into the film material at an initial velocity of 100-3000 m/s. The metallized film is placed with the metal coating facing up on a suitable surface.

The septum of the needle-free syringe containing the powder is brought into contact with this film and fired. Use a suitable solvent to remove the remaining powder on the metallized film. Adopt GS-700 type BioRad imaging densitometer to scan this metal-coated film, use a personal computer with SCSI interface and install MultiAnalyst (BioRad) and Matlad (version 5.1, The Math Works, Inc.) to analyze the density The reading of the meter is used to measure the osmotic energy. The densitometer is scanned using the transmittance or reflectance method, and a program is used to process the readings from the densitometer scan. The osmotic energy of the spray-coated powder should be equivalent to or better than that of mannitol particles of the same size treated in the same way (by freeze-drying, compressing , grinding and sieving to obtain the mannitol particles, which are incorporated herein by reference).

The term "subject" means any individual of the subphylum cordata, including, but not limited to, humans and other primates, including animals other than humans such as chimpanzees and other apes and Animals of the genus Monkey; domestic animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; experimental animals including rodents such as mice, rats and guinea pigs; birds including domestic and wild birds such as chickens, turkeys and others Quail, chicken, duck, goose, etc. The term "subject" does not specify a specific age and thus can include both adult and newborn individuals. The methods described herein can be used with any of the vertebrates mentioned above, since the immune systems of all these vertebrates work in a similar manner.

The term "transdermal administration" includes both dermal and transmucosal routes of administration, ie, administration through the skin or mucosal tissue. See, e.g., "Transdermal Drug Delivery: Developmental Issues and Research Initiatives", Hadgraft and Guy (Eds.), Marcel Dekker, Inc., (1989); Controlled Drug Delivery Controlled Drug Delivery: Fundamentals and Applications, Robinson and Lee (Eds.), Marcel Dekker, Inc., (1987); and Transdermal Delivery of Drugs, Volumes 1-3, Kydonieus and Berner (eds.), CRC Press, (1987).

B. General approach

The present invention relates to a gel-forming and free-flowing powder suitable for use as a vaccine. The powder is suitable for transdermal administration using a needle-free injector delivery device. In this regard, the particles making up the powder composition must have sufficient mechanical strength to withstand sudden acceleration to velocities several times the speed of sound, but also to withstand impact from and pass through skin and tissue. Such particles may be prepared by spray-drying or spray-lyophilizing an aqueous suspension which, in certain embodiments, comprises or consists essentially of:

(a) 0.1-0.95% by weight of an aluminum or calcium salt adjuvant that has adsorbed the antigen;

(b) 0.5-6% by weight of sugar;

(c) 0.1-2% by weight of an amino acid or a salt thereof; and

(d) 0.02-1% by weight of colloidal substances.

The aqueous suspension contains, as component (a), less than 1% by weight of the adjuvant to which the antigen is adsorbed. Preferably, the aqueous suspension contains 0.2 or 0.3 to 0.6 or 0.75% by weight of the adsorbed antigen, more preferably 0.2 to 0.4% by weight of the adjuvant. Aluminum salt adjuvants are usually aluminum hydroxide or aluminum phosphate. Alternatively, the adjuvant may be aluminum sulfate or calcium phosphate.

Any suitable antigen as defined herein may be used. An antigen can be a viral antigen. Therefore, the antigen can be any of the following families of viruses: Picornaviridae (such as picornavirus, etc.); Caliciviridae; Togaviridae (such as rubella virus, dengue virus, etc.); Flaviviridae; Coronavirus Family; Reoviridae; Birnaviridae; Rhabdoviridae (such as rabies virus, etc.); Filoviridae; Paramyxoviridae (such as mumps virus, measles virus, respiratory syncytial virus, etc.); Orthomyxoviridae (e.g. influenza A, B, and C viruses, etc.); Bunyanviridae; Arenaviridae; Retroviridae (e.g. HTLV-I; HTLV-II; HIV-1 and HIV-2); Simian Immunodeficiency Virus (SIV), etc.

Alternatively, viral antigens may be from papillomaviruses (such as HPV); herpesviruses; hepatitis viruses such as hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), hepatitis E virus (HEV) or hepatitis virus (HGV); and tick-borne encephalitis virus. For a description of the above-mentioned viruses see, for example, Virology, Third Edition, (ed. W.K. Joklik, 1988); Fundamental Virology, Second Edition (B.N. Fields and D.M. Knipe, edited, 1991) .

Bacterial antigens used in the present invention may be derived from organisms capable of causing diphtheria, cholera, tuberculosis, tetanus, pertussis, meningitis, and other morbid conditions including, for example, meningococci types A, B, and C, Haemophilus influenza type B (HIB), Helicobacter pylori, Vibrio cholerae, Escherichia coli, Campylobacter, Salmonella, Streptococcus, and Gluconococcus. Combinations of various bacterial antigens can be provided, for example diphtheria, pertussis and tetanus antigens. Suitable pertussis antigens are pertussis toxin and/or filamentous hemagglutinin and/or pertactin, also known as P69. Antiparasitic antigens are available from organisms that cause malaria and Lyme arthritis.

Antigens used in the present invention can be prepared by various methods known to those skilled in the art. In particular, antigens can be isolated directly from natural sources using common purification techniques. Alternatively, completely killed, attenuated or inactivated bacteria, viruses, parasites or other microorganisms may be used. In addition, antigens can be produced recombinantly using known techniques. See, eg, Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, Volumes I and II (D.N. Glover et al., 1985).

According to the published amino acid sequence, the antigen used in the present invention can be synthesized by chemical polymer synthesis method such as solid phase peptide synthesis method. Such methods are known to those skilled in the art. See, e.g., J.M.Stewart and J.D.Young, "Solid Phase Peptide Synthesis (Solid Phase Peptide Synthesis)", Second Ed., Pierce Chemical Co., Rockford, IL (1984); G.Barany and R.B.Merrifield, "Synthesis of Peptides Analysis, Synthesis, and Biology (The Petides: Analysis, Synthesis, Biology), eds. E. Gross and J. Meienhofer, Volume II, Academic Press, New York, (1980), pp. 3-254, on solid phase shrinkage Synthetic technology of amino acid; M.Bodansky's "Principles of Peptide Synthesis (Principles of Peptide Synthesis)", Springer-Verlag Berlin (1984); and "Analysis and Synthesis of Peptides" edited by E.Gross and J.Meienhofer and Biology (The Petides: Analysis, Synthesis, Biology), see above, Vol. 1, on traditional solution synthesis.

The aqueous suspension may contain one or more sugars as component (b). The sugar content is typically 1.5-5% by weight, preferably 2-4% by weight. Sugars can be monosaccharides such as glucose, xylose, galactose, fructose, D-mannose, or sorbose; disaccharides such as lactose, maltose, sucrose, trehalose, or sucrose; or sugar alcohols such as mannitol, sorbitol , xylitol, glycerol, erythritol, or arabitol.

The aqueous suspension may contain one or more amino acids or amino acid salts as component (c). Any physiologically acceptable amino acid salt can be used. The salt may be an alkali metal or alkaline earth metal salt, such as a sodium, potassium or magnesium salt. Amino acids can be acidic, neutral or basic amino acids. Suitable amino acids are glycine, alanine, glutamic acid, arginine, lysine and histidine. Monosodium glutamate is a suitable amino acid salt. The aqueous suspension generally contains 0.5-1.5% by weight, more preferably 0.75-1.25% by weight, of amino acids and/or amino acid salts.

A colloidal substance (d) is a dispersed substance that cannot pass through a semipermeable membrane, the colloidal substance consists of particles that exist in suspension or solution without settling out. EP-B-0130619 discloses suitable colloidal substances. Component (d) may be selected from polysaccharides such as dextran or maltose-dextran complexes; hydrogels such as gelatin or agarose; or proteins such as human serum albumin. The colloidal material may have a molecular weight of 500-80,000 or more, for example from 1000 or 2000 to 30,000, or from 5,000 to 25,000. The content of component (d) in the aqueous suspension is generally 0.05-0.5% by weight, preferably 0.07-0.3% by weight.

Suspend the antigen-adsorbed adjuvant, sugar, amino acid or its salt, and colloidal substance in water. The aqueous suspension is spray dried or spray lyophilized. The conditions of spray drying or spray lyophilization are chosen such that the desired particles are produced. The input temperature of the air, the output temperature of the air, the feed rate of the aqueous suspension, the air flow rate, etc. can be varied as desired. The size of the nozzle can be changed if necessary. The specific conditions of spray freeze-drying will be described in detail below.

It is thus possible to provide a powder suitable as a vaccine which forms a gel and is free flowing. By adjusting the composition of the suspension used for spray-drying or spray-lyophilization, the proportions of the powder components can be adjusted. However, the powder typically comprises, or in certain embodiments consists essentially of:

(i) 5-60% of the antigen is adsorbed, for example 7-50%, such as 10-30% by weight of aluminum salt or calcium salt adjuvant;

(ii) 25-90%, such as 30-80%, and such as 40-70% by weight of sugar;

(iii) 4.5-40%, such as 7-30%, and such as 10-20% by weight of amino acids or salts thereof; and

(iv) 0.5-10%, eg 0.8-6%, eg 1-3% by weight of colloidal substances.

The present invention generally relates to powders suitable for use as vaccines, which powders are formed by spray-lyophilization of aqueous suspensions containing antigen-adsorbed aluminum or calcium salt adjuvants. The powder is suitable for transdermal administration using a needle-free syringe delivery device. In this regard, the particles making up the powder composition must have sufficient mechanical strength to be able to withstand sudden accelerations to velocities several times the speed of sound and to withstand impact from and pass through skin and tissue.

Preferably, the aqueous suspension contains less than 10% by weight, such as less than 5% by weight and preferably less than 3% by weight of antigen-adsorbed salt adjuvant before spray-lyophilization. The aqueous suspension typically contains at least 0.05% by weight, such as at least 0.1% or at least 0.6% by weight of the adjuvant to which the antigen is adsorbed. More preferably, the aqueous 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 the antigen-adsorbed adjuvant. When the concentration of adjuvant salt is higher than about 10% by weight, the viscosity of the aqueous suspension becomes very high. This limits the ability to spray the suspension into a mist.

It should be understood that the preferred upper limit of adjuvant concentration applies to the aqueous suspension prior to spray lyophilization. The amount of the antigen-adsorbed adjuvant may be 50% by weight or more of the powder of the present invention.

Adjuvants are usually aluminum salts, such as aluminum hydroxide or aluminum phosphate. Alternatively, the adjuvant may be aluminum sulfate or calcium phosphate.

Likewise, any suitable antigen as defined herein may be employed. An antigen can be a viral antigen. Therefore, the antigen can be any of the following families of viruses: Picornaviridae (such as picornavirus, etc.); Caliciviridae; Togaviridae (such as rubella virus, dengue virus, etc.); Flaviviridae; Coronavirus Family; Reoviridae; Birnaviridae; Rhabdoviridae (such as rabies virus, etc.); Filoviridae; Paramyxoviridae (such as mumps virus, measles virus, respiratory syncytial virus, etc.); Orthomyxoviridae (e.g. influenza A, B, and C viruses, etc.); Bunyanviridae; Arenaviridae; Retroviridae (e.g. HTLV-I; HTLV-II; HIV-1 and HIV-2); Simian Immunodeficiency Virus (SIV), etc.

Alternatively, viral antigens may be from papillomaviruses (such as HPV); herpesviruses; hepatitis viruses such as hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), hepatitis E virus (HEV) or hepatitis virus (HGV); and tick-borne encephalitis virus. For a description of the above-mentioned viruses see, for example, Virology, Third Edition, (ed. W.K. Joklik, 1988); Fundamental Virology, Second Edition (B.N. Fields and D.M. Knipe, edited, 1991) .

Bacterial antigens used in the present invention may be derived from organisms capable of causing diphtheria, cholera, tuberculosis, tetanus, pertussis, meningitis, and other morbid conditions including, for example, meningococci types A, B, and C, Haemophilus influenza type B (HIB), Helicobacter pylori, Vibrio cholerae, Escherichia coli, Campylobacter, Salmonella, Streptococcus, and Gluconococcus. Combinations of various bacterial antigens can be provided, for example diphtheria, pertussis and tetanus antigens. Suitable pertussis antigens are pertussis toxin and/or filamentous hemagglutinin and/or pertactin, also known as P69. Antiparasitic antigens are available from organisms that cause malaria and Lyme arthritis.

Antigens used in the present invention can be prepared by various methods known to those skilled in the art. In particular, antigens can be isolated directly from natural sources using common purification techniques. Alternatively, completely killed, attenuated or inactivated bacteria, viruses, parasites or other microorganisms may be used. In addition, antigens can be produced recombinantly using known techniques. See, eg, Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, Volumes I and II (D.N. Glover et al., 1985).

According to the published amino acid sequence, the antigen used in the present invention can be synthesized by chemical polymer synthesis method such as solid phase peptide synthesis method. Such methods are known to those skilled in the art. See, e.g., J.M.Stewart and J.D.Young, "Solid Phase Peptide Synthesis (Solid Phase Peptide Synthesis)", Second Ed., Pierce Chemical Co., Rockford, IL (1984); G.Barany and R.B.Merrifield, "Synthesis of Peptides Analysis, Synthesis, and Biology (The Petides: Analysis, Synthesis, Biology), eds. E. Gross and J. Meienhofer, Volume II, Academic Press, New York, (1980), pp. 3-254, on solid phase shrinkage Synthetic technology of amino acid; M.Bodansky's "Principles of Peptide Synthesis (Principles of Peptide Synthesis)", Springer-Verlag Berlin (1984); and "Analysis and Synthesis of Peptides" edited by E.Gross and J.Meienhofer and Biology (The Petides: Analysis, Synthesis, Biology), see above, Vol. 1, on traditional solution synthesis.

The aqueous suspension may consist essentially of water and an adjuvant to which the antigen is adsorbed, or the suspension may further contain additives. Any substantially nontoxic and pharmacologically inert additive can be used. We have found that the spray freeze-drying method works very well when using aqueous suspensions containing various additives, and have also found that the method of the invention and the powders obtained therefrom are completely independent of its formulation.

Typically, such aqueous suspensions contain suitable excipients as well as protecting agents, solvents, salts, surfactants, buffers and the like. Suitable excipients may include free-flowing solid particles that do not viscoseize or aggregate when in contact with water, are not toxic when administered to a subject, and do not react significantly with the pharmaceutical agent to alter the activity of the drug . Examples of commonly used excipients include, but are not limited to, monosaccharides such as glucose, xylose, galactose, fructose, D-mannose or sorbose, disaccharides such as lactose, maltose, sucrose, trehalose or sucrose , sugar alcohols such as mannitol, sorbitol, xylitol, glycerol, erythritol or arabitol, polymers such as dextran, starch, cellulose, high molecular weight polyethylene glycol (PEG), amino acids or salts thereof such as glycine, alanine, alanine salts, arginine, lysine or histidine, or alkali or alkaline earth metal salts of the above amino acids such as sodium, potassium or magnesium, or sodium or calcium phosphate , calcium carbonate, calcium sulfate, sodium citrate, citric acid, tartaric acid and combinations thereof. Suitable solvents include, but are not limited to, dichloromethane, acetone, methanol, ethanol, isopropanol and water. Typically, water is used as the solvent. Pharmaceutically acceptable salts are generally employed at molar concentrations of about 1 mM to 2M. Pharmaceutically acceptable salts include, for example, inorganic acid salts such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and the like; and organic acid salts such as acetate, propionate, malonate, benzoate and the like. "REMINGTON'SPHARMACEUTICAL SCINECES" (REMINGTON'SPHARMACEUTICAL SCINECES) (Mack Pub.Co., N.J.1991) has done a detailed description to pharmaceutically acceptable excipients, vehicles and auxiliary agents, and is hereby quoted for reference.

Preferred excipients for aqueous suspensions include sugars, amino acids or salts thereof, and polymers. Typically, the aqueous suspension contains one or more sugars, such as a combination of mannitol and trehalose. The sugar content is typically 0.5-30% by weight. Amino acid salts such as arginine, glutamate or aspartate in amounts of 0.1-30% by weight and/or polymers such as dextran typically in amounts of 0-30% by weight. A typical composition of excipients includes one or more sugars and polymers and is substantially free of amino acid salts. The content of excipients in the aqueous suspension is typically 0-50%, more preferably 10-30%.

The method for forming the particles of the present invention is that the adjuvant adsorbed with the antigen and any required additives are first dissolved in water, and then the aqueous suspension is sprayed and freeze-dried. The spray lyophilization step can be performed using any technique known in the art (for example, Mumenthaler et al. in "International Journal of Pharmaceutical Sciences (Int.J.Pharmaceutics)" (1991) pp. 72, 97-110, and Maa et al. in "Pharmaceutical Research (Phar .Res.)" (1999) the method described in Vol. 16, p. 249). A typical spray freeze-drying technique involves the step of spraying an aqueous suspension into a mist of liquid nitrogen under stirring. The liquid nitrogen containing the frozen particles is then stored at a low temperature such as -60°C to -20°C, followed by vacuum drying preferably at a pressure of 20 to 500 mT (2.666 to 66.65 Pa) and a low temperature of -50°C to 0°C. Typically a two-step drying process is employed, namely primary drying and secondary drying. The primary drying time is typically 4 to 24 hours and the secondary drying time is typically 6-24 hours. You can gradually increase the temperature, but keep the low pressure until it reaches room temperature.

The technique involves the step of rapidly freezing an aqueous suspension into small droplets. The subsequent drying step removes the ice by sublimation without the need for high air temperatures. Powders can be collected using any known technique. The exact conditions employed for spray lyophilization can be chosen according to the desired properties of the resulting particles. Thus, temperature, pressure and other conditions can be changed as necessary.

The powders of the invention are generally free-flowing. The powder contains little or no aggregated adjuvant salts and thus forms a gel when resuspended in water. Typically, there is substantially no sedimentation upon resuspension. After adding the powder to distilled water (1:500 by weight) and shaking for 3 minutes, a gel-like suspension without any precipitation is typically obtained. After 3 hours no precipitation was observed. On standing overnight, for example 12 hours, no precipitate will form.

Typically, the presence of precipitates and the degree of agglomeration in a reconstituted gel formulation can be assessed by the ability of the reconstituted gel formulation to diffract a light beam. The extent of aggregation can also be assessed quantitatively using standard light microscopy and/or precipitation methods. Another suitable method for determining particle agglomeration may be to measure the particle size before and after rehydration using any standard particle sizing technique, such as measuring laser or dimming of light.

The particle size of the present invention is suitable for high-speed transdermal administration to a subject, typically through the stratum corneum or mucous membranes. The mass median aerodynamic diameter (MMAD) of the particles is about 0.1-250 μm. MMAD may be 5-100 μm or 10-100 μm, preferably 10-70 μm or 20-70 μm. Typically, less than 10% by weight of particles have a diameter of at least 5 μm above the MMAD or at least 5 μm below the MMAD. Preferably, there is no more than 5% by weight of particles having a diameter of 5 μm or more above the MMAD. Also preferably, no more than 5% by weight of particles having a diameter of 5 μm or more below the MMAD.

The bulk density of the particles is 0.1-25 g/cm ³ , preferably 0.8-1.5 g/cm ³ . Although individual particles may vary in shape when viewed under a microscope, spherical particles are preferred. The average value of the ratio of the major axis to the minor axis is typically from 3:1 to 1:1, for example from 2:1 to 1:1.

The individual particles of the powder are substantially spherical in aerodynamic shape with a substantially uniform non-porous surface. The particles also have a particle osmotic energy suitable for transdermal administration using a needle-free injector device.

As described herein, the prior art has specified needle-free injector devices useful with the present invention. Such injectors are known as needle-free injector devices and are represented by the cutaneous PowderJect needle-free injector device and the oral PowderJect needle-free injector device (PowderJect Technologies Limited, Oxford, UK). Using such devices an effective amount of a powder of the invention can be administered to a subject. An effective amount refers to the amount necessary to administer sufficient of the desired antigen to a subject. The amount administered will vary depending on the nature of the antigen and can be readily determined from clinical trials based on the known activity of the administered antigen. The "Physicians Desk Reference" and "Goodman and Gilman's The Pharmacological Basis of Therapeutics" are useful in determining this required amount.

A needle-free injector for administering particles is first described in co-owner US Patent No. 5,630,796 to Bellhouse et al., incorporated herein by reference. Although there are a variety of needle-free injectors with special configurations, these injectors are typically pen-shaped devices that move from top to bottom in a linear sequence, including cylinders, pellet boxes or packets, and supersonic nozzles with sound-absorbing media. A suitable powder (referred to herein as a spray-dried or spray-lyophilized powder according to the invention) is placed in a suitable container, such as a box formed of two rupturable polymer films welded into a gasket-like partitions, thus forming an independent airtight unit. The membrane material is chosen so that it can reach the pressure at which it opens and bursts in a certain way, which determines the conditions for supersonic flow. In operation, the device is activated, releasing compressed gas from the cylinder into the expansion chamber in the device. This released gas comes into contact with the pellet cartridge, and when sufficient pressure builds up, the membrane of the pellet cartridge bursts, blowing the pellets into a supersonic nozzle for subsequent dosing. Nozzles should be designed with specific gas velocities and flow patterns in order to deliver a certain amount of particles to the intended surface in a given area. The silencer is used to attenuate the noise generated by the rupture of the membrane.

International Publication WO 96/20022 by co-owners describes another needle-free injector device for particle administration. This drug delivery device also uses compressed gas as energy to accelerate and deliver the powder composition; however, unlike US Patent No. 5,630,796, it uses shock waves rather than airflow to accelerate the particles. More specifically, a shock wave emanating from the back of the flexible dome creates a momentary increase in pressure that impinges on the back of the dome, causing the flexible dome to suddenly invert in the direction of the desired surface, displacing the powder composition (located on The exterior of the dome) is ejected with sufficient velocity so that sufficient momentum is generated to penetrate target tissue, such as oral mucosal tissue. The powder composition is released when the inversion of the dome reaches its highest point. The dome also acts as a complete containment of the high-pressure airflow, thereby preventing the high-pressure airflow from coming into contact with the tissue. Since no gas is released during this administration, the device itself is noiseless. This design could also be used in other closed or sensitive applications, for example, to deliver particles to mildly diseased surgical sites.

In yet another aspect of the invention, the powders of the invention may be presented prior to use in unit-dose or multi-dose containers, including sealed containers containing the appropriate dose of the powder. Sterile formulations of the powder can be filled into containers, therefore, the design of the sealed container should maintain the sterility of the formulation prior to administration. Such containers may, if desired, be adapted for direct application to the needle-free injector devices described above.

Thus, the powders of the present invention are packaged as single doses for needle-free injector administration. A "single dose" as used herein refers to a dosage container containing a therapeutically effective amount of a powder of the invention. The dosage container is typically mounted in a needle-free injector so that transdermal administration can be performed through the injector. Such containers may be capsules, foil pouches, sachets, boxes and the like.

The container containing the powder can in turn be labeled to identify its composition and provide information on dosage. In addition, a precautionary notice may be placed on the container in a manner prescribed by an official agency such as the Food and Drug Administration, which notice states that the manufacture, use, and sale of the contained powder for human administration is in accordance with federal regulations. The law was approved by the agency.

The actual depth to which the applied particles penetrate the target surface depends on the particle size (e.g., the nominal diameter of the particle assuming a roughly spherical particle geometry), particle density, the initial velocity of the particle impacting the surface, and the density and movement of the target skin tissue speed. In this regard, preferred particle densities for needle-free injection are generally between about 0.1 g/cm ³ and 25 g/cm ³ , for example between about 0.8 g/cm ³ and 1.7 g/cm ³ , preferably between Between about 0.9g/cm ³ and 1.5g/cm ³ . Injection speeds are typically between about 100 and 3,000 meters/second. Under appropriate gas pressure, particles with an average diameter of 10-70 μm can be accelerated to supersonic speed close to the driving gas flow through the nozzle.

If desired, needle-free injector devices may be pre-filled containing appropriate doses of powders of the invention. The syringe containing the powder can be filled into a sealed container which can then be labeled as described above.

To characterize the performance of needle-free injector devices, various new test methods have been developed, or existing ones modified. These tests include characterization of powder compositions, measurement of airflow and particle acceleration, impact on man-made or biological targets, and testing of overall device performance. One, several or all of the following test methods may be used to evaluate the physical properties and functional suitability of the powders of the present invention for use in needle-free injector devices.

Tests for Effects on Artificial Membrane Targets

A functional test that simultaneously measures multiple aspects of a powder injector device is known as a "metallization" or "penetration energy" (PE) test. The test is based on the damage that particles can do to a thin layer of metal supported by a plastic film substrate, which has a precise thickness. The conditions of destruction are related to the dynamic energy and certain other properties of the particles. The more intense the response tested (ie, the more severe the damage/rupture of the membrane), the higher the energy imparted to the particle by the device. Measuring the change in electrical resistance or measuring the imaging density in reflection or transmission is a reliable, controllable and repeatable means of evaluating the performance of the device or formulation.

It has been found that the membrane test bed is sensitive to changes in all major parameters of the device during drug delivery, including pressure, dose, particle size distribution and material, and that the membrane test bed is insensitive to gas. The membrane used in this test, about 350 Angstroms of aluminum coated on a 125 μm thick polyester substrate, was used to test syringe devices operating at up to 60 bar.

Testing of Impact Effects on Engineered Foam Targets

Another method of testing the performance of the particles when administered from a needle-free injector device is to measure the performance of the particles on rigid polymethylimide foam (Rohacell 5 IIG, density 52 kg/m ³ , Rohm Tech Inc., Malden, Mass. state) impact. The setup for this experiment was similar to that used for the metallized film tests. The depth of penetration was measured using precision calipers. For each experiment, a treated mannitol standard was used as a control, and all other parameters such as apparatus pressure, particle size range, etc. were kept constant. The data show that the method is also sensitive to differences in particle size and pressure. In preclinical experiments, it has been confirmed that processed mannitol standards as pharmaceutical excipients can be distributed throughout the body, so in the foam penetration test, the test of the relevant performance has a practical in vivo basis. Those powders that exhibit equal or better penetration than mannitol are expected to have appropriate performance in preclinical or clinical studies. This simple and rapid test is a relative method of evaluating powders and should not be considered in isolation.

Particle Abrasion Test

Another indicator of particle performance is the ability of each candidate composition to withstand the forces associated with high velocity particle injection techniques, i.e., the force of a high velocity gas stream in sudden contact with a stationary particle, when the powder The force of the particle-to-particle collision as it passes through the needle-free injector, and the particle-to-device collision force as the powder passes through the device. Accordingly, a simple particle attrition experiment was designed to measure the change in particle size distribution of the initial composition as well as the composition after administration through a needle-free injector device.

The test method is to load the above-mentioned needle-free syringe with the granular composition, and then fill the flask with the carrier fluid, the granular composition is insoluble in the fluid (for example, mineral oil, silicone oil, etc.) . The carrier stream is collected and the particle size of the initial composition and the composition discharged into the carrier stream is calculated using a suitable particle sizing device, such as a Model 780 AccuSizer Optical Particle Sizer. Compositions having less than about 50%, more preferably less than about 20% reduction in mass median diameter (as measured by the AccuSizer device) after impacting the device are considered suitable for use in the needle-free injector devices described herein.

Topical administration to human skin and transepidermal water loss

In order to bring the performance testing of the powders closer to the final practical application, the alternative powder compositions were injected into full thickness skin grafts taken from the human abdomen with a dermotomy. Following injection, the same skin flap can be placed on a modified Franz diffusion dish filled with water, saline or buffer at 32°C. Additives such as surfactants can be used to prevent the skin sheet from sticking to certain parts of the diffusion vessel. Two tests were performed to evaluate the performance of the formulation in the skin.

To measure the physical effect, ie the effect of particle injection on the skin barrier function, the transepidermal water loss (TEWL) can be measured. A Tewameter TM 210 (Courage & Khazaka Co., Cologne, Germany) was placed on the top cover of the diffusion vessel, which acts like a chimney of about 12 mm, and tested at equilibrium (about 1 hour). TEWL values also increased proportionally for larger particles injected in vitro and higher injection pressures, and the results were shown to correlate with those of the in vivo tests. After in vitro particle injection, the TEWL value increased from about 7 to about 27 (g/m ² h) depending on particle size and helium pressure. Injecting helium without particles has no effect. When tested in vivo, TEWL returned to pre-treatment values within about 1 hour for most powder particle sizes, indicating that the skin barrier function quickly returned to normal levels. For the largest particles, 53-75 [mu]m, 50% recovery of the flakes was shown within 1 hour and complete recovery within 24 hours.

In vitro drug administration to human skin and drug diffusion rate

To test the in vitro performance of formulations, the antigenic components of candidate powders are collected for chemical analysis using HPLC or other appropriate analytical techniques by fully or partially displacing the solution in Franz dish receivers at predetermined time intervals. Concentration data can be used to develop dosing curves and calculate steady state permeation rates. Prior to in vivo studies, this technique can be used to screen for formulations that show early signs of antigen binding to the skin, antigen dispersion, and efficiency of particle penetration through the stratum corneum.

The above and other qualitative and quantitative testing methods can be used to evaluate the physical properties and functional suitability of the powders for high-speed particle injection devices of the present invention. Preferably, but not necessarily, the particles of the powder have the following properties: substantially spherical (e.g., the ratio of major axis to minor axis is as close to 1 as possible); smooth surface; suitable effective loading; particle size after particle abrasion test The reduction of is less than 20%; the bulk density is as close as possible to the true density of the composition (eg, greater than about 0.8 g/ml); the particle size distribution is narrow, with a mass median aerodynamic diameter of about 20-70 μm. Typically the composition is free flowing (eg, free flowing after storage at 50% relative humidity for 8 hours and storage at 40% relative humidity for 24 hours). All of the above-mentioned parameters can be determined by the method described above, and further detailed descriptions can be found in the following references, which are hereby incorporated by reference. Etzler et al. (1995), Part.Part.Syst.Charact. 12 :217; Ghadiri et al. (1992), IFPRI Final Report, FRR 16-03 University of Surrey, UK; Bellhouse et al. (1997), Using Shockwaves and Ultrasound "Needleless delivery of drugs in dry powder form, using shockwaves and supersonic gas flow", Plenary Lecture 6, ^21st International Symposium on Shock Waves, Australia; and Kwon et al. (1998 ), "Pharm.Sci.Suppl.1" (1), 103.

Alternatively, the subject may be immunized by other routes using the powder of the invention. For this purpose, the powder can be mixed with a suitable carrier or diluent such as water for injection or physiological saline. The resulting vaccine composition is typically administered by injection, eg subcutaneously or intramuscularly.

Regardless of the route of administration chosen, an effective amount of the antigen needs to be administered to the subject to be immunized. Typically the amount of antigen to generate an immune response is from 50 ng to 1 mg, more preferably from 1 μg to about 50 μg. The exact amount required will vary depending on the age and general condition of the subject being tested, the particular antigen or antigens selected, the site of administration, and other factors. An appropriate effective amount can be readily determined by those skilled in the art.

Dosage therapy can be in a single-dose course or in multiple-dose courses. A multi-dose regimen is such that 1-10 individual doses may be used for the initial phase of immunity, with additional doses given at subsequent intervals, with the option to maintain and/or boost the immune response, e.g. a second dose at 1-4 months and, if As needed, take subsequent doses over several months. The dosing schedule, or at least a portion thereof, will also be determined according to the needs of the subject and will also depend on the judgment of the physician. Of course, immunity usually occurs prior to initial infection by a pathogen, which requires protective measures.

c. experiment

The following are examples of specific embodiments of the invention. The examples are provided for the purpose of illustration only and are not intended to limit the scope of the invention in any way.

Efforts have been made to ensure accuracy of quoted data (eg, amounts, temperature, etc.), but some experimental errors and deviations should, of course, be accounted for.

Reference Example 1

A spray-dried immediate release immunological formulation was obtained according to the method described in US Patent No. 5,902,565. A formulation containing 5% by weight of mannitol and 5% by weight of aluminum phosphate (phosphate adjuvant) was spray dried using a bench top spray dryer (Buchi 190). The conditions for spray drying were: inlet temperature = 130 °C; outlet temperature = 70 °C, liquid feed rate = 3 ml/min; spray air flow rate = 500 l/hr; and full scale drying air. The resulting free-flowing powder had a particle size of about 10 μm. The powder was rehydrated in distilled water (weight ratio 1:500). The solution containing the suspended particles was left to stand for 15 minutes and no gel was formed. Observed by optical microscopy, the rehydrated particles maintained their shape and size, which indicated that the alum remained aggregated rather than dispersed.

Example 1

Mix the ingredients listed in the table below with 15ml of distilled water to get the following formula: formula Aluminum salt Mannitol Glycine Dextran 1 (comparison) 14.5g aluminum glue ¹⁾ 322mg 131mg 17.5mg 2 (the present invention) 2.5g aluminum glue1 ⁾ 693mg 130mg 18mg 3 (comparison) 15g phosphate adjuvant ²⁾ 438mg 173mg 16.9mg 4 (comparison) 7.7 g phosphate adjuvant ²⁾ 882mg 172mg 16.2mg

1) Aluminum glue: aluminum hydroxide of 3% by weight

2) Phosphate adjuvant: 2% aluminum phosphate by weight

The above formulation was spray dried using a Buchi 190 Mini-Spin dryer under the following conditions: air inlet temperature = 130°C; air outlet temperature = 70°C; Q liquid feed rate: 5 steps; and Q spray air flow rate: 500 l/hr . Set Dry Air to the maximum setting. A free-flowing powder was obtained. The yields are as follows: formula Powder output (g) % yield MMAD 1 0.52 68.4 8-10μm 2 0.48 53.9 8-10μm 3 0.91 74.1 8-10μm 4 0.38 31.0 8-10μm The resulting powder composition is related to the solids content of the suspension for spray drying as follows: Al(OH) ₃ Mannitol Glycine Dextran Total solids Formula 1 Solid content (%) in the suspension for spray drying Content in the powder 2.948.3% 2.135.0% 0.915.0% 0.11.7% 6 Formula 2 is used for the solid content (%) in the powder of the suspension of spray-drying 0.58.2% 4.675.4% 0.914.8% 0.11.6% 6.1 Formulation 3 is used for the solid content (%) in the powder of spray-dried suspension 448.8% 2.935.4% 1.214.6% 0.11.2% 8.2 Formula 4 is used for the solid content (%) in the powder of the suspension of spray-drying 112.3% 5.972.8% 1.113.6% 0.11.2% 8.1

The spray dried powder was resuspended in distilled water. Specifically, each powder was added to distilled water (1:500 by weight) and shaken for 3 minutes. The coagulation of the resulting suspension was determined. Only formulation 2 according to the invention formed a gel-like suspension without precipitation. The results are as follows: - Formulation 1 32.59 mg of spray-dried powder was added to 1 ml of distilled water. The resulting suspension

A white precipitate formed after standing overnight. - Formulation 2 Add 37.1mg of spray-dried powder to 1ml of distilled water. Formed off-white

A gel-like suspension. No sedimentation was observed after the resulting suspension stood overnight

Lake. - Formulation 3 44.34 mg of spray-dried powder was added to 1 ml of distilled water. The resulting suspension

A white precipitate formed after standing overnight. - Formulation 4 Add 29.4mg of spray-dried powder to 1ml of distilled water. The resulting suspension

A white precipitate formed after standing overnight.

Example 2

Two vaccine formulations were prepared as follows:

Formulation A:

Concentrated alum-HBsAg suspensions were prepared as follows: the alum adsorbed HBsAg vaccine from Rhein American S.A. containing 500 μg of alum (approximately 1500 μg of aluminum hydroxide) was first washed with deionized distilled water to remove buffer salts , the alum adsorbed 20 μg of HBsAg (about the dose of 1 person). The alum gel was placed in a 250ml Nalgene narrow-mouthed square polycarbonate bottle and allowed to stand overnight at 2-8°C. The supernatant (150ml) was removed, and the same volume of water was added to the precipitate with stirring. Repeat this step once.

Weigh out 100 g of the washed Alum-HBsAg formulation in a Nalgene square bottle and let stand overnight at 2-8°C. After removing 90 ml of the supernatant, the remaining suspension was transferred to a 50 ml polypropylene centrifuge tube and centrifuged at 200 rpm for 4 minutes using a tabletop centrifuge (Allegra 6R, Beckman). Furthermore, the supernatant was removed to obtain 3.369 g of a concentrated alum-HBsAg suspension. Then 315.24 mg mannitol, 81.73 mg glycine, 101.91 mg dextran and placebo alum gel (2% Al ₂ O ₃ ) were added to this suspension to obtain an alum concentration of 3% Alum-HBsAg liquid formula.

Formulation B:

Wash the alum-HBsAg suspension as described in Recipe A. 20.79 g of this suspension was weighed out in a 50 ml centrifuge tube and allowed to stand overnight at 2-8°C. After removing 17 ml of supernatant, the remaining concentrated suspension (3.572 g) was mixed with 113.06 mg of mannitol, 47.31 mg of glycine and 23.22 mg of dextran to obtain a liquid formulation with an alum concentration of 0.6%.

The two formulations were dried using the techniques listed in Table 1 below:

Table 1: Drying Techniques powder formula drying technology 1 (comparison) A freeze-dried 2 (the present invention) A Spray freeze drying 3 (the present invention) B Spray freeze drying 4 (comparison) A Lyophilization followed by compression/grinding/sieving (fraction <20 μm used) 5 (comparison) A Lyophilization followed by compression/grinding/sieving (38-45 μm fraction was used) 6 (comparison) A Lyophilization followed by compression/grinding/sieving (53-75 μm fraction was used)

Freeze-dried:

According to the freeze-drying steps in Table 2, the alum formula with adsorbed HBsAg was freeze-dried using a Dura-Stop freeze-dryer (FTS System, Stone Ridge, New York).

Table 2: Lyophilization steps Step/Cycle condition freezing Pre-cooler temperature (ST) = 0°C, the cooling rate is 1.0°C/min, until ST = -55°C, keep for 15 minutes Wait until product temperature (PT) = -48°C for 120 minutes primary drying When the condensing temperature reaches -40°C, turn the condensing/vacuum (C/V) switch to "ON", turn on the vacuum pump and wait for the vacuum in the cabin to reach 150mT (20.0Pa) and wait for the vacuum of the foreline to reach 100mT (13.3 Pa) Raise the temperature at a rate of 1.0°C/min to ST=-25°C and keep it for 18 hours secondary drying Raise the temperature at a rate of 1.0°C/min to ST=10°C and keep it for 4 hours Raise the temperature at a rate of 1.0°C/min to ST=20°C and keep it for 11 hours

A vacuum of 100 mT (13.3 Pa) is maintained throughout the primary and secondary drying process.

Spray freeze-drying:

Each suspension was sprayed into a stirred stainless steel pan with liquid nitrogen using an ultrasonic nebulizer (Sono Tek Corporation, Milton, New York) at a nozzle frequency of 60 kHz. The acoustic power used for nebulization was set at 5.0 watts. The liquid feed rate of the MasterFlex C/L peristaltic pump is 1.5ml/min. The liquid nitrogen tray containing the frozen particles was loaded into a Dura-lyophilizer pre-cooled to -50°C, and then freeze-dried according to the conditions in Table 3.

Table 3: Lyophilization steps Step/Cycle condition freezing Pre-cooler temperature (ST) = -50°C The cooling rate is 1.0°C/min, until ST = -55°C, keep for 15 minutes Wait until product temperature (PT) = -48°C for 120 minutes primary drying When the condensing temperature reaches -40°C, turn the condensing/vacuum (C/V) switch to "ON", turn on the vacuum pump and wait for the vacuum degree in the cabin to reach 150mT (20.0Pa) and wait for the vacuum degree of the pre-extraction pipeline to reach 100mT (13.3Pa) Raise the temperature at a rate of 1.0°C/min to ST=-25°C and keep it for 18 hours secondary drying Raise the temperature to ST=20°C at a rate of 1.0°C/min and keep it for 9 hours

A vacuum of 200mT (16.6Pa) is maintained throughout the primary and secondary drying process.

Compression/grinding/sieving:

The lyophilized material is granulated using the compression, grinding and sieving ("C/G/S") technique. Specifically, the lyophilized material was compressed at 12,000 psi for 5-10 minutes in a 13 mm diameter stainless steel die (Carver Press, Wabash, Indiana). Grind the biscuits by hand with a mortar and pestle. The milled powder was manually sieved with a nest of sieves (3 inches in diameter) and separated into three diameter fractions: 53-75 μm, 38-53 μm and 20-38 μm.

Experiment 1: Effect of the drying process on the degree of coagulation

Powders 1 to 3 were reconstituted at a ratio of 1:500 w/w and examined by light microscopy following standard techniques. Particles were analyzed visually using an optical microscope (Model DMR, Leica, Germany) with a 10x eyepiece and a 5x objective. The microscope was equipped with a Polaroid camera for image output. The degree of coagulation of alum was qualitatively analyzed by optical microscopic examination. In this experiment, powder 1 formed large agglomerates after rehydration, while powder 2 only slightly agglomerated. Powder 3 had almost no agglomeration at all.

The particle size of the rehydrated powder was also quantitatively determined. Stir/sonicate the rehydrated powder sample to obtain a homogeneous suspension. The suspension was then added to a glass container of a particle size analyzer (AccuSizer 780, Particle Sizing Systems, Santa Barbara, California) to determine its particle size distribution. The measurement results of powders 2 and 3 before and after spray freeze-drying are shown in Fig. 1 . Figure 2 shows a similar comparison of the particle size of Powder 1 before and after spray freeze-drying. These results showed that powders 2 and 3 had similar particle size distributions before and after drying, suggesting that little or no alum agglomeration occurred during lyophilization. In contrast, the particle size of Powder 1 increased significantly after drying, indicating significant alum aggregation.

Experiment 2: Effect of condensation on the stability of alum containing hepatitis B vaccine

We investigated the effect of alum coacervation on the immunogenicity of alum-adsorbed hepatitis B vaccine. As mentioned earlier, agglomeration was severe when the HBV vaccine (containing alum) was dried by lyophilization, but no agglomeration occurred in the spray-lyophilized HBV vaccine. In this mouse experiment, we compared the immunogenicity of lyophilized and spray-lyophilized HBV vaccines. To determine which fraction of the vaccine is more immunogenic, we compared the immunogens of unfractionated and different fractions (<20, 38-45 and 53-75 μm in diameter) of lyophilized HBV vaccine sex. The experimental design is shown in Table 4.

Table 4: Experimental design for mouse immunogenicity studies group formula* drying technology particle size Injection route (reconstituted) 1 A freeze-dried unrated peritoneum 2 A freeze-dried ＜20μm peritoneum 3 A freeze-dried 38-45μm peritoneum 4 A freeze-dried 53-75μm peritoneum 5 A Spray freeze drying 10-75μm peritoneum 6 B Spray freeze drying 10-75μm peritoneum 7 unprocessed liquid alum vaccine - peritoneum

*See above for details of recipes A and B.

The powder was rehydrated with distilled water and used to immunize Balb/C mice (female, 8 per group, 5-7 weeks old at the start of the experiment). The reconstituted vaccine was administered by intraperitoneal injection using a 23 1/5 needle. Each injection contains 200 l of a solution containing 2 μg of HBsAg adsorbed by alum. A control group of mice was immunized with untreated liquid hepatitis B vaccine. After the initial immunization period (0 day) and the immunization booster period (28 days), enzyme-linked immunosorbent assay (ELISA) was performed on the serum collected on the 42nd day to measure the immune response to hepatitis B vaccine. Antibody titers were determined against a reference serum.

As shown in Figure 3, the results of these experiments clearly showed that alum aggregation caused by lyophilization resulted in a reduction or even loss of immunogenicity of the hepatitis B vaccine. The immunogenicity of the freeze-dried HBV vaccine (Group 1) was reduced compared to the untreated liquid vaccine. The immunogenicity of the lyophilized particles was inversely proportional to the size of the particles (Groups 2, 3 and 4). Fractions with larger particle sizes are less immunogenic than smaller sized particles. This clearly shows that the immunity of the large size particles formed by the agglomeration is lost. The spray-lyophilized hepatitis B vaccine maintained its immunogenicity compared to untreated vaccine (Groups 5 and 6). The content of alum in the total dry matter (50% or 12%) had no effect on the immunity of the dry powder. Neither of the spray-lyophilized powders had agglomeration problems. It is important that the spray lyophilized formulation preserves the immunity of the alum salt adjuvant at very high concentrations (3% by weight).

Based on the above data, it can be concluded that the condensation of alum by freeze-drying technology is related to the loss of immunity of alum vaccine. It is believed that those agglomerated large-sized particles do not dissolve in the body, that they cannot be processed by cells of the immune system, and thus are not immune. More importantly, the method of the present invention can produce a stable dry powder in which the vaccine contains alum without causing agglomeration. The step of rapid freezing in liquid nitrogen used in the spray freeze-drying method is believed to be critical in preventing agglomeration, which preserves immunity.

Experiment 3: Effects of excipients and drying methods on the stability of spray-lyophilized hepatitis B vaccine

In this experiment, the effects of excipients and different spray-lyophilization methods on the stability of alum vaccines were investigated. The hepatitis B surface antigen (HbsAg) adsorbed by aluminum hydroxide was used as the model antigen. Furthermore, the immunogenicity of the spray-lyophilized powder in mice was investigated by two different immunization routes. The aforementioned two immunization routes are intramuscular injection with a needle and epidermal powder immunization with a needle-free powder syringe. The excipients of the spray-lyophilized formulation are shown in Table 5. In this case, the spray-lyophilized formulation used a combination of two sugars and one polymer. No amino acids/amino acid salts are employed. The conditions for spray lyophilization were the same as in Table 3, but no compression/grinding/sieving steps were used. The particle size distribution of the spray freeze-dried powder is also shown in Table 5.

Table 5: Comparison of spray-lyophilized formulations formula vaccine excipient method Particle size, μm (Aerosizer) Dv10 Dy50 Dv95 SFD-C 2 μg HBsAg/50 μg alum Trehalose/Mannitol/PEG (3:4:3) Spray freeze drying twenty three 38 57 SFD-D 2 μg HBsAg/50 μg alum Trehalose/mannitol/37kD dextran (3:4:3) Spray freeze drying 26 39 59 SFD-E 2 μg HBsAg/50 μg alum Trehalose/mannitol/10kD dextran (3:4:3) Spray freeze drying twenty four 36 56

The immunogenicity of the spray-lyophilized formulation was studied in mice. Balb/C mice (female, 8 per group, 5-7 weeks old at the start of the experiment) were used. The design of the experiment is shown in Table 6. For intramuscular injection (IM), the powder is rehydrated in distilled water and 200 l of this solution containing 2 μg of HBsAg adsorbed by alum is injected into the quadriceps muscle with a 23 1/5 needle. For transdermal (EPI) powder immunization, the abdominal skin of shaved mice was administered using a refillable powder delivery device. A control group of mice was immunized with untreated hepatitis B vaccine by intramuscular injection. After the initial immunization period (0 day) and the immunization booster period (28 days), enzyme-linked immunosorbent assay (ELISA) was performed on the serum collected on the 42nd day to measure the immune response to hepatitis B vaccine. Antibody titers were determined against a reference serum.

Table 6: Experimental design for mouse immunization studies group formula Rehydration way 1 SFD-C yes IM 2 SFD-D yes IM 3 SFD-E yes IM 4 SFD-C no EPI 5 SFD-D no EPI 6 SFD-E no EPI 7 unprocessed not applicable IM

The results of this study are shown in Figure 4, which clearly show that the three spray-lyophilized hepatitis B vaccines are immunogenic in mice, whether administered intramuscularly after reconstitution or transdermally as a powder. sex. These formulations used different excipients, but there was no significant difference in the immunogenicity of these formulations. All of these formulations had no problems with coagulation upon rehydration (data not shown). This further demonstrates that the quick freezing step in the spray freeze-drying method is a critical step to stabilize alum. The role of excipients is less critical. This study also showed that alum-adsorbed spray-lyophilized vaccines are useful for immunization by different routes, such as reconstituted intramuscular injection or transdermal administration in powder form.

Experiment 4: Immunogenicity of spray-lyophilized diphtheria-tetanus toxoid vaccine

To determine that other vaccines containing alum can be prepared stably by spray lyophilization, we prepared a spray lyophilized powder using diphtheria-toxoid vaccine from CSL Limited (Australia). The commercial vaccine contains 5% w/v of aluminum phosphate which adsorbs diphtheria toxoid and tetanus toxoid at concentrations of 563Lf/mL, respectively. Spray freeze-dried diphtheria-tetanus-toxoid vaccine was prepared according to the conditions described in Table 3, followed by compression/grinding/sieving to obtain particles with an average particle size of 20-38 μm and 38-53 μm

The formulation information is summarized in Table 7. Observation by light microscopy after rehydration revealed no problems with aggregation of these particles (data not shown).

Table 7 HBsAg-aluminophosphate (mg) Trehalose dihydrate (mg) Glycine (mg) Dextran (mg) Total solid content (%) Diphtheria-Tetanus (DT) Dosage 250 292.9 66.1 86.6 4 3 Lf/1-mg powder

The immunogenicity of the spray lyophilized diphtheria-tetanus-toxoid vaccine was determined in guinea pigs (Charles River). Immunization was carried out on the abdominal skin of guinea pigs (4 animals/group) using a powder drug delivery device on day 0 and day 28. Each guinea pig received 0.5 mg of powder containing 1.5 Lf of diphtheria toxoid and 1.5 Lf of tetanus toxoid adsorbed by 250 μg of aluminum phosphate. A control group of guinea pigs was immunized by intramuscular injection of untreated vaccine using a 23 1/2 needle. Serum antibody responses to diphtheria toxoid and tetanus toxoid were measured by enzyme-linked immunosorbent assay using sera collected on day 42.

The results of the immunogenicity study are shown in FIG. 5 . Transdermal powder immunization with alum-adsorbed spray-lyophilized diphtheria toxoid elicited antibody responses to each vaccine component with antibody titers similar to those elicited by transdermal injection of untreated vaccine. The size of the spray-lyophilized powders does not appear to have a significant effect on immunogenicity, as these powders do not have aggregation problems in vivo. The smaller sized fraction of the spray-lyophilized formulation particles resulted in slightly lower titers against diphtheria toxoid than the larger sized fraction. This study again demonstrates that the spray freeze-drying method preserves immunity in dry solid dose forms of alum-containing vaccines.

We therefore describe new spray-lyophilized powder compositions and methods for their preparation. Although the invention has been described in detail with respect to a preferred embodiment, it should be understood that significant changes may be made therein without departing from the spirit and scope of the invention as defined in the appended claims.

Claims (49)

1. A powder suitable for use as a vaccine, said powder forming a gel and free-flowing, said powder being formed by spray-drying or spray-lyophilizing an aqueous suspension comprising: (a) 0.1-0.95% by weight of an aluminum or calcium salt adjuvant that has adsorbed the antigen; (b) 0.5-6% by weight of sugar; (c) 0.1-2% by weight of an amino acid or a salt thereof; and (d) 0.02-1% by weight of colloidal substances. 2. Powder according to claim 1, wherein the adjuvant is aluminum hydroxide or aluminum phosphate. 3. Powder according to claim 1, wherein 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 sugar is a monosaccharide, a disaccharide or a sugar alcohol. 6. The powder according to any one of claims 1 to 4, wherein the sugar is selected from the group consisting of glucose, xylose, galactose, fructose, D-mannose, sorbose, lactose, maltose, sucrose, trehalose, sucrose, Group consisting of mannitol, sorbitol, xylitol, 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. 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, glutamic acid, arginine, lysine, histidine and glutamic acid monosodium salt Group. 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. Powder according to claim 9, wherein said substance is selected from the group consisting of dextran, maltose-dextran complex, gelatin, agarose and human serum albumin. 11. Powder according to any one of the preceding claims, wherein the aqueous suspension contains 0.2 to 0.4% by weight of an adjuvant with adsorbed antigen, 2 to 4% by weight of sugar, 0.75% to 1.25% by weight of amino acid or a salt thereof , and 0.07% to 0.3% by weight of colloidal substances. 12. A powder according to any one of the preceding claims, comprising: (i) Adjuvant with 7-50% weight of antigen adsorbed; (ii) 30-80% by weight sugar; (iii) 7-30% by weight of amino acids or salts thereof; and (iv) 0.8-6% by weight of colloidal substances. 13. A powder according to any one of the preceding claims, said powder having a mass median aerodynamic diameter of 10 to 100 μm and a bulk density of 0.8 to 1.5 g/ ^cm3 . 14. A powder according to any one of the preceding claims, wherein the powder forms a gel-like suspension without forming a precipitate after being added to distilled water (1:500 by weight) and shaken for 3 minutes. 15. A process for the preparation of a powder suitable for use as a vaccine, said powder being gel-forming and free-flowing, said process comprising the step of spray-drying or spray-lyophilizing an aqueous suspension comprising: (a) 0.1-0.95% by weight of an aluminum or calcium salt adjuvant that has adsorbed the antigen; (b) 0.5-6% by weight of sugar; (c) 0.1-2% by weight of an amino acid or a salt thereof; and (d) 0.02-1% by weight of colloidal substances. 16. The method according to claim 15, wherein the aqueous suspension contains 0.2-0.4% by weight of the adjuvant that has adsorbed the antigen, 2-4% by weight of sugar, 0.75-1.25% by weight of amino acid or its salt, and 0.07- 0.3% by weight of colloidal substances. 17. The method according to claim 15 or 16, wherein the obtained powder is added to distilled water (1:500 by weight) and shaken for 3 minutes, forming a suspension similar to a gel without forming a precipitate. 18. A dosage container for a needle-free injector containing an effective amount of a powder as defined in any one of claims 1 to 14, which powder is gel-forming and free-flowing. 19. The container according to claim 18, wherein the container is selected from the group consisting of capsules, foil pouches, sachets and boxes. 20. A needle-free injector containing a powder as defined in any one of claims 1 to 14 which is gel-forming and free-flowing. 21. A vaccine composition comprising a pharmaceutically acceptable carrier or diluent and a powder as defined in any one of claims 1 to 14 which is gel-forming and free-flowing. 22. A method of immunizing a subject, wherein the method comprises the step of administering to said subject a powder as defined in any one of claims 1 to 14, said powder being gel-forming and free flowing. 23. The method of claim 22, wherein the powder is administered using a needle-free syringe. 24. A method according to claim 22, wherein the powder is formulated with a pharmaceutically acceptable carrier or diluent. 25. The 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, said powder comprising: (i) 5-60% by weight of an aluminum or calcium adjuvant that has absorbed the antigen; (ii) 25-90% by weight sugar; (iii) 4.5-40% by weight of amino acids or salts thereof; and (iv) 0.5-10% by weight of colloidal substances. 27. A powder according to claim 26, comprising: (i) Adjuvant with 7-50% weight of antigen adsorbed; (ii) 30-80% by weight sugar; (iii) 7-30% by weight of amino acids or salts thereof; and (iv) 0.8-6% by weight of colloidal substances. 28. The powder according to claim 26 or 27, wherein the powder forms a gel-like suspension without forming a precipitate after being added to distilled water (1:500 by weight) and shaken for 3 minutes. 29. A powder suitable for use as a vaccine obtained by spray lyophilization of an aqueous suspension containing an adjuvant of aluminum or calcium salts to which the antigen is adsorbed. 30. A powder according to claim 29, wherein the adjuvant is aluminum hydroxide, aluminum phosphate, aluminum sulfate or calcium phosphate. 31. 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 contains less than 10% by weight of the adjuvant to which the antigen has been adsorbed. 33. A powder according to any one of claims 29 to 32, said powder having a mass median aerodynamic diameter of 1 to 100 μm and a bulk density of 0.8 to 1.5 g/ ^cm3 . 34. Powder according to any one of claims 29 to 33, wherein the suspension further comprises amorphous sugars, crystalline sugars and optionally polymers and/or amino acids or salts thereof. 35. The powder according to any one of claims 29 to 34, wherein the powder forms a gel-like suspension without forming a precipitate after being 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, said process comprising the step of spray-lyophilizing an aqueous suspension containing an adjuvant of aluminum or calcium salt to which the antigen is adsorbed. 37. The method according to claim 36, wherein the adjuvant is aluminum hydroxide, aluminum phosphate, aluminum sulfate or calcium phosphate. 38. A method according to claim 36 or 37, wherein the antigen is a bacterial or viral antigen. 39. A method according to any one of claims 36 to 38, wherein the aqueous suspension contains less than 10% by weight of the adjuvant with adsorbed antigen. 40. A method according to any one of claims 36 to 39, wherein the suspension further comprises amorphous sugars, crystalline sugars and optionally polymers and/or amino acids or salts thereof. 41. The method according to any one of claims 36 to 40, wherein the spray-lyophilized powder obtained is added to distilled water (1:500 by weight) and shaken for 3 minutes to form a suspension similar to a gel liquid without forming a precipitate. 43. A dosage container for a needle-free injector containing an effective amount of a powder as defined in any one of claims 29 to 35. 44. The container according to claim 43, wherein the container is selected from the group consisting of capsules, foil pouches, sachets and boxes. 45. A needle-free injector filled 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 immunizing a subject, wherein the method comprises the step of administering to said subject an effective amount of a powder as defined in any one of claims 29 to 35. 48. The method of claim 47, wherein the powder is administered using a needle-free syringe. 49. The method according to claim 47, wherein the powder is formulated with a pharmaceutically acceptable carrier or diluent. 50. The method according to claim 49, wherein the formulation is administered subcutaneously or intramuscularly.

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