Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/08—Solutions
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/095—Sulfur, selenium, or tellurium compounds, e.g. thiols
  • A61K31/10—Sulfides; Sulfoxides; Sulfones
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/13—Amines
  • A61K31/131—Amines acyclic
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/13—Amines
  • A61K31/14—Quaternary ammonium compounds, e.g. edrophonium, choline
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K33/00—Medicinal preparations containing inorganic active ingredients
  • A61K33/06—Aluminium, calcium or magnesium; Compounds thereof, e.g. clay
  • A61K33/08—Oxides; Hydroxides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
  • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
  • A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/20—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing sulfur, e.g. dimethyl sulfoxide [DMSO], docusate, sodium lauryl sulfate or aminosulfonic acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
  • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55505—Inorganic adjuvants

Definitions

• the invention relates to a method for preserving an aluminium salt adjuvant during freezing or drying, typically during freezing or drying of a vaccine preparation comprising an aluminium salt adjuvant and one or more vaccine antigens.
• Aluminium salt adjuvants are currently the most widely used adjuvants for human and veterinary vaccines.
• Aluminium adjuvant compounds include aluminium salts such as aluminium phosphate (A1P0 4 ) and aluminium hydroxide (Al(OH) 3 ) which are generically referred to in the field of vaccine adjuvants as "alum ".
• aluminium salts such as aluminium phosphate (A1P0 4 ) and aluminium hydroxide (Al(OH) 3 ) which are generically referred to in the field of vaccine adjuvants as "alum ".
• Al(OH) 3 aluminium hydroxide
• alum adjuvants act as an immune system stimulus as well as providing a depot of antigen at the site of administration (e.g. by injection) thereby providing a gradual and continuous release of antigen to stimulate antibody production.
• Aluminium adjuvants in their natural form are commonly known as gels, which are particulate suspensions in aqueous media.
• Freeze-drying is a process frequently used to improve long-term stability of various protein preparations. Nevertheless, commercial vaccine compositions containing aluminium salt adjuvants cannot be freeze-dried without causing damage to the adjuvant structure. Freeze-drying causes the collapse of the gel structure of the adjuvant resulting in aggregation and precipitation of the adjuvant salt on resuspension in water. The effect is to significantly reduce the immunogenicity of the vaccine.
• WO 01/93829 describes a method of preparing an adjuvanted vaccine comprising spray-drying or spray freeze-drying an aqueous solution comprising: (a) from 0.1 to 0.95% by weight of an aluminium salt or calcium salt adjuvant having an antigen adsorbed therein;
• WO 2008/118691 describes a method of preparing an immunologically-active adjuvant-bound dried vaccine composition comprising (a) combining at least one aluminium-salt adjuvant, at least one buffer system, at least one glass-forming agent and at least one antigen to create a liquid vaccine formulation; (b) freezing the liquid vaccine formulation to create a frozen vaccine formulation; and (c) lyophilizing the frozen vaccine formulation to create a dried vaccine composition.
• the glass-forming agent is preferably trehalose.
• an aluminium salt adjuvant can be reduced by freezing or drying, in particular freeze- drying, the adjuvant in the presence of a compound of formula (I) or (II) or a physiologically acceptable salt or ester thereof.
• the additional presence of one or more sugars can lead to a further reduction in the structural damage to the adjuvant during freezing or drying.
• the present invention provides a method for preserving an aluminium-salt adjuvant during freezing or drying comprising freezing or drying an aqueous suspension or solution comprising:
• R ⁇ represents hydrogen or C 1-6 alkyl
• - R represents hydrogen
• R 2 represents hydrogen, C ]-6 alkyl or -(CH 2 ) 2-5 NHC(0)(CH 2 ) 5-15 CH 3 ;
• R 3 represents Ci -6 alkyl; or a compound of formula (II) or a physiologically acceptable salt or ester thereof
• - X represents -S(0) 2 - or -S + (Rc)- ;
• R a and R b independently represent C 1-6 alkyl
• Rc represents C 1-6 alkyl substituted with a carboxylate anion and with an
• the present invention also provides:
• an excipient comprising (i) a compound of formula (I) or (II) of the invention or a physiologically acceptable salt or ester thereof and (ii) optionally, one or more sugars, for preserving an aluminium salt adjuvant during freezing or drying; - a vaccine composition comprising: an aluminium-salt adjuvant; one or more antigens; a compound of formula (I) or (II) of the invention or a physiologically acceptable salt or ester thereof; and optionally, one or more sugars.
• a vaccine composition obtainable by the method of the invention; and use of an excipient comprising (i) a compound of formula (I) or (II) of the invention or a physiologically acceptable salt or ester thereof and (ii) optionally one or more sugars, as a resuspension agent for a vaccine composition.
• the frozen or dried vaccine compositions facilitate appropriate storage and maximize the shelf-life of the compositions.
• the compositions can be stock piled for prolonged periods of time.
• the immunogenicity, potency and efficacy of the vaccines can thus be maintained.
• the compound of formula (I) or (II) or physiologically acceptable salt or ester thereof and the optional sugar(s) act as cryoprotectants and protect the aluminium salt adjuvants against the stresses encountered during freezing and also as a lyoprotectant during freeze-drying.
• Figure 1 shows the results of analysing adjuvants microscopically in the Reference Examples after freezing the aluminium hydroxide gel.
• Panel A shows an example of normal undamaged structure and panel B shows damaged agglomerated crystalline structure post-freezing of the aluminium hydroxide adjuvant.
• Figure 2 shows the results of an adjuvant agglomeration assay after freezing an aluminium hydroxide gel in the presence of various concentrations of sucrose and dimethylglycine (DMG) in Example 1.
• Figure 3 shows recovery of adjuvant (Al(OH) 3 ) after freeze-thaw in the formulations described in Example 2 containing sucrose and/or trimethylglycine (TMG) as assessed using an agglomeration assay.
• Figure 4 shows recovery of adjuvant (Al(OH) 3 ) after freeze-thaw in the formulations described in Example 2 containing sucrose and/or S-methyl-L- methionine (SMM) or methylsulfonylmethane (MSM) as assessed by an
• Figure 5 shows results of an adjuvant agglomeration assay after freeze-drying of an aluminium hydroxide gel in the presence of various concentrations of sucrose and dimethylglycine (DMG), trimethylglycine (TMG), S-methyl methionine (SMM) or sarcosine in Example 3.
• Figure 6 shows the percentage of BSA bound to the adjuvant in Example 6 compared to the control.
• Figure 7 shows the concentration of BSA bound to the adjuvant in Example 6.
• Figure 8 shows the dot blot results from Example 7.
• Figure 8A shows the dot blot of the samples set out in Table 19 stored at 4°C.
• Figure 8B shows the dot blot of the samples set out in Table 19 stored at -80°C
• Figure 9 shows more dot blot results from Example 7.
• Figure 9 A shows the dot blot of the samples set out in Table 20 stored at 4°C.
• Figure 9B shows the dot blot of the samples set out in Table 20 stored at -80°C.
• the present invention relates to the reduction and/or prevention of structural damage to aluminium salt vaccine adjuvants when frozen or dried, especially freeze- dried. Such structural damage is reduced or prevented by freezing or drying the adjuvant in the presence of a compound of formula (I) or (II) or physiologically acceptable salt or ester thereof and optionally (ii) one or more sugars.
• the aluminium salt adjuvant on which typically at least one antigen is adsorbed, is contacted with the compound of formula (I) or (II) or physiologically acceptable salt or ester thereof in aqueous solution.
• the resulting aqueous solution is contacted with the compound of formula (I) or (II) or physiologically acceptable salt or ester thereof in aqueous solution.
• composition in which one or more sugars may also be present, is then frozen or dried.
• the method is a method of preparing a vaccine
• composition comprising an aluminium salt adjuvant and at least one antigen.
• a vaccine preparation comprising the aluminium adjuvant can be thawed or
• the invention enables the structure and function of the aluminium adjuvant to be preserved during the freezing or drying step.
• the immunogenicity of aluminium adjuvanted vaccines following freezing or drying can consequently be maintained.
• Aluminium salt adjuvant
• aluminium salt suitable for use as an adjuvant may be used in the invention.
• the aluminium salt may be aluminium hydroxide (Al(OH) 3 ), aluminium phosphate (A1P0 4 ), aluminium hydrochloride, alumimum sulphate, ammonium alum, potassium alum or alumimum silicate.
• the aluminium salt adjuvant used is aluminium hydroxide or aluminium phosphate.
• the aluminium salt adjuvant is aluminium hydroxide (Al(OH) 3 ).
• the aluminium salt adjuvant takes the form of a hydrated gel made from an aluminium salt, the hydrated gel being a particulate suspension in aqueous media.
• aluminium-salt adjuvants are well known to those skilled in the art.
• alumimum hydroxide and aluminium phosphate adjuvants are generally prepared by exposing aqueous solutions of alumimum ions (typically as sulfates or chlorides) to alkaline conditions in a well-defined and controlled chemical environment, as known to those skilled in the art.
• alumimum ions typically as sulfates or chlorides
• Such methods can be used for example, to prepare an aluminium hydroxide or aluminium phosphate hydrated geL
• an antigen suitable for use in the invention includes any immunogenic component of a vaccine.
• the antigen may be a protein, bacterial-specific protein, mucoprotein, glycoprotein, peptide, lipoprotein, polysaccharide,
• peptidoglycan peptidoglycan, nucleoprotein or fusion protein.
• the antigen may be derived from a microorganism (such as a bacterium, virus or fungus), a protozoan, a tumour, a malignant cell, a plant, an animal, a human, or an allergen.
• the antigen is a protein but excludes a whole virus or virion.
• the antigen may be synthetic, for example as derived using recombinant DNA techniques.
• the antigen may be a disease-related antigen such as a pathogen-related antigen, tumour-related antigen, allergy-related antigen, neural defect-related antigen, cardiovascular disease antigen, rheumatoid arthritis-related antigen.
• the antigen may be an inactivated or attenuated/detoxifed toxin (toxoid).
• the pathogens from which the vaccine immunogen is derived may include human papilloma viruses (HPV), HIV, HSV2/HSV1, influenza virus (types A, B and C), para influenza virus, polio virus, RSV virus, rhinoviruses, rotaviruses, hepaptitis A virus, norwalk virus, enteroviruses, astroviruses, measles virus, mumps virus, varicella-zoster virus, cytomegalovirus, epstein-barr virus, adenoviruses, rubella virus, human T-cell lymphoma type I virus (HTLV-I), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus, poxvirus, vaccinia virus, Salmonella, Neisseria, Borrelia, Chlamydia, Clostridium such as C.
• HPV human papilloma viruses
• HIV HIV
• HSV2/HSV1 influenza virus
• Bordetella such as Bordetella pertussis
• Corynebacterium such as C. diptheriae
• Plasmodium Plasmodium
• Coxoplasma Pneumococcus
• Meningococcus Cryptococcus
• Streptococcus Vibriocholerae, Staphylococcus, Haemophilus, Bacillus such as Bacillus anthracis (anthrax), Escherichia, Candida, Aspergillus, Entamoeba, Giardia and Trypan asoma.
• the vaccine may further be used to stimulate a suitable immune response against numerous veterinary diseases.
• the vaccine antigen may therefore be derived from a foot and mouth disease virus (including serotypes O, A, C, SAT-1, SAT-2, SAT-3 and Asia-1), coronavirus, bluetongue virus, feline leukaemia virus, avian influenza virus, hendra and nipah virus, pestivirus such as bovine viral diarrhoea virus and canine parvovirus.
• Tumor-associated antigens include for example, melanoma-associated antigens, mammary cancer-associated antigens, colorectal cancer-associated antigens or prostate cancer-associated antigens
• An allergen-related antigen includes any allergen antigen suitable for use in a vaccine to stimulate suppression of an allergic reaction in an individual to which the vaccine is administered (e.g. antigens derived from pollens, dust mites, insects, food allergens, dust, poisons, toxins, venoms and parasites).
• allergens derived from pollens, dust mites, insects, food allergens, dust, poisons, toxins, venoms and parasites.
• the compound of formula (I) and (II) may be present as a physiologically acceptable salt or ester thereof.
• the salt is typically a salt with a physiologically acceptable acid and thus includes those formed with an inorganic acid such as hydrochloric or sulphuric acid or an organic acid such as citric, tartaric, malic, maleic, mandelic, fumaric or
• hydrochloride salt is preferred.
• the ester is typically a Ci -6 alkyl ester, preferably a Cj_4 alkyl ester.
• the ester may therefore be the methyl, ethyl, propyl, isopropyl, butyl, isobutyl or tert-butyl ester.
• the ethyl ester is preferred.
• a Ci -6 alkyl group is preferably a C alkyl group.
• Preferred alkyl groups are selected from methyl, ethyl, propyl, isopropyl, butyl, isobutyl and tert-butyl. Methyl and ethyl are particularly preferred.
• the definitions of compounds of formula (I) and formula (II) also include compounds in which the carboxylate anion is protonated to give -COOH and the ammonium or sulfonium cation is associated with a
• Ri represents hydrogen or C 1-6 alkyl and R4 represents hydrogen.
• R 2 represents hydrogen or C 1-6 alkyl.
• P represents hydrogen or Ci_6 alkyl
• R4 represents hydrogen and R 2 represents hydrogen or C 1-6 alkyl.
• the compound of formula (I) is an N-Ci- alkyl-, N,N-di(Ci-6 alkyl)- or N,N,N-tri(Ci-6 alkyl)-glycine or physiologically acceptable salt or ester thereof.
• the alkyl group is typically a C 1-4 alkyl group.
• Preferred alkyl groups are selected from methyl, ethyl, propyl, isopropyl, butyl, isobutyl and tert-butyl. Methyl and ethyl are particularly preferred.
• Preferred compound of formula (I) are N-methylglycine, N,N-dimethylglycine or ⁇ , ⁇ , ⁇ -trimethylglycine or physiologically acceptable salts or esters thereof.
• N- Methyl-glycine is also called sarcosine.
• ⁇ , ⁇ -Dimethylglycine is also termed dimethylglycine (DMG) or 2-(dimethylamino)-acetic acid.
• ⁇ , ⁇ , ⁇ -trimethylglycine is termed trimethylglycine (TMG).
• the compound of formula (I) is typically a glycine derivative of formula (IA) or a physiologically acceptable salt or ester thereof:
• R 5 and 3 ⁇ 4 independently represent C 1-6 alkyl, for example C alkyl such as methyl or ethyl; and R represents C 1-6 alkyl, for example C 1-4 alkyl such as methyl or ethyl, or -(CH 2 ) 2-5 NHC(0)(CH 2 ) 5- i 5 CH3.
• Preferred compounds of formula (IA) are trimethylglycine (TMG) and cocamidopropyl betaine (CAPB) or physiologically acceptable salts or esters thereof.
• the compound of formula (I) is typically a proline derivative of formula (IB) or a physiologically acceptable salt or ester thereof:
• R 9 independently represent Ci- alkyl, for example C 1-4 alkyl such as methyl or ethyl.
• the compound of formula (IB) is an S-proline derivative.
• R 3 ⁇ 4 and R 9 both represent methyl; this compound is known as proline betaine.
• S-proline betaine or physiologically acceptable salt or ester thereof is particularly preferred:
• the compound of formula (I) is N, N-dimethylglycine or physiologically acceptable salt or ester thereof.
• Rc is attached to the same carbon atom of the R c alkyl moiety.
• R c is a C 2-4 or C 2-3 alkyl moiety.
• the compound of formula (II) is typically a sulfone compound of formula (IIA) or a physiologically acceptable salt or ester thereof:
• Rc and R d independently represent Ci -6 alkyl, for example C alkyl such as methyl or ethyl.
• a preferred sulfone compound is methylsulfonylmethane (MSM), which is also known as dimethylsulfone (DMS0 2 ).
• the compound of formula (II) is typically a compound of formula (IIB) or a physiologically acceptable salt or ester thereof:
• Re and R f independently represent Ci -6 alkyl, for example C alkyl such as methyl or ethyl, and R g represents C 1-6 alkyl, for example C alkyl such as methyl or ethyl, substituted with a carboxylate anion and with an amine (-N3 ⁇ 4) moiety.
• a preferred compound of formula (IIB) is S-methyl-L-methionine (SMM) or a physiologically acceptable salt or ester thereof.
• Sugars suitable for use in the present invention include reducing sugars such as glucose, fructose, glyceraldehydes, lactose, arabinose and maltose; and preferably non-reducing sugars such as sucrose and raffinose.
• the sugar may be a
• sugar alcohols examples include sugar alcohols.
• Monosaccharides such as galactose and mannose; dissaccharides such as sucrose, lactose and maltose; trisaccharides such as raffinose and tetrasaccharides such as stachyose are envisaged.
• Trehalose, umbelliferose, verbascose, isomaltose, cellobiose, maltulose, turanose, melezitose and melibiose are also suitable for use in the present invention.
• a suitable sugar alcohol is mannitol.
• sugars may be present. Two, three or four sugars may be used. When one or more sugars are present in the aqueous suspension that is frozen or freeze-dried, preferably sucrose or sucrose and raffinose are present. Sucrose is a disaccharide of glucose and fructose. Raffinose is a trisaccharide composed of galactose, fructose and glucose. Aqueous suspension to be frozen or dried
• the aqueous suspension or solution to be frozen or dried can be prepared by admixing the aluminium salt adjuvant with an aqueous solution of the compound of formula (I) or (II) or physiologically acceptable salt or ester thereof.
• the compound of formula (I) or (II) or physiologically acceptable salt or ester thereof may in particular be selected from dimethylglycine, S-methyl-L-methionine,
• methylsulfonylmethane, sarcosine and trimethylglycine and may for example be dimethylglycine, S-methyl-L-methionine, methylsulfonylmethane or trimethylglycine.
• Any suitable aqueous solution may be used.
• the solution may be buffered.
• the solution may be a HEPES, Tris-buffered, phosphate-buffered or pure water solution.
• one or more sugars is dissolved in the aqueous solution prior to admixture with the adjuvant.
• the sugar(s) can be admixed with the suspension of the adjuvant in the aqueous solution of the compound of formula (I) or (II) or physiologically acceptable salt or ester thereof.
• the antigen(s) are generally adsorbed onto the adjuvant prior to admixture of the adjuvant with the aqueous solution of the compound of formula (I) or (II) or physiologically acceptable salt or ester thereof.
• the adjuvants can be prepared in the form of a hydrated gel and the antigen adsorbed into the hydrated gel.
• Antigen adsorption can be carried out using techniques well known to those skilled in the art. For example, for certain protein antigens, adsorption may best be carried out at a pH interval where the adjuvant and antigen will have opposite electrical charges, facilitating electrostatic attraction and adsorption. Protein adsorption for a particular antigen-adjuvant combination will depend on the nature of the antigen and the chemical environment (pH, ionic strength, presence of surfactants etc).
• concentrations of the compound of formula (I) or (II) or physiologically acceptable salt or ester thereof and of the or each sugar in the aqueous suspension or solution to be frozen or can be determined by routine experimentation. Optimised concentrations can thus be selected.
• the compound of formula (I) or (II) or physiologically acceptable salt or ester thereof can act synergistically with the sugar(s) to improve stability.
• concentration of the compound of formula (I) or (II) or physiologically acceptable salt or ester thereof in the aqueous suspension or solution is typically in the range of 0.001 or more, preferably in the range of 0.01M or more and more preferably 0.1M or more, for example from 0.1M to 5.0M.
• concentration that is employed will depend on several factors including, where present, the nature of the antigen; the particular compound of formula (I) or (II) or physiologically acceptable salt or ester thereof being used; whether one or more sugar is being used and if so the identity of the sugar(s); and the particular freezing or drying procedure that is adopted.
• the concentration of a compound of formula (I) or a compound of formula (IA) or formula (IB), such as TMG, or a physiologically acceptable salt or ester thereof is preferably from 0.01M to 5M, from 0.1M to 5M, from 0.2M to 5.0M or from 0.1M to 1M.
• concentration of a compound of formula (II) in which X represents - S(0) 2 - or a compound of formula (IIA), such as MSM, or a physiologically acceptable salt or ester thereof is preferably from 0.01M to 4M, from 0.05M to 2M or from 0.07M to 1M or even to 0.53M.
• concentration of a compound of formula (II) in which X represents - S + (Rc)- or a compound of formula (IIB), such as S-methyl-L-methionine, or a physiologically acceptable salt or ester thereof is preferably from 0.01M to 5M, from 0.1M to 5M, from 0.2M to 3M or from 0.1M to 1M.
• the concentration of a compound of formula (I) which is a N,N-di(Ci-6 alkyl)- , N,N,N-tri(Ci -6 alkyl)-, or N-Cj.6 alkyl-glycine, such as ⁇ , ⁇ -dimethylglycine, ⁇ , ⁇ , ⁇ - trimethylglycine, or N-methylglycine, or a physiologically acceptable salt or ester thereof is typically 0.01 M or more and preferably 0.1 M or more, for example from 0.1M to 5.0M, from 0.33M to 5.0M, from 0.5M to 4M or from 0.5M to 3M.
• the concentration of a compound of formula (I) which N,N-dimethylglycine (DMG) or a physiologically acceptable salt or ester thereof is typically 0.01M or more and preferably 0.1M or more, for example from 0.1M to 5.0M, from 0.33M to 5.0M, from 0.5M to 4M or from 0.5M to 3M. Less DMG or DMG salt or ester can be employed when one or more sugars are present.
• concentration of sugar in the aqueous suspension or solution that is to be frozen or dried is typically 1M or less or 0.7M or less, for example 0.5M or less or 0.29M or less.
• a 10% w/v sucrose solution has a sucrose concentration of 0.29M.
• the sugar concentration or the total concentration may be down to 0.1 mM, to 0.5mM, to 0.073M or to 0.146M.
• the concentration of sugar, if present, in the aqueous suspension or solution for freezing or drying is typically 1M or less or 0.7M or less, for example 0.5M or less or 0.29M or less.
• a 10% w/v sucrose solution has a sucrose concentration of 0.29M.
• the concentration of the sugar such as sucrose or raffinose or, if more than one sugar is present the total concentration of sugar is 0.5M or less, 0.2M or less,
• the minimum concentration of the sugar if present or, if more than one sugar is present, the minimum total concentration of sugar may be 0.01M, 0.1M or 0.2M.
• the sugar concentration for example the concentration of sucrose or raffinose, or the total concentration if more than one sugar is present may thus be from 0.01M to 0.7M, from 0.029M to 0.5M, from 0.058M to 0.3M or from 0.1M to 0.3M.
• the concentration of sucrose is preferably from 0.01 to 0.2M and the concentration of DMG or salt or ester thereof is preferably from 0.2 to 2M.
• the particular concentration that is employed will depend on several factors including the nature of the antigen, the particular the compound of formula (I) or (II) or physiologically acceptable salt or ester thereof being used and the particular freezing or drying procedure that is adopted.
• the sugar concentration or the total concentration may be from 0.1 mM to 0.7M, from 5mM to 0.7M, from 0.073M to 0.5M, or from 0.146M to 0.389M.
• the sugar is mannitol
• the mannitol concentration is typically 0.2 to 1M or 0.2 to 0.8M, preferably 0.25 to 0.6 or 0.4 to 0.8M, for example 0.5 to 0.6M.
• the most effective concentration of the compound of formula (I) or (II) or physiologically acceptable salt or ester thereof will depend on the particular type of compound used, whether it is used in combination with a sugar and the type of aluminium salt adjuvant that is used e.g. whether an aluminium hydroxide or aluminium phosphate adjuvant is used.
• the inventors Using a mixture of a compound of formula (I) or (II) or physiologically acceptable salt or ester thereof together with a sugar, the inventors have demonstrated that lower concentrations of each component can be used to achieve the same level of protection of the adjuvant as that obtained when each component is used separately.
• the invention has the advantage that lower concentrations of sugars can be used when in combination with a compound of formula (I) or (II) or physiologically acceptable salt or ester thereof. As a result, when such vaccine preparations are reconstituted or thawed, the concentration of sugar is reduced and the likelihood of site-specific reaction is minimised. Freezing Drying
• Freezing is conducted by any suitable method. Freezing may thus be carried out by immersing in liquid nitrogen or liquid nitrogen vapour, placing in a freezer at a temperature of from - °C to -80°C or using a dry ice and alcohol freezing bath. At atmospheric pressure, temperatures such as -4°C or below, -10°C or below, -15°C or below, -20°C or below, -25°C or below may be used.
• drying is achieved by freeze-drying, vacuum drying, spray-drying, spray freeze-drying or fluid bed drying. Freeze-drying is preferred.
• Freeze-drying is preferred.
• a composition which incorporates the viral particles.
• a matrix incorporating the viral particles is thus produced.
• the composition is typically an amorphous solid.
• a solid matrix, generally an amorphous solid matrix, is thus generally formed.
• amorphous is meant non-structured and having no observable regular or repeated organization of molecules (i.e. non-crystalline).
• the drying procedure can be effected to form an amorphous cake e.g. by freeze-drying.
• Freeze-drying can be carried out according to standard procedures. There are three main stages: freezing, primary drying and secondary drying. Freezing is typically performed using a freeze-drying machine. In this step, it is important to cool the biological material below its eutectic point, the lowest temperature at which the solid and liquid phase of the material can coexist. This ensures that sublimation rather than melting will occur in the following steps. Alternatively, amorphous materials do not have a eutectic point, but do have a critical point, below which the product must be maintained to prevent melt-back or collapse during primary and secondary drying.
• a cold condenser chamber and/or condenser plates provide surfaces on which the water vapour is trapped by
• the vacuum can either be broken with an inert gas such as nitrogen prior to sealing or the material can be sealed under vacuum.
• drying is carried out using vacuum desiccation at around 1300Pa.
• vacuum desiccation is not essential to the invention and in other embodiments, the preservation mixture contacted with the viral particle is spun (i.e. rotary desiccation) or freeze-dried (as further described below).
• the method of the invention further comprises subjecting the preservation mixture containing the viral particle to a vacuum.
• the vacuum is applied at a pressure of 20,000Pa or less, preferably 10,000Pa or less.
• the vacuum is applied for a period of at least 10 hours, preferably 16 hours or more. As known to those skilled in the art, the period of vacuum application will depend on the size of the sample, the machinery used and other parameters.
• drying is achieved by spray-drying or spray freeze- drying the viral particles admixed with the preservation mixture of the invention.
• spray-drying or spray freeze- drying are well known to those skilled in the art and involve a method of drying a liquid feed through a gas e.g. air, oxygen-free gas or nitrogen or, in the case of spray freeze-drying, liquid nitrogen.
• the liquid feed is atomized into a spray of droplets.
• the droplets are then dried by contact with the gas in a drying chamber or with the liquid nitrogen.
• drying is achieved by fluid bed drying the viral particles admixed with the preservation mixture of the invention.
• This technique is well known to those skilled in the art and typically involves passing a gas (e.g. air) through a product layer under controlled velocity conditions to create a fluidized state.
• the technique can involve the stages of drying, cooling, agglomeration, granulation and coating of particulate product materials.
• Heat may be supplied by the fluidization gas and/or by other heating surfaces (e.g. panels or tubes) immersed in the fluidized layer. Cooling can be achieved using a cold gas and/or cooling surfaces immersed in the fluidized layer.
• the steps of agglomeration and granulation are well known to those skilled in the art and can be performed in various ways depending on the product properties to be achieved.
• Coating of particulate products such as powders, granules or tablets can be achieved by spraying a liquid on the fluidized particles under controlled conditions.
• the composition that is produced by the freezing or drying is typically a solid matrix having a low residual moisture content.
• a level of residual moisture content is achieved which offers long term preservation of vaccine activity at temperatures greater than refrigeration temperatures, e.g. from 4°C to 56°C or more, or lower than refrigeration temperatures, e.g. from 0°C to -70°C or below.
• the composition that is produced according to the invention may thus have a residual moisture content of 5% or less, 2% or less or 1% or less by weight.
• the composition has residual moisture content of from 0.1 to 5% or from 0.5 to 5%.
• the composition can be obtained in dry powder form.
• a cake resulting from the drying, e.g. freeze-drying step can be milled to powder form.
• a solid composition according to the invention thus may take the form of free-flowing particles.
• the solid composition is typically provided as a powder in a sealed vial, ampoule or syringe. If for inhalation the powder can be provided in a dry powder inhaler.
• the solid matrix can alternatively be provided as a patch.
• a powder may be compressed into tablet form.
• composition may consist, or consist essentially, of: the aluminium-salt adjuvant; one or more antigens; the compound of formula (I) or (II) or a
• compositions of the invention are physiologically acceptable salt or ester thereof; and optionally one or more sugars.
• compositions of the invention are physiologically acceptable salt or ester thereof; and optionally one or more sugars.
• the frozen or dried vaccine compositions are converted into liquid form (aqueous solution) prior to administration to a patient.
• a frozen composition is thawed and diluted as necessary with e.g. phosphate-buffered saline or Water for Injections.
• a dried composition is reconstituted as an aqueous solution, for example by phosphate-buffered saline or Water for Injections.
• the resulting aqueous solution can then be administered, e.g. by injection, to a patient in need of vaccination.
• the compound of formula (I) or (II) or a physiologically acceptable salt or ester thereof and, optionally, one or more sugars typically acts as a resuspension agent for the vaccine composition, for example when it is converted into liquid form (aqueous solution) prior to administration to a patient.
• Aluminium salt adjuvants in their natural form are commonly in the form of gels that are particulate suspensions in aqueous media. Freezing or drying often causes structural alterations typified by an increased particle size with corresponding increased sedimentation rates and tighter packing of the sedimented solid compounds. Using the present invention, however, damage in the form of increased particle size, increased sedimentation rate and/or tighter packing of sedimented solids as a result of freezing or freeze-drying can be reduced.
• Structural damage in the form of increased particle size with corresponding increased sedimentation rates and tighter packing of the sedimented solid compounds can been assessed using the adjuvant agglomeration assay described in Example 1.
• Other analytical methods for assessing the physiochemical characteristics of aluminium adjuvants before and after freezing or freeze-drying may also be used.
• particle size distributions of the aluminium gel particles can be obtained using laser diffraction analysis, X-ray diffraction or infrared spectroscopy.
• Microscopy can also be used to visualise structural changes.
• the following Examples illustrate the invention. A Reference Example is also provided.
• Aluminium hydroxide gel (Al(OH) 3 ) was obtained from Sigma (A8222) as a 13mg/ml solution (with a pH of 6.8).
• the adjuvant was frozen by being placed in a laboratory freezer where it was left overnight at -20°C. It was then allowed to thaw at and equilibrate to room temperature (approximately 20°C).
• Adjuvants were examined microscopically at a magnification of lOOx.
• Photograph A shows the evenly distributed particulate suspension of undamaged adjuvant compared with photograph B which shows the formation of large agglomerated flat crystal structures typical of freeze-damaged adjuvant.
• the aluminium hydroxide adjuvant was obtained from Sigma (A8222) as a 13mg/ml solution at pH 6.8. Initially, 50 ⁇ 1 volumes of the aluminium hydroxide were added to ⁇ volumes of sucrose and/or a further excipient diluted in Dulbecco's phosphate buffered saline (PBS) in wells of 96 well flat bottomed microplates. The further excipient was DMG. A list of final concentrations of DMG and sucrose before freezing can be seen in Table 1 below. The adjuvants were frozen at -20°C. After approximately 18 hours samples containing Al(OH) 3 were thawed and assessed for sediment levels as described using the adjuvant agglomeration assay described below.
• PBS Dulbecco's phosphate buffered saline
• the amount of agglomeration was assessed by taking up samples from each well into ⁇ micropipettes, allowing resettling to occur for 1 hour at room temperature and then measuring the height of the sedimented gel as a percentage of the total height of the solution in the pipette.
• the height of the sedimented gel as a percentage of the total height of the solution in the pipette was expressed as % gel volume. The greater the % gel volume, the more structurally intact is the adjuvant.
• the aluminium hydroxide adjuvant was obtained from Sigma (A8222) as a
• the further excipients were S-methyl-L-methionine, MSM and TMG.
• the adjuvants were frozen at -20°C.
• a list of final concentrations of sucrose and the further excipient before freezing can be seen in Table 2 below.
• the aluminium hydroxide adjuvant was obtained from Sigma (A8222) as a 13mg/ml solution at pH 6.8. A volume of adjuvant was centrifuged to form a pellet which was subsequently washed in 40m HEPES + 25mM NaCl at pH 7.9 (twice) and re-suspended in half the original volume, resulting in an approximately 26 mg/ml solution. Into each vial was added 75 ⁇ of 26 mg/ml adjuvant solution and 225 ⁇ of relevant excipient (adjusted in concentration to account for added adjuvant volume) to equal the appropriate concentration, with each vial containing a final adjuvant concentration of 6.5 mg/ml. A list of final concentrations of excipients are set out in Table 4 below.
• Samples were freeze dried by the VirTis Advantage freeze dryer, using the drying cycles shown in Table 3 below, lasting for approximately 3 days. Samples were frozen at -40°C for 2 hours before a vacuum was applied, initially at 300 milliTorre with a Thermo Savant VLP pump (Thermofisher, UK). Shelf temperature and vacuum were adjusted throughout the process and the condenser was maintained at -80°C. Step 11 was extended until the samples were stoppered before releasing the vacuum.
• the vials containing freeze-dried adjuvant were reconstituted into 300 ⁇ of purified water and vortexed.
• the amount of agglomeration was assessed by taking up samples from each well into ⁇ micropipettes, allowing resettling to occur for 90 minutes at room temperature and then measuring the height of the sedimented gel as a percentage of the total height of the solution in the pipette.
• the height of the sedimented gel as a percentage of the total height of the solution in the pipette was expressed as % gel volume. The greater the % gel volume, the more structurally intact is the adjuvant.
• Bovine serum albumin (BSA) is commonly used as a model in experiments where, for example, protein adsorption onto an adjuvant is to be measured.
• Alhydrogel (supplied at 2% stock (w/v)) was added to PBS containing BSA to equal a final 10 ml volume with concentration of 0.52% Alhydrogel and 200 ⁇ g/ml BSA.
• the protein adsorption step was incubated by gently rocking at room temperature before placing overnight at +4 °C.
• the adjuvant was mixed with the excipient concentration in a 1 :1 ratio (2ml + 2ml) to create half concentration of excipients above, 0.26 % Alhydrogel and 100 ⁇ g/ml BSA. This was incubated at +4 °C for 12 hours before being split off into 300 ⁇ volumes which were either (a) frozen (-80°C), (b) lyophilised as set out in Table 15 below or (c) held at +4 °C as liquid.
• the liquid, frozen and lyophilised vials were then placed at room temperature to equilibrate/thaw whilst the lyophilised vials were reconstituted in 300 ⁇ of purified water and vortexed until complete reconstitution was observed.
• each of the excipient combinations was run in duplicate with a duplicate counterpart blank (i.e. no protein).
• the liquid, lyophilised and frozen samples were run on separate plates.
• To standardise protein concentrations each plate was run with a standard curve starting with BSA at 200 ⁇ g/ml serially diluted down to 6.25 ⁇ g/ml.
• a volume of 50 ⁇ of adjuvant sample was added to each well before adding 125 ⁇ of Bradford solution (equilibrated to room temperature).
• the plates were transferred to the plate reader which was set on the plate shake mode (to keep adjuvant in suspension) for 5 minutes before each plate was read at an absorbance at 595nm.
• This experiment compares a mannitol base with DMG, TMG, and SMM at levels that have previously been shown to protect adjuvant structure. It compares the antigenicity of the antibody bound to the alum both when the alum antibody has been kept at 4°C and when it has been freeze thawed, using a dot blot to probe the activity of the antibody in both storage methods.
• Mouse antibody adsorbed onto alum was freeze thawed and kept at 4°C in the presence of various excipients. This was assayed using a dot blot to see if the mouse antibody had retained its antigenicity.
• a nitrocellulose membrane was cut to the required size and 2 ⁇ 1 of samples applied as dots. This was allowed to dry and then incubated in 10ml PBS +0.05% Tween 20+ 5% milk for lhour at room temperature on a rocker. This solution was then removed and the membrane then incubated in 10ml of anti-mouse-HRP
• Figure 8 shows the layout of samples tested in Figure 8) shows that in both the liquid ( Figure 8A) and freeze-thawed ( Figure 8B) samples, all samples not containing antibody are negative as expected and the positive control of antibody only is strongly positive. In the liquid samples all the dots are similar at the same dilutions. The frozen samples are less consistent, especially between the samples in excipient and the PBS control sample. The PBS sample is weaker at the 1 :500 dilution then the samples in the different excipients.
• Figure 9 (Table 20 shows the layout of samples tested in Figure 9) shows results consistent with this. All the negative controls without antibody are negative, including the excipient only controls which show that the excipients are not interfering with the assay. The PBS samples are again weaker than the liquid samples when frozen, especially when compared to samples in excipient at 1 :300 and 1 :500.
• Mouse mAb- 12% 5. Mouse mAb- 12% mannitol- 6. Mouse mAb- 12% mannitol- mannitol-0.26% alum 0.26% alum 0.26% alum
• Mouse mAb- 12% 8. Mouse mAb- 12% mannitol- 9. Mouse mAb- 12% mannitol-0.8M DMG- 0.8M DMG-0.26% alum 0.8M DMG-0.26% alum 0.26% alum 1 :100 1 :300 1 :500
• Mouse mAb- 12% 11. Mouse mAb- 12% mannitol- 12. Mouse mAb- 12% mannitol- mannitol-0.8M TMG- 0.8M TMG-0.26% alum 0.8M TMG-0.26% alum 0.26% alum 1 : 100 1 :300 1 :500
• excipients are offering protection to the antibody with alum when compared to

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