Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/66—Microorganisms or materials therefrom
  • A61K35/76—Viruses; Subviral particles; Bacteriophages
• • 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
  • A61K39/12—Viral antigens
• • 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
  • 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
  • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
  • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/08—Antiallergic agents
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
  • C12N7/02—Recovery or purification
• • 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
• • 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
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
  • C12N2710/00011—Details
  • C12N2710/10011—Adenoviridae
  • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
  • C12N2710/10351—Methods of production or purification of viral material

Definitions

• the invention relates to the stabilisation of viral particles.
• Some biological molecules are sufficiently stable that they can be isolated, purified and then stored in solution at room temperature. However, this is not possible for many materials and techniques involving storage at low temperature, addition of stabilizers or cryoprotectants, freeze-drying, vacuum-drying and air-drying have been tried to ensure shelf preservation.
• Freeze drying of biopharmaceuticals involves freezing solutions or suspensions of thermosensitive biomaterials, followed by primary and secondary drying. The technique is based on sublimation of water at subzero temperature under vacuum without the solution melting. Freeze-drying represents a key step for manufacturing solid protein and vaccine pharmaceuticals. The rate of water vapour diffusion from the frozen biomaterial is very low and therefore the process is time-consuming. Additionally, both the freezing and drying stages introduce stresses that are capable of unfolding or denaturing proteins.
• WO 90/05182 describes a method of protecting proteins against denaturation on drying. The method comprises the steps of mixing an aqueous solution of the protein with a soluble cationic polyelectrolyte and a cyclic polyol and removing water from the solution. Diemylaniinoethyldextran (DEAE-dextran) and chitosan are the DAE-dextran and chitosan.
• WO-A-2006/0850082 reports a desiccated or preserved product comprising a sugar, a charged material such as a histone protein and a desiccation- or thermo- sensitive biological component.
• the sugar forms an amorphous solid matrix.
• the histone may have immunological consequences if the preserved biological component is administered to a human or animal.
• WO 2008/114021 describes a method for preserving viral particles.
• the method comprises drying an aqueous solution of one or more sugars, a
• the aqueous solution contains the polyemyleneimine at a concentration of 15 ⁇ or less based on the number-average molar mass (M n ) of the polyemyleneimine and the sugar concentration or, if more than one sugar is present, total sugar concentration is greater than 0.1M.
• viral preparations are preserved stably by compounds of formula (I) and/or (II) as defined herein or physiologically acceptable salts or esters thereof and optionally one or more sugars during drying.
• Virus activity was preserved following subsequent heat challenge.
• Virus activity was also preserved during long-term stability tests.
• Virus activity may also be preserved in the aqueous solution prior to drying. The viruses were protected against damage caused by freezing, freeze-drying and thawing.
• the present invention provides a method for preserving viral particles comprising:
• - Ri represents hydrogen or Ci-6 alkyl
• - R4 represents hydrogen
• - R 2 represents hydrogen, CM, alkyl or -(CH2) 2- 5NHC(0)(CH2)5- 15 CH3;
• R3 represents Ci-6 alkyl
• - X represents -S(0) 2 - or -S + (Rc)- ;
• - Rc represents Q-6 alkyl substituted with a carboxylate anion and with an amine (- H2) moiety;
• the invention further provides:
• composition which comprises a compound of formula (I) or a
• physiologically acceptable salt or ester thereof and/or a compound of formula (II) or a physiologically acceptable salt or ester thereof and optionally one or more sugars and which incorporates viral particles;
• a vaccine comprising a composition of the invention which incorporates noninfectious viral particles and optionally an adjuvant;
• a method of preparing a vaccine which incorporates viral particles comprises:
• composition or dry powder which comprises viral particles or non-infectious viral particles and which is obtainable by a method of the invention
• a method for preserving viral particles prior to drying comprising: (a) providing an aqueous solution of (i) viral particles, (ii) optionally one or more sugars, and (iii) a compound of formula (I) of the invention or a
• a bulk aqueous solution of (i) viral particles, (ii) optionally one or more sugars, and (iii) a compound of formula (I) or a physiologically acceptable salt or ester thereof of the invention and/or a compound of formula (II) of the invention or a physiologically acceptable salt or ester thereof, which solution is provided in a sealed container and is stored prior to drying in a refrigerator or freezer;
• physiologically acceptable salt or ester thereof of the invention and, optionally, one or more sugars for preserving viral particles in an aqueous solution which comprises said viral particles, prior to drying;
• Figure 1 shows the results obtained in Example 1. The ability of an excipient to help adenovirus withstand cycling between 37°C and -20°C was assessed.
• Dimethylsulfone also called methylsulfonylmethane, MSM
• MSM methylsulfonylmethane
• Figure 2A shows the temperature set for the shelf temperature of the VirTis Advantage freeze dryer used in various of the Examples.
• Figure 2B shows the condenser temperature of the VirTis Advantage freeze dryer used in various of the Examples.
• Figure 3 shows the results of the experiment of Example 2 that investigated the effect of sugars and MSM on preservation of adenovirus during freeze drying.
• Figure 4 shows the results obtained in Example 3 of adenovirus infectivity tested immediately after thawing as well as those of samples lyophilised after formulation with TMG (trimethylglycine) with or without sugars.
• Figure 5 demonstrates the lyophilisation conditions used in Example 4.
• Figure 7 shows the results obtained in Example 5 for adenovirus samples which were tested immediately after thawing ("Pre-Lyophilisation") as well as those of samples which were formulated in PBS (phosphate buffered saline) at DMG concentrations of 0.00M, 0.07M, 0.23M and 0.70M with and without sugars and which were subsequently lyophilised.
• Figure 8 shows the lyophilisation conditions used in Examples 5 and 6.
• Figure 9 shows the results obtained in Example 6 for adenovirus samples tested immediately after defrosting as well as those of samples lyophilised after formulation with DMG with or without sugars and subsequently thermochallenged.
• A Adenovirus activity after lyophilisation and storage at +4°C for 7 days.
• Figure 10 shows the shelf temperatures, condenser temperatures and vacuum conditions during freeze drying in the VirTis Advantage freeze-dryer in Example 7.
• Figure 11 shows the results obtained in Example 7.
• Figure 12 shows the appearance of the freeze-dried cakes obtained in Example
• Figure 13 reports the results obtained in Example 9.
• Figure 14 shows the results obtained in Example 10 in which the ability of eleven formulations to stabilise adenovirus through freeze-drying and thermal challenge was assessed.
• Figure 15 shows the results obtained in Example 11 in which the ability of eleven formulations to stabilise MVA through freeze-drying and thermal challenge was assessed.
• Figure 16 shows a 3D representation of the design space in Example 12. Balls represent formulations within the design space that were tested. This design is a three factor, full factorial screening design.
• Figures 17 and 18 show the freeze-drying program used in Example 12 and temperature readings from sensors during that program.
• Figure 19 shows a residual normal probability plot for data from formulations containing DMG in Example 12.
• Figure 20 shows retained coefficients (effects) of the modelled data from formulations containing DMG in Example 12. Error bars indicate significance if not crossing the origin.
• Figure 21 shows retained coefficients (effects) of the modelled data from formulations containing SMM in Example 12. Error bars indicate significance if not crossing the origin.
• Figure 22 shows a residual normal probability plot for data from formulations containing SMM in Example 12.
• Figure 23 shows retained coefficients (effects) of the modelled data from formulations containing SMM in Example 12 after inclusion of a non-specific 2 nd order term. Error bars indicate significance if not crossing the origin.
• Figure 24 shows a residual normal probability plot for data from formulations containing SMM in Example 12.
• Figure 25 shows retained coefficients (effects) of the modelled data from formulations containing TMG in Example 12. Error bars indicate significance if not crossing the origin.
• Figure 26 shows a residual normal probability plot for data from formulations containing TMG in Example 12.
• Figure 27 shows a 3D representation of the design space in Example 13.
• Spheres represent formulations within the design space that were tested. This design is a Doehlert RSM design.
• Figure 28 shows the freeze-drying program used in Example 13.
• Figure 29 summarises various statistics for the model derived from the data in Example 13.
• Figure 30 shows terms retained in the model in Example 13 after fine tuning.
• Figure 32 shows the settings and outputs from an optimum prediction based on the model of the data in Example 13 generated using Monte-Carlo simulations.
• Figures 33 A and 33B show an optimum region plot from the Example 13 data.
• Figure 33A is a contour plot where a cross marks the predicted optimum.
• Figure 33B is an identical graph region highlighting region of the model where predicted recovered viral activity is greater than or equal to initial activity.
• Figure 34 shows the freeze-drying program used in Example 14.
• Figure 35 shows recovered virus activity in Example 14 as a percentage of
• Figure 36 shows recovered virus activity over time at the accelerated stability temperature (+25°C) in Example 14.
• Figure 37 shows recovered virus activity over time at the stress testing temperature (+37°C) in Example 14.
• Figure 38 shows a 3D representation of the design space in Example 15. Spheres represent formulations within the design space that are tested. This design is a Doehlert RSM design.
• Figure 39 shows the lyophilisation conditions used in Example 15.
• Figure 40 summarises the statistics of the model in Example 15 used to represent the data.
• Figure 41 shows terms retained in the model in Example 15 after fine tuning. Error bars not crossing the origin indicate a significant factor at the 95% C.I.
• Figure 42 shows contours plot of recovered viral titre (TCID50/ml) with varying formulations in Example 15.
• Figure 43 shows a representation of the design space in Example 16.
• Numbered circles represent formulations within the design space that are tested.
• Figure 44 shows the lyophilisation conditions used in Example 16.
• Figure 45 summarises the statistics of the model in Example 16 used to represent the data.
• Figure 46 shows terms retained in the model in Example 16 after fine tuning. Error bars not crossing the origin indicate a significant factor at the 95% C.I.
• Figure 47 shows a surface response plot of the predicted recovered viral titre in formulations of DMG and mannitol using the model of Example 16.
• Figure 48 shows a screen capture of the settings and outputs from the optimum predictions based on the model of the data in Example 16, generated using Monte- Carlo simulations. Iteration 48 highlighted in grey is the optimum formulation (1.0107M DMG).
• the present invention relates to the preservation of viral particles by a compound of formula (I) or a physiologically acceptable salt or ester thereof and/or a compound of formula (II) or a physiologically acceptable salt or ester thereof and optionally one, two or more sugars.
• the viral particles are contacted with the compound of formula (I) or a physiologically acceptable salt or ester thereof and/or compound of formula (II) or a physiologically acceptable salt or ester thereof and optionally one or more sugars in an aqueous solution and the resulting solution in which the viral particles are present is then dried to form a composition incorporating the viral particles.
• the viral particles may therefore be admixed with an aqueous solution ( "preservation mixture ") of the compound of formula (I) or a physiologically acceptable salt or ester thereof and/or compound of formula (II) or a physiologically acceptable salt or ester thereof and optionally one or more sugars.
• the resulting solution is then dried to form a composition incorporating the viral particles.
• the dried composition may take the form of a cake or powder. The cake can be milled to a powder if required.
• the invention enables virus structure and function to be preserved during the drying step. Virus activity following drying can thus be maintained.
• a compound of formula (I) or a physiologically acceptable salt or ester thereof and/or a compound of formula (II) or a physiologically acceptable salt or ester thereof alone allows preservation of viral activity. Further improvements in preservation of viral activity can be achieved by use of one or more sugars in combination with a compound of formula ( ⁇ ) or a physiologically acceptable salt or ester thereof and/or a compound of formula (II) or a physiologically acceptable salt or ester thereof.
• the preserved viral particles demonstrate improved thermal resistance allowing extension of shelf life, ease of storage and transport and obviating the need for a cold chain for distribution.
• the invention can thus provide protection as a cryoprotectant (protection against freeze damage), lyoprotectant (protection during freeze-drying) and/or a thermoprotectant (protection against temperatures higher or lower than 4°C).
• the viral particles are preserved in the aqueous solution prior to the drying step. This allows the aqueous solution to be stored after preparation, until such time as the drying step can be carried out, without undue loss of viral activity.
• the viral particles used in the present invention may be whole viruses such as live viruses, killed viruses, live attenuated viruses, inactivated viruses such as chemically inactivated viruses or virulent or non-virulent viruses.
• a live virus is capable of infecting and replicating within the host cell.
• a killed virus is inactivated and is unable to replicate within the host cell.
• the particles may be virus-like particles (VLPs) or nucleocapsids.
• the virus may be infectious to prokaryotic or eukaryotic cells.
• the virus may be a human or animal virus.
• the viral particle may be, or may be derived from, a dsDNA virus, a ssDNA virus, a dsR A virus, a (+)ssRNA virus, a (-)ssR A virus, a ssRNA-RT virus or a dsDNA-RT virus.
• the viral particle can be, or can be derived from, a virus of the following families:
• Adenoviridae such as a human adenovirus or non-human adenovirus, for example human adenovirus A, B, C, D, E or F including human Ad5, Ad2, Ad4, Ad6, Ad7, Adl 1, Adl4, Ad24, Ad26, Ad35 and Ad36 serotypes;
• Caliciviridae such as the norwalk virus
• Coronaviridae such as human coronavirus 299E or OC43 and SARS- coronavirus;
• Filoviridae such as ebola virus
• Flaviviridae such as yellow fever virus, west nile virus, dengue virus, hepatitis C virus;
• Hepadnaviridae such as hepatitis B virus
• Herpesviridae such as herpes simplex virus e.g. HSV1 or HSV2, human herpesvirus 1, 3, 4, 5 or 6
• Herpesviridae such as herpes simplex virus e.g. HSV1 or HSV2, human herpesvirus 1, 3, 4, 5 or 6
• HSV1 or HSV2 herpes simplex virus
• human herpesvirus 1 1, 3, 4, 5 or 6
• Orthomyxoviridae such as influenzavirus A, B, C including but not limited to influenza A virus serotypes H1N1, H2N2, H3N2, H5N1, H7N7, H1N2, H9H2, H7N2, H7 3 and N10N7;
• Papillomaviridae such as human papilloma virus
• Paramyxoviridae such as human parainfluenza virus 1 , measles virus and mumps virus;
• Parvoviridae such as adeno-associated virus
• Picornaviridae such as human poliovirus, foot and mouth disease virus
• Poxviridae such as vaccinia virus, variola virus and avian poxvirus (fowlpox); Reoviridae such as bluetongue virus group;
• Retrovir idae such as lentivirus including human immunodeficiency virus 1 and 2;
• Togaviridae such as rubella virus.
• the viral particle can be or can be derived from an Adenoviridae, Orthomyxoviridae, Paramyxoviridae, Parvoviridae, Picornaviridae or Poxviridae virus.
• the viral particle can be or can be derived from an adenovirus, vaccinia virus, influenza virus, or measles virus.
• the virus can be Modified Vaccinia Virus Ankara (MVA) or a viral particle derived
• VLPs include viral proteins derived from the structural proteins of a virus, but lack viral nucleic acid. When overexpressed, these viral structural proteins spontaneously self-assemble into particles. VLPs are replication incompetent. In some embodiments, the VLPs are viral proteins embedded vvdthin a lipid bilayer. Examples of VLPs includes phage-derived VLPs, human
• Viral particles can be prepared using standard techniques well known to those skilled in the art. For example, a virus may be prepared by infecting cultured host cells with the virus strain that is to be used, allowing infection to progress such that the virus replicates in the cultured cells and can be released by standard methods known in the art for harvesting and purifying viruses.
• the compounds 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 C ⁇ alkyl ester, preferably a C alkyl ester.
• the ester may therefore be the methyl, ethyl, propyl, isopropyl, butyl, isobutyl or tert-butyl ester.
• the ethyl ester is preferred.
• a d-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 ⁇ alkyl.
• Ri represents hydrogen or d-6 alkyl
• R represents hydrogen and R 2 represents hydrogen or C 1-6 alkyl.
• Ri represents hydrogen or Ci_6 alkyl
• R4 represents hydrogen and R 2 represents C ⁇ alkyl.
• the compound of formula (I) is an N-Cj-6 alkyl-, N,N-di(d ⁇ alkyl)- or N,N,N-tri(Ci.6 alkyl)-glycine or physiologically acceptable salt or ester thereof, more preferably an 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 compounds 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:
• R5 and R $ independently represent Ci-6 alkyl, for example C alkyl such as methyl or ethyl; and R 7 represents Ci-6 alkyl, for example C ⁇ alkyl such as methyl or ethyl, or -(CH 2 ) 2-5 NHC(0)(CH 2 ) 5- isCH 3 .
• Preferred compounds of formula (IA) are trimethylglycine (TMG) and cocamidopropyl betaine (CAPB) or physiologically acceptable salts or esters thereof. Trimethyglycine is preferred.
• the compound of formula (I) is typically a proline derivative of formula (IB) or a physiologically acceptable salt or ester thereof:
• 3 ⁇ 4 and R independently represent Ci-6 alkyl, for example C alkyl such as methyl or ethyl.
• the compound of formula (IB) is an S-proline derivative.
• 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 N, N, N- trimethylglycine or physiologically acceptable salt or ester thereof. Most preferably, 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.
• Rc 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: wherein Rc and Rd independently represent Ci-6 alkyl, for example C alkyl.
• Preferred alkyl groups are selected from methyl, ethyl, propyl, isopropyl, butyl, isobutyl and tert-butyl. Methyl and ethyl are particularly preferred.
• 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 Rf independently represent Ci_6 alkyl, for example C alkyl such as methyl or ethyl, and Rg represents C 1-6 alkyl, for example C alkyl such as methyl or ethyl, substituted with a carboxylate anion and with an amine (-NH 2 ) 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, more preferably sucrose.
• the sugar may be a monosaccharide, disaccharide, trisaccharide, or other
• sugar alcohol includes sugar alcohols. In one embodiment, therefore, use of a non-reducing sugar or a sugar alcohol is preferred. 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. When mannitol is used, cakes of improved appearance can be obtained on freeze-drying.
• sugar may act to improve stability.
• sugar may also provide other benefits such as an altered lyophilisation cake and improved solubility for faster reconstitution.
• one or more sugars is present when freeze-drying is used.
• the sugar is preferably sucrose or mannitol, more preferably mannitol.
• Preservation of viral activity is particularly effective when two or more sugars are used in the preservation mixture. Two, three or four sugars may be used.
• the aqueous solution is a solution of sucrose and raffinose.
• Sucrose is a disaccharide of glucose and fructose.
• Raffinose is a trisaccharide composed of galactose, fructose and glucose.
• an aqueous solution comprising the viral particles, optionally one or more sugars and a compound of formula (I) or a physiologically acceptable salt or ester thereof and/or a compound of formula (II) or a physiologically acceptable salt or ester thereof is dried.
• Any suitable aqueous solution may be used.
• the solution may be buffered.
• the solution may be a HEPES, phosphate-buffered, Tris-buffered or pure water solution.
• the solution may have a pH of from 2 to about 12 and may be buffered.
• the solution may be buffered with HEPES buffer, phosphate-buffer, Tris-buffer, sodium citrate buffer, bicine buffer (i.e. N,N-bis(2-hydroxyethyl) glycine buffer) or MOPS buffer (i.e. 3-(N-morpholino) propanesulfonic acid buffer).
• the solution may or may not contain NaCl.
• the solution may thus be a saline sodium citrate (SSC) buffered solution.
• SSC saline sodium citrate
• the preservation mixture may itself be buffered. It may be a HEPES, phosphate-buffered, Tris-buffered or pure water solution.
• the aqueous solution may typically consist, or consist essentially, of viral particles, a compound of formula (I) or a physiologically acceptable salt or ester thereof and/or a compound of formula (II) or a physiologically acceptable salt or ester thereof, and optionally one or more sugars.
• concentrations of the compound of formula (I) or a physiologically acceptable salt or ester thereof and/or a compound of formula (II) or a physiologically acceptable salt or ester thereof and of each optional sugar can be deterrnined by routine experimentation. Optimised concentrations which result in the best stability can thus be selected.
• the compound of formula (I) or a physiologically acceptable salt or ester thereof and/or a compound of formula (II) or a physiologically acceptable salt or ester thereof compound may act synergistically to improve stability.
• the concentration of sugar when present in the aqueous solution for drying is at least 0.01M, typically up to saturation.
• the sugar concentration when present is at least 0.1M, at least 0.2M or at least 0.5M up to saturation e.g. saturation at room temperature or up to 3M, 2.5M or 2M.
• the sugar concentration may therefore range from, for example, 0.1 to 3M or 0.2M to 2M.
• a sugar is present.
• the sugar concentration or the total sugar concentration if more than one sugar is present may therefore range from 0.08M to 3M, from 0.15M to 2M or from 0.2M to 1M.
• a suitable range is from 0.05 to 1M.
• sucrose When more than one sugar is present, preferably one of those sugars is sucrose.
• the sucrose may be present at a concentration of from 0.05M, 0.1M, 0.25M or 0.5M up to saturation e.g. saturation at room temperature or up to 3M, 2.5M or 2M.
• the ratio of the molar concentration of sucrose relative to the molar concentration of the other sugar(s) is typically from 1 : 1 to 20 : 1 such as from 5 : 1 to 15:1.
• the ratio of molar concentrations of sucrose is typically from 1 :1 to 20:1 such as from 5:1 to 15:1 and preferably about 10:1.
• the concentration of each compound of formula (I) or physiologically acceptable salt or ester thereof or compound of formula (II) or physiologically acceptable salt or ester thereof in the aqueous solution for drying is generally in the range of from 0.001M to 2.5M and more especially from 0.01M to 2.5M.
• the concentration range may be from 0.1M to 2.5M.
• the concentration of each compound of formula (I) or physiologically acceptable salt or ester thereof or compound of formula (II) or physiologically acceptable salt or ester thereof in the aqueous solution for drying is generally in the range of O.lmM to 3M or from ImM to 2M.
• the concentration may be from ImM to 1.5M or from 5mM to 1M or from 0.07M to 0.7M.
• Preferred concentrations are from 7mM to 1.5M or from 0.07M to 1.2M.
• Another further preferred range is 0.5 to 1.5M, particularly when the compound of formula (I) is an N-alkylated glycine derivative such as DMG.
• composition of compound of formula (I) or physiologically acceptable salt or ester thereof or compound of formula (II) or physiologically acceptable salt or ester thereof that is employed will depend on several factors including the type of viral particle to be preserved; the particular compound being used; whether one, two more sugars are present and the identity of the sugar(s); and the drying procedure and conditions. Thus:
• 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 0.2mM to 1M such as from
• 0.35mM to 1M from 3.5mM to 0.5M, from 0.035M to 0.5M or from 0.035M to 0.25M.
• 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 used at a concentration from 0.01M to 2M such as from 0.07M to 2M, from 0.2M to 1.5M, from 0.23M to 1.5M or from 0.07M to 0.7M.
• 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 0.005M to 2M such as from 0.007M to 2M, from 0.02M to 2M, from 0.023M to 1.5M or
• concentration of a compound of formula (I), such as N,N-dimethylglycine (DMG) or a physiologically acceptable salt or ester thereof, when no sugar is present are from 5mM to 1.5M or from 70mM to 1.5M or to 1.2M or from 7mM to 1M. More preferred concentrations are from 0.023M to 0.7M or 1M, or from 0.07M to 0.7M or 1M, such as about 0.7M
• concentration of a compound of formula (I), such as N,N-dimethylglycine (DMG) or a physiologically acceptable salt or ester thereof, when one or more sugars are present are generally lower and in the range of from lmM to 1M or 1.5M or from 5mM to 1M. More preferred concentrations are from 0.007M to 0.7M or 1M such as about 0.007M. A particularly preferred range is 0.5 to 1.5M.
• the compounds can be present in amounts that result in synergy.
• the compounds can be present in amounts that result in synergy.
• the concentration of the N-alkylated glycine derivative or salt or ester thereof in the aqueous solution for drying is generally in the range of 0.1 mM to 3M or from lmM to 2M.
• the concentration may be from lmM to 1.5M or from 5mM to 1M.
• Preferred concentrations are from 0.1M to 1.5M or from 0.5M to 1.25M.
• the concentration of the sulfone compound of formula (IIA) or (IIC) in the aqueous solution for drying is generally in the range of 0. lmM to 3M, from lmM to 2M or from 0.2mM to 1M.
• the concentration may be from 0.1M to 1.5M or from 0.5M to 1.25M.
• drying is achieved by freeze drying, vacuum drying, fluid bed drying or spray-drying. Freeze-drying is preferred.
• Freeze-drying is preferred.
• composition which incorporates the viral particles.
• a matrix incorporating the viral particles is 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 sugar or sugars when present provide the amorphous matrix in the dried composition.
• the compound of formula (I) or a physiologically acceptable salt or ester thereof and/or a compound of formula (II) or physiologically acceptable salt or ester thereof is dispersed in the sugar matrix.
• the compound of formula (I) or a physiologically acceptable salt or ester thereof and/or compound of formula (II) or physiologically acceptable salt or ester thereof is thus incorporated within the sugar matrix.
• the viral particles are incorporated within the sugar matrix too.
• the drying procedure can thus be effected e.g. by freeze-drying to form an amorphous cake within which the viral particles are incorporated.
• the drying step is generally performed as soon as the aqueous solution has been prepared or shortly afterwards. Alternatively, the aqueous solution is typically stored prior to the drying step.
• the viral particle in the aqueous solution is preserved by the compound of formula (I) or a physiologically acceptable salt or ester thereof and/or a compound of formula (II) or physiologically acceptable salt or ester thereof and, optionally, one or more sugars during storage.
• the aqueous solution, or bulk intermediate solution is generally stored for up to 5 years, for example up to 4 years, 3 years, 2 years or 1 year.
• the solution is stored for up to 6 months, more preferably up to 3 months or up to 2 months, for example 1 day to 1 month or 1 day to 1 week.
• the solution Prior to drying, the solution is typically stored in a refrigerator or in a freezer.
• the temperature of a refrigerator is typically 2 to 8 °C, preferably 4 to 6°C, or for example about 4°C.
• the temperature of a freezer is typically -10 to -80°C, preferably -10 to - 30°C, for example about -20°C.
• the solution is typically stored in a sealed container, preferably a sealed inert plastic container, such as a bag or a bottle.
• the container is typically sterile.
• the volume of the bulk intermediate solution is typically 0.1 to 100 litres, preferably 0.5 to 100 litres, for example 0.5 to 50 litres, 1 to 20 litres or 5 to 10 litres.
• the container typically has a volume of 0.1 to 100 litres, preferably 0.5 to 100 litres, for example 0.5 to 50 litres, 1 to 20 litres or 5 to 10 litres.
• the stored bulk intermediate solution is to be freeze-dried, it is typically poured into a freeze-drying tray prior to the drying step.
• Stable storage of the solution increases the flexibility of the manufacturing process.
• the solution can be easily stored, shipped and later dried.
• Freeze-drying is a dehydration process typically used to preserve perishable material or make the material more convenient for transport. Freeze-drying represents a key step for manufacturing solid protein and vaccine pharmaceuticals. However, biological materials are subject to both freezing and drying stresses during the procedure, which are capable of unfolding or denaturing proteins. Furthermore, the rate of water vapour diffusion from the frozen biological material is very low and therefore the process is time-consuming. The preservation technique of the present invention enables biological materials to be protected against the desiccation and/or thermal stresses of the freeze-drying procedure.
• Freezing is typically performed using a freeze-drying machine. In this step, it is important to cool the biological material below its eutectic point, (Teu) in the case of simple crystalline products or glass transition temperature (Tg') in the case of amorphous products, i.e. below 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 primary drying stage.
• 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,OOOPa 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.
• 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. Dried composition
• a composition having a low residual moisture content can be obtained.
• a level of residual moisture content is achieved which offers long term preservation at greater than refrigeration temperatures e.g. within the range from 4°C to 56°C or more, or lower than refrigeration temperatures e.g. within the range from 0 to -70°C or below.
• the dried composition may thus have residual moisture content of 10% or less, 5% or less, 2% or less or 1% or less by weight.
• the residual moisture content is 0.5% or more 1% or more.
• a dried composition has residual moisture content of from 0.5 to 10% by weight and preferably from 1 to 5% by weight.
• the composition can be obtained in a dry powder form.
• a cake resulting from e.g. freeze-drying can be milled into 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 typically consist, or consist essentially, of viral particles, a compound of formula (I) or a physiologically acceptable salt or ester thereof and/or a compound of formula (II) or a physiologically acceptable salt or ester thereof, and optionally one or more sugars.
• the admixture comprising viral particles is dried onto a solid support.
• the solid support may comprise a bead, test tube, matrix, plastic support, microtitre dish, microchip (for example, silicon, silicon-glass or gold chip), or membrane.
• microchip for example, silicon, silicon-glass or gold chip
• preservation in relation to viral particles refers to resistance of the viral particle to physical or chemical degradation and/or loss of biological activity such as nucleic acid or protein degradation, loss of transfection efficiency, loss of ability to stimulate a cellular or humoral immune response, loss of viral infectivity, loss of immunogenicity, loss of virus titre, loss of host cell response or loss of vaccine potency, under exposure to conditions of desiccation, freezing, temperatures below 0°C or below -25°C, freeze-drying, room temperature, temperatures above 0°C, above 25°C or above 30 °C.
• preservation according to the present invention comprises cryoprotection (protection against freeze damage), lyoprotection
• thermoprotection protection against temperatures higher or lower than 4°C.
• Methods of assaying for viral activity such as infectivity and/or
• immunogenicity are well known to those skilled in the art and include but are not limited to growth of a virus in a cell culture, detection of virus-specific antibody in blood, ability to elicit T and/or B cell responses, detection of viral antigens, detection of virus encoded DNA or RNA, or observation of virus particles using a microscope.
• a virus gives rise to morphological changes in the host cell, which can be measured to give an indication of viral activity. Detectable changes such as these in the host cell due to viral infection are known as cytopathic effect. Cytopathic effects may consist of cell rounding, disorientation, swelling or shrinking, death and detachment from the surface. Many viruses induce apoptosis (programmed cell death) in infected cells, measurable by techniques such as the TU EL (Terminal uridine deoxynucleotidyl transferase dUTP nick end labelling) assay and other techniques well known to those skilled in the art.
• TU EL Terminal uridine deoxynucleotidyl transferase dUTP nick end labelling
• Viruses may also affect the regulation of expression of the host cell genes and these genes can be analysed to give an indication of whether viral activity is present or not. Such techniques may involve the addition of reagents to the cell culture to complete an enzymatic or chemical reaction with a viral expression product.
• the viral genome may be modified in order to enhance detection of viral infectivity.
• the viral genome may be genetically modified to express a marker that can be readily detected by phase contrast microscopy, fluorescence microscopy or by radioimaging.
• the marker may be an expressed fluorescent protein such as GFP (Green Fluorescent Protein) or an expressed enzyme that may be involved in a colourimetric or radiolabelling reaction.
• the marker could also be a gene product that interrupts or inhibits a particular function of the cells being tested.
• An assay for plaque-forming units can be used to measure viral infectivity and to indicate viral titre.
• suitable host cells are grown on a flat surface until they form a monolayer of cells covering a plastic bottle or dish.
• the selection of a particular host cell will depend on the type of virus. Examples of suitable host cells include but are not limited to CHO, BHK, MDCK, 10T1/2, WEHI cells, COS, BSC 1, BSC 40, BMT 10, VERO, WI38, MRC5, A549, HT1080, 293, B-50, 3T3, NIH3T3, HepG2, Saos-2, Huh7, HEK293 and HeLa cells. The monolayer of host cells is then infected with the viral particles.
• a plaque is produced when a virus particle infects a cell, replicates, and then kills that cell.
• a plaque refers to an area of cells in the monolayer which display a cytopathic effect, e.g. appearing round and darker than other cells under the microscope, or as white spots when visualized by eye; the plaque center may lack cells due to virus-induced lysis.
• the newly replicated virus infects surrounding cells and they too are killed. This process may be repeated several times.
• the cells are then stained with a dye such as methylene blue, which stains only living cells. The dead cells in the plaque do not stain and appear as unstained areas on a coloured background.
• plaques are the result of infection of one cell by one virus followed by replication and spreading of that virus.
• viruses that do not kill cells may not produce plaques.
• a plaque refers to an area of cells in a monolayer which display a cytopathic effect, e.g. appearing round and darker than other cells under the microscope, or as white spots when visualized by eye; the plaque center may lack cells due to virus-induced lysis.
• An indication of viral titre is given by measuring "plaque-forming units" (PFU).
• PFU plaque-forming units
• Levels of viral infectivity can be measured in a sample of biological material preserved according to the present invention and compared to control samples such as freshly harvested virus or samples subjected to desiccation and/or thermal variation without addition of the preservation mixture of the present invention.
• viral particles of the invention such as viral proteins, VLPs, or some inactivated viruses do not have the ability to form plaques in the plaque assay.
• preservation can be measured by other methods such as methods for deterrnining immunogenicity which are well known to those skilled in the art.
• in vivo and in vitro assays for measuring antibody or cell-mediated host immune responses are known in the art and suitable for use in the present invention.
• an antibody based immune response may be measured by comparing the amount, avidity and isotype distribution of serum antibodies in an animal model, before and after immunization using the preserved viral particle of the invention.
• the preserved viral particles of the present invention may find use as a vaccine.
• preserved viral particles such as whole killed virus, live attenuated virus, chemically inactivated virus, VLPs or live viral vectors are suitable for use as a vaccine.
• the preserved viral particles of the invention may be used as antigens or to encode antigens such as viral proteins for the treatment or prevention of a number of conditions including but not limited to viral infection, sequelae of viral infection including but not limited to viral-induced toxicity, cancer and allergies.
• antigens contain one or more epitopes that will stimulate a host's immune system to generate a humoral and/or cellular antigen-specific response.
• the preserved vaccine of the invention may be used to prevent or treat infection by viruses such as human papilloma viruses (HPV), HTV, 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 and vaccinia virus.
• viruses such as human papilloma viruses (HPV), HTV, HSV2/HSV1, influenza virus (types A, B and C), para influenza virus, polio virus, RSV virus
• the vaccine may further be used to provide a suitable immune response against numerous veterinary diseases, such as foot and mouth disease (including serotypes O, A, C, SAT-1, SAT-2, SAT-3 and Asia-1), coronavirus, bluetongue, feline leukaemia virus, avian influenza, hendra and nipah virus, pestivirus, canine parvovirus and bovine viral diarrhoea virus.
• the vaccine is a subunit, conjugate or multivalent vaccine.
• the preserved vaccine of the invention may be used to treat infection by two or more different types of virus such as measles, mumps and rubella (e.g. MMR vaccine).
• the vaccine compositions of the present invention comprise viral particles admixed with the preservation mixture of the invention containing one or more sugars and a sulfoxide, sulfone, sulfonium, thetin or betaine compound.
• the vaccine composition may further comprise appropriate buffers and additives such as antibiotics, adjuvants or other molecules that enhance presentation of vaccine antigens to specific cells of the immune system.
• adjuvants well known in the art can be used in order to increase potency of the vaccine and/or modulate humoral and cellular immune responses.
• Suitable adjuvants include, but are not limited to, mineral salts (e.g., apatinium hydroxide (“alum"), duminium phosphate, calcium phosphate), particulate adjuvants (e.g., virosomes, ISCOMS (structured complex of saponins and lipids)), microbial derivatives (e.g., MPL(monophosphoryl lipid A), CpG motifs, modified toxins including TLR adjuvants such as flagellin), plant derivatives (e.g., saponins (QS-21)) and endogenous irnmunostimulatory adjuvants (e.g., cytokines and any other substances that act as immunostimulating agents to enhance the effectiveness of the vaccine).
• mineral salts e.g., apatinium hydroxide (“alum”)
• the vaccine composition of the present invention can be in a freeze-dried (lyophilised) form in order to provide for appropriate storage and maximize the shelf- life of the preparation. This will allow for stock piling of vaccine for prolonged periods of time and help maintain immunogenicity, potency and efficacy.
• the preservation mixture of the present invention is particularly suited to preserve viral substances against desiccation and thermal stresses encountered during freeze- drying/lyophilisation protocols. Therefore, the preservation mixture is suitable for adding to the virus or viral particle soon after harvesting and before subjection of the sample to the freeze-drying procedure.
• the potency of the vaccine can be measured using techniques well known to those skilled in the art.
• the generation of a cellular or humoral immune response can be tested in an appropriate animal model by monitoring the generation of antibodies or immune cell responses to the vaccine.
• the ability of vaccine samples prepared in accordance with the method of the present invention to trigger an immune response may be compared with vaccines not subjected to the same preservation technique.
• a virus or viral vector preserved according to the method of the present invention can be used to transfer a heterologous gene or other nucleic acid sequence to target cells.
• the heterologous sequence i.e. transgene
• the heterologous sequence encodes a protein or gene product which is capable of being expressed in the target cell.
• Suitable transgenes include desirable reporter genes, therapeutic genes and genes encoding immunogenic polypeptides (for use as vaccines).
• Gene therapy an approach for treatment or prevention of diseases associated with defective gene expression, involves the insertion of a therapeutic gene into cells, followed by expression and production of the required proteins. This approach enables replacement of damaged genes or inhibition of expression of undesired genes.
• the preserved virus or viral vector may be used in gene therapy to transfer a therapeutic transgene or gene encoding immunogenic polypeptides to a patient.
• the preserved viral particle is a live viral vector.
• live viral vector is meant a live viral vector that is non-pathogenic or of low pathogenicity for the target species and in which has been inserted one or more genes encoding antigens that stimulate an immune response protective against other viruses or microorganisms, a reporter gene or a therapeutic protein.
• nucleic acid is introduced into the viral vector in such a way that it is still able to replicate thereby expressing a polypeptide encoded by the inserted nucleic acid sequence and in the case of a vaccine, eliciting an immune response in the infected host animal.
• the live viral vector is an attenuated live viral vector i.e. is modified to be less virulent (disease-causing) than wildtype virus.
• recombinant viruses as potential vaccines involves the incorporation of specific genes from a pathogenic organism into the genome of a nonpathogenic or attenuated virus.
• the recombinant virus can then infect specific eukaryotic cells either in vivo or in vitro, and cause them to express the recombinant protein.
• Live viral vector vaccines derived by the insertion of genes encoding sequences from disease organisms may be preferred over live attenuated vaccines, inactivated vaccines, subunit or DNA approaches.
• One of the most important safety features of live viral vectors is that the recipients may be immunized against specific antigens from pathogenic organisms without exposure to the disease agent itself. Safety is further regulated by the selection of a viral vector that is either attenuated for the host or unable to replicate in the host although still able to express the
• heterologous antigen of interest A vaccine strain that has a history of safety in the target species offers an additional safety feature.
• Several systems have been developed in which the vector is deleted of essential genes and preparation of the vaccine is carried out in cell systems that provide the missing function.
• a variety of vectors such as retroviral, lentiviral, herpes virus, poxvirus, adenoviral and adeno-associated viral vectors can be used for the delivery of heterologous genes to target cells.
• the heterologous gene of interest may be inserted into the viral vector.
• the viral vectors of the invention may comprise for example a virus vector provided with an origin of replication, optionally a promoter for the expression of the heterologous gene and optionally a regulator of the promoter.
• adenoviruses useful in the practice of the present invention can have deletions in the El and/or E3 and /or E4 region, or can otherwise be maximized for receiving heterologous DNA.
• the viral vector may comprise a constitutive promoter such as a
• CMV cytomegalovirus
• MLP mouse mammary tumour virus LTR promoter
• SV40 early promoter adenovirus promoters
• adenovirus promoters such as the adenovirus major late promoter (Ad MLP)
• HSV promoters such as the HSV IE promoters
• HPV promoters such as the HPV upstream regulatory region (URR) or rous sarcoma virus promoter together with other viral nucleic acid sequences operably linked to the heterologous gene of interest.
• Tissue- specific or inducible promoters can also be used to control expression of the heterologous gene of interest. Promoters may also be selected to be compatible with the host cell for which expression is designed.
• the viral vector may also comprise other transcriptional modulator elements such as enhancers.
• Enhancers are broadly defined as a cis-acting agent, which when operably linked to a promoter/gene sequence, will increase transcription of that gene sequence. Enhancers can function from positions that are much further away from a sequence of interest than other expression control elements (e.g. promoters) and may operate when positioned in either orientation relative to the sequence of interest. Enhancers have been identified from a number of viral sources, including polyoma virus, BK virus, cytomegalovirus (CMV), adenovirus, simian virus 40 (SV40),
• CMV cytomegalovirus
• SV40 simian virus 40
• Suitable enhancers include the SV40 early gene enhancer, the enhancer/promoter derived from the long terminal repeat (LTR) of the Rous Sarcoma Virus, and elements derived from human or murine CMV, for example, elements included in the CMV intron A sequence.
• LTR long terminal repeat
• the viral vector containing a heterologous gene of interest may then be preserved according to the method of the invention before storage, subjecting to further preservation techniques such as lyophilisation, or administration to a patient or host cell.
• Nucleic acids encoding for polypeptides known to display antiviral activity immunomodulatory molecules such as cytokines (e.g. TNF-alpha, interleukins such as IL-6, and IL-2, interferons, colony stimulating factors such as GM-CSF), adjuvants and co-stimulatory and accessory molecules may be included in the viral vector of the invention.
• immunomodulatory molecules such as cytokines (e.g. TNF-alpha, interleukins such as IL-6, and IL-2, interferons, colony stimulating factors such as GM-CSF)
• cytokines e.g. TNF-alpha, interleukins such as IL-6, and IL-2, interferons, colony stimulating factors such as GM-CSF
• adjuvants and co-stimulatory and accessory molecules may be included in the viral vector of the invention.
• such polypeptides may be provided separately, for example in the preservation mixture of the invention or may be administrated simultaneously,
• the preserved viral vector of the invention may be introduced into suitable host cells using a variety of viral techniques that are known in the art, such as for example infection with recombinant viral vectors such as retroviruses, herpes 5 simplex virus and adenoviruses.
• administration of the preserved viral vector of the invention containing a gene of interest is mediated by viral infection of a target cell.
• Retroviral vectors may be based upon the Moloney murine leukaemia virus (Mo-MLV). In a retroviral vector, one or more of the viral genes
• a number of adenovirus vectors are known.
• Adenovirus subgroup C serotypes 2 and 5 are commonly used as vectors.
• the wild type adenovirus genome is approximately 35kb of which up to 30kb can be replaced with foreign DNA.
• Adenovirus vectors may have the El and/or E3 gene inactivated. The missing gene(s) may then be supplied in trans either by a helper virus, plasmid or integrated into a helper cell genome.
• Adenovirus vectors may use an E2a temperature sensitive mutant or an E4 deletion.
• Minimal adenovirus vectors may contain only the inverted terminal 5 repeats (ITRs) & a packaging sequence around the transgene, all the necessary viral genes being provided in trans by a helper virus. Suitable adenoviral vectors thus include Ad5 vectors and simian adenovirus vectors.
• Viral vectors may also be derived from the pox family of viruses, including vaccinia viruses and avian poxvirus such as fowlpox vaccines.
• modified pox virus including fowlpox virus, modified
• MVA vaccinia virus Ankara
• AAV adeno-associated virus
• HSV herpes simplex virus
• an excipient for the preservation of viral particles comprises (a) optionally one or more sugars such as sucrose, raffinose, stachyose, trehalose, or a sugar alcohol or any combination thereof; and (b) a compound of formula (I) or a physiologically acceptable salt or ester thereof and/or a compound of formula (II) or a physiologically acceptable salt or ester thereof.
• sugars such as sucrose, raffinose, stachyose, trehalose, or a sugar alcohol or any combination thereof
• a compound of formula (I) or a physiologically acceptable salt or ester thereof and/or a compound of formula (II) or a physiologically acceptable salt or ester thereof Preferably one or more sugars is present.
• the excipient consists, or consists essentially of these components.
• excipient is meant an inactive substance used as a carrier for the viral particles of the invention (for example when the viral particles are used as a vaccine).
• the viral particles e.g. for use as a vaccine
• an excipient may also comprise other preservatives such as antioxidants, lubricants and binders well known in the art, as long as those ingredients do not significantly reduce the effectiveness of the preservation mixture of the present invention.
• Preserved viral particles stored on a solid support may be used for diagnostic purposes or to monitor a vaccination regime.
• a patient sample such as bodily fluid (blood, urine, saliva, phlegm, gastric juices etc) may be preserved according to the methods described herein by drying an admixture comprising the patient sample and preservation mixture of the present invention onto a solid support.
• Preserved patient samples may then be tested for the presence of viral antigens/epitopes in the sample using anti- viral antibodies (for example using
• viral particles of interest may be preserved according to the methods described herein by drying an aclmixture comprising the viral particles and preservation mixture of the present invention onto a solid support.
• Patient samples may be tested for the presence of anti-viral antibodies by contacting the patient sample with a solid support onto which the viral particles of interest are attached.
• the formation of antigen-antibody complexes can elicit a measurable signal.
• the presence and/or amount of viral particle antigen-antibody complexes in a sample may be used to indicate the presence of a viral infection or progress of a vaccination regime in a patient.
• Preserved vaccines or viral particles according to the present invention may be administered, in some instances after reconstitution of a dried or freeze-dried product, to a subject in vivo using a variety of known routes and techniques.
• the preserved vaccines can be provided as an injectable solution, suspension or emulsion and administered via parenteral, subcutaneous, oral, epidermal, intradermal, intramuscular, interarterial, intraperitoneal, intravenous injection using a conventional needle and syringe, or using a liquid jet injection system.
• Preserved vaccines may be adrninistered topically to skin or mucosal tissue, such as nasally, intratrachealy, intestinal, sublingually, rectally or vaginally, or provided as a finely divided spray suitable for respiratory or pulmonary aclministration.
• the method of the invention further comprises the step of processing the mixture into a formulation suitable for administration as a liquid injection.
• the method further comprises the step of processing the mixture into a formulation suitable for administration via ingestion or via the pulmonary route.
• the preserved product is adrninistered to a subject in an amount that is compatible with the dosage formulation and that will be prophylactically and/or therapeutically effective.
• the administration of the preserved product or vaccine of the invention may be for either "prophylactic " or "therapeutic " purpose.
• therapeutic or “treatment” includes any of the following: the prevention of infection or reinfection; the reduction or elimination of symptoms; and the reduction or complete elimination of a pathogen. Treatment may be effected prophylactically (prior to infection) or therapeutically (following infection).
• the compound of formula (I) or physiologically acceptable salt or ester thereof and/or compound of formula (II) or physiologically acceptable salt or ester thereof and, optionally, one or more sugars typically acts as a resuspension agent for a dried or freeze-dried product comprising viral particles, preferably a product of the invention, for example when it is converted into liquid form (aqueous solution) prior to administration to a patient.
• HEK-293 cells (ECACC 85120602)
• MSM Dimethylsulfone
• Adenovirus GFP (Vector Biolabs cat. 1060)
• Measles virus strains 3A and 1 A (a kind gift provided by P. Christian at NIBSC) Dulbecco' s Modified Eagles Medium (DMEM) (Sigma D5796, Lot R BB 1139) Foetal Bovine Serum (FBS) (Sigma F7524, Lot 109K3395)
• DMEM Dulbecco' s Modified Eagles Medium
• FBS Foetal Bovine Serum
• Samples were freeze dried by the VirTis Advantage freeze dryer, using the pre-programmed protocol lasting for approximately 3 days. Samples were frozen at - 40°C for 1 hour before a vacuum was applied, initially at 200 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 8 was extended until the samples were stoppered before releasing the vacuum. The drying cycle used is shown below:
• the shelf temperature is raised to -32°C from - 45°C.
• the secondary drying phase included a ramp to 20°C until the drying was completed.
• the condenser temperature was set to stay at a constant -80°C. Probes recorded shelf temperatures and condenser temperatures (see Figures 2 A and 2B).
• Model validity (MV) "a test of diverse model problems”.
• Model validity ⁇ 0.25 indicator of statistically significant model problems e.g. outliers, incorrect model / transformation.
• Reproducibility measure of variation between replicates compared to over all variability. Reproducibility > 0.5 implies significance.
• each type of excipient was made up as a stock and 250 ⁇ 1 added to appropriately labelled 5ml glass vials. 50 ⁇ 1 of adenovirus was then added to each vial. After vortexing, vials were loaded onto the VirTis Advantage freeze drier and freeze dried according to the protocol given in the general experimental techniques section above.
• 96 flat bottomed cell culture dishes (Jencons, UK) were seeded with HEK 293 cells (ECACC 85120602) at 10 5 cells per ml ( ⁇ per well) and maintained at 37°C with 5% C0 2 .
• vials containing the adenovirus plus excipient were reconstituted in 300 ⁇ 1 PBS.
• a 1 in 10 dilution step was then taken by taking 20 ⁇ 1 from the reconstituted vial and adding to 180 ⁇ 1 of Dulbecco's Modified Eagle Medium (DMEM).
• DMEM Dulbecco's Modified Eagle Medium
• a further 1 in 100 dilution was performed by taking 20 ⁇ 1 of the 1 inlO dilution and adding it to 180 ⁇ 1 of DMEM.
• GFP Green Fluorescent Protein
• excipients containing dimethylsulfone showed little deterioration following heat and freeze challenge compared to control samples.
• thermoprotection during the FD process is essential as inadequate excipients such as sugars alone or PBS fail to produce any significant virus titre following FD.
• virus titre remains close to that of the original titre even during freeze thaw cycles.
• each type of excipient plus virus was made up as a stock in PBS and 250 ⁇ 1 added to appropriately labelled 5ml glass vials. All vials were prepared in triplicate. 50 ⁇ 1 of adenovirus was added to each vial. After vortexing, rubber bungs were partially inserted and vials were loaded onto the VirTis advantage and freeze- dried (FD) according to the freeze drying protocol given in the general experimental techniques section above. Following freeze drying, samples were assessed for virus titre using the adenovirus assay described in Example 1.
• Titration was carried out by weighing vials containing the dried excipient mixture using a balance (Kern, Germany). 1ml of liquid (hydranal methanol rapid and hydranol methanol composite, Fluka) from the chamber is transferred from the titration chamber to the glass vial using a 5ml syringe and needle. Once the excipient has dissolved the liquid is then taken back up into the syringe and the liquid injected into the titration chamber. The vial was reweighed and the difference in weight (the weight of the excipient) was inputted into the titrator. The titrator then calculated the residual moisture.
• liquid hydroanal methanol rapid and hydranol methanol composite, Fluka
• a mixture of excipient plus virus was prepared and processed as described in Example 2.
• the excipient contained TMG and optionally sugars.
• the final concentration of each component in the excipient before drying is shown in Table 4 below. All vials were prepared in triplicate.
• TMG Trimethylglycine
• Example 4 describes experimentation to elucidate the interaction between S- methyl-L-methionine (SMM), sucrose and raffinose as excipients in a freeze dried formulation of adenovrius.
• SMM S- methyl-L-methionine
• Recombinant adenovirus (Vector Biolabs) expressing enhanced GFP under a CMV promoter, and with a titre (pre-freeze) of 2x10 6 pfu ml, was removed from storage at -80°C and allowed to thaw. 50 ⁇ 1 aliquots of the virus were diluted to 300 ⁇ 1 in PBS containing a variable concentration of each of the excipients. A full list of excipient formulations tested can be seen in Table 5.
• 96 flat bottomed cell culture dishes (VWR, UK) were seeded with HEK 293 (ECA CC 85120602) cells at 10 5 cells per ml ( 1 ⁇ per well) and maintained at 37°C with 5% C0 2 .
• HEK 293 EAA CC 85120602
• After achieving 90% confluence vials containing the adenovirus plus excipient were reconstituted in 300 ⁇ 1 of PBS.
• the reconstituted samples were serially diluted l :10 and 1 :100 in DMEM plus 5% FBS. ⁇ of each of the resulting diluted virus samples were then added to individual wells of the plate. After a further 48 hours, the number of GFP cells per well were counted using fluorescent microscopy.
• S-methyl-L-methionine alone shows a concentration dependent protection of adenovirus during lyophilisation. Increased S-memyl-L-methionine in the formulation gave an increase in the recovered viral infectivity from reconstituted samples in this concentration range. Co-formulation of S-memyl-L-methionine with the low concentration treatment of sugars (0.1M Sucrose, lOmM Raffinose) did not significantly alter this relationship. However, co-formulation of S-methyl-L- methionine with the high sugar treatment (1.0M Sucrose, lOOmM Raffmose) did significantly enhance the recovery of viral infectivity at low S-methyl-L-methionine concentrations.
• Recombinant adenovirus (Vector Biolabs) expressing enhanced GFP (Green Fluorescent Protein) under a CMV promoter was formulated with excipient mixtures so that, after lyophilisation, levels of recovered infectious adenovirus could easily be assayed.
• Each type of excipient plus virus (see Table 6 below) was made up as a stock in PBS and 300 ⁇ 1 added to appropriately labelled 5ml glass vials. After vortexing, rubber bungs were partially inserted and vials were loaded onto the VirTis Advantage freeze dryer and freeze-dried (FD) as according to the freeze-drying protocol given above. Following freeze drying, samples virus titre was assessed in an adenovirus assay as described below.
• Samples were freeze dried by the VirTis Advantage freeze dryer according to the protocol given in the general experimental techniques section above. Following freeze-drying, the samples were assayed in an adenovirus assay as described in Example 1.
• the experiment in this Example expands on the capacity of DMG to protect adenovirus during lyophilisation in conjunction with raffinose and sucrose, by exploring the capability of DMG to protect adenovirus during thermal challenge.
• a strain of adenovirus expressing GFP was formulated with the excipient mixtures so that, after lyophilisation and thermal treatment, levels of recovered infectious adenovirus could easily be assayed.
• Adenovirus was formulated with the excipients and lyophilized before storage at +4°C and +37°C for one week. Samples were subsequently inoculated to HEK293 cells and recovered virus assessed by counting the number of GFP-expressing cells at 48 hours post-inoculation.
• Recombinant adenovirus (Vector Biolabs) expressing enhanced GFP under a CMV promoter, and with a titre (pre-freeze) of 2x10 6 pfu/ml, was removed from storage at -80°C and allowed to thaw. 50 ⁇ 1 aliquots of the virus were diluted to 300 ⁇ 1 in PBS containing a variable concentration of each of the excipients. A full list of excipient formulations tested can be seen in Table 7 below. Each treatment was made up in 6 replicate 5ml vials. Rubber bungs were partially inserted, and after vortexing were loaded onto the VirTis Advantage freeze-dryer and lyophilised on program 10 (see Figure 8).
• DMG for the protection of adenovirus during lyophilisation in this experiment appears to be 0.07M or greater.
• recovered titres of between 7.5-
• Coformulation of adenovirus with the same lower concentration of sugars and DMG at 0.07M or above was at least as good as the equivalent DMG concentrations in the absence of any sugars and possibly gave a slight enhancement of the protective effect.
• Recombinant adenovirus (Vector Biolabs) expressing enhanced GFP under a CMV promoter was formulated with excipient mixtures so that, after lyophilisation, levels of recovered infectious adenovirus could easily be assayed.
• excipient See Table 8 below
• Table 8 Each type of excipient was made up as a stock and 250 ⁇ 1 added to
• Samples were freeze dried by the VirTis Advantage freeze dryer, using the pre-programmed protocol lasting for approximately 3 days. Samples were frozen at -40°C for 1 hour 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 9 was extended until the samples were stoppered before releasing the vacuum. The drying cycle used is shown in Table 9 below:
• the shelf temperature is held at -34°C.
• the secondary drying phase included a ramp to +20°C until the drying was completed.
• the condenser temperature was set to stay at a constant -80°C. Probes recorded shelf temperatures and condenser temperatures (see Figure 10).
• 96 flat bottomed cell culture dishes (Jencons, UK) were seeded with HEK 293 cells (ECACC 85120602) at 10 s cells per ml ( ⁇ per well) and maintained at 37 ° C with 5% C0 2 .
• vials containing the adenovirus plus excipient were reconstituted in 1ml of Dulbecco's Minimum Essential Medium (DMEM) plus 5% Foetal Bovine Serum (FBS).
• DMEM Dulbecco's Minimum Essential Medium
• FBS Foetal Bovine Serum
• a 1:10 dilution step was carried out by taking ⁇ from the reconstituted vial and adding to 900 ⁇ 1 of DMEM.
• ⁇ of the resulting diluted virus was then added to the first row on the plate and a 1 :2 dilution ran down the plate. The process was repeated with the next excipient.
• the number of GFP cells per well were counted using fluorescent microscopy.
• Figure 11 shows the benefit of a combination of mannitol and DMG on the preservation of adenovirus titre following freeze drying. Following freeze drying there was approximately half a log drop in virus titre when DMG was used as an excipient on its own. When mannitol was the sole excipient the loss in titre was more significant than DMG with virus titre being reduced by 2 logs. When however both mannitol and DMG were used, there was no significant loss in titre and the
• Example 8 was conducted in the same manner as Example 7 except that a broader panel of excipients mixes were employed. Each type of excipient (see Table 10 below) was made up as a stock and 300 ⁇ 1 added to appropriately labelled 2ml glass vials. After vortexing, vials were loaded onto the VirTis Advantage freeze drier which was run according to the freeze drying protocol given in Table 9. Following freeze drying, samples were photographed and assessed for cake formation.
• Example 9 was conducted in the same manner as Example 7 except that only two types of excipients were prepared.
• the first excipient was the adenovirus in PBS made up to a final volume of 300ul.
• the second excipient was mannitol (0.58M) and DMG (0.7M) with the adenovirus in 2ml glass vials. After vortexing, vials were loaded onto the VirTis Advantage freeze drier and freeze-dried according to the freeze drying protocol given in Table 9. Following freeze drying, samples were either assayed for virus titre or heat treated for one week at +37°C and then assayed.
• Example 10 Stablisation of adenovirus
• Recombinant human adenovirus Ad5 (Vector Biolabs) expressing enhanced GFP (Green Fluorescent Protein) under a CMV promoter, and with a titre (pre-freeze) of6.7xl0 5 pfu/rnl in SSC, was removed from storage at -80 o C and allowed to thaw. 50 ⁇ 1 aliquots were added to 2 ml freeze-drying vials. To these 50 ⁇ 1 virus samples was added 250 ⁇ 1 of a formulation mixture composed of DMG, MSM and optionally sucrose. Each formulation mixture was made up in SSC. The concentration of DMG, MSM and sucrose in each formulation after addition to the virus sample is shown in Table 11:
• vials were immediately capped, removed, crimped and then placed at 37°C for thermal challenge. Thermal challenge was for 7 days, after which all the vials were returned to 4°C until it was practical to assay them.
• Vials containing adenovirus plus excipient were reconstituted in 300 ⁇ 1 SSC.
• a 1 in 10 dilution step was then taken by taking 20 ⁇ 1 from the reconstituted vial and adding to 180 ⁇ 1 of Dulbecco's Modified Eagle Medium (DMEM).
• a further 1 in 100 dilution was performed by taking 20 ⁇ 1 of the 1 in 10 dilution and adding it to 180 ⁇ 1 of DMEM.
• ⁇ of each of the resultant dilution (1 in 10 and 1 in 100) was then added to wells of the plate containing HEK 293 cells.
• a further sample of adenovirus from the same source and with the same titre (on storage at -80°C) used in the excipient treatments, was thawed and used to produce a 1 in 10 dilution series (in DMEM + 10% FBS). Dilutions ranging from 1 in 10 to 1 in 10 6 were also added to individual wells containing HEK 293s. At 48 hours post inoculation, the number GFP (Green Fluorescent Protein) cells per well were counted using fluorescent microscopy, and this was subsequently converted to pfu ml of the treated samples taking into account the volume applied and dilution of the inoculum. Results
• MVA was recovered from storage at -80°C and thawed. 50 ⁇ 1 aliquots were added to 2 ml freeze-drying vials. To these virus samples was added 250 ⁇ 1 of a formulation mixture listed in Table 11 above. Rubber bungs were partially inserted. After vortexing, the vials were loaded onto a Virtis Advantage Plus EL85 freeze-dryer and lyophilised on program 4 as described in Example 10.
• vials were immediately capped, removed, crimped and then placed at 37°C for thermal challenge. Thermal challenge was for 7 days, after which all the vials were returned to 4°C until it was practical to assay them.
• MVA plus excipient were reconstituted in 300ml of SSC. The reconstituted samples were diluted and assayed.
• Assay plates (96 wells) were seeded with BHK-21 cells ( ⁇ per well, 10 s cells/ml). Cells were diluted in DMEM supplemented with 10% FBS, and 1% PS.
• the plates were placed at +37°C, + 5% C0 2 for 1 to 2 hours.
• a dilution series of the formulated MVA samples was prepared (in the same growth media) ranging from 10 " to 10 .
• Each dilution series was prepared 4 times. 35 ⁇ 1 of each dilution was applied to individual wells containing BHK-21 cells and the wells were topped up with a further 65 ⁇ of media.
• CPE cytopathic effect
• Ad-CMV-GFP BHK-21 cell line ECACC CB2857 HEK 293 ECACC 85120602
• MODDE 9.0 was used to generate a three factor, two level full factorial screening design (see Figure 16 showing coded values, and Table 13 showing actual concentrations applied). This design involves testing combinations of the excipients at the high and low levels of the tested range as well as replicated centre points. The replicated centre points give an indication of error in the experiment.
• the design can model 1 st order effects of each tested factor (excipient) and interactions between them, that is, determine the impact of the presence of the exipients to the formulation. It cannot model 2 nd order of higher effects but can give an indication of whether they are present (curvature in the data). Second order effects result from covariance within the data, that is, two or more variables are dependent upon one another. Though 2 nd order effects are expected, the intent is to use this simple screening study, with minimal treatments, in order to detect any effect of the excipient and then take forward any excipient that have an effect into a more sophisticated study that can model the effects more accurately.
• Recombinant Adenovirus expressing enhanced GFP under a CMV promoter with a titre (pre-freeze) of 6.7x10 s pfu/ml in saline sodium citrate (SSC), was removed from storage at -80°C and allowed to thaw at room temperature.
• SSC saline sodium citrate
• Rubber bungs were partially inserted, and after vortexing were loaded onto a VirTis Advantage Freeze Dryer and lyophilised on program 4 (see Figure 17). After lyophilisation samples were immediately capped under vacuum, removed, crimped and placed at 37°C for thermal challenge. Thermal challenge was for 7 days, after which all the vials were held at 4°C until it was practical to assay them. Freeze-dried samples were reconstituted in 300 ⁇ 1 SSC immediately prior to assay.
• HEK 293 cells were prepared in 96 well flat bottomed cell culture dishes for inoculation by seeding at 10 5 cells per ml ( ⁇ per well) and maintained at 37°C with 5% C0 2 . After 2 hours cells were inoculated as follows.
• Thermo-challenged virus samples were diluted 1 in 10, and 1 in 100 in DMEM +10% FBS. ⁇ of each of the resulting diluted virus samples were then added to individual wells of the assay plate. Additionally, a second aliquot of the original adenovirus in SSC was thawed from -80°C and a 10 fold dilution series (from 1 in 10 to 1 in 100,000) also prepared in DMEM +10% FBS. Two repeats of this positive control dilution series was inoculated to each 96 well plate used. After a further 48 hours, the number of GFP cells per well were counted using fluorescent microscopy.
• the lowest response was below the detection threshold of the assay.
• the response in these cases was assigned the level of the detection threshold, which in this case (taking to account the countable level and then allowing for dilution factors etc.) is 6xl0 2 pfu/ml.
• Table 15 Further modelling analyses are set out in Table 15 to 17. Table 15 - model assessment parameters from models where a non-specific 2nd order interaction is allowed
• NE novel excipient
• Sue sucrose
• Raff raffinose (all 1 st order effects).
• NExS interaction between NE and sucrose.
• NExR interaction between NE and raffinose.
• SxR interaction between sucrose and raffinose.
• Curvature indication of 2 nd order effect.
• NE novel excipient
• Sue sucrose
• Raff raffinose (all 1st order effects).
• NExS interaction between NE and sucrose.
• NExR interaction between NE and raffinose.
• SxR interaction between sucrose and raffinose.
• Curvature indication of 2nd order effect.
• 2nd order term a 2nd order effect predicted by curvature in the data, that strengthens the models.
• the experimental design is unable to identify specific 2nd order effects.
• Figure 22 shows evidence of curvature in the model.
• a new model was developed with the inclusion of a 2 nd order effect. As in previous examples the specific 2 nd order effect cannot be identified with this experimental design.
• the new model scored more highly on all four model assessment parameters. This model identified sucrose, and raffinose as 1st order effects as well as an interaction between SMM and sucrose and the putative 2 nd order effect of one excipient. (see Figure 23). This new model showed no evidence of curvature within the model (see Figure 24).
• Doehlert designs are response surface modelling designs constructed from regular simplexes. They are easily extendable in different directions and new factors can be added to an existing design. Unlike regular formulation designs nonsignificant factors can be eliminated from the analysis and so do not become a confounding factor. Furthermore, different factors within the design are tested at a different number of levels, so it is possible to allocate more test levels to factors that are suspected of greater importance. Thus the excipients were tested at 7 levels, whilst sucrose was tested at 5 levels and raffinose at only 3 levels. This model retains the ability to model for second order effects and interactions. The design included 3 factors and 3 replicate centre-points resulting in 15 test samples.
• Sucrose was tested between 0 and 1M.
• the upper level of sucrose was set at 1M because it has proved close to the limit for acceptable freeze-drying. It has also proved to be a highly successful level in prior studies, and in general higher sucrose concentrations are undesirable in parenterals.
• the lowest level of Sucrose was set at 0 M.
• Raffinose was tested over a range of 0 to 300mM although the nature of the Doehlert design meant that tested levels did not include OmM, instead the following concentrations were tested; 27.5, 150.0, and 272.5mM.
• DMG was tested over a linear range of 0 to 2M. It was possible to limit this range based on previous experiments in which the optimum concentration was frequently between 0.5 and 1.5M in a freeze-dried setting.
• Recombinant Adenovirus expressing enhanced GFP under a CMV promoter with a titre (pre-freeze) of 6.7x10 5 pfu/ml in SSC, was removed from storage at -80°C and allowed to thaw. Subsequently, 50 ⁇ 1 aliquots of virus were added to 15, 2ml, glass freeze-drying vials. To each vial 250 ⁇ 1 of an excipient blend was admixed. The excipient blend formulations once mixed with virus are described in Table 18 and were made up in SSC. Table 18
• HEK 293 cells were prepared in 96 well flat bottomed cell culture dishes for inoculation by seeding at 10 s cells per ml ( ⁇ per well) and maintained at 37°C with 5% C0 2 . After 2 hours cells were inoculated as follows.
• Thermo-challenged virus samples were diluted 1 in 10, and 1 in 100 in DMEM +10% FBS. ⁇ of each of the resulting diluted virus samples were then added to individual wells of the assay plate. Additionally, a second aliquot of the original Adenovirus in SSC was thawed from -80°C and a 10 fold dilution series (from 1 in 10 to 1 in 100,000) also prepared in DMEM +10% FBS. Two repeats of this positive control dilution series was inoculated to each 96 well plate used. After a further 48 hours, the number of GFP cells per well were counted using fluorescent microscopy.
• the model identified (see Figure 30) 1st order effects of both sucrose and DMG as well as a 2nd order effect of DMG. No 1st or 2nd order effects of raffinose were observed. However, raffinose does have an interaction with DMG and thus the 1 st order raffinose coefficient must be retained in the model to preserve the models hierarchical structure. Furthermore, the 2nd order raffinose effect was retained as it resulted in a stronger model (as assessed by the indicators shown in Figure 29 and discussed above). In any case the 2nd order raffinose effect was close to significance at the 90% C.I. and may be a genuine effect that simply cannot be conclusively detected over the range tested.
• Figure 31 shows a series of 3D plots of recovered virus activity (Y-Axis) against varied sucrose (X-axis) and DMG (Z-axis) concentrations.
• Low denotes a raffinose concentration of OmM
• Mod denotes a raffinose concentration of 150mM
• High denotes a raffinose concentration of 300mM.
• Figure 33 a The predicted optimum is shown on a contour plot ( Figure 33 a) which puts the optimum into context.
• the model predicts whole regions of the design space in which formulations would yield 100% or greater recovered virus activity. This region needs to be viewed as a plateau in the data within which close to zero loss of virus activity would be expected.
• Figure 33b highlights this region. The figure shows that as raffinose concentration is increased the region moves down the Y-axis (DMG concentration) and up the X-axis (sucrose concentration).
• a formulation of DMG, sucrose and raffinose has been identified with significant potential for the preservation of adenovirus through lyophilisation and heat challenge.
• sucrose concentration is beyond the tested range and also likely beyond other constraints of sucrose concentration.
• Raffinose does not appear to be a critical factor in this model.
• a long-term stability testing temperature of +4°C ⁇ 3 was selected. This is broadly consistent with standard industry guidelines for long-term testing of products intended for refrigerated storage (+5°C ⁇ 3).
• An accelerated stability temperature of +25°C was adopted and a thermal challenge of +37°C was adopted to represent a stress testing temperature, or a further elevated accelerated thermal stability temperature.
• the samples at 25°C and 37°C were tested 1, 2, 5 and 15 weeks post lyophilisation.
• the samples at +4°C were tested at 15 weeks post lyophilisation.
• Recombinant adenovirus expressing enhanced GFP under a CMV promoter with a titre (pre-freeze) of 6.7x10 5 pfu/ml in SSC, was removed from storage at -80°C and allowed to thaw. Subsequently, 50 ⁇ 1 aliquots of virus were added to 2ml glass freeze-drying vials. To each vial 250 ⁇ 1 of an excipient blend was admixed.
• excipient blend formulations used were as described above, namely (i) buffer alone (SSC), (ii) sugars (0.5M Sucrose, 150mM Raffinose in SSC), and (iii) a putative optimal formulation (0.5M Sucrose, 150mM Raffinose, 1M DMG, also in SSC).
• HEK 293 cells were prepared in 96 well flat bottomed cell culture dishes for inoculation by seeding at 10 5 cells per ml ( ⁇ per well) and maintained at 37°C with 5% C0 2 . After 2 hours cells were inoculated as follows.
• Thermo-challenged virus samples were recovered from thermo challenge as described above diluted 1 in 10, and 1 in 100 in DMEM +10% FBS. ⁇ of each of the resulting diluted virus samples were then added to individual wells of the assay plate.
• a second aliquot of the original adenovirus in SSC was thawed from -80°C and a 10 fold dilution series (from 1 in 10 to 1 in 100,000) also prepared in DMEM +10% FBS. Two repeats of this positive control dilution series was inoculated to each 96 well plate used. After a further 48 hours, the number of GFP cells per well were counted using fluorescent microscopy.
• Figures 36 and 37 further support these findings. At both, +25°C and +37°C, no virus activity is recovered from samples stored in buffer alone at any time-point. Those formulated in sugars alone retain some activity throughout. Their activity declines by a slightly greater degree and slightly more rapidly at the higher temperature (+37°C). In the putative optimal formulation there is a steeper decline in viral activity at +37°C but both temperatures decline to similar levels over time.
• Sucrose was tested between 0 and 1M.
• Raffinose was tested over a range of 0 to 300mM, although the nature of the Doehlert design meant that tested levels did not include OmM. Instead the following ranges were tested: 27.5, 150.0, and 272.5mM.
• TMG was tested over a linear range of 0 to 2M.
• Rubber bungs were partially inserted, and after vortexing were loaded onto a Virtis advantage freeze-dryer and lyophilised as described in Figure 39. After lyophilisation samples were immediately capped under vacuum, removed, crimped and placed at 37°C for thermal challenge. Thermal challenge was for 7 days, after which all the vials were returned to the control vials and held at 4°C until it was practical to assay them. Freeze-dried samples were reconstituted in 300 ⁇ 1 SSC immediately prior to assay.
• Assay plates (96 well) were seeded with BHK-21 cells ( ⁇ per well, 10 5 cells/ml). Cells were diluted in DMEM supplemented with 10% FBS, and 1% PS. The plates were placed at +37°C, + 5% C0 2 for 1-2 hours.
• a 10 fold dilution series of the formulated MVA samples was prepared (in the same growth media) ranging from 1 in 10 to 1 in 10,000. Each dilution series was prepared 5 times. ⁇ of each dilution was applied to individual wells containing BHK-21 cells (described above).
• Figure 42 shows a contour plot of the model. The optimum TMG
• CCF Central Composite Face-Centred
• RBM Response Surface Modelling
• MVA was recovered from storage at -80°C and thawed. 50 ⁇ 1 aliquots of the MVA were added to 2ml glass freeze-drying vials. Subsequently 250 ⁇ 1 of an excipient blend was added to each vial.
• the excipient blend formulations once mixed with virus are described in Table 20 and were made up in SSC. Table 20
• Rubber bungs were partially inserted, and after vortexing were loaded onto a Virtis advantage freeze-dryer and lyophilised as described in Figure 44. After lyophilisation samples were immediately capped under vacuum, removed, crimped and placed at 37°C for thermal challenge. Thermal challenge was for 7 days, after which all the vials were returned to the control vials and held at 4°C until it was practical to assay them. Freeze-dried samples were reconstituted in 300 ⁇ 1 SSC immediately prior to assay.
• Assay plates (96 well) were seeded with BHK-21 cells ( ⁇ per well, 10 5 cells/ml). Cells were diluted in DMEM supplemented with 10% FBS, and 1% PS. The plates were placed at +37°C, + 5% C0 2 for 1 -2 hours.
• a 10 fold dilution series of the formulated MVA samples was prepared in the same growth media ranging from 1 in 10 to 1 in 10,000. Each dilution series was prepared 5 times. ⁇ of each dilution was applied to individual wells containing BHK-21 cells (described above). On 6 d p.i. the wells were scored for presence or absence of CPE and TCID50 calculated. These were then used to estimate the concentration of infectious MVA per ml in the thermo-challenged vials.
• Figure 47 shows the RSM model generated. It is effectively a simple DMG dose response curve that is not altered by mannitol within the tested concentration range.
• the dose response curve identifies a clear optimum DMG concentration, as do monte-carlo simulations (see Figure 48).
• the predicted optimum DMG concentration is 1.00M and predicted recovery of viral activity is 117% of starting titre.

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• 2011
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