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  • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
• • 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
  • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
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• • A—HUMAN NECESSITIES
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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/08—Solutions
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • 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
  • A61K9/141—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers
  • A61K9/146—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers with organic macromolecular compounds
• • 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
  • A61K9/141—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers
  • A61K9/148—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers with compounds of unknown constitution, e.g. material from plants or animals
• • 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
  • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
  • A61K9/1605—Excipients; Inactive ingredients
  • A61K9/1629—Organic macromolecular compounds
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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
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  • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/04—Preserving or maintaining viable microorganisms
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  • 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
  • C12N2710/10321—Viruses as such, e.g. new isolates, mutants or their genomic sequences
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  • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
  • C12N2710/00011—Details
  • C12N2710/16011—Herpesviridae
  • C12N2710/16711—Varicellovirus, e.g. human herpesvirus 3, Varicella Zoster, pseudorabies
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  • C12N2720/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsRNA viruses
  • C12N2720/00011—Details
  • C12N2720/12011—Reoviridae
  • C12N2720/12021—Viruses as such, e.g. new isolates, mutants or their genomic sequences
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  • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
  • C12N2760/00011—Details
  • C12N2760/18011—Paramyxoviridae
  • C12N2760/18021—Viruses as such, e.g. new isolates, mutants or their genomic sequences
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  • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
  • C12N2760/00011—Details
  • C12N2760/18011—Paramyxoviridae
  • C12N2760/18611—Respirovirus, e.g. Bovine, human parainfluenza 1,3
  • C12N2760/18621—Viruses as such, e.g. new isolates, mutants or their genomic sequences

Definitions

• the present invention relates to virus stabilizers, gelatin hydrolysates for virus stabilizers, and virus-containing compositions.
• Patent Document 1 discloses a refrigerator-stable influenza virus composition that is stable at refrigeration temperatures (eg, 2-8°C).
• Patent Document 1 discloses that a composition containing 0.5 to 2% gelatin hydrolyzate and the like achieves stable storage and transportation of influenza virus strains at refrigeration temperatures. However, Patent Document 1 does not disclose whether or not stable storage and transportation of the virus strain can be achieved at room temperature around 25°C. According to Patent Document 1, since the gelatin hydrolyzate is obtained from porcine type A gelatin having an isoelectric point of about 9, the gelatin hydrolyzate strongly interacts with specific proteins on the virus surface. It is presumed that this makes it difficult to stably store and transport the virus at room temperature.
• the present invention provides a virus-containing formulation, etc., without the need for a refrigeration apparatus, etc., by suppressing the decline in the ability of the virus at both the refrigeration temperature (2 to 8 ° C.) and the room temperature around 25 ° C.
• An object of the present invention is to provide a virus stabilizer, a gelatin hydrolyzate for the virus stabilizer, and a virus-containing composition that enable storage and transportation of the virus.
• the present inventors arrived at the present invention as a result of diligent studies. That is, attention was first focused on a gelatin hydrolyzate obtained by hydrolyzing gelatin having an isoelectric point of pH around 5 so as to have a predetermined weight-average molecular weight. Furthermore, when the virus is contained in a preparation containing the gelatin hydrolyzate at a predetermined concentration, the reduction in virus ability such as infectivity is suppressed both at refrigerated temperatures (2 to 8°C) and at room temperature around 25°C. The inventors have found that the virus-containing preparations can be stably stored and transported without the need for a freezer or the like, and have completed the present invention.
• the present invention has the following features.
• the virus stabilizer according to the present invention is a virus stabilizer comprising a gelatin hydrolyzate and an aqueous medium, and the virus stabilizer contains the gelatin hydrolyzate in an amount of 1% by mass or more and 20% by mass. % or less, the gelatin hydrolyzate has a weight average molecular weight of 10000 or less, and the gelatin hydrolyzate has an isoelectric point pH of 4.0 or more and 7.0 or less.
• the gelatin hydrolyzate preferably has a weight-average molecular weight of 6,000 or less.
• the gelatin hydrolyzate is preferably a hydrolyzate of alkali-treated gelatin.
• the virus stabilizer preferably contains more than 2% by mass and 10% by mass or less of the gelatin hydrolyzate.
• the aqueous medium preferably contains a salt having a buffering action.
• the aqueous medium preferably contains at least one sugar selected from the group consisting of sucrose, lactose, sorbitol, inositol, trehalose, mannitol, maltitol, xylitol, erythritol and glycerol.
• the aqueous medium preferably contains at least one amino acid selected from the group consisting of methionine, arginine, tryptophan, glutamine and glutamic acid.
• the hydrolyzate of gelatin for a virus stabilizer according to the present invention has a weight average molecular weight of 10000 or less and an isoelectric point pH of 4.0 or more and 7.0 or less.
• the gelatin hydrolyzate for virus stabilizer is preferably liquid or powder.
• a virus-containing composition according to the present invention comprises the virus stabilizer and a virus.
• the virus preferably contains at least one selected from the group consisting of enveloped DNA viruses, non-enveloped DNA viruses, enveloped RNA viruses and non-enveloped RNA viruses.
• the virus is preferably gene-introduced.
• the virus-containing composition has a difference of 1 between the logarithmic reduction in virus titer when stored at 25°C for 21 days and the logarithmic reduction in virus titer when stored at -80°C for 21 days. 0.5 log or less is preferred.
• the virus-containing composition has a difference of 1 between the logarithmic reduction in virus titer when stored at 4°C for 210 days and the logarithmic reduction in virus titer when stored at -80°C for 210 days. .0 log or less is preferred.
• the virus-containing composition preferably exhibits a logarithmic reduction in virus titer of 0.3 log or less when freeze-thaw is repeated three times.
• Virus-stabilizing agents, gelatin hydrolysates for virus-stabilizing agents, and virus-containing compositions can be provided.
• the notation of the format “A to B” means the upper and lower limits of the range (that is, A to B or less), and when there is no unit description in A and only a unit is described in B , the units of A and B are the same.
• virus titer refers to the lowest concentration at which the virus in the sample (herein, “virus-containing composition”) can infect cells, ie, the highest dilution factor.
• “reduction” in virus titer means that the above-mentioned dilution rate is reduced, and means that the above-mentioned minimum concentration at which infection is possible is increased.
• gelatin may be used when referring to the substance name, gelatin gel, and gelatin solution, respectively.
• gelatin hydrolyzate may also be used to refer to a gelatin hydrolyzate solution in the same manner as the gelatin described above.
• the virus stabilizer according to this embodiment is a virus stabilizer comprising a gelatin hydrolyzate and an aqueous medium.
• the virus stabilizer contains 1% by mass or more and 20% by mass or less of the gelatin hydrolyzate.
• the gelatin hydrolyzate has a weight average molecular weight of 10,000 or less.
• the gelatin hydrolyzate has an isoelectric point pH of 4.0 or more and 7.0 or less.
• the virus stabilizers include gelatin hydrolysates as described above.
• gelatin hydrolyzate refers to a peptide aggregate (hydrolyzate) obtained by hydrolyzing both or either one of gelatin and collagen. That is, “gelatin hydrolyzate” means the equivalent of an assembly of peptides commonly referred to as collagen peptides or collagen hydrolysates.
• the gelatin hydrolyzate contained in the virus stabilizer according to the present embodiment has the weight average molecular weight and isoelectric point pH as described above.
• the gelatin hydrolyzate means an assembly of peptides as described above, it has the same characteristics as collagen and gelatin, such as having a primary structure in which glycine is repeated every three residues in the amino acid sequence that constitutes the peptide chain. have.
• the term "gelatin” means a polypeptide in which the triple helical structure of collagen is unwound by heat denaturation, acid denaturation, or the like, chemical modifications thereof, and pharmaceutically acceptable salts thereof.
• collagen derived from at least one selected from the group consisting of Groups 1 to 6 below is subjected to conventionally known treatments such as degreasing treatment, decalcification treatment, acid or alkali treatment, and hot water extraction treatment. It can be obtained by applying the treatment of Gelatin may be a polypeptide obtained by fermentation using microorganisms, a recombinant polypeptide obtained by chemical synthesis or genetic recombination, or a synthesized polypeptide.
• collagen refers to proteins derived from the extracellular matrix in the skin of vertebrates, which are classified into Groups 1 to 6 below.
• Collagen has a right-handed helical structure consisting of three peptide chains, and the amino acid residues constituting the peptide chain have a primary structure in which glycine residues are repeated every three residues (so-called collagen-like sequence). have.
• Group 1 Group consisting of hide, skin, bone, cartilage and tendon of cattle
• Group 2 Group consisting of hide, skin, bone, cartilage and tendon of pig
• Group 3 Hide, skin, bone, cartilage and tendon of sheep
• Group 4 Group consisting of chicken skin, skin, bones, cartilage and tendons
• Group 5 Group consisting of ostrich skin, skin, bones, cartilage and tendons
• Group 6 Fish bones, skin and scales The group consisting of
• the "chemically modified form" of the above polypeptide (gelatin) means a polypeptide in which the amino group, carboxyl group, hydroxy group, thiol group, etc. of the amino acid residues constituting gelatin are chemically modified.
• Chemically modified gelatin can change its solubility in water, isoelectric point, and the like. Specifically, chemical modifications such as O-acetylation can be carried out on the hydroxy groups of hydroxyproline residues in gelatin. Chemical modifications such as esterification and amidation can be performed on the ⁇ -carboxyl group of glycine residues in gelatin.
• Chemical modification of the hydroxy group of a hydroxyproline residue can be carried out, for example, by O-acetylation, such as by reacting acetic anhydride in an aqueous or non-aqueous solvent.
• O-acetylation such as by reacting acetic anhydride in an aqueous or non-aqueous solvent.
• esterification can be carried out, such as by bubbling dry hydrogen chloride gas after suspension in methanol.
• amidation can be carried out by the action of carbodiimide or the like.
• the "derivatives" of the above polypeptide include gelatin derivatives obtained by introducing functional groups into gelatin, copolymers of gelatin with lactic acid, glycolic acid, etc., and copolymers of gelatin with polyethylene glycol and propylene glycol. etc. may be included.
• gelatin derivatives include derivatives obtained by introducing functional groups such as guanidyl groups, thiol groups, amino groups, carboxyl groups, sulfate groups, phosphoric acid groups, alkyl groups, acyl groups, phenyl groups, and benzyl groups into gelatin. be able to.
• “Pharmaceutically acceptable salt” of the above polypeptide (gelatin) means a salt that is pharmaceutically acceptable and has the desired activity (eg, gelling ability) of the original polypeptide (gelatin).
• Pharmaceutically acceptable salts include inorganic acid salts such as hydrochlorides, sulfates, phosphates and hydrobromides, acetates, methanesulfonates, benzenesulfonates, p-toluenesulfonates. , organic acid salts such as succinate, oxalate, fumarate and maleate; inorganic base salts such as sodium, potassium and calcium salts; and organic base salts such as triethylammonium salt. can.
• a specific peptide in gelatin can be converted into a pharmaceutically acceptable salt according to a conventional method.
• gelatin is a polypeptide derived from collagen that many organisms possess, it has excellent biocompatibility. Therefore, a gelatin hydrolyzate obtained by hydrolyzing the above collagen and gelatin also has excellent biocompatibility and is suitable as one component of a virus stabilizer for medical use.
• the term "sol” means a dispersion system comprising a dispersoid and a dispersion medium, in which the dispersion medium is liquid.
• “Gel” means a state in which the dispersoids form a crosslinked structure in a dispersion system composed of dispersoids and a dispersion medium, and the dispersion system as a whole loses fluidity.
• a gelatin hydrolyzate is obtained by hydrolyzing either or both gelatin and collagen as described above.
• “hydrolysis” includes hydrolysis using acids, hydrolysis using bases, hydrolysis using enzymes, and hydrolysis using heat. From the viewpoint of preventing contamination with impurities, the gelatin hydrolyzate is preferably obtained by hydrolysis using heat.
• the enzyme include collagenase, thiol protease, serine protease, acid protease, alkaline protease, metalloprotease, and the like. The above enzymes can be used singly or in combination.
• Examples of the thiol protease include plant-derived chymopapain, papain, bromelain, ficin, animal-derived cathepsin, calcium-dependent protease, and the like.
• Examples of the serine protease include trypsin, cathepsin D, and the like.
• Examples of the acid protease include pepsin and chymotrypsin.
• Non-pathogenic microorganisms from which the above enzymes are derived include Bacillus iicheniforms, Bacillus subtillis, Aspergillus oryzae, Streptomyces, Bacillus amyloliquefaciens, and the like.
• an enzyme derived from one of the above-described non-pathogenic microorganisms may be used, or a plurality of types of enzymes derived from the above-described non-pathogenic microorganisms may be used in combination.
• a conventionally known method may be used as a specific method for enzymatic treatment.
• the enzyme is preferably not collagenase. This is because when the above gelatin is hydrolyzed with collagenase alone, a specific peptide having an amino acid sequence with glycine at the N-terminus may be the main component (more than 50% by mass).
• the above-mentioned gelatin hydrolyzate is an aggregate composed of two or more peptides, and the above-mentioned specific peptide is less than 50% by mass in its composition.
• a gelatin hydrolyzate can be obtained as a liquid by hydrolyzing both or either one of gelatin and collagen by the method described above, followed by purification. Furthermore, it is also possible to obtain a powder by heat-drying or freeze-drying the above liquid by a known means.
• the virus stabilizer contains 1% by mass or more and 20% by mass or less of the gelatin hydrolyzate.
• concentration of the gelatin hydrolyzate contained in the virus stabilizer is less than 1% by mass, a decrease in virus titer is suppressed when a virus-containing composition containing the virus stabilizer and virus is constructed. Since the action becomes insignificant, it becomes difficult to achieve the desired effect. If the concentration of the gelatin hydrolyzate contained in the virus stabilizer exceeds 20% by mass, the virus stabilizer may deteriorate in operability.
• the virus stabilizer preferably contains more than 2% by mass and 10% by mass or less of the gelatin hydrolyzate from the viewpoint of further suppressing a decrease in virus titer.
• concentration of the gelatin hydrolyzate in the virus stabilizer can be measured by a known method such as hydroxyproline determination.
• the gelatin hydrolyzate has a weight average molecular weight of 10,000 or less.
• the gelatin hydrolyzate preferably has a weight-average molecular weight of 6,000 or less. More preferably, the gelatin hydrolyzate has a weight-average molecular weight of 5,000 or less.
• the virus stabilizer can maintain a sol state in an environment of about 2 to 30°C.
• the gelatin hydrolyzate in the composition can suppress virus aggregation, thereby suppressing a decrease in virus titer. can do.
• the gelatin hydrolyzate can protect the surface of the virus by covering it because it is possible to interact with the surface of the virus with moderate strength regardless of the presence or absence of the virus. be.
• the gelatin hydrolyzate has a weight-average molecular weight of more than 10,000, it may gel at 2-8°C.
• the lower limit of the weight-average molecular weight of the gelatin hydrolyzate is not particularly limited, but is, for example, 75, which is the molecular weight of glycine.
• the weight average molecular weight of the gelatin hydrolyzate can be determined by performing gel filtration chromatography under the following measurement conditions.
• Equipment high performance liquid chromatography (HPLC) (manufactured by Tosoh Corporation) Column: TSKGel® G2000SW XL Column temperature: 30°C Eluent: 40 wt% acetonitrile (containing 0.05 wt% TFA) Flow rate: 0.5mL/min Injection volume: 10 ⁇ L Detection: UV220nm
• Molecular weight marker Use the following three types Cytochrom C Mw: 12384 Aprotinin Mw: 6512 Bacitracin Mw: 1423.
• the weight-average molecular weight of the gelatin hydrolyzate can be obtained by measuring the substance to be measured under the gel filtration chromatography conditions described above.
• the gelatin hydrolyzate has an isoelectric point pH of 4.0 or more and 7.0 or less.
• the pH of the isoelectric point of the gelatin hydrolyzate is preferably from 4.0 to 5.5, more preferably from 4.0 to 4.8.
• a gelatin hydrolyzate having an isoelectric point pH in such a range is obtained by hydrolyzing alkali-treated collagen or gelatin obtained from alkali-treated collagen (so-called alkali-treated gelatin). It can be efficiently obtained by hydrolysis. That is, the gelatin hydrolyzate is preferably a hydrolyzate of alkali-treated gelatin.
• the gelatin hydrolyzate is preferably a hydrolyzate of alkali-treated gelatin having an isoelectric point of about pH 4.8 to 5.5.
• gelatin obtained by treating collagen with an inorganic acid is called acid-treated gelatin
• gelatin obtained by treating collagen with an inorganic base is called alkali-treated gelatin.
• Alkali-treated gelatin can be specifically obtained by treating collagen with an inorganic base such as sodium hydroxide, calcium hydroxide or potassium hydroxide.
• Acid-treated gelatin has an isoelectric point of pH 8-9.
• alkali-treated gelatin has an isoelectric point pH of 4.0 to 7.0. Examples of such alkali-treated gelatin include pig skin-derived alkali-treated gelatin (trade name: "beMatrix (registered trademark) gelatin LS-H", manufactured by Nitta Gelatin Co., Ltd.).
• a virus-containing composition is composed of a virus stabilizer containing a gelatin hydrolyzate having an isoelectric point pH within the above range and a virus
• the gelatin hydrolyzate in the composition causes the virus to aggregate. can be suppressed, thereby suppressing a decrease in virus titer.
• the gelatin hydrolyzate having an isoelectric point pH in such a range has relatively more negative charges than positive charges in the molecule, so as a whole Although it exhibits a negative charge, it will have both positively charged and negatively charged sites. Therefore, the gelatin hydrolyzate exhibits a weak electrostatic interaction between the positively charged site and the negatively charged capsid protein, spike protein, envelope protein on the virus surface, etc. in the virus.
• the gelatin hydrolyzate repels each other with the virus and other peptide chains that make up the gelatin hydrolyzate at the negatively charged sites. From the above, it is presumed that the gelatin hydrolyzate can protect the virus surface by covering it while suppressing virus aggregation.
• a gelatin hydrolyzate obtained from an acid-treated gelatin having an isoelectric point of pH around 9 (for example, the gelatin hydrolyzate disclosed in Patent Document 1 above) has a positive charge, so exhibits a negative charge. There is a possibility that viruses aggregate by exhibiting strong electrostatic interactions with capsid proteins and the like. It is presumed that the gelatin hydrolyzate obtained from acid-treated gelatin exhibits a marked effect of coagulating viruses, especially in a temperature environment of around 25°C.
• the pH of the isoelectric point of the gelatin hydrolyzate can be obtained by measuring the pH of the isoelectric point of either or both of gelatin and collagen, which are raw materials of the gelatin hydrolyzate, using a conventionally known method. However, it is preferable to use the following method for measuring the isoelectric point using the zeta potential as an index, since the isoelectric point value can be obtained more accurately. First, a gelatin hydrolyzate to be measured is dissolved in an acetate buffer (pH 4.0 to 5.5) to obtain a 0.4 w/v % solution to be measured.
• the solution to be measured is filtered through a 0.22 ⁇ m filter (manufactured by Merck), and then 0.8 mL of the solution to be measured is filled into a capillary cell while preventing air bubbles from entering.
• the capillary cell filled with the solution to be measured is set in a zeta potential measuring device (manufactured by Malvern Panalytical) to measure the zeta potential at each pH at 25°C.
• the pH value at which the zeta potential becomes 0 can be obtained as the isoelectric point of the solution to be measured (the gelatin hydrolyzate to be measured).
• Virus stabilizers include aqueous media as described above.
• aqueous medium refers to a medium that dissolves or disperses the gelatin hydrolyzate, and means a medium that can contain components other than water, such as amino acids, sugars, and salts with buffering properties, which will be described later.
• the aqueous medium may be a buffer solution containing a buffering salt.
• the aqueous medium may be GTS buffer. That is, the aqueous medium preferably contains a salt having a buffering action. Accordingly, when a virus-containing composition is formed by containing a virus, the virus-stabilizing agent can contribute to the stabilization of the virus by the above-mentioned buffering action.
• salts having a buffering action examples include sodium phosphate, potassium phosphate, calcium phosphate, magnesium phosphate, sodium hydrogen phosphate, potassium hydrogen phosphate, calcium hydrogen phosphate, magnesium hydrogen phosphate, sodium chloride, potassium chloride, and the like. can do.
• the aqueous medium may contain one of the above salts having a buffering action, or may contain two or more of them in combination.
• Examples of the aqueous medium containing a salt having a buffering action include the GTS buffer, PBS buffer, Tris buffer, HEPES buffer, citrate buffer, and the like.
• the composition of the GTS buffer can be, for example, 2.5 mass % glycerol, 20 mM Tris pH 8, and 25 mM NaCl.
• the aqueous medium preferably contains at least one amino acid selected from the group consisting of methionine, arginine, tryptophan, glutamine and glutamic acid.
• the aqueous medium may contain one type of amino acid selected from these groups alone, or may contain two or more types in combination. More preferably, the aqueous medium contains methionine and/or arginine amino acids.
• the virus-stabilizing agent can further contribute to the stabilization of the virus when the virus-containing composition is constituted by containing the virus.
• the aqueous medium preferably contains at least one sugar selected from the group consisting of sucrose, lactose, sorbitol, inositol, trehalose, mannitol, maltitol, xylitol, erythritol and glycerol.
• the aqueous medium may contain one type of saccharide selected from these groups alone, or may contain two or more types in combination. More preferably, the aqueous medium contains at least one of sucrose, lactose or sorbitol.
• the virus-stabilizing agent can further contribute to the stabilization of the virus when the virus-containing composition is constituted by containing the virus.
• the compounds included in "sugars” include not only organic compounds generally classified as sugars, but also organic compounds classified as sugar alcohols.
• Organic compounds classified as sugar alcohols in the above group of sugars are the above sorbitol, mannitol, maltitol, xylitol, erythritol and glycerol.
• the aqueous medium may contain other components as long as the effects of the present invention are exhibited.
• Other ingredients include growth factors, differentiation factors, hormones, chemokines, cytokines, cell adhesion molecules, chemotactic factors, enzymes, enzyme inhibitors, coenzymes (vitamins), minerals, fats, lipids, stabilizers and preservatives. etc. can be mentioned.
• the virus stabilizer according to the present embodiment comprises an aqueous medium and gelatin hydrolyzate contained at a concentration of 1% by mass or more and 20% by mass or less, as described above.
• the gelatin hydrolyzate has a weight average molecular weight of 10000 or less and an isoelectric point pH of 4.0 or more and 7.0 or less.
• the gelatin hydrolyzate for a virus stabilizer has a weight average molecular weight of 10000 or less and an isoelectric point pH of 4.0 or more and 7.0 or less.
• the gelatin hydrolyzate for virus stabilizer preferably has a weight-average molecular weight of 6000 or less, or an isoelectric point of pH of 4.0 to 5.5.
• the gelatin hydrolyzate for a virus stabilizer has the same characteristics as described in the ⁇ Gelatin hydrolyzate> section, so redundant description will not be repeated.
• the gelatin hydrolyzate for virus stabilizer has a previously unknown attribute of the gelatin hydrolyzate, i.e., an unknown attribute.
• the gelatin hydrolyzate for virus stabilizer can be used as a virus stabilizer, and the virus-containing composition can be frozen at both refrigeration temperature (2 to 8 ° C.) and room temperature around 25 ° C. can be stably stored and transported without requiring
• the gelatin hydrolyzate for the virus stabilizer is preferably liquid or powder.
• the virus-containing composition can be easily prepared by adding the virus together with the aqueous medium.
• the gelatin hydrolyzate for virus stabilizer is powder, the virus stabilizer is prepared by dissolving or dispersing it in an aqueous medium, and the virus is added to the virus stabilizer to easily prepare the virus-containing composition. can be done.
• a gelatin hydrolyzate for a virus stabilizer can be prepared as a liquid by hydrolyzing gelatin and/or collagen followed by purification.
• the gelatin hydrolyzate for a virus stabilizer can be prepared as a powder by heat-drying or freeze-drying the liquid gelatin hydrolyzate for a virus stabilizer prepared as described above by a known method. can.
• the virus-containing composition according to this embodiment contains the virus stabilizer and a virus.
• the virus-containing composition can suppress aggregation of the virus in the composition, thereby suppressing a decrease in virus titer. Therefore, the virus-containing composition can be stably stored and transported both at a refrigeration temperature (2 to 8° C.) and at room temperature around 25° C. without the need for a freezer or the like.
• the virus stabilizer contained in the virus-containing composition specifically has the characteristics as described in the [Virus Stabilizer] section, so redundant description will not be repeated.
• a virus-containing composition comprises a virus as described above.
• the virus preferably contains at least one selected from the group consisting of enveloped DNA viruses, non-enveloped DNA viruses, enveloped RNA viruses and non-enveloped RNA viruses. That is, the type of virus that constitutes the virus-containing composition is not limited to use.
• the virus-containing composition may contain one type of virus selected from the above group alone, or may contain two or more types of virus in combination. Even in such a case, the virus-containing composition contains a virus stabilizer so that the virus can be stably stored and transported both at a refrigeration temperature (2 to 8°C) and at room temperature around 25°C. can be done.
• viruses used as live or inactivated vaccines such as influenza virus and rotavirus, oncolytic viruses that have the effect of lysing tumors, herpes simplex virus, vaccinia virus, adenovirus, and coxsackievirus. , poxvirus, paramyxovirus, picornavirus, rapdovirus, reovirus, parvovirus, bracken virus, orthomyxovirus, retrovirus, and the like.
• the virus may be gene-introduced.
• the virus-containing composition may contain other components as long as the effects of the present invention are exhibited.
• Other ingredients include growth factors, differentiation factors, hormones, chemokines, cytokines, cell adhesion molecules, chemotactic factors, enzymes, enzyme inhibitors, coenzymes (vitamins), minerals, fats, lipids, stabilizers and preservatives. etc. can be mentioned.
• the virus-containing composition is the logarithmic reduction value of the virus titer when stored at 25° C. for 21 days (hereinafter also referred to as “LRV”) and the virus titer LRV when stored at ⁇ 80° C. for 21 days.
• the difference is 1.5 log or less, more preferably, the difference is 1.0 log or less, and the virus-containing composition preferably has a lower limit of 0 for the difference.
• the virus-containing composition preferably has a difference of 1.0 log or less between the LRV of the virus titer when stored at 4°C for 210 days and the LRV of the virus titer when stored at -80°C for 210 days. . More preferably, the difference is 0.5 log or less.
• the virus-containing composition preferably has a lower limit of 0 for the above difference.
• the virus-containing composition has the LRV characteristics of the virus titer as described above, it can stably store and transport the virus both at refrigerated temperatures (2-8°C) and at room temperature around 25°C. Ability is guaranteed.
• the virus-containing composition has a difference of 1.5 log or less between the LRV of the virus titer pair when stored at 25°C for 21 days and the LRV of the virus titer when stored at -80°C for 21 days. Also preferably, the difference between the LRV of the virus titer when stored at 4° C. for 21 days and the LRV of the virus titer when stored at ⁇ 80° C. for 21 days is 1.0 log or less.
• the virus-containing composition preferably has a logarithmic reduction in virus titer of 0.3 log or less when freeze-thaw is repeated three times. As a result, the virus-containing composition can maintain its virus titer even after repeated freezing and thawing up to three times. This allows flexibility in the use of virus-containing compositions, which is extremely convenient.
• the LRV of virus titer in a virus-containing composition can be determined using conventional methods of measuring virus titer, eg, by determining the TCID50 of the virus in the virus-containing composition.
• TCID50 refers to a culture dish such as a test tube or a 96-well flat-bottom plate in which cells are cultured and attached in advance, and inoculated with a virus dilution (in this specification, a dilution of the "virus-containing composition"). In some cases, it refers to the dilution of virus at which 50% infection is confirmed. The infection can be confirmed microscopically, for example, using cytopathic effect (CPE) as an indicator.
• CPE cytopathic effect
• the virus titer LRV can be calculated based on TCID50 according to the formula of the Beherens-Karber method.
• a method for measuring the LRV of the virus titer can be as follows. Specifically, DMEM medium can be used as a diluent for diluting the virus-containing composition after first storing it for a predetermined period of time. For cell culture, 100 ⁇ L/well (approximately 10 4 cells/well) of a predetermined cell suspension was spread on a 96-well flat-bottom plate (manufactured by TPP) and incubated overnight in a 37° C. CO 2 incubator (CO 2 concentration 5%). It can be carried out by incubating.
• DMEM medium can be used as a diluent for diluting the virus-containing composition after first storing it for a predetermined period of time.
• 100 ⁇ L/well (approximately 10 4 cells/well) of a predetermined cell suspension was spread on a 96-well flat-bottom plate (manufactured by TPP) and incubated overnight in a 37° C. CO 2 incubator (CO 2 concentration
• the virus-containing composition is then serially diluted 10-fold in DMEM medium. 50 ⁇ L/well of the diluted virus was added to predetermined cells (various cells corresponding to the virus species contained in the virus-containing composition (eg, LCC-MK2 cells, MDBK cells, HEK293 cells, etc.)) in a 96-well flat-bottom plate. Then, cultured in a CO 2 incubator (CO 2 concentration 5%) at 37 ° C. On the 7th day, the virus titer (TCID50) was determined by using CPE as an index and according to the above-mentioned calculation formula, and the TCID50 value was LRV can be calculated based on
• the LRV of the virus titer can be calculated as follows. That is, the diluted virus was added to Vero cells in a 24-well flat-bottom plate at 50 ⁇ L/well, cultured in a CO 2 incubator (CO 2 concentration 5%) at 37° C. for 24 to 48 hours, and the cells were added to 0.25 mass. Recover with % trypsin EDTA (manufactured by SIGMA-Aldrich). Then wash with DMEM medium and PBS.
• the above cells are suspended in 0.5 mass % formalin-added PBS, the number of infected cells is counted by FCM analysis or fluorescence microscopy, and the virus titer (FFU/mL) is determined according to the above-mentioned formula. LRV can then be calculated based on the virus titer (FFU/mL).
• the virus stabilizer according to this embodiment can be preferably obtained by the following method. That is, by going through the step of preparing a gelatin hydrolyzate (first step) and the step of preparing a virus stabilizer comprising the gelatin hydrolyzate and an aqueous medium (second step), the virus stabilizing agent can be obtained.
• the first step is to prepare a gelatin hydrolyzate.
• the gelatin hydrolyzate is, as described above, gelatin whose isoelectric point has a pH of 4.0 or more and 7.0 or less, or gelatin whose isoelectric point has a pH of 4.0 or more and 7.0 or less by alkali treatment. It can be prepared by hydrolyzing both or either one of the collagens obtained so as to have a weight average molecular weight of 10,000 or less.
• As gelatin having an isoelectric point pH of 4.0 or more and 7.0 or less alkali-treated gelatin is preferably used.
• a gelatin hydrolyzate can also be prepared from a commercially available product (for example, trade name: "beMatrix (registered trademark) gelatin HG" manufactured by Nitta Gelatin Co., Ltd.).
• the second step is to prepare a virus stabilizer from the above gelatin hydrolyzate and aqueous medium.
• the virus stabilizer can be prepared by mixing the gelatin hydrolyzate and the aqueous medium at a mass ratio such that the concentration of the gelatin hydrolyzate is 1% by mass or more and 20% by mass or less.
• a conventionally known method can be used as a specific mixing method.
• the aqueous medium can also be prepared by a conventionally known method such as adding a salt having a buffering action to deionized water so as to have a predetermined concentration.
• a virus-containing composition can be prepared by adding a virus to the virus stabilizer.
• a virus can be added to the virus stabilizer by a conventionally known method.
• the virus can be added to the virus stabilizer generally so that the final virus titer (TCID50) is 10 5 /mL or more.
• TCID50 final virus titer
• Sample preparation Samples (Samples 1 to 17) of virus-containing compositions used in various experiments (Experiments 1 to 6) described below were prepared as described below. Samples 1 to 4, 9 to 12 and 14 to 17 are examples, and samples 5 to 8 and 13 are comparative examples.
• Example 1 Alkaline-treated gelatin “beMatrix (registered trademark) gelatin LS-H (manufactured by Nitta Gelatin Co., Ltd.)” derived from swine skin with an isoelectric point pH of 5 is hydrolyzed by heat so that the weight average molecular weight becomes 3800. to obtain a gelatin hydrolyzate. Then, the virus stabilizer was obtained by dissolving in GTS buffer, which is an aqueous medium, so as to contain 5% by mass of the gelatin hydrolyzate.
• GTS buffer which is an aqueous medium
• a virus-containing composition was obtained by adding bovine herpes virus (BHV-1), which is an enveloped DNA virus, to the virus stabilizer so that the TCID50 was 2 ⁇ 10 8.1 /mL.
• the virus-containing composition was further divided into 7 specimens and stored at 4°C for 21 days, 56 days and 210 days, 25°C for 21 days, and -80°C for 21 days, 56 days and 210 days, respectively.
• Example 2 Alkali-treated gelatin “beMatrix (registered trademark) gelatin LS-H (manufactured by Nitta Gelatin Co., Ltd.)” derived from pigskin with an isoelectric point pH of 5 is hydrolyzed by heat so that the weight average molecular weight becomes 650. to obtain a gelatin hydrolyzate. Then, the virus stabilizer was obtained by dissolving the gelatin hydrolyzate in the GTS buffer so as to contain 5% by mass. Then, a virus-containing composition was obtained by adding a virus to the virus stabilizer by the same method as for sample 1. The virus-containing composition was divided into four specimens and stored at 4°C for 56 days, 25°C for 21 days, and -80°C for 21 days and 56 days, respectively.
• Example 3 Alkali-treated gelatin “beMatrix (registered trademark) gelatin LS-H (manufactured by Nitta Gelatin Co., Ltd.)” derived from swine skin with an isoelectric point pH of 5 is hydrolyzed by heat so that the weight average molecular weight becomes 1980. to obtain a gelatin hydrolyzate. Then, the virus stabilizer was obtained by dissolving the gelatin hydrolyzate in the GTS buffer so as to contain 5% by mass. Then, a virus-containing composition was obtained by adding a virus to the virus stabilizer by the same method as for sample 1. The virus-containing composition was divided into four specimens and stored at 4°C for 56 days, 25°C for 21 days, and -80°C for 21 days and 56 days, respectively.
• Example 4 Alkali-treated gelatin “beMatrix (registered trademark) gelatin LS-H (manufactured by Nitta Gelatin Co., Ltd.)” derived from swine skin with an isoelectric point pH of 5 is hydrolyzed by heat so that the weight average molecular weight becomes 9890. to obtain a gelatin hydrolyzate. Then, the virus stabilizer was obtained by dissolving the gelatin hydrolyzate in the GTS buffer so as to contain 5% by mass. Then, a virus-containing composition was obtained by adding a virus to the virus stabilizer by the same method as for sample 1. The virus-containing composition was divided into four specimens and stored at 4°C for 56 days, 25°C for 21 days, and -80°C for 21 days and 56 days, respectively.
• Example 5 Alkali-treated gelatin “beMatrix (registered trademark) gelatin LS-H (manufactured by Nitta Gelatin Co., Ltd.)” derived from swine skin with an isoelectric point pH of 5 is hydrolyzed by heat so that the weight average molecular weight becomes 59,800. to obtain a gelatin hydrolyzate. Then, the virus stabilizer was obtained by dissolving the gelatin hydrolyzate in the GTS buffer so as to contain 5% by mass. Then, a virus-containing composition was obtained by adding a virus to the virus stabilizer by the same method as for sample 1. The virus-containing composition was further divided into four specimens and stored at 4°C for 56 days, 25°C for 21 days, and -80°C for 21 days and 56 days, respectively.
• Example 7 Alkaline-treated gelatin “beMatrix (registered trademark) gelatin LS-H (manufactured by Nitta Gelatin Co., Ltd.)” derived from swine skin with an isoelectric point pH of 5 is hydrolyzed by heat so that the weight average molecular weight becomes 3800. to obtain a gelatin hydrolyzate.
• a virus-containing composition was obtained in the same manner as for Sample 1, except that the gelatin hydrolyzate was dissolved in the GTS buffer so as to contain 0.1 mass % of the gelatin hydrolyzate to obtain the virus stabilizer.
• the virus-containing composition was divided into four specimens and stored at 4°C for 56 days, 25°C for 21 days, and -80°C for 21 days and 56 days, respectively.
• Example 8 Alkaline-treated gelatin “beMatrix (registered trademark) gelatin LS-H (manufactured by Nitta Gelatin Co., Ltd.)” derived from swine skin with an isoelectric point pH of 5 is hydrolyzed by heat so that the weight average molecular weight becomes 3800. to obtain a gelatin hydrolyzate.
• a virus-containing composition was obtained in the same manner as for Sample 1, except that the gelatin hydrolyzate was dissolved in the GTS buffer to contain 0.5% by mass of the gelatin hydrolyzate to obtain the virus stabilizer.
• the virus-containing composition was divided into four specimens and stored at 4°C for 56 days, 25°C for 21 days, and -80°C for 21 days and 56 days, respectively.
• Example 9 Alkaline-treated gelatin “beMatrix (registered trademark) gelatin LS-H (manufactured by Nitta Gelatin Co., Ltd.)” derived from swine skin with an isoelectric point pH of 5 is hydrolyzed by heat so that the weight average molecular weight becomes 3800. to obtain a gelatin hydrolyzate.
• a virus-containing composition was obtained in the same manner as in Sample 1, except that the gelatin hydrolyzate was dissolved in the GTS buffer so as to contain 1% by mass of the gelatin hydrolyzate to obtain the virus stabilizer.
• the virus-containing composition was divided into four specimens and stored at 4°C for 56 days, 25°C for 21 days, and -80°C for 21 days and 56 days, respectively.
• Example 10 Alkaline-treated gelatin “beMatrix (registered trademark) gelatin LS-H (manufactured by Nitta Gelatin Co., Ltd.)” derived from swine skin with an isoelectric point pH of 5 is hydrolyzed by heat so that the weight average molecular weight becomes 3800. to obtain a gelatin hydrolyzate.
• a virus-containing composition was obtained in the same manner as in Sample 1, except that the gelatin hydrolyzate was dissolved in the GTS buffer to contain 2.5% by mass of the gelatin hydrolyzate to obtain the virus stabilizer.
• the virus-containing composition was divided into four specimens and stored at 4°C for 56 days, 25°C for 21 days, and -80°C for 21 days and 56 days, respectively.
• Example 11 Alkaline-treated gelatin “beMatrix (registered trademark) gelatin LS-H (manufactured by Nitta Gelatin Co., Ltd.)” derived from swine skin with an isoelectric point pH of 5 is hydrolyzed by heat so that the weight average molecular weight becomes 3800. to obtain a gelatin hydrolyzate.
• a virus-containing composition was obtained in the same manner as for Sample 1, except that the gelatin hydrolyzate was dissolved in the GTS buffer so as to contain 10% by mass of the gelatin hydrolyzate to obtain the virus stabilizer.
• the virus-containing composition was divided into four specimens and stored at 4°C for 56 days, 25°C for 21 days, and -80°C for 21 days and 56 days, respectively.
• Example 12 Alkaline-treated gelatin “beMatrix (registered trademark) gelatin LS-H (manufactured by Nitta Gelatin Co., Ltd.)” derived from swine skin with an isoelectric point pH of 5 is hydrolyzed by heat so that the weight average molecular weight becomes 3800. to obtain a gelatin hydrolyzate.
• a virus-containing composition was obtained in the same manner as in Sample 1, except that the gelatin hydrolyzate was dissolved in the GTS buffer so as to contain 20% by mass of the gelatin hydrolyzate to obtain the virus stabilizer.
• the virus-containing composition was divided into four specimens and stored at 4°C for 56 days, 25°C for 21 days, and -80°C for 21 days and 56 days, respectively.
• a virus stabilizer was obtained by dissolving recombinant human albumin "Recombumin (registered trademark) Elite" (manufactured by Albumedix) in the GTS buffer so as to contain 1% by mass.
• a virus-containing composition was obtained by adding the virus to the reagent, and the virus-containing composition was divided into four specimens and incubated at 4°C for 56 days, 25°C for 21 days, and -80°C for 21 days. days and 56 days Sample 13 is a known virus stabilizer.
• Example 14 A virus-containing composition was prepared in the same manner as in Sample 1, except that parainfluenza virus (PI-3), which is an enveloped RNA virus, was added to the virus stabilizer so that the TCID50 was 2 ⁇ 10 5.5 /mL. Obtained.
• the virus-containing composition was divided into 7 specimens and stored at 4° C. for 21 days, 56 days and 210 days, 25° C. for 21 days, and ⁇ 80° C. for 21 days, 56 days and 210 days, respectively.
• Example 15 A virus-containing composition was prepared in the same manner as in Sample 1, except that reovirus (Reo-3), an RNA virus having no envelope, was added to the virus stabilizer so that the TCID50 was 2 ⁇ 10 6.5 /mL. got The virus-containing composition was divided into 7 specimens and stored at 4° C. for 21 days, 56 days and 210 days, 25° C. for 21 days, and ⁇ 80° C. for 21 days, 56 days and 210 days, respectively.
• Reo-3 reovirus
• Example 16 A virus-containing composition was obtained in the same manner as in Sample 1, except that adenovirus (Ad5), which is a DNA virus without an envelope, was added to the virus stabilizer so that the TCID50 was 2 ⁇ 10 8 /mL. rice field.
• the virus-containing composition was divided into 7 specimens and stored at 4° C. for 21 days, 56 days and 210 days, 25° C. for 21 days, and ⁇ 80° C. for 21 days, 56 days and 210 days, respectively.
• Example 17 A paramyxovirus (Newcastle disease virus: rNDV-GFP), which is an oncolytic virus with an enveloped RNA virus into which GFP is introduced, is added to the virus stabilizer so that the FFU contains 4 ⁇ 10 6 /mL.
• a virus-containing composition was obtained in the same manner as for sample 1, except that it was added. The virus-containing composition was divided into four specimens and stored at 4°C for 56 days, 25°C for 21 days, and -80°C for 21 days and 56 days, respectively. Table 1 shows a list of samples 1 to 17.
• the pH of the isoelectric point of the gelatin hydrolyzate contained in the virus stabilizers of Samples 1 to 4, Samples 9 to 12, and Samples 14 to 17 is the isoelectric point with the above-mentioned zeta potential as an index.
• sample 1 sample 2, sample 3, sample 4 and sample 5
• the LRV of the virus titer in each sample stored at 25 ° C. for 21 days and the virus titer in each sample stored at -80 ° C. for 21 days was calculated as the difference between the LRV of Table 2 shows the results.
• Table 3 regarding samples 1, 2, 3 and 4, the LRV of the virus titer in each sample stored at 4°C for 56 days and the LRV of the virus titer in each sample stored at -80°C for 56 days and shows the result of calculating the difference.
• Sample 1, Sample 2, Sample 3 and Sample 4 have a difference in the LRV of 1.0 log or less and 1.5 log or less, respectively, so 4°C (refrigeration temperature) and 25°C. It can be evaluated that the virus can be stably stored and transported at room temperature. Sample 5 gelled so that the above LRV difference exceeded 1.5 log at 25°C. Sample 5 was not evaluated at 4°C for 56 days because of gelation.
• Sample 1, Sample 2, Sample 3 and Sample 4 have a difference in the LRV of 1.0 log or less and 1.5 log or less, respectively, so 4°C (refrigeration temperature) and 25°C. It can be evaluated that the virus can be stably stored and transported at room temperature. For sample 6, the LRV difference exceeded 1.5 log at 25°C and exceeded 1.0 log at 4°C.
• Sample 1 Sample 9
• Sample 10 Sample 11 and Sample 12 had differences in the LRV of 1.0 log or less and 1.5 log or less, respectively. ) and 25° C. (room temperature), the virus can be stably stored and transported.
• Both Sample 7 and Sample 8 exceeded 1.5 log difference in LRV at 25°C and exceeded 1.0 log at 4°C.
• Table 8 shows the results. Furthermore, in Table 9, for samples 1 and 13, the difference between the LRV of the virus titer in each sample stored at 4°C for 56 days and the LRV of the virus titer in each sample stored at -80°C for 56 days was calculated. Show the results.
• Sample 1 can suppress the decrease in virus titer at 4°C (refrigeration temperature) and 25°C (room temperature) compared to Sample 13.
• Sample 14 Sample 15 and Sample 16
• the TCID50 of each specimen stored at 4°C for 21 days, 56 days and 210 days was determined, and based on the TCID50 value, the virus titer at 4°C LRV was calculated.
• Sample 14 Sample 15, and Sample 16
• the TCID50 of each specimen stored at -80°C for 21 days, 56 days, and 210 days was also determined, and the virus titer at -80°C was determined based on the TCID50 values. was calculated.
• Sample 1, Sample 14, Sample 15, Sample 16 and Sample 17 have a difference in the LRV of 1.0 log or less and 1.5 log or less, respectively, so the refrigeration temperature ) and 25° C. (room temperature), the virus can be stably stored and transported.
• each example of Samples 1 to 4, Samples 9 to 12, and Samples 14 to 17 stably contained viruses for 21 days in an environment of 4°C. It can be evaluated as being storable and transportable.

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