Virus stabilizer, gelatin hydrolyzate for virus stabilizer, and virus-containing composition

Abstract

The virus stabilizer of the present invention is composed of gelatin hydrolyzate and an aqueous medium, wherein the virus stabilizer contains more than 1% by mass and less than 20% by mass of the gelatin hydrolyzate, the weight average molecular weight of the gelatin hydrolyzate is less than 10,000, and the isoelectric point pH of the gelatin hydrolyzate is more than 4.0 and less than 7.0.

Description

Technical Field

[0001] The invention relates to a virus stabilizer, a gelatin hydrolyzate for the virus stabilizer and a virus-containing composition. Background Art

[0002] As for the storage and transportation of viruses, in order to maintain the ability of the virus such as infectivity, or to prevent the reduction of these abilities, an environment of -80 to -60°C is usually required. Alternatively, when the virus is stored and transported at room temperature, the preparation containing the virus needs to be made into a freeze-dried product. As such, the storage and transportation of known viruses have always been complicated. In contrast, Japanese Patent Publication No. 2013-035861 (Patent Document 1) discloses a refrigerator-stable influenza virus composition that is stable at refrigerated temperatures (e.g., 2 to 8°C).

[0003] Prior art literature

[0004] Patent Literature

[0005] Patent Document 1: Japanese Patent Application Publication No. 2013-035861 Summary of the invention

[0006] Problems to be solved by the invention

[0007] The above-mentioned patent document 1 discloses the fact that the stable storage and transportation of influenza virus strains at refrigerated temperature are achieved by a composition containing 0.5 to 2% gelatin hydrolysate, etc. However, in the above-mentioned patent document 1, whether the stable storage and transportation of the above-mentioned virus strains can be achieved at room temperature of about 25°C is not disclosed. According to the above-mentioned patent document 1, it is presumed that since the above-mentioned gelatin hydrolysate is obtained from porcine type A gelatin with an isoelectric point of about 9, the gelatin hydrolysate strongly interacts with specific proteins on the surface of the virus, making stable storage and transportation at room temperature difficult. In addition, it is known that in order to provide a preparation containing a virus stored in an environment of -80 to -60°C for use, it is solubilized by heating, and when it is returned to the refrigerated temperature (2 to 8°C) or room temperature, the reduction of viral ability such as infectivity cannot be avoided. Therefore, a material that can store and transport a preparation containing a virus without reducing viral ability such as infectivity at both the refrigerated temperature (2 to 8°C) and the room temperature of about 25°C without complicated operations has not yet been realized, and its development is expected.

[0008] In view of the above situation, the object of the present invention is to provide a virus stabilizer, a gelatin hydrolyzate for a virus stabilizer, and a virus-containing composition, wherein the virus stabilizer inhibits the reduction of the ability of the virus at both refrigerated temperature (2 to 8°C) and room temperature of about 25°C, thereby being able to store and transport preparations containing the virus without the need for a refrigeration device, etc.

[0009] Solutions for solving problems

[0010] The inventors of the present invention have conducted intensive research and have achieved the present invention. That is, they first focused on a gelatin hydrolyzate obtained by hydrolyzing gelatin having an isoelectric point of pH 5 in a manner to achieve a predetermined weight average molecular weight. Furthermore, they discovered the following fact, which led to the completion of the present invention: when a virus is contained in a preparation containing the gelatin hydrolyzate at a predetermined concentration, the reduction in viral ability such as infectivity can be suppressed at both a refrigerated temperature (2 to 8°C) and a room temperature of about 25°C, thereby enabling the preparation containing the virus to be stably stored and transported without the need for a refrigeration device or the like.

[0011] The present invention has the following features.

[0012] [1] The virus stabilizer of the present invention is composed of gelatin hydrolyzate and an aqueous medium, wherein the virus stabilizer contains more than 1% by mass and less than 20% by mass of the gelatin hydrolyzate, the weight average molecular weight of the gelatin hydrolyzate is less than 10,000, and the isoelectric point of the gelatin hydrolyzate has a pH of more than 4.0 and less than 7.0.

[0013] [2] Preferably, the weight average molecular weight of the gelatin hydrolyzate is 6,000 or less.

[0014] [3] Preferably, the gelatin hydrolyzate is a hydrolyzate of alkali-treated gelatin.

[0015] [4] Preferably, the virus stabilizer contains more than 2% by mass and less than 10% by mass of the gelatin hydrolyzate.

[0016] [5] Preferably, the aqueous medium contains a salt having a buffering effect.

[0017] [6] Preferably, the aqueous medium contains at least one sugar selected from the group consisting of sucrose, lactose, sorbitol, inositol, trehalose, mannitol, maltitol, xylitol, erythritol and glycerol.

[0018] [7] Preferably, the aqueous medium contains at least one amino acid selected from the group consisting of methionine, arginine, tryptophan, glutamine and glutamic acid.

[0019] [8] The gelatin hydrolyzate for virus stabilizer of the present invention has a weight average molecular weight of 10,000 or less and an isoelectric point of pH 4.0 or more and 7.0 or less.

[0020] [9] Preferably, the gelatin hydrolyzate used as the virus stabilizer is in the form of liquid or powder.

[0021]

[10] The virus-containing composition of the present invention comprises the above-mentioned virus stabilizer and a virus.

[0022]

[11] Preferably, the virus comprises at least one selected from the group consisting of a DNA virus having an envelope, a DNA virus not having an envelope, an RNA virus having an envelope, and an RNA virus not having an envelope.

[0023]

[12] Preferably, the above-mentioned virus is introduced with a gene.

[0024]

[13] Preferably, the difference between the logarithmic decrease in virus titer of the virus-containing composition when stored at 25°C for 21 days and the logarithmic decrease in virus titer when stored at -80°C for 21 days is 1.5 log or less.

[0025]

[14] Preferably, the difference between the logarithmic decrease in virus titer of the virus-containing composition when stored at 4°C for 210 days and the logarithmic decrease in virus titer when stored at -80°C for 210 days is 1.0 log or less.

[0026]

[15] Preferably, the logarithmic decrease value of the virus titer when the virus-containing composition is frozen and thawed three times is 0.3 log or less.

[0027] Effects of the Invention

[0028] Based on the above contents, the following virus stabilizers, gelatin hydrolysates for virus stabilizers, and virus-containing compositions can be provided. The virus stabilizers inhibit the reduction of viral ability at both refrigerated temperatures (2 to 8°C) and room temperature of around 25°C, and can store and transport preparations containing viruses without the need for refrigeration equipment, etc. DETAILED DESCRIPTION

[0029] Hereinafter, an embodiment of the present invention (hereinafter also referred to as "the present embodiment") is described in further detail. Here, in the present specification, expressions in the form of "A to B" refer to the upper and lower limits of the range (i.e., greater than A and less than B). When there is no unit recorded in A and only the unit is recorded in B, the unit of A is the same as the unit of B. In the present specification, "virus titer" refers to the minimum concentration of the virus in the specimen (the "virus-containing composition" in the present specification) that can infect cells, that is, the highest dilution ratio. Moreover, "reduction" in virus titer means a decrease in the above-mentioned dilution ratio, which means an increase in the above-mentioned minimum concentration that can infect. In the present specification, the term "gelatin" is sometimes used to describe the substance name, gelatin gel, and gelatin solution, respectively. In addition, the term "gelatin hydrolyzate" is sometimes used to describe the solution of gelatin hydrolyzate, similar to the above-mentioned gelatin.

[0030] 〔Virus stabilizer〕

[0031] The virus stabilizer of the present embodiment is a virus stabilizer composed of a gelatin hydrolyzate and an aqueous medium. The above-mentioned virus stabilizer contains more than 1% by mass and less than 20% by mass of the above-mentioned gelatin hydrolyzate. Moreover, the weight average molecular weight of the above-mentioned gelatin hydrolyzate is less than 10,000. The pH of the isoelectric point of the above-mentioned gelatin hydrolyzate is more than 4.0 and less than 7.0. By having such characteristics, the virus stabilizer of the present embodiment can inhibit the reduction of virus titer at both refrigerated temperature (2 to 8°C) and room temperature of about 25°C, so that when constituting a virus-containing composition comprising a virus stabilizer and a virus, the virus-containing composition can be stably stored and transported without the need for a refrigeration device or the like.

[0032] <Gelatin hydrolysate>

[0033] The above-mentioned virus stabilizer contains gelatin hydrolyzate as described above. In this specification, "gelatin hydrolyzate" refers to a collection of peptides (hydrolyzate) obtained by hydrolyzing both or either of gelatin and collagen. That is, "gelatin hydrolyzate" refers to a substance equivalent to a collection of peptides generally referred to as collagen peptides or collagen hydrolyzate. Among them, the gelatin hydrolyzate contained in the virus stabilizer of the present embodiment has a weight-average molecular weight and isoelectric pH as described above. Moreover, gelatin hydrolyzate refers to a collection of peptides as described above, and therefore has the same characteristics as collagen and gelatin in the amino acid sequence constituting the peptide chain, which refers to a structure in which glycine is repeated once every three residues, etc.

[0034] In this specification, "gelatin" refers to a polypeptide whose triple helical structure of collagen is untied by heat modification, acid modification, etc., a chemically modified body thereof, and a pharmaceutically acceptable salt thereof. Specifically, it can be obtained by subjecting a collagen derived from at least one of the groups selected from the following first to sixth groups to a previously known treatment such as defatting, deashing, acid or alkali treatment, hot water extraction treatment, etc. Gelatin can be a polypeptide obtained by a fermentation method using a microorganism, a recombinant polypeptide obtained by chemical synthesis or genetic recombination operation, or a synthetic polypeptide. In addition, "collagen" refers to a protein derived from the extracellular matrix in the skin of vertebrates, etc., which are classified into the following first to sixth groups. Collagen has a right-handed helical structure composed of three peptide chains, and the amino acid residues constituting the peptide chain have a structure in which glycine residues are repeated once every three residues (the so-called collagen-like sequence).

[0035] Group 1: A group consisting of cow hide, skin, bones, cartilage and tendons.

[0036] The second group: a group consisting of pig skin, bones, cartilage and tendons.

[0037] Group 3: A group consisting of sheep's hide, skin, bones, cartilage and tendons.

[0038] Group 4: A group consisting of chicken skin, bones, cartilage and tendons.

[0039] Group 5: A group consisting of ostrich skin, bones, cartilage and tendons.

[0040] Group 6: The group composed of fish bones, skin and scales.

[0041] Here, the "chemically modified form" of the above-mentioned polypeptide (gelatin) refers to a polypeptide obtained by chemically modifying the amino group, carboxyl group, hydroxyl group or thiol group in the amino acid residues constituting gelatin. The chemically modified gelatin can change the solubility in water, isoelectric point, etc. Specifically, the hydroxyl group of the hydroxyproline residue in gelatin can be subjected to chemical modifications such as O-acetylation. The α-carboxyl group of the glycine residue in gelatin can be subjected to chemical modifications such as esterification and amidation. The α-amino group of the proline residue in gelatin can be subjected to chemical modifications such as polypeptideization, succinylation, maleoylation, acetylation, deamination, benzoylation, alkylsulfonylation, allylsulfonylation, dinitrophenylation, trinitrophenylation, carbamylation, phenylcarbamylation, thiolation, etc.

[0042] The specific method and treatment conditions for chemical modification of gelatin can be applied to conventionally known chemical modification methods. Regarding the chemical modification of the hydroxyl group of hydroxyproline residues, for example, O-acetylation can be carried out by reacting acetic anhydride in an aqueous solvent or a non-aqueous solvent. Regarding the chemical modification of the α-carboxyl group of glycine residues, for example, esterification can be carried out by suspending in methanol and then passing dry hydrogen chloride gas. Regarding the chemical modification of the α-carboxyl group of glycine residues, amidation can be carried out by reacting carbodiimide or the like.

[0043] Furthermore, the "derivatives" of the above-mentioned polypeptide (gelatin) sometimes include gelatin derivatives obtained by introducing functional groups into gelatin, copolymers of gelatin with lactic acid, glycolic acid, etc., copolymers of gelatin with polyethylene glycol, propylene glycol, etc. Examples of gelatin derivatives include derivatives obtained by introducing functional groups such as guanidine groups, thiol groups, amino groups, carboxyl groups, sulfate groups, phosphoric acid groups, alkyl groups, acyl groups, phenyl groups, benzyl groups, etc. into gelatin.

[0044] The "pharmaceutically acceptable salt" of the above-mentioned polypeptide (gelatin) refers to a salt that is pharmaceutically acceptable and has the desired activity (e.g., gelling ability) of the polypeptide (gelatin) as a raw material. As pharmaceutically acceptable salts, for example, inorganic acid salts such as hydrochloride, sulfate, phosphate, and hydrobromide, organic acid salts such as acetate, methanesulfonate, benzenesulfonate, p-toluenesulfonate, succinate, oxalate, fumarate, and maleate, inorganic base salts such as sodium salt, potassium salt, and calcium salt, organic base salts such as triethylammonium salt, etc. can be exemplified. The specific peptide in gelatin can be made into a pharmaceutically acceptable salt according to conventional methods.

[0045] Gelatin is a polypeptide derived from collagen that is present in many organisms and therefore has excellent biocompatibility. Therefore, the gelatin hydrolyzate obtained by hydrolyzing the above collagen and gelatin also has excellent biocompatibility and is suitable as a component of a virus stabilizer for medical use.

[0046] When gelatin hydrolyzate is dissolved in a solvent (e.g., water), it will not gel at room temperature of about 25°C, let alone at 2 to 8°C, and will maintain a sol state. Therefore, the above-mentioned virus stabilizer does not require heating operation for solubilization when used at room temperature of about 25°C. In the present embodiment, "sol" refers to a dispersion system in which the dispersion medium is liquid in a dispersion system composed of a dispersoid and a dispersion medium. "Gel" refers to a state in which the dispersoid forms a cross-linked structure in a dispersion system composed of a dispersoid and a dispersion medium, and the dispersion system as a whole loses fluidity.

[0047] As described above, gelatin hydrolyzate is obtained by hydrolyzing both or either of gelatin and collagen. In the "hydrolysis" of this case, hydrolysis using acid, hydrolysis using alkali, hydrolysis using enzymes, and hydrolysis using heat are all included. From the viewpoint of preventing contamination by impurities, gelatin hydrolyzate is preferably obtained by hydrolysis using heat. Moreover, in the case of using enzymes to hydrolyze both or either of gelatin and collagen, as the above-mentioned enzymes, for example, collagenase, thiol protease, serine protease, acid protease, alkaline protease, metalloprotease, etc. can be listed. With regard to the above-mentioned enzymes, a variety of these enzymes can be used alone or in combination. As the above-mentioned thiol protease, papain, bromelain, ficin derived from plants, tissue proteases, calcium-dependent proteases, etc. can be listed. As the above-mentioned serine protease, trypsin, cathepsin D, etc. can be listed. As the above-mentioned acidic protease, pepsin, chymotrypsin, etc. can be listed.

[0048] As the enzyme used, if gelatin hydrolyzate is considered to be used in medicine, it is preferred to use enzymes other than enzymes derived from pathogenic microorganisms (e.g., enzymes derived from non-pathogenic microorganisms). As non-pathogenic microorganisms from which the above-mentioned enzymes are derived, Bacillus licheniforms, Bacillus subtillis, Aspergillus oryzae, Streptomyces, Bacillus amyloliquefaciens, etc. can be listed. The above-mentioned enzyme can use an enzyme derived from the above-mentioned non-pathogenic microorganisms, or a combination of multiple enzymes derived from the above-mentioned non-pathogenic microorganisms. The specific method of enzyme treatment can use a previously known method.

[0049] It should be noted that, when a "gelatin hydrolyzate" is obtained from the above-mentioned gelatin by hydrolysis using only one enzyme, the enzyme is preferably not collagenase. This is because when the above-mentioned gelatin is hydrolyzed using collagenase alone, a specific peptide whose N-terminus of the amino acid sequence is glycine may become the main component (more than 50% by mass). The above-mentioned gelatin hydrolyzate is an aggregate composed of two or more peptides, but in its composition, the above-mentioned specific peptide is less than 50% by mass.

[0050] The gelatin hydrolyzate can be obtained in the form of a liquid by hydrolyzing either or both of gelatin and collagen by the above method and then purifying the hydrolyzate. Alternatively, the gelatin hydrolyzate can be obtained in the form of a powder by heating and drying or freeze-drying the liquid by a known method.

[0051] (concentration)

[0052] The virus stabilizer contains the above-mentioned gelatin hydrolyzate in an amount of more than 1% by mass and less than 20% by mass. When the concentration of the above-mentioned gelatin hydrolyzate contained in the virus stabilizer is less than 1% by mass, when a virus-containing composition comprising the virus stabilizer and a virus is formed, the effect of inhibiting the reduction in virus titer is extremely small, and therefore it is difficult to exert the desired effect. When the concentration of the above-mentioned gelatin hydrolyzate contained in the virus stabilizer exceeds 20% by mass, the operability of the virus stabilizer may deteriorate. From the viewpoint of further inhibiting the reduction in virus titer, the virus stabilizer preferably contains the above-mentioned gelatin hydrolyzate in an amount of more than 2% by mass and less than 10% by mass. The concentration of the above-mentioned gelatin hydrolyzate in the virus stabilizer can be measured by known methods such as hydroxyproline quantification.

[0053] (Weight average molecular weight)

[0054] The weight average molecular weight of the gelatin hydrolyzate is 10,000 or less. The weight average molecular weight of the gelatin hydrolyzate is preferably 6,000 or less. The weight average molecular weight of the gelatin hydrolyzate is further preferably 5,000 or less. Thus, the virus stabilizer can maintain a sol state in an environment of about 2 to 30°C. Moreover, in the case of a virus-containing composition comprising a virus stabilizer and a virus, in the above composition, the gelatin hydrolyzate can inhibit viral aggregation, thereby inhibiting the reduction in viral titer. This is because, although the detailed mechanism is unclear, it is presumed that: when the weight average molecular weight of the gelatin hydrolyzate is within the above range, both the hydrophilic region and the hydrophobic region in the molecule are exposed on the surface side (outside) of the molecule, and can interact with the surface of the virus with appropriate strength regardless of the presence or absence of a capsule, so that the gelatin hydrolyzate can be protected by coating the surface of the virus.

[0055] When the weight average molecular weight of the gelatin hydrolyzate exceeds 10,000, gelation may occur at 2 to 8° C. The lower limit of the weight average molecular weight of the gelatin hydrolyzate is not particularly limited, and is, for example, 75, which is the molecular weight of glycine.

[0056] The weight average molecular weight of the gelatin hydrolyzate can be determined by subjecting the gel filtration chromatography to the following measurement conditions.

[0057] Equipment: High Performance Liquid Chromatography (HPLC) (manufactured by Tosoh Corporation).

[0058] Chromatographic column: TSKGel (registered trademark) G2000SW XL .

[0059] Column temperature: 30°C.

[0060] Eluent: 40 mass% acetonitrile (containing 0.05 mass% TFA).

[0061] Flow rate: 0.5mL / min.

[0062] Injection volume: 10 μL.

[0063] Detection: UV220nm.

[0064] Molecular weight markers: The following three were used.

[0065] Cytochrome C (Cytochrom C) Mw: 12384.

[0066] Aprotinin Mw:6512.

[0067] Bacitracin Mw:1423.

[0068] Specifically, a virus stabilizer equivalent to 0.5 g of the above-mentioned gelatin hydrolyzate is added to about 100 ml of distilled water, stirred, and filtered using a 0.2 μm filter to prepare a sample (substance to be measured) for measuring the weight average molecular weight. The weight average molecular weight of the above-mentioned gelatin hydrolyzate can be obtained by measuring the substance to be measured based on the conditions of the above-mentioned gel filtration chromatography.

[0069] (Isoelectric point)

[0070] The isoelectric point of the gelatin hydrolyzate is preferably 4.0 to 5.5, and more preferably 4.0 to 4.8. The gelatin hydrolyzate having an isoelectric point of pH in such a range can be efficiently obtained by hydrolyzing collagen subjected to alkali treatment or hydrolyzing gelatin obtained from collagen subjected to alkali treatment (so-called alkali-treated gelatin). That is, the gelatin hydrolyzate is preferably a hydrolyzate of alkali-treated gelatin. It is particularly preferred that the gelatin hydrolyzate is a hydrolyzate of alkali-treated gelatin having an isoelectric point of about pH 4.8 to 5.5. Generally, gelatin obtained by treating collagen with an inorganic acid is called acid-treated gelatin, and gelatin obtained by treating collagen with an inorganic base is called alkali-treated gelatin. Specifically, alkali-treated gelatin can be obtained by treating collagen with an inorganic base such as sodium hydroxide, calcium hydroxide or potassium hydroxide. The isoelectric point of acid-treated gelatin is pH 8 to 9. In contrast, the isoelectric point of alkali-treated gelatin is pH 4.0 to 7.0. As such alkali-treated gelatin, for example, alkali-treated gelatin derived from pig skin (trade name: "beMatrix (registered trademark) gelatin LS-H", manufactured by Nitta Gelatin Co., Ltd.) can be exemplified.

[0071] In the case where a virus stabilizer and virus of a gelatin hydrolyzate with an isoelectric point of pH in the above range constitute a composition containing a virus, in the above composition, the gelatin hydrolyzate can inhibit viral aggregation, thereby inhibiting the reduction of viral titer. Although the detailed mechanism is unknown, for gelatin hydrolyzates with an isoelectric point of pH in such a range, there are many negative charges in the molecule relative to positive charges, so as a whole, negative charges are displayed, but there are both sites showing positive charges and sites showing negative charges. Therefore, gelatin hydrolyzates can bind to viruses by showing weak electrostatic interactions between sites showing positive charges and capsid proteins (capsid protein), spike proteins (spike protein) and envelope proteins (envelope protein) on the surface of viruses that show negative charges. On the other hand, gelatin hydrolyzates repel each other at sites showing negative charges with other peptide chains constituting viruses and gelatin hydrolyzates. According to the above, it is inferred that: for gelatin hydrolyzates, the gelatin hydrolyzate can be protected by inhibiting viral aggregation and coating the surface of the virus. On the other hand, gelatin hydrolysates obtained from acid-treated gelatin with an isoelectric point of pH around 9 (e.g., gelatin hydrolysates disclosed in Patent Document 1) have positive charges and thus strongly exhibit electrostatic interactions with negatively charged capsid proteins, etc., which may cause virus aggregation. It is presumed that gelatin hydrolysates obtained from acid-treated gelatin have a significant effect of causing virus aggregation, particularly under a temperature environment of around 25°C.

[0072] The pH of the isoelectric point of the gelatin hydrolysate can be determined by measuring the pH of the isoelectric point of both or either of the gelatin and collagen as the raw materials of the gelatin hydrolysate using a conventionally known method, but the isoelectric point determination method using the following Zeta potential as an indicator can more accurately determine the value of the isoelectric point, so it is preferred. That is, first, the gelatin hydrolysate to be measured is dissolved in an acetic acid buffer solution (pH 4.0-5.5) to obtain a 0.4w / v% solution to be measured. Then, the solution to be measured is filtered using a 0.22μm filter (Merck), and then 0.8mL of the solution to be measured is filled into a capillary sample cell in a manner that does not allow bubbles to enter. Next, the capillary sample cell filled with the above-mentioned solution to be measured is placed in a Zeta potential measuring device (Malvern Panalytical) to measure the Zeta potential at each pH at 25°C. At this time, the pH value at which the zeta potential is 0 can be determined as the isoelectric point of the solution to be measured (gelatin hydrolyzate to be measured).

[0073] <Aqueous medium>

[0074] The virus stabilizer contains an aqueous medium as described above. In this specification, "aqueous medium" refers to a medium that dissolves or disperses gelatin hydrolyzate, and refers to a medium that can contain components other than water such as amino acids, sugars, and salts with buffering effects described later. For example, the aqueous medium is sometimes a buffer containing salts with buffering effects. Specifically, the aqueous medium is sometimes a GTS buffer. That is, the above-mentioned aqueous medium preferably contains salts with buffering effects. Therefore, when the virus stabilizer constitutes a virus-containing composition by containing viruses, it can contribute to the stabilization of the virus through the above-mentioned buffering effect.

[0075] (Salt with buffering effect)

[0076] As salts with a buffering effect, there can be exemplified: sodium phosphate, potassium phosphate, calcium phosphate, magnesium phosphate, sodium hydrogen phosphate, potassium hydrogen phosphate, calcium hydrogen phosphate, magnesium hydrogen phosphate, sodium chloride, potassium chloride, etc. The aqueous medium can contain one of the above salts with a buffering effect alone, or two or more of them can be combined. As aqueous media containing the above salts with a buffering effect, there can be exemplified the above GTS buffer, PBS buffer, Tris buffer, HEPES buffer, citrate buffer, etc. As the composition of the GTS buffer, for example, 2.5 mass% glycerol, 20mM Tris pH8, and 25mM NaCl can be used.

[0077] (Amino Acid)

[0078] 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 amino acid selected from the group consisting of these amino acids alone, or may contain two or more in combination. The aqueous medium more preferably contains both or any one of methionine and arginine. Thus, the virus stabilizer can contribute to the stabilization of the virus when the virus-containing composition is formed by containing the virus.

[0079] (carbohydrate)

[0080] The above-mentioned aqueous medium preferably includes at least one saccharide selected from the group consisting of sucrose, lactose, sorbitol, inositol, trehalose, mannitol, maltitol, xylitol, erythritol and glycerol. The aqueous medium may contain a saccharide selected from these groups alone, or may contain two or more in combination. The aqueous medium more preferably contains at least any of sucrose, lactose or sorbitol. Thus, the virus stabilizer can contribute to the stabilization of the virus when a virus-containing composition is formed by containing the virus. In this specification, the compound contained in "sugar" includes not only organic compounds that are usually classified as sugars, but also organic compounds that are classified as sugar alcohols. The organic compounds classified as sugar alcohols in the sugars of the above-mentioned group are the above-mentioned sorbitol, mannitol, maltitol, xylitol, erythritol and glycerol.

[0081] (Other ingredients)

[0082] As long as the effects of the present invention can be exerted, the aqueous medium may also contain other components. Examples of other components include growth factors, differentiation factors, hormones, chemokines, cytokines, cell adhesion molecules, chemotactic factors, enzymes, enzyme inhibitors, coenzymes (vitamins), minerals, fats, lipids, stabilizers, and preservatives.

[0083] <Effects>

[0084] The virus stabilizer of the present embodiment, as described above, is composed of an aqueous medium and a gelatin hydrolyzate contained at a concentration of 1% by mass or more and 20% by mass or less. The weight average molecular weight of the gelatin hydrolyzate is less than 10,000, and the isoelectric point pH is greater than 4.0 and less than 7.0. In the case where the virus stabilizer of the present embodiment constitutes a virus-containing composition described later by containing a virus, the aggregation of the virus in the virus-containing composition can be suppressed, and adverse effects on the infectivity of the virus, etc. can be prevented. Thereby, the reduction in virus titer can be suppressed, and the virus-containing composition can be stably stored and transported at both refrigerated temperature (2 to 8°C) and room temperature of about 25°C.

[0085] [Gelatin hydrolysate for virus stabilizer]

[0086] The weight average molecular weight of the gelatin hydrolyzate for virus stabilizer of the present embodiment is 10,000 or less, and the pH of the isoelectric point is 4.0 or more and 7.0 or less. The weight average molecular weight of the gelatin hydrolyzate for virus stabilizer is 6,000 or less, or the pH of the isoelectric point is preferably 4.0 to 5.5. Specifically, regarding the gelatin hydrolyzate for virus stabilizer, as a gelatin hydrolyzate, it has the characteristics described in the section <Gelatin Hydrolyzate>, so it will not be repeatedly described. As a previously unknown property of gelatin hydrolyzate, that is, an unknown property, when the virus-containing composition described later is formed by including a virus, the gelatin hydrolyzate for virus stabilizer has the effect of inhibiting the aggregation of viruses in the virus-containing composition. Thus, the gelatin hydrolyzate for virus stabilizer can be used as a virus stabilizer, and the above-mentioned virus-containing composition can be stably stored and transported at both refrigerated temperatures (2 to 8°C) and room temperature of about 25°C without the need for a refrigeration device or the like.

[0087] The gelatin hydrolyzate for virus stabilizer is preferably a liquid or a powder. Thus, when the gelatin hydrolyzate for virus stabilizer is a liquid, a virus-containing composition can be easily prepared by adding the virus together with the aqueous medium. When the gelatin hydrolyzate for virus stabilizer is a powder, the virus stabilizer is prepared by dissolving or dispersing the virus in an aqueous medium, and the virus-containing composition can be easily prepared by adding the virus thereto. The gelatin hydrolyzate for virus stabilizer can be prepared into a liquid by hydrolyzing both or either of gelatin and collagen and purifying them. The gelatin hydrolyzate for virus stabilizer can be prepared into a powder by heating and drying or freeze-drying the liquid of the gelatin hydrolyzate for virus stabilizer prepared as described above using a known method.

[0088] [Composition containing virus]

[0089] The virus-containing composition of the present embodiment includes the above-mentioned virus stabilizer and a virus. By including a virus stabilizer, the virus-containing composition can inhibit the aggregation of the virus in the above-mentioned composition, thereby inhibiting the reduction of the virus titer. Thus, the virus-containing composition can be stably stored and transported at both refrigerated temperatures (2 to 8°C) and room temperature of about 25°C without the need for a refrigeration device or the like. Here, the virus stabilizer contained in the virus-containing composition specifically has the characteristics described in the section [Virus Stabilizer], so the repeated description will not be repeated.

[0090] <Virus>

[0091] The virus-containing composition contains viruses as described above. The above-mentioned viruses preferably include at least one selected from the group consisting of DNA viruses with an envelope, DNA viruses without an envelope, RNA viruses with an envelope, and RNA viruses without an envelope. That is, the viruses constituting the virus-containing composition are used according to their types and are not limited. Furthermore, the virus-containing composition may contain a single virus selected from the above-mentioned group, or may contain two or more viruses in combination. Even in such a case, the virus-containing composition can stably store and transport the virus at both refrigerated temperatures (2 to 8°C) and room temperature of about 25°C by containing a virus stabilizer.

[0092] Specifically, the above-mentioned viruses can be exemplified by viruses used as live vaccines or inactivated vaccines such as influenza virus and rotavirus, oncolytic viruses having the effect of dissolving tumors, herpes simplex virus, vaccinia virus, adenovirus, coxsackie virus, poxvirus, paramyxovirus, picornavirus, rhabdovirus, reovirus, parvovirus, Brackenvirus, myxovirus, retrovirus, etc. Furthermore, the above-mentioned viruses can be introduced with genes.

[0093] <Other ingredients>

[0094] As long as the effects of the present invention can be exerted, the virus-containing composition may also contain other components. Examples of other components include growth factors, differentiation factors, hormones, chemokines, cytokines, cell adhesion molecules, chemotactic factors, enzymes, enzyme inhibitors, coenzymes (vitamins), minerals, fats, lipids, stabilizers, and preservatives.

[0095] <Logarithmic reduction value (LRV) of virus titer>

[0096] The difference between the logarithmic reduction value (hereinafter also referred to as "LRV") of the virus titer when the virus-containing composition is stored at 25°C for 21 days and the LRV of the virus titer when stored at -80°C for 21 days is preferably 1.5 log or less. The above difference is more preferably 1.0 log or less. For the virus-containing composition, the lower limit of the above difference is preferably 0.

[0097] The difference between the LRV of the virus titer when the virus-containing composition is stored at 4°C for 210 days and the LRV of the virus titer when stored at -80°C for 210 days is preferably 1.0 log or less. The above difference is more preferably 0.5 log or less. For the virus-containing composition, the lower limit of the above difference is preferably 0.

[0098] With the above-mentioned LRV-related characteristics of virus titer, the virus-containing composition will ensure the ability to stably store and transport the virus at both refrigerated temperatures (2 to 8°C) and room temperature around 25°C.

[0099] Furthermore, the difference between the LRV of the virus titer when the virus-containing composition is stored at 25°C for 21 days and the LRV of the virus titer when stored at -80°C for 21 days is preferably 1.5 log or less, and 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 preferably 1.0 log or less.

[0100] The logarithmic decrease value of the virus titer of the virus-containing composition when the freezing and thawing are repeated three times is preferably 0.3 log or less. Thus, the virus titer of the virus-containing composition can be maintained even when the freezing and thawing are repeated three times. Thus, the virus-containing composition can be used flexibly, which is very convenient.

[0101] (Method for determining the logarithmic reduction value (LRV) of virus titer)

[0102] In a virus-containing composition, the LRV of the virus titer can be obtained using an existing method for measuring the virus titer, for example, by obtaining the TCID50 of the virus in the virus-containing composition. "TCID50" refers to the dilution of the virus at which 50% infection is confirmed when a virus dilution (a dilution of the "virus-containing composition" in this specification) is inoculated on a test tube or a culture dish such as a 96-well flat-bottom plate to which cells have been pre-cultured and attached. The above-mentioned infection can be confirmed, for example, by a microscope using the cell modification effect (CPE) as an indicator. The LRV of the virus titer can be calculated based on the TCID50 calculated according to the Beherens-Karber method.

[0103] The method for determining the LRV of the virus titer can be as follows. Specifically, first, in the dilution of the virus-containing composition after storage for a predetermined period, DMEM medium can be used as a diluent. Cell culture can be performed by adding a predetermined cell suspension 100 μL / well (about 10 4 The cells were spread in a 96-well flat-bottom plate (manufactured by TPP) and heated at 37°C with CO 2 Constant temperature box (CO 2 Concentration 5%) was cultivated overnight.

[0104] After that, the virus-containing composition was serially diluted 10-fold using DMEM medium. The diluted virus was added to the specified cells (various cells corresponding to the type of virus contained in the virus-containing composition (e.g., LCC-MK2 cells, MDBK cells, HEK293 cells, etc.)) in a 96-well flat-bottom plate at 50 μL / well and incubated at 37°C under CO 2 Constant temperature box (CO 2 The cells were cultured in a 5% concentration (TCID50) medium and on the seventh day, CPE was used as an indicator and the virus titer (TCID50) was calculated according to the above calculation formula. LRV was calculated based on the TCID50 value.

[0105] When the virus type contained in the virus-containing composition is rNDV-GFP, the LRV of the virus titer can be calculated as follows. That is, the diluted virus is added to Vero cells in a 24-well flat-bottom plate at 50 μL / well and incubated at 37°C under CO 2 Constant temperature box (CO 2 The cells were cultured in PBS (concentration 5%) for 24 to 48 hours, and the cells were recovered using 0.25% by mass trypsin EDTA (manufactured by SIGMA-ALDRICH). Thereafter, the cells were washed with DMEM medium and PBS. Furthermore, the cells were suspended in PBS containing 0.5% by mass formalin, and the number of infected cells was calculated using FCM analysis or fluorescence microscopy, and the virus titer (FFU / mL) was calculated according to the above calculation formula. Thereafter, the LRV can be calculated based on the virus titer (FFU / mL).

[0106] <Method for preparing virus stabilizer>

[0107] The virus stabilizer of this embodiment can be preferably obtained by the following method: That is, the virus stabilizer can be obtained through a step of preparing a gelatin hydrolyzate (first step) and a step of preparing a virus stabilizer composed of the gelatin hydrolyzate and an aqueous medium (second step).

[0108] (First process)

[0109] The first step is a step of preparing a gelatin hydrolyzate. As described above, the gelatin hydrolyzate can be prepared by hydrolyzing either gelatin having an isoelectric point of pH 4.0 to 7.0 or collagen having an isoelectric point of pH 4.0 to 7.0 by alkali treatment so that the weight average molecular weight is 10,000 or less. As the gelatin having an isoelectric point of pH 4.0 to 7.0, alkali-treated gelatin is preferably used. Furthermore, a commercially available product (for example, trade name: "beMatrix (registered trademark) gelatin LS-H", manufactured by Nitta Gelatin Co., Ltd.) can also be used to prepare the gelatin hydrolyzate.

[0110] (Second process)

[0111] The second step is a step of preparing a virus stabilizer from the gelatin hydrolyzate and an aqueous medium. The virus stabilizer can be prepared by mixing the gelatin hydrolyzate and the aqueous medium at a mass ratio of 1 mass % to 20 mass % at a concentration of the gelatin hydrolyzate. A specific mixing method can use a conventionally known method. The aqueous medium can also be prepared by a conventionally known method such as adding a salt having a buffering effect to ion exchange water in a manner of a prescribed concentration.

[0112] <Method for preparing virus-containing composition>

[0113] The preparation of the virus-containing composition can be carried out by adding the virus to the above-mentioned virus stabilizer. The virus can be added to the above-mentioned virus stabilizer using a conventionally known method. The virus can usually be added to the above-mentioned virus stabilizer with a virus titer (TCID50) of 10 5 / mL or more is added to the above-mentioned virus stabilizer. In summary, the virus-containing composition of this embodiment can be obtained.

[0114] Example

[0115] Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.

[0116] 〔Sample preparation〕

[0117] The samples (samples 1 to 17) of the virus-containing composition used in various experiments (experiments 1 to 6) described below were prepared as follows. Samples 1 to 4, samples 9 to 12, and samples 14 to 17 are examples, and samples 5 to 8 and sample 13 are comparative examples.

[0118] <Sample 1>

[0119] Alkaline-treated gelatin derived from pig skin, "beMatrix (registered trademark) gelatin LS-H (produced by Nitta Gelatin Co., Ltd.)" with an isoelectric point of pH 5, was hydrolyzed by heat to a weight average molecular weight of 3800, thereby obtaining a gelatin hydrolyzate. Next, the gelatin hydrolyzate was dissolved in a GTS buffer as an aqueous medium in a manner containing 5% by mass, thereby obtaining a virus stabilizer. Bovine herpes virus (BHV-1), a DNA virus having an envelope, was added to the virus stabilizer at a TCID50 of 2×10 8.1The virus-containing composition was then divided into seven specimens and stored at 4°C for 21 days, 56 days and 210 days, at 25°C for 21 days, and at -80°C for 21 days, 56 days and 210 days, respectively.

[0120] <Sample 2>

[0121] Alkaline-treated gelatin "beMatrix (registered trademark) Gelatin LS-H (produced by Nitta Gelatin Co., Ltd.)" derived from pig skin and having an isoelectric point of pH 5 was hydrolyzed by heat to a weight average molecular weight of 650, thereby obtaining a gelatin hydrolyzate. Next, the gelatin hydrolyzate was dissolved in the GTS buffer solution in an amount of 5% by mass, thereby obtaining a virus stabilizer. Thereafter, the virus was added to the virus stabilizer in the same manner as in Sample 1, thereby obtaining a virus-containing composition. The virus-containing composition was divided into four specimens, which were stored at 4°C for 56 days, at 25°C for 21 days, and at -80°C for 21 days and 56 days, respectively.

[0122] <Sample 3>

[0123] Alkaline-treated gelatin "beMatrix (registered trademark) Gelatin LS-H (produced by Nitta Gelatin Co., Ltd.)" derived from pig skin and having an isoelectric point of pH 5 was hydrolyzed by heat to a weight average molecular weight of 1980, thereby obtaining a gelatin hydrolyzate. Next, the gelatin hydrolyzate was dissolved in the GTS buffer solution in a manner containing 5% by mass, thereby obtaining a virus stabilizer. Thereafter, the virus was added to the virus stabilizer in the same manner as sample 1, thereby obtaining a virus-containing composition. The virus-containing composition was divided into four specimens, which were stored at 4°C for 56 days, at 25°C for 21 days, and at -80°C for 21 days and 56 days, respectively.

[0124] <Sample 4>

[0125] Alkaline-treated gelatin "beMatrix (registered trademark) Gelatin LS-H (produced by Nitta Gelatin Co., Ltd.)" derived from pig skin and having an isoelectric point of pH 5 was hydrolyzed by heat to a weight average molecular weight of 9890, thereby obtaining a gelatin hydrolyzate. Next, the gelatin hydrolyzate was dissolved in the GTS buffer solution in a manner containing 5% by mass, thereby obtaining a virus stabilizer. Thereafter, the virus was added to the virus stabilizer in the same manner as sample 1, thereby obtaining a virus-containing composition. The virus-containing composition was divided into four specimens, which were stored at 4°C for 56 days, at 25°C for 21 days, and at -80°C for 21 days and 56 days, respectively.

[0126] <Sample 5>

[0127] Alkaline-treated gelatin "beMatrix (registered trademark) Gelatin LS-H (produced by Nitta Gelatin Co., Ltd.)" derived from pig skin and having an isoelectric point of pH 5 was hydrolyzed by heat to a weight average molecular weight of 59,800, thereby obtaining a gelatin hydrolyzate. Next, the gelatin hydrolyzate was dissolved in the GTS buffer solution in a manner containing 5% by mass, thereby obtaining a virus stabilizer. Thereafter, the virus was added to the virus stabilizer in the same manner as sample 1, thereby obtaining a virus-containing composition. Furthermore, the virus-containing composition was divided into four specimens, which were stored at 4°C for 56 days, at 25°C for 21 days, and at -80°C for 21 days and 56 days, respectively.

[0128] <Sample 6>

[0129] A gelatin hydrolyzate (manufactured by Nitta Gelatin Co., Ltd.) obtained from acid-treated gelatin derived from pig skin with an isoelectric point of pH 9 was hydrolyzed by heat in a manner having a weight average molecular weight of 2060, thereby obtaining a gelatin hydrolyzate. Next, the gelatin hydrolyzate was dissolved in the GTS buffer in a manner containing 5% by mass, thereby obtaining a virus stabilizer. Thereafter, a virus was added to the virus stabilizer in the same manner as sample 1, thereby obtaining a virus-containing composition. The virus-containing composition was divided into four specimens, which were stored at 4°C for 56 days, at 25°C for 21 days, and at -80°C for 21 days and 56 days, respectively.

[0130] <Sample 7>

[0131] Alkaline-treated gelatin "beMatrix (registered trademark) gelatin LS-H (produced by Nitta Gelatin Co., Ltd.)" derived from pig skin and having an isoelectric point of pH 5 was hydrolyzed by heat in a manner to a weight average molecular weight of 3800, thereby obtaining a gelatin hydrolyzate. Next, the above-mentioned gelatin hydrolyzate was dissolved in the above-mentioned GTS buffer in a manner containing 0.1% by mass, thereby obtaining a virus stabilizer, and a virus-containing composition was obtained by the same method as sample 1. The above-mentioned virus-containing composition was divided into four specimens, which were stored at 4°C for 56 days, at 25°C for 21 days, and at -80°C for 21 days and 56 days, respectively.

[0132] <Sample 8>

[0133] Alkaline-treated gelatin "beMatrix (registered trademark) gelatin LS-H (produced by Nitta Gelatin Co., Ltd.)" derived from pig skin and having an isoelectric point of pH 5 was hydrolyzed by heat in a manner having a weight average molecular weight of 3800, thereby obtaining a gelatin hydrolyzate. Next, the above-mentioned gelatin hydrolyzate was dissolved in the above-mentioned GTS buffer in a manner containing 0.5% by mass, thereby obtaining a virus stabilizer, and a virus-containing composition was obtained by the same method as sample 1. The above-mentioned virus-containing composition was divided into four specimens, which were stored at 4°C for 56 days, at 25°C for 21 days, and at -80°C for 21 days and 56 days, respectively.

[0134] <Sample 9>

[0135] Alkaline-treated gelatin "beMatrix (registered trademark) gelatin LS-H (produced by Nitta Gelatin Co., Ltd.)" derived from pig skin and having an isoelectric point of pH 5 was hydrolyzed by heat in a manner to a weight average molecular weight of 3800, thereby obtaining a gelatin hydrolyzate. Next, the above-mentioned gelatin hydrolyzate was dissolved in the above-mentioned GTS buffer in a manner containing 1% by mass, thereby obtaining a virus stabilizer, and a virus-containing composition was obtained by the same method as sample 1. The above-mentioned virus-containing composition was divided into four specimens, which were stored at 4°C for 56 days, at 25°C for 21 days, and at -80°C for 21 days and 56 days, respectively.

[0136] <Sample 10>

[0137] Alkaline-treated gelatin "beMatrix (registered trademark) gelatin LS-H (produced by Nitta Gelatin Co., Ltd.)" derived from pig skin and having an isoelectric point of pH 5 was hydrolyzed by heat in a manner to a weight average molecular weight of 3800, thereby obtaining a gelatin hydrolyzate. Next, the above-mentioned gelatin hydrolyzate was dissolved in the above-mentioned GTS buffer in a manner containing 2.5% by mass, thereby obtaining a virus stabilizer, and a virus-containing composition was obtained by the same method as sample 1. The above-mentioned virus-containing composition was divided into four specimens, which were stored at 4°C for 56 days, at 25°C for 21 days, and at -80°C for 21 days and 56 days, respectively.

[0138] <Sample 11>

[0139] Alkaline-treated gelatin "beMatrix (registered trademark) gelatin LS-H (produced by Nitta Gelatin Co., Ltd.)" derived from pig skin and having an isoelectric point of pH 5 was hydrolyzed by heat in a manner having a weight average molecular weight of 3800, thereby obtaining a gelatin hydrolyzate. Next, the above-mentioned gelatin hydrolyzate was dissolved in the above-mentioned GTS buffer in a manner containing 10% by mass, thereby obtaining a virus stabilizer, and a virus-containing composition was obtained by the same method as sample 1. The above-mentioned virus-containing composition was divided into four specimens, which were stored at 4°C for 56 days, at 25°C for 21 days, and at -80°C for 21 days and 56 days, respectively.

[0140] <Sample 12>

[0141] Alkaline-treated gelatin "beMatrix (registered trademark) gelatin LS-H (produced by Nitta Gelatin Co., Ltd.)" derived from pig skin and having an isoelectric point of pH 5 was hydrolyzed by heat in a manner having a weight average molecular weight of 3800, thereby obtaining a gelatin hydrolyzate. Next, the above-mentioned gelatin hydrolyzate was dissolved in the above-mentioned GTS buffer in a manner containing 20% ​​by mass, thereby obtaining a virus stabilizer, and a virus-containing composition was obtained by the same method as sample 1. The above-mentioned virus-containing composition was divided into four specimens, which were stored at 4°C for 56 days, at 25°C for 21 days, and at -80°C for 21 days and 56 days, respectively.

[0142] <Sample 13>

[0143] The recombinant human albumin "Recombumin (registered trademark) Elite (manufactured by Albumedix)" was dissolved in the above-mentioned GTS buffer in a manner containing 1% by mass, thereby obtaining a virus stabilizer. Thereafter, the virus was added to the above-mentioned virus stabilizer by the same method as sample 1, thereby obtaining a virus-containing composition. Furthermore, the above-mentioned virus-containing composition was divided into four specimens, which were stored at 4°C for 56 days, at 25°C for 21 days, and at -80°C for 21 days and 56 days, respectively. Sample 13 is a conventionally known virus stabilizer.

[0144] <Sample 14>

[0145] Parainfluenza virus (PI-3), an enveloped RNA virus, was added to a virus stabilizer at a TCID50 of 2×10 5.5 / mL, a virus-containing composition was obtained by the same method as Sample 1. The virus-containing composition was divided into seven specimens and stored at 4°C for 21 days, 56 days and 210 days, at 25°C for 21 days, and at -80°C for 21 days, 56 days and 210 days, respectively.

[0146] <Sample 15>

[0147] Reovirus (Reo-3), a RNA virus without an envelope, was added to the virus stabilizer at a TCID50 of 2×10 6.5 / mL, and a virus-containing composition was obtained by the same method as Sample 1. The virus-containing composition was divided into seven specimens and stored at 4°C for 21 days, 56 days and 210 days, at 25°C for 21 days, and at -80°C for 21 days, 56 days and 210 days, respectively.

[0148] <Sample 16>

[0149] Adenovirus (Ad5), a DNA virus without an envelope, was added to a virus stabilizer at a TCID50 of 2×10 8 / mL, a virus-containing composition was obtained by the same method as Sample 1. The virus-containing composition was divided into seven specimens and stored at 4°C for 21 days, 56 days and 210 days, at 25°C for 21 days, and at -80°C for 21 days, 56 days and 210 days, respectively.

[0150] <Sample 17>

[0151] Paramyxovirus (Newcastle disease virus: rNDV-GFP) was added to the virus stabilizer at an FFU of 4×10 6 / mL was added in a manner including, the paramyxovirus is an oncolytic virus into which GFP is gene-introduced, and is an RNA virus with an envelope. In addition, a virus-containing composition was obtained by the same method as sample 1. The above-mentioned virus-containing composition was divided into four specimens, which were stored at 4°C for 56 days, at 25°C for 21 days, and at -80°C for 21 days and 56 days. An overview of samples 1 to 17 is shown in Table 1. Here, the pH of the isoelectric point of the gelatin hydrolyzate contained in the virus stabilizer of samples 1 to 4, samples 9 to 12, and samples 14 to 17 represents the value measured by the above-mentioned isoelectric point measurement method using Zeta potential as an indicator.

[0152] [Table 1]

[0153] Table 1

[0154]

[0155] ※In sample 13, the virus stabilizer was composed of albumin and an aqueous medium.

[0156] [Experiment 1: Study on the appropriate weight-average molecular weight of gelatin hydrolysate]

[0157] For sample 1, sample 2, sample 3, sample 4, and sample 5, the TCID50 of each specimen stored at 4°C for 56 days and at 25°C for 21 days was calculated, and the LRV of the virus titer at 4°C and the LRV of the virus titer at 25°C were calculated based on the TCID50 value. Further, for sample 1, sample 2, sample 3, sample 4, and sample 5, the TCID50 of each specimen stored at -80°C for 21 days and 56 days was calculated, and the LRV of the virus titer at -80°C was calculated based on the TCID50 value. Based on these, for sample 1, sample 2, sample 3, sample 4, and sample 5, the difference between the LRV of the virus titer of each specimen stored at 25°C for 21 days and the LRV of the virus titer of each specimen stored at -80°C for 21 days was calculated. The results are shown in Table 2. Furthermore, Table 3 shows the results of calculating the difference between the LRV of the virus titer of each sample stored at 4°C for 56 days and the LRV of the virus titer of each sample stored at -80°C for 56 days for Sample 1, Sample 2, Sample 3, and Sample 4.

[0158] [Table 2]

[0159] Table 2

[0160]

[0161] [Table 3]

[0162] Table 3

[0163]

[0164] According to Tables 2 and 3, the differences in the LRV of each of Sample 1, Sample 2, Sample 3, and Sample 4 were 1.0 log or less and 1.5 log or less, respectively, and therefore it can be evaluated that the virus can be stably stored and transported at 4°C (refrigerated temperature) and 25°C (room temperature). In Sample 5, the difference in the LRV exceeded 1.5 log at 25°C due to gelation. It should be noted that for Sample 5, the evaluation at 4°C for 56 days was not performed due to gelation.

[0165] [Experiment 2: Study on the appropriate isoelectric point of gelatin hydrolysate]

[0166] For sample 1, sample 2, sample 3, sample 4, and sample 6, the TCID50 of each specimen stored at 4°C for 56 days and at 25°C for 21 days was calculated, and the LRV of the virus titer at 4°C and the LRV of the virus titer at 25°C were calculated based on the TCID50 value. Further, for sample 1, sample 2, sample 3, sample 4, and sample 6, the TCID50 of each specimen stored at -80°C for 21 days and 56 days was calculated, and the LRV of the virus titer at -80°C was calculated based on the TCID50 value. Based on these, for the virus-containing compositions of sample 1, sample 2, sample 3, sample 4, and sample 6, the difference between the LRV of the virus titer of each specimen stored at 25°C for 21 days and the LRV of the virus titer of each specimen stored at -80°C for 21 days was calculated. The results are shown in Table 4. Furthermore, Table 5 shows the results of calculating the difference between the LRV of the virus titer of each sample stored at 4°C for 56 days and the LRV of the virus titer of each sample stored at -80°C for 56 days for Sample 1, Sample 2, Sample 3, Sample 4, and Sample 6.

[0167] [Table 4]

[0168] Table 4

[0169]

[0170] [Table 5]

[0171] Table 5

[0172]

[0173] According to Tables 4 and 5, the differences in the LRV of each of Sample 1, Sample 2, Sample 3, and Sample 4 were 1.0 log or less and 1.5 log or less, and thus it was evaluated that the virus could be stably stored and transported at 4°C (refrigerated temperature) and 25°C (room temperature). In Sample 6, the difference in the LRV exceeded 1.5 log at 25°C and exceeded 1.0 log at 4°C.

[0174] [Experiment 3: Study on the appropriate concentration of gelatin hydrolysate in virus stabilizer]

[0175] For sample 1, sample 7, sample 8, sample 9, sample 10, sample 11, and sample 12, the TCID50 of each sample stored at 4°C for 56 days and at 25°C for 21 days was determined, and the LRV of the virus titer at 4°C and the LRV of the virus titer at 25°C were calculated based on the TCID50 value. Further, for sample 1, sample 7, sample 8, sample 9, sample 10, sample 11, and sample 12, the TCID50 of each sample stored at -80°C for 21 days and 56 days was determined, and the LRV of the virus titer at -80°C was calculated based on the TCID50 value. Based on these, for sample 1, sample 7, sample 8, sample 9, sample 10, sample 11, and sample 12, the difference between the LRV of the virus titer of each sample stored at 25°C for 21 days and the LRV of the virus titer of each sample stored at -80°C for 21 days was calculated. The results are shown in Table 6. Furthermore, in Table 7, for Sample 1, Sample 7, Sample 8, Sample 9, Sample 10, Sample 11, and Sample 12, the results obtained by calculating the difference between the LRV of the virus titer of each specimen stored at 4°C for 56 days and the LRV of the virus titer of each specimen stored at -80°C for 56 days are shown.

[0176] [Table 6]

[0177] Table 6

[0178]

[0179] [Table 7]

[0180] Table 7

[0181]

[0182] According to Tables 6 and 7, the differences in the LRV of each of Sample 1, Sample 9, Sample 10, Sample 11, and Sample 12 were 1.0 log or less and 1.5 log or less, and thus it was evaluated that the virus could be stably stored and transported at 4°C (refrigerated temperature) and 25°C (room temperature). In Samples 7 and 8, the differences in the LRV exceeded 1.5 log at 25°C and exceeded 1.0 log at 4°C.

[0183] [Experiment 4: Comparison with previous virus stabilizers]

[0184] For sample 1 and sample 13, the TCID50 of each specimen stored at 4°C for 56 days and at 25°C for 21 days was calculated, and the LRV of the virus titer at 4°C and the LRV of the virus titer at 25°C were calculated based on the TCID50 value. Further, for sample 1 and sample 13, the TCID50 of each specimen stored at -80°C for 21 days and 56 days was calculated, and the LRV of the virus titer at -80°C was calculated based on the TCID50 value. Based on these, for sample 1 and sample 13, the difference between the LRV of the virus titer of each specimen stored at 25°C for 21 days and the LRV of the virus titer of each specimen stored at -80°C for 21 days was calculated. The results are shown in Table 8. Furthermore, Table 9 shows, for Sample 1 and Sample 13, the results of calculating the difference between the LRV of the virus titer of each sample stored at 4°C for 56 days and the LRV of the virus titer of each sample stored at -80°C for 56 days.

[0185] [Table 8]

[0186] Table 8

[0187]

[0188] [Table 9]

[0189] Table 9

[0190]

[0191] According to Tables 8 and 9 above, Sample 1 can be evaluated as being able to suppress the decrease in virus titer at 4°C (refrigerated temperature) and 25°C (room temperature) as compared to Sample 13.

[0192] [Experiment 5: Study on the effect of virus stabilizer for each virus type]

[0193] For sample 1, sample 14, sample 15, sample 16 and sample 17, the TCID50 of each specimen stored at 25°C for 21 days was calculated, and the LRV of the virus titer at 25°C was calculated based on the value of the TCID50. Further, for sample 1, sample 14, sample 15, sample 16 and sample 17, the TCID50 of each specimen stored at -80°C for 21 days was calculated, and the LRV of the virus titer at -80°C was calculated based on the value of the TCID50. Based on these, for sample 1, sample 14, sample 15, sample 16 and sample 17, the difference between the LRV of the virus titer of each specimen stored at 25°C for 21 days and the LRV of the virus titer of each specimen stored at -80°C for 21 days was calculated. It should be noted that for sample 17, FFU was calculated instead of TCID. The results are shown in Table 10.

[0194] Furthermore, for sample 1, 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 calculated, and the LRV of the virus titer at 4°C was calculated based on the value of the TCID50. For sample 1, 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 calculated, and the LRV of the virus titer at -80°C was calculated based on the value of the TCID50. Based on these, for sample 1, sample 14, sample 15, and sample 16, the difference between the LRV of the virus titer of each specimen stored at 4°C for 21 days, 56 days, and 210 days and the LRV of the virus titer of each specimen stored at -80°C for 21 days, 56 days, and 210 days was calculated. The results are shown in Table 11.

[0195] [Table 10]

[0196] Table 10

[0197]

[0198] [Table 11]

[0199] Table 11

[0200]

[0201] According to Tables 10 and 11, the differences in the LRV of each of Sample 1, Sample 14, Sample 15, Sample 16, and Sample 17 are 1.0 log or less and 1.5 log or less, and therefore it can be evaluated that the virus can be stably stored and transported at 4°C (refrigerated temperature) and 25°C (room temperature). It should be noted that according to Tables 10 and 11, Samples 1, 14 to 16, which maintained activity for 3 weeks at 25°C, can maintain activity for 30 weeks at 4°C, and therefore it is estimated that Sample 17 is also likely to maintain activity for 30 weeks at 4°C (refrigerated temperature).

[0202] Considering the results of Experiments 1 to 5, it can be evaluated that the samples 1 to 4, 9 to 12, and 14 to 17 can stably store and transport the virus in an environment of 4° C. for 21 days.

[0203] [Experiment 6: Effect of repeated freezing and thawing of virus stabilizer]

[0204] Sample 1 was frozen at -80°C and then thawed in ice water. This operation was repeated three times. The virus titer of sample 1 was measured each time it was thawed (the first, second, and third freeze-thaw), and the logarithmic reduction value (LRV) was calculated for comparison with the virus titer of sample 1 before freezing and thawing. The results are shown in Table 12.

[0205] [Table 12]

[0206] Table 12

[0207]

[0208] According to Table 12, Sample 1 maintained the LRV at approximately 0 in all of the 1st to 3rd freeze-thaw cycles, and thus it can be evaluated that the virus titer can be maintained even when freeze-thaw is performed.

[0209] As mentioned above, although embodiment and Example of this invention were demonstrated, it is also expected from the beginning that the structure of each embodiment and Example mentioned above can be suitably combined.

[0210] The embodiments and examples disclosed herein are illustrative in all aspects and should not be construed as restrictive. The scope of the present invention is indicated by the claims rather than the above description, and is intended to include all modifications within the meaning and scope equivalent to the claims.

Claims

1. A virus stabilizer, wherein: The virus stabilizer is composed of gelatin hydrolyzate and aqueous medium. The virus stabilizer contains 1% by mass or more and 20% by mass or less of the gelatin hydrolyzate, The weight average molecular weight of the gelatin hydrolyzate is less than 10,000, The isoelectric point of the gelatin hydrolyzate has a pH of 4.0 or more and less than 7.

0.

2. The virus stabilizer according to claim 1, wherein The weight average molecular weight of the gelatin hydrolyzate is less than 6000.

3. The virus stabilizer according to claim 1 or 2, wherein The gelatin hydrolysate is a hydrolysate of alkali-treated gelatin.

4. The virus stabilizer according to claim 1 or 2, wherein The virus stabilizer contains more than 2 mass % and 10 mass % or less of the gelatin hydrolyzate.

5. The virus stabilizer according to claim 1 or 2, wherein The aqueous medium contains salts which have a buffering effect.

6. The virus stabilizer according to claim 1 or 2, wherein The aqueous medium contains at least one selected from the group consisting of sucrose, lactose, sorbitol, inositol, trehalose, mannitol, maltitol, xylitol, erythritol and glycerol.

7. The virus stabilizer according to claim 1 or 2, wherein The aqueous medium contains at least one amino acid selected from the group consisting of methionine, arginine, tryptophan, glutamine, and glutamic acid.

8. A gelatin hydrolyzate for virus stabilizer, wherein: The gelatin hydrolyzate for virus stabilizer has a weight average molecular weight of 6000 or less and an isoelectric point pH of 4.0 or more and less than 7.

0.

9. The gelatin hydrolyzate for virus stabilizer according to claim 8, wherein The gelatin hydrolyzate used as virus stabilizer is in the form of liquid or powder.

10. A virus-containing composition, wherein: The virus-containing composition comprises the virus stabilizer according to any one of claims 1 to 7 and a virus.

11. The virus-containing composition according to claim 10, wherein The virus comprises at least one selected from the group consisting of a DNA virus having an envelope, a DNA virus not having an envelope, an RNA virus having an envelope, and an RNA virus not having an envelope.

12. The virus-containing composition according to claim 10 or 11, wherein The virus is introduced with a gene.

13. The virus-containing composition according to claim 10 or 11, wherein The difference between the logarithmic reduction value of the virus titer when the virus-containing composition is stored at 25° C. for 21 days and the logarithmic reduction value of the virus titer when the composition is stored at −80° C. for 21 days is 1.5 log or less.

14. The virus-containing composition according to claim 10 or 11, wherein The difference between the logarithmic decrease value of the virus titer when the virus-containing composition is stored at 4° C. for 210 days and the logarithmic decrease value of the virus titer when the composition is stored at −80° C. for 210 days is 1.0 log or less.

15. The virus-containing composition according to claim 10 or 11, wherein When the virus-containing composition was frozen and thawed three times, the logarithmic decrease value of the virus titer was less than 0.3 log.

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