Virus stabilizers, gelatin hydrolysates for virus stabilizers, and virus-containing compositions

Abstract

A virus stabilizer comprising a gelatin hydrolysate and an aqueous medium, in which the gelatin hydrolysate is contained in an amount of 1 to 20% by mass inclusive in the virus stabilizer, the gelatin hydrolysate has a weight average molecular weight of 10000 or less, and the gelatin hydrolysate has a pH value at an isoelectric point of 4.0 to 7.0 inclusive.

Description

Technical Field

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

Background Art

[0002] For the storage and transportation of viruses, in order to maintain the abilities of viruses such as infectivity or to prevent their decline, an environment of generally -80 to -60°C is generally required. Alternatively, when storing and transporting viruses at room temperature, it is necessary to make the preparation containing the virus a lyophilized product. Thus, it has been conventionally known that the storage and transportation of viruses are complicated. In contrast, Japanese Patent Application Laid-Open No. 2013-035861 (Patent Document 1) discloses a refrigerator-stable influenza virus composition having stability at refrigeration temperatures (for example, 2 to 8°C).

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] Patent Document 1 discloses that a composition containing 0.5-2% hydrolyzed gelatin achieved stable storage and transport of influenza virus strains at refrigeration temperatures. However, Patent Document 1 does not disclose whether stable storage and transport of the virus strains can be achieved at room temperature around 25°C. According to Patent Document 1, since the hydrolyzed gelatin is obtained from porcine type A gelatin, which has an isoelectric point of around 9, it is presumed that the hydrolyzed gelatin strongly interacts with specific proteins on the virus surface, making stable storage and transport of the virus at room temperature difficult. Furthermore, it is known that when a preparation containing a virus stored in an environment of -80 to -60°C is heated to a sol state for use and returned to refrigeration temperature (2-8°C) or room temperature, a decrease in the virus's capabilities, such as infectivity, is unavoidable. Therefore, a material that eliminates the need for complicated procedures and enables the storage and transport of virus-containing preparations without reducing the infectivity or other capabilities of the virus at both refrigerated temperatures (2-8°C) and room temperatures around 25°C has not yet been realized, and its development is urgently needed.

[0005] In view of the above, the present invention aims to provide a virus stabilizer, a gelatin hydrolysate for virus stabilizers, and a virus-containing composition that suppress the decrease in viral activity at both refrigerated temperatures (2-8°C) and room temperatures around 25°C, thereby enabling the storage and transportation of virus-containing preparations without the need for refrigeration equipment. [Means for solving the problem]

[0006] The present inventors arrived at the present invention after diligent research. Specifically, they first focused on gelatin hydrolysates obtained by hydrolyzing gelatin, which has an isoelectric point pH of around 5, to a predetermined weight-average molecular weight. Furthermore, they discovered that when a virus is added to a formulation containing the gelatin hydrolysate at a predetermined concentration, the decrease in the virus's capabilities, such as infectivity, can be suppressed at both refrigerated temperatures (2-8°C) and room temperature of around 25°C. Thus, formulations containing the virus can be stably stored and transported without the need for refrigeration equipment, and thus completed the present invention.

[0007] The present invention has the following features. [1] The virus stabilizer according to the present invention is a virus stabilizer comprising a gelatin hydrolysate and an aqueous medium, wherein the virus stabilizer contains 1% by mass or more and 20% by mass or less of the gelatin hydrolysate, the weight-average molecular weight of the gelatin hydrolysate is 10,000 or less, and the pH of the isoelectric point of the gelatin hydrolysate is 4.0 or more and 7.0 or less. [2] The above gelatin hydrolysate preferably has a weight-average molecular weight of 6000 or less. [3] The above gelatin hydrolysate is preferably a hydrolysate of alkali-treated gelatin. [4] The above virus stabilizer preferably contains more than 2% by mass and 10% by mass or less of the above gelatin hydrolysate. [5] The aqueous medium preferably contains a salt that has a buffering effect. [6] 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. [7] The aqueous medium preferably contains at least one amino acid selected from the group consisting of methionine, arginine, tryptophan, glutamine, and glutamic acid. [8] The gelatin hydrolysate for virus stabilization according to the present invention has a weight-average molecular weight of 10,000 or less and an isoelectric point pH of 4.0 or more and 7.0 or less. [9] The above-mentioned gelatin hydrolysate for virus stabilization is preferably in liquid or powder form.

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

[11] Preferably, the above-mentioned virus includes at least one selected from the group consisting of enveloped DNA viruses, non-enveloped DNA viruses, enveloped RNA viruses, and non-enveloped RNA viruses.

[12] The above virus is preferably genetically modified.

[13] It is preferable that the difference between the logarithmic decrease in viral titer when the above virus-containing composition is stored at 25°C for 21 days and the logarithmic decrease in viral titer when it is stored at -80°C for 21 days is 1.5 log or less.

[14] It is preferable that the difference between the logarithmic decrease in viral titer when the above virus-containing composition is stored at 4°C for 210 days and the logarithmic decrease in viral titer when it is stored at -80°C for 210 days is 1.0 log or less.

[15] The above virus-containing composition is preferably such that the logarithmic decrease in viral titer after three freeze-thaw cycles is 0.3log or less. [Effects of the Invention]

[0008] According to the above, it is possible to provide a virus stabilizer, a gelatin hydrolysate for virus stabilizers, and a virus-containing composition that suppress the decrease in viral activity at both refrigerated temperatures (2-8°C) and room temperature of around 25°C, thereby enabling the storage and transportation of virus-containing preparations without the need for refrigeration equipment. [Modes for carrying out the invention]

[0009] The embodiments of the present invention (hereinafter also referred to as "this embodiment") will be described in more detail below. In this specification, the notation in the form of "A~B" means the upper and lower limits of a range (i.e., A or greater and B or less), and if there is no unit specified for A and a unit is specified only for B, the units for A and B are the same. In this specification, "viral titer" means the lowest concentration at which the virus in the sample (hereinafter referred to as "virus-containing composition") can infect cells, i.e., the highest dilution ratio. Furthermore, a "decrease" in viral titer means a decrease in the above dilution ratio, which means an increase in the above lowest concentration at which infection is possible. In this specification, the term "gelatin" may be used to refer to the substance name, gelatin gel, and gelatin solution, respectively. Similarly, the term "gelatin hydrolysate" may be used to refer to a solution of gelatin hydrolysate, in the same way as gelatin.

[0010] [Virus stabilizer] The virus stabilizer according to this embodiment is a virus stabilizer comprising a gelatin hydrolysate and an aqueous medium. The virus stabilizer contains 1% to 20% by mass of the gelatin hydrolysate. Furthermore, the gelatin hydrolysate has a weight-average molecular weight of 10,000 or less. The gelatin hydrolysate has an isoelectric point pH of 4.0 to 7.0. By having these characteristics, the virus stabilizer according to this embodiment can suppress the decrease in viral titer at both refrigerated temperatures (2 to 8°C) and room temperature of around 25°C. Thus, when a virus-containing composition containing the virus stabilizer and a virus is formed, the virus-containing composition can be stably stored and transported without the need for a refrigeration device or the like.

[0011] <Hydrolyzed gelatin> The above-mentioned virus stabilizer contains gelatin hydrolysate as described above. In this specification, "gelatin hydrolysate" refers to a peptide aggregate (hydrolysate) obtained by hydrolyzing either or both gelatin and collagen. In other words, "gelatin hydrolysate" means something equivalent to a peptide aggregate generally called collagen peptide or collagen hydrolysate. Among these, the gelatin hydrolysate contained in the virus stabilizer according to this embodiment has the weight-average molecular weight and isoelectric point pH as described above. Furthermore, since gelatin hydrolysate means a peptide aggregate 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 constituting the peptide chain.

[0012] In this specification, "gelatin" means a polypeptide in which the triple helix structure of collagen has been unraveled by thermal denaturation, acid denaturation, etc., its chemically modified products, and pharmaceutically acceptable salts thereof. Specifically, collagen derived from at least one selected from the following groups 1 to 6 can be obtained by subjecting it to conventionally known treatments such as degreasing, demineralization, acid or alkali treatment, and hot water extraction. Gelatin may be a polypeptide obtained by fermentation using microorganisms, a recombinant polypeptide obtained by chemical synthesis or genetic recombination, or a synthesized polypeptide. Furthermore, "collagen" refers to a protein derived from the extracellular matrix of vertebrates such as the skin, which are classified into the following groups 1 to 6. Collagen has a right-handed helical structure consisting of three peptide chains, and the amino acid residues constituting these peptide chains have a primary structure (so-called collagen-like sequence) in which glycine residues are repeated every three residues. Group 1: A group consisting of cowhide, skin, bone, cartilage, and tendons. Group 2: A group consisting of pig skin, skin, bones, cartilage, and tendons. Group 3: A group consisting of sheepskin, skin, bone, cartilage, and tendons. Group 4: A group consisting of chicken skin, skin, bones, cartilage, and tendons. Group 5: A group consisting of ostrich skin, bones, cartilage, and tendons. Group 6: A group consisting of fish bones, skin, and scales.

[0013] Here, the "chemically modified form" of the above polypeptide (gelatin) means a polypeptide in which an amino group, carboxyl group, hydroxy group, or thiol group, etc. in the amino acid residues constituting gelatin is chemically modified. Gelatin subjected to chemical modification can change its solubility in water, isoelectric point, etc. Specifically, for the hydroxy group of hydroxyproline residues in gelatin, chemical modifications such as O-acetylation can be performed. For the α-carboxyl group of glycine residues in gelatin, chemical modifications such as esterification and amidation can be performed. For the α-amino group of proline residues in gelatin, chemical modifications such as polypeptidylation, succinylation, maleylation, acetylation, deamination, benzoylation, alkylsulfonylation, allylsulfonylation, dinitrophenylation, trinitrophenylation, carbamylation, phenylcarbamylation, and thiolation can be performed.

[0014] For the specific means and treatment conditions of chemical modification of gelatin, conventionally known chemical modification methods can be applied. For the chemical modification of the hydroxy group of hydroxyproline residues, O-acetylation, for example, can be performed by allowing acetic anhydride to act in an aqueous solvent or a non-aqueous solvent. For the chemical modification of the α-carboxyl group of glycine residues, esterification, for example, can be performed by suspending in methanol and then passing dry hydrogen chloride gas. For the chemical modification of the α-carboxyl group of glycine residues, amidation can be performed by allowing carbodiimide, etc. to act.

[0015] Furthermore, the "derivatives" of the above polypeptide (gelatin) may include gelatin derivatives obtained by introducing functional groups into gelatin, copolymers of gelatin and lactic acid, glycolic acid, etc., copolymers of gelatin and polyethylene glycol, propylene glycol, etc. Examples of gelatin derivatives include derivatives obtained by introducing functional groups such as guanidyl group, thiol group, amino group, carboxyl group, sulfate group, phosphate group, alkyl group, acyl group, phenyl group, benzyl group, etc. into gelatin.

[0016] The "pharmaceutically acceptable salts" of the above polypeptide (gelatin) mean salts that are pharmaceutically acceptable and have the desired activity (e.g., gelling ability) of the original polypeptide (gelatin). Examples of pharmaceutically acceptable salts include 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, and organic base salts such as triethylammonium salt. Specific peptides in gelatin can be made into pharmaceutically acceptable salts according to conventional methods.

[0017] Since gelatin is a polypeptide derived from collagen possessed by many organisms, it has excellent biocompatibility. Therefore, the above collagen and gelatin hydrolysates obtained by hydrolyzing gelatin also have excellent biocompatibility and are suitable as a component of a virus stabilizer for pharmaceutical use.

[0018] When dissolved in a solvent (e.g., water), gelatin hydrolysates do not gel at room temperature around 25°C, nor at environments between 2 and 8°C, but maintain a sol state. Therefore, when using the above virus stabilizer at room temperature around 25°C, heating for sol formation is unnecessary. In this embodiment, "sol" refers to a dispersion system consisting of a dispersed phase and a dispersion medium in which the dispersion medium is in a liquid state. "Gel" refers to a dispersion system consisting of a dispersed phase and a dispersion medium in which the dispersed phase forms a cross-linked structure, and the dispersion system as a whole loses its fluidity.

[0019] Gelatin hydrolysates are obtained by hydrolyzing either or both gelatin and collagen, as described above. "Hydrolysis" in this context includes hydrolysis using acids, hydrolysis using bases, hydrolysis using enzymes, and hydrolysis using heat. Gelatin hydrolysates are preferably obtained by hydrolysis using heat, from the viewpoint of preventing contamination by impurities. Furthermore, when hydrolyzing either or both gelatin and collagen using enzymes, examples of such enzymes include collagenase, thiol protease, serine protease, acid protease, alkaline protease, and metalloprotease. These enzymes can be used individually or in combination. Examples of thiol proteases include plant-derived chymopapain, papain, bromelain, and ficin, while animal-derived cathepsin and calcium-dependent proteases are also used. Examples of serine proteases include trypsin and cathepsin D. Examples of acid proteases include pepsin and chymotrypsin.

[0020] Considering the pharmaceutical use of gelatin hydrolysates, it is preferable to use enzymes other than those derived from pathogenic microorganisms (for example, enzymes derived from non-pathogenic microorganisms). Examples of non-pathogenic microorganisms from which the above enzymes can be derived include Bacillus Iicheniforms, Bacillus subtillis, Aspergillus oryzae, Streptomyces, and Bacillus amyloliquefaciens. The above enzymes may be derived from one of the above-mentioned non-pathogenic microorganisms, or a combination of enzymes derived from multiple of the above-mentioned non-pathogenic microorganisms may be used. The specific method of enzyme treatment can be any conventionally known method.

[0021] Furthermore, when obtaining "gelatin hydrolysate" from the above-mentioned gelatin by hydrolysis using a single enzyme, it is preferable that the enzyme is not collagenase. This is because when the above-mentioned gelatin is hydrolyzed with collagenase alone, a specific peptide whose N-terminus is glycine may become the main component (more than 50% by mass). The above-mentioned gelatin hydrolysate is an aggregate of two or more peptides, but the above-mentioned specific peptide is less than 50% by mass in its composition.

[0022] Gelatin hydrolysates can be obtained as liquids by hydrolyzing both or either gelatin and collagen using the method described above, followed by purification. Furthermore, it is possible to obtain powders from the liquid by heating and drying or freeze-drying using known means.

[0023] (concentration) The virus stabilizer contains 1% to 20% by mass of the above-mentioned gelatin hydrolysate. If the concentration of the above-mentioned gelatin hydrolysate in the virus stabilizer is less than 1% by mass, when a virus-containing composition is formed with the virus stabilizer and the virus, the effect of suppressing the decrease in viral titer will be minimal, making it difficult to achieve the desired effect. If the concentration of the above-mentioned gelatin hydrolysate in the virus stabilizer exceeds 20% by mass, the operability of the virus stabilizer may deteriorate. From the viewpoint of further suppressing the decrease in viral titer, it is preferable that the virus stabilizer contains more than 2% to 10% by mass of the above-mentioned gelatin hydrolysate. The concentration of the above-mentioned gelatin hydrolysate in the virus stabilizer can be measured by known methods such as hydroxyproline quantification.

[0024] (Weight average molecular weight) The above gelatin hydrolysate has a weight-average molecular weight of 10,000 or less. Preferably, the above gelatin hydrolysate has a weight-average molecular weight of 6,000 or less. More preferably, the above gelatin hydrolysate has a weight-average molecular weight of 5,000 or less. This allows the virus stabilizer to maintain a sol state in an environment of about 2 to 30°C. Furthermore, when a virus-containing composition is formed that includes the virus stabilizer and a virus, the gelatin hydrolysate in the above composition can suppress the aggregation of the virus, thereby suppressing the decrease in viral titer. Although the detailed mechanism is unknown, it is presumed that when the weight-average molecular weight of the gelatin hydrolysate is within the above range, both the hydrophilic and hydrophobic regions in the molecule are exposed on the molecular surface side (outside), and it becomes possible to interact with the surface of the virus with an appropriate strength regardless of the presence or absence of an envelope, so the gelatin hydrolysate can protect the surface of the virus by coating it.

[0025] If the weight-average molecular weight of the above-mentioned gelatin hydrolysate exceeds 10,000, there is a risk of gelation at 2-8°C. There is no particular lower limit to the weight-average molecular weight of the gelatin hydrolysate, but for example, it is 75, which is the molecular weight of glycine.

[0026] The weight-average molecular weight of the above gelatin hydrolysate 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(registered trademark) G2000SW XL Column temperature: 30℃ Eluent: 40% by mass acetonitrile (containing 0.05% by mass TFA) Flow rate: 0.5mL / min Injection volume: 10μL Detection: UV220nm Molecular weight markers: The following three types are used. Cytochrom C Mw:12384 Aprotinin Mw:6512 Bacitracin Mw:1423.

[0027] Specifically, a sample (substance to be measured) for weight-average molecular weight measurement is prepared by adding 0.5 g of the virus stabilizer containing the above-mentioned gelatin hydrolysate to approximately 100 ml of distilled water, stirring, and then filtering through a 0.2 μm filter. By measuring this substance under the conditions of gel filtration chromatography described above, the weight-average molecular weight of the gelatin hydrolysate can be determined.

[0028] (isoelectric point) The above-mentioned gelatin hydrolysate has an isoelectric point pH of 4.0 to 7.0. Preferably, the isoelectric point pH of the above-mentioned gelatin hydrolysate is 4.0 to 5.5, and more preferably 4.0 to 4.8. Gelatin hydrolysate having an isoelectric point pH in such a range can be efficiently obtained by hydrolyzing alkali-treated collagen, or by hydrolyzing gelatin obtained from alkali-treated collagen (so-called alkali-treated gelatin). In other words, the above-mentioned gelatin hydrolysate is preferably a hydrolysate of alkali-treated gelatin. In particular, the above-mentioned gelatin hydrolysate is preferably a hydrolysate 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. Acid-treated gelatin has an isoelectric point pH of 8-9. In contrast, alkali-treated gelatin has an isoelectric point pH of 4.0-7.0. An example of such alkali-treated gelatin is porcine-derived alkali-treated gelatin (product name: "beMatrix® Gelatin LS-H", manufactured by Nitta Gelatin Co., Ltd.).

[0029] When a virus-containing composition is constructed from a virus stabilizer containing a gelatin hydrolysate having an isoelectric point pH within the above range, and a virus, the gelatin hydrolysate in the composition can suppress viral aggregation, thereby suppressing a decrease in viral titer. Although the detailed mechanism is unknown, gelatin hydrolysate having an isoelectric point pH within such a range has relatively more negative charges than positive charges within the molecule. Therefore, although it exhibits a negative charge overall, it has both positively charged and negatively charged sites. For this reason, the gelatin hydrolysate can bind to the virus by exhibiting weak electrostatic interactions between the positively charged sites and the negatively charged capsid protein, spike protein, and envelope protein on the virus surface. On the other hand, the gelatin hydrolysate repels the virus and other peptide chains constituting the gelatin hydrolysate at the negatively charged sites. Based on the above, it is presumed that the gelatin hydrolysate can protect the virus by coating its surface while suppressing viral aggregation. On the other hand, gelatin hydrolysates obtained from acid-treated gelatin with an isoelectric point pH of around 9 (for example, the gelatin hydrolysate disclosed in Patent Document 1 above) have a positive charge and therefore may agglutinate viruses by exhibiting strong electrostatic interactions with negatively charged capsid proteins and the like. It is presumed that the virus agglutination effect of gelatin hydrolysates obtained from acid-treated gelatin is particularly pronounced in temperature environments around 25°C.

[0030] The pH of the isoelectric point of a gelatin hydrolysate can be determined by measuring the pH of the isoelectric points of both or either the gelatin and collagen that are the raw materials for the gelatin hydrolysate using conventionally known methods. However, it is preferable to use the following method of measuring the isoelectric point using zeta potential as an indicator, as this allows for a more accurate determination of the isoelectric point value. Specifically, first, the gelatin hydrolysate to be measured is dissolved in acetate buffer (pH 4.0-5.5) to obtain a 0.4 w / v% solution to be measured. Next, the solution to be measured is filtered through a 0.22 μm filter (Merck), and then 0.8 mL of the solution to be measured is packed into a capillary cell, taking care to avoid air bubbles. Subsequently, the capillary cell filled with the solution to be measured is set in a zeta potential measuring device (Malvern Panalytical), and the zeta potential is measured at various pH values ​​at 25°C. At this time, the pH value at which the zeta potential becomes 0 can be determined as the isoelectric point of the solution to be measured (the gelatin hydrolysate to be measured).

[0031] <Water-based medium> The virus stabilizer contains an aqueous medium as described above. In this specification, "aqueous medium" refers to a medium for dissolving or dispersing gelatin hydrolysates, and means a medium that may contain components other than water, such as amino acids, sugars, and buffering salts, as described later. For example, the aqueous medium may be a buffer solution containing a buffering salt. Specifically, the aqueous medium may be a GTS buffer. That is, it is preferable that the aqueous medium contains a buffering salt. As a result, when the virus stabilizer contains a virus to constitute a virus-containing composition, it can contribute to the stabilization of the virus through the buffering action described above.

[0032] (A buffering salt) Examples of buffering salts include sodium phosphate, potassium phosphate, calcium phosphate, magnesium phosphate, sodium hydrogen phosphate, potassium hydrogen phosphate, calcium hydrogen phosphate, magnesium hydrogen phosphate, sodium chloride, and potassium chloride. The aqueous medium may contain one of the above buffering salts alone or a combination of two or more. Examples of aqueous mediums containing the above buffering salts include the above GTS buffer, PBS buffer, Tris buffer, and HEPES buffer. -na Examples include the following. The composition of the GTS buffer can be, for example, 2.5% by mass glycerol, 20 mM Tris pH 8, and 25 mM NaCl.

[0033] (amino acid) 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 these groups alone, or two or more in combination. It is more preferable that the aqueous medium contains both or either methionine and arginine. This allows the virus stabilizer to contribute more to the stabilization of the virus when it constitutes a virus-containing composition by containing a virus.

[0034] (Sugars) 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 sugar selected from these groups alone, or two or more in combination. It is more preferable that the aqueous medium contains at least one of sucrose, lactose, or sorbitol. This allows the virus stabilizer to contribute more to the stabilization of the virus when it constitutes a virus-containing composition by containing a virus. In this specification, the compounds included in "sugars" include not only organic compounds generally classified as sugars, but also organic compounds classified as sugar alcohols. The organic compounds classified as sugar alcohols in the above group of sugars are sorbitol, mannitol, maltitol, xylitol, erythritol, and glycerol.

[0035] (Other ingredients) The aqueous medium may contain other components insofar as the effects of the present invention are achieved. 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.

[0036] <Effects and Effects> The virus stabilizer according to this embodiment consists of an aqueous medium and a gelatin hydrolysate contained in a concentration of 1% to 20% by mass, as described above. The gelatin hydrolysate has a weight-average molecular weight of 10,000 or less and an isoelectric point pH of 4.0 to 7.0. When the virus stabilizer according to this embodiment contains a virus to form a virus-containing composition as described later, it can suppress the aggregation of the virus in the virus-containing composition and prevent adverse effects on the infectivity of the virus. As a result, the decrease in viral titer can be suppressed, and the virus-containing composition can be stably stored and transported at both refrigerated temperatures (2-8°C) and room temperature around 25°C.

[0037] [Hydrolyzed gelatin for virus stabilization] The gelatin hydrolysate for virus stabilization according to this embodiment has a weight-average molecular weight of 10,000 or less and an isoelectric point pH of 4.0 to 7.0. Preferably, the gelatin hydrolysate for virus stabilization has a weight-average molecular weight of 6,000 or less, or an isoelectric point pH of 4.0 to 5.5. Specifically, the gelatin hydrolysate for virus stabilization has the characteristics described in the section on <Gelatin Hydrolysate>, so we will not repeat the same explanation. The gelatin hydrolysate for virus stabilization has an attribute not previously known for gelatin hydrolysates, i.e., an unknown attribute, which is that when it contains a virus to form a virus-containing composition as described later, it has the effect of suppressing the aggregation of the virus in the virus-containing composition. Thus, the gelatin hydrolysate for virus stabilization can be used as a virus stabilizer, and the virus-containing composition can be stably stored and transported at both refrigerated temperatures (2 to 8°C) and room temperature around 25°C without the need for a refrigeration device.

[0038] The above-mentioned gelatin hydrolysate for virus stabilization is preferably in liquid or powder form. If the gelatin hydrolysate for virus stabilization is liquid, a virus-containing composition can be easily prepared by adding the virus together with an aqueous medium. If the gelatin hydrolysate for virus stabilization is powder, a virus-containing composition can be easily prepared by dissolving or dispersing it in an aqueous medium to prepare a virus stabilizer, and then adding the virus to this stabilizer. The gelatin hydrolysate for virus stabilization can be prepared as a liquid by hydrolyzing both or either gelatin and collagen, followed by purification. The gelatin hydrolysate for virus stabilization can be prepared as a powder by heating and drying or freeze-drying the liquid gelatin hydrolysate for virus stabilization prepared as described above using a known method.

[0039] [Virus-containing composition] The virus-containing composition according to this embodiment comprises the above-mentioned virus stabilizer and a virus. By including the virus stabilizer, the virus-containing composition can suppress the aggregation of the virus within the composition, thereby suppressing a decrease in the viral titer. This allows the virus-containing composition to be stably stored and transported at both refrigerated temperatures (2-8°C) and room temperature around 25°C without the need for a refrigeration device. The virus stabilizer contained in the virus-containing composition has the characteristics described in the section on [Virus Stabilizer], so a redundant explanation will not be repeated.

[0040] <virus> The virus-containing composition contains a virus as described above. Preferably, the virus 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. In other words, the type of virus used to constitute the virus-containing composition is not limited. Furthermore, the virus-containing composition may contain one virus selected from the above group alone, or it may contain a combination of two or more viruses. Even in such cases, the virus-containing composition can be stably stored and transported at both refrigerated temperatures (2-8°C) and room temperature around 25°C by containing a virus stabilizer.

[0041] The viruses mentioned above can specifically include viruses used as live or inactivated vaccines such as influenza virus and rotavirus, oncolytic viruses that have tumor-lysing properties, herpes simplex virus, vaccinia virus, adenovirus, coxsackievirus, poxvirus, paramyxovirus, picornavirus, rapdovirus, reovirus, parvovirus, bracken virus, orthomyxovirus, retrovirus, and the like. Furthermore, the viruses mentioned above may be genetically modified.

[0042] <Other ingredients> The virus-containing composition may contain other components as long as the effects of the present invention are achieved. 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.

[0043] <Logarithmic reduction in viral titer (LRV)> The virus-containing composition preferably has a difference of 1.5 log or less between the logarithmic decrease in viral titer (hereinafter also referred to as "LRV") when stored at 25°C for 21 days and the LRV of the viral titer when stored at -80°C for 21 days. More preferably, the above difference is 1.0 log or less. The lower limit of the above difference for the virus-containing composition is preferably 0.

[0044] The virus-containing composition preferably has a difference of 1.0 log or less between the LRV (Liquid Resistance Value) of the virus titer after 210 days of storage at 4°C and the LRV after 210 days of storage at -80°C. More preferably, the above difference is 0.5 log or less. The lower limit of the above difference for the virus-containing composition is preferably 0.

[0045] When a virus-containing composition possesses the characteristics of LRV with the viral titer described above, it is ensured that the virus can be stably stored and transported at both refrigerated temperatures (2-8°C) and room temperatures around 25°C.

[0046] Furthermore, it is preferable that the virus-containing composition has a difference of 1.5 log or less between the paired LRV of the viral titer when stored at 25°C for 21 days and the LRV of the viral titer when stored at -80°C for 21 days, and also a difference of 1.0 log or less between the LRV of the viral titer when stored at 4°C for 21 days and the LRV of the viral titer when stored at -80°C for 21 days.

[0047] Preferably, the above-mentioned virus-containing composition has a logarithmic decrease in viral titer of 0.3log or less after three freeze-thaw cycles. This allows the virus-containing composition to maintain its viral titer even after up to three freeze-thaw cycles. This makes the virus-containing composition extremely convenient as it allows for flexible use.

[0048] (Method for measuring the logarithmic decrease in viral titer (LRV)) In a virus-containing composition, the viral titer LRV can be determined using conventional methods for measuring viral titer, for example, by determining 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 (dilution of the "virus-containing composition" in this specification) is inoculated onto a culture dish such as a test tube or 96-well flat plate to which cells have been previously cultured and attached. The above infection can be confirmed by microscopy, for example, using cytopathic effect (CPE) as an indicator. The viral titer LRV can be calculated based on the TCID50 calculated according to the Beherens-Karber formula.

[0049] The method for measuring the viral titer of LRV can be as follows. Specifically, DMEM medium can be used as a diluent to dilute the virus-containing composition after it has been stored for a predetermined period. Cell culture is performed using 100 μL / well (approximately 10) of the predetermined cell suspension. 4 This can be done by sowing (1 / well) into a 96-well flat-bottom plate (manufactured by TPP) and incubating it overnight in a 37°C CO2 incubator (CO2 concentration 5%).

[0050] Subsequently, the virus-containing composition is serially diluted 10-fold in DMEM medium. The diluted virus is added at a rate of 50 μL / well to designated cells in a 96-well flat-bottom plate (various cells corresponding to the virus species contained in the virus-containing composition, such as LCC-MK2 cells, MDBK cells, HEK293 cells, etc.), cultured in a 37°C CO2 incubator (CO2 concentration 5%), and the viral titer (TCID50) is determined on the 7th day using CPE as an indicator and according to the calculation formula described above. Based on this TCID50 value, the LRV can be calculated.

[0051] Furthermore, if the virus species contained in the virus-containing composition is rNDV-GFP, the viral titer LRV can be calculated as follows: Diluted virus is added to Vero cells in a 24-well flat-bottom plate at a volume of 50 μL / well, cultured in a 37°C CO2 incubator (CO2 concentration 5%) for 24-48 hours, and the cells are recovered using 0.25% by mass trypsin EDTA (SIGMA Aldrich). The cells are then washed with DMEM medium and PBS. The cells are then suspended in 0.5% by mass formalin-added PBS, the number of infected cells is measured by FCM analysis or fluorescence microscopy, and the viral titer (FFU / mL) is determined according to the above-mentioned formula. The LRV can then be calculated based on this viral titer (FFU / mL).

[0052] <Method for preparing virus stabilizers> The virus stabilizer according to this embodiment can preferably be obtained by the following method: a first step of preparing a gelatin hydrolysate and a second step of preparing a virus stabilizer consisting of the gelatin hydrolysate and an aqueous medium.

[0053] (1st step) The first step is to prepare a gelatin hydrolysate. As described above, the gelatin hydrolysate can be prepared by hydrolyzing either or both gelatin having an isoelectric point pH of 4.0 to 7.0, or collagen whose isoelectric point pH has been adjusted to 4.0 to 7.0 by alkali treatment, so that the weight-average molecular weight is 10,000 or less. It is preferable to use alkali-treated gelatin as the gelatin having an isoelectric point pH of 4.0 to 7.0. Furthermore, commercially available products (for example, product name: "beMatrix(registered trademark) gelatin") can also be used. LS-H Gelatin hydrolysate can also be prepared using a product manufactured by Nitta Gelatin Co., Ltd.

[0054] (2nd process) The second step is to prepare a virus stabilizer from the above-mentioned gelatin hydrolysate and aqueous medium. The virus stabilizer can be prepared by mixing the above-mentioned gelatin hydrolysate and aqueous medium in a mass ratio such that the concentration of the gelatin hydrolysate is 1% by mass or more and 20% by mass or less. A conventionally known method can be used for the specific mixing method. The aqueous medium can also be prepared by a conventionally known method, such as adding a buffering salt to deionized water to a predetermined concentration.

[0055] <Method for preparing a virus-containing composition> The virus-containing composition can be prepared by adding a virus to the above-mentioned virus stabilizer. The virus can be added to the above-mentioned virus stabilizer by conventionally known methods. The virus is usually added to the above-mentioned virus stabilizer until the viral titer (TCID50) is ultimately 10 5 It can be added in a quantity of 1 / mL or more. Thus, the virus-containing composition according to this embodiment can be obtained. [Examples]

[0056] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0057] [Preparation of the sample] The virus-containing compositions used in the various experiments described later (Experiments 1 to 6) were prepared as follows (Samples 1 to 17). Samples 1 to 4, 9 to 12, and 14 to 17 are examples, while samples 5 to 8 and 13 are comparative examples.

[0058] <Sample 1> Gelatin hydrolysate was obtained by thermally hydrolyzing "beMatrix® Gelatin LS-H (manufactured by Nitta Gelatin Co., Ltd.)", an alkali-treated gelatin derived from pigskin with an isoelectric point pH of 5, to a weight-average molecular weight of 3800. A virus stabilizer was then obtained by dissolving the above gelatin hydrolysate in GTS buffer, an aqueous medium, to a concentration of 5% by mass. Bovine herpesvirus (BHV-1), an enveloped DNA virus, was added to the above virus stabilizer, resulting in a TCID50 of 2 × 10⁻⁶. 8.1 A virus-containing composition was obtained by adding the virus to a concentration of / mL. The virus-containing composition was then divided into seven samples 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.

[0059] <Sample 2> Gelatin hydrolysate was obtained by thermally hydrolyzing "beMatrix® Gelatin LS-H (manufactured by Nitta Gelatin Co., Ltd.)", an alkali-treated gelatin derived from pigskin with an isoelectric point pH of 5, to a weight-average molecular weight of 650. Next, a virus stabilizer was obtained by dissolving the above gelatin hydrolysate in the above GTS buffer to a concentration of 5% by mass. Subsequently, a virus-containing composition was obtained by adding the virus to the above virus stabilizer using the same method as for sample 1. The above virus-containing composition was divided into four samples and stored at 4°C for 56 days, at 25°C for 21 days, and at -80°C for 21 days and 56 days, respectively.

[0060] <Sample 3> Gelatin hydrolysate was obtained by thermally hydrolyzing "beMatrix® Gelatin LS-H (manufactured by Nitta Gelatin Co., Ltd.)", an alkali-treated gelatin derived from pigskin with an isoelectric point pH of 5, to a weight-average molecular weight of 1980. Next, a virus stabilizer was obtained by dissolving the above gelatin hydrolysate in the above GTS buffer to a concentration of 5% by mass. Subsequently, a virus-containing composition was obtained by adding the virus to the above virus stabilizer using the same method as for sample 1. The above virus-containing composition was divided into four samples and stored at 4°C for 56 days, at 25°C for 21 days, and at -80°C for 21 days and 56 days, respectively.

[0061] <Sample 4> Gelatin hydrolysate was obtained by thermally hydrolyzing "beMatrix® Gelatin LS-H (manufactured by Nitta Gelatin Co., Ltd.)", an alkali-treated gelatin derived from pigskin with an isoelectric point pH of 5, to a weight-average molecular weight of 9890. Next, a virus stabilizer was obtained by dissolving the above gelatin hydrolysate in the above GTS buffer to a concentration of 5% by mass. Subsequently, a virus-containing composition was obtained by adding the virus to the above virus stabilizer using the same method as for sample 1. The above virus-containing composition was divided into four samples and stored at 4°C for 56 days, at 25°C for 21 days, and at -80°C for 21 days and 56 days, respectively.

[0062] <Sample 5> Gelatin hydrolysate was obtained by thermally hydrolyzing "beMatrix® Gelatin LS-H (manufactured by Nitta Gelatin Co., Ltd.)", an alkali-treated gelatin derived from pigskin with an isoelectric point pH of 5, to a weight-average molecular weight of 59800. Next, a virus stabilizer was obtained by dissolving the above gelatin hydrolysate in the above GTS buffer to a concentration of 5% by mass. Subsequently, a virus-containing composition was obtained by adding the virus to the above virus stabilizer using the same method as for sample 1. Furthermore, the above virus-containing composition was divided into four samples and stored at 4°C for 56 days, at 25°C for 21 days, and at -80°C for 21 days and 56 days, respectively.

[0063] <Sample 6> Gelatin hydrolysate was obtained by hydrolyzing gelatin hydrolysate (manufactured by Nitta Gelatin Co., Ltd.) obtained from acid-treated porcine skin gelatin with an isoelectric point pH of 9, by heat, so that the weight-average molecular weight was 2060. Next, a virus stabilizer was obtained by dissolving the above gelatin hydrolysate in the above GTS buffer to a concentration of 5% by mass. Subsequently, a virus-containing composition was obtained by adding the virus to the above virus stabilizer using the same method as for sample 1. The above virus-containing composition was divided into four samples and stored at 4°C for 56 days, at 25°C for 21 days, and at -80°C for 21 days and 56 days, respectively.

[0064] <Sample 7> Gelatin hydrolysate was obtained by thermally hydrolyzing "beMatrix® Gelatin LS-H (manufactured by Nitta Gelatin Co., Ltd.)", an alkali-treated gelatin derived from pigskin with an isoelectric point pH of 5, to a weight-average molecular weight of 3800. A virus-containing composition was then obtained by the same method as for sample 1, except that a virus stabilizer was obtained by dissolving the above gelatin hydrolysate in the above GTS buffer to a concentration of 0.1% by mass. The above virus-containing composition was divided into four samples and stored at 4°C for 56 days, at 25°C for 21 days, and at -80°C for 21 days and 56 days, respectively.

[0065] <Sample 8> Gelatin hydrolysate was obtained by thermally hydrolyzing "beMatrix® Gelatin LS-H (manufactured by Nitta Gelatin Co., Ltd.)", an alkali-treated gelatin derived from pigskin with an isoelectric point pH of 5, to a weight-average molecular weight of 3800. A virus-containing composition was then obtained by the same method as for sample 1, except that a virus stabilizer was obtained by dissolving the above gelatin hydrolysate in the above GTS buffer to a concentration of 0.5% by mass. The above virus-containing composition was divided into four samples and stored at 4°C for 56 days, at 25°C for 21 days, and at -80°C for 21 days and 56 days, respectively.

[0066] <Sample 9> Gelatin hydrolysate was obtained by thermally hydrolyzing "beMatrix® Gelatin LS-H (manufactured by Nitta Gelatin Co., Ltd.)", an alkali-treated gelatin derived from pigskin with an isoelectric point pH of 5, so that its weight-average molecular weight was 3800. A virus-containing composition was then obtained by the same method as for sample 1, except that a virus stabilizer was obtained by dissolving the above gelatin hydrolysate in the above GTS buffer to a concentration of 1% by mass. The above virus-containing composition was divided into four samples and stored at 4°C for 56 days, at 25°C for 21 days, and at -80°C for 21 days and 56 days, respectively.

[0067] <Sample 10> Gelatin hydrolysate was obtained by thermally hydrolyzing "beMatrix® Gelatin LS-H (manufactured by Nitta Gelatin Co., Ltd.)", an alkali-treated gelatin derived from pigskin with an isoelectric point pH of 5, so that its weight-average molecular weight was 3800. A virus-containing composition was then obtained by the same method as for sample 1, except that a virus stabilizer was obtained by dissolving the above gelatin hydrolysate in the above GTS buffer to a concentration of 2.5% by mass. The above virus-containing composition was divided into four samples and stored at 4°C for 56 days, at 25°C for 21 days, and at -80°C for 21 days and 56 days, respectively.

[0068] <Sample 11> Gelatin hydrolysate was obtained by thermally hydrolyzing "beMatrix® Gelatin LS-H (manufactured by Nitta Gelatin Co., Ltd.)", an alkali-treated gelatin derived from pigskin with an isoelectric point pH of 5, so that its weight-average molecular weight was 3800. A virus-containing composition was then obtained by the same method as for sample 1, except that a virus stabilizer was obtained by dissolving the above gelatin hydrolysate in the above GTS buffer to a concentration of 10% by mass. The above virus-containing composition was divided into four samples and stored at 4°C for 56 days, at 25°C for 21 days, and at -80°C for 21 days and 56 days, respectively.

[0069] <Sample 12> Gelatin hydrolysate was obtained by thermally hydrolyzing "beMatrix® Gelatin LS-H (manufactured by Nitta Gelatin Co., Ltd.)", an alkali-treated gelatin derived from pigskin with an isoelectric point pH of 5, so that its weight-average molecular weight was 3800. A virus-containing composition was then obtained by the same method as for sample 1, except that a virus stabilizer was obtained by dissolving the above gelatin hydrolysate in the above GTS buffer to a concentration of 20% by mass. The above virus-containing composition was divided into four samples and stored at 4°C for 56 days, at 25°C for 21 days, and at -80°C for 21 days and 56 days, respectively.

[0070] <Sample 13> A virus stabilizer was obtained by dissolving recombinant human albumin "Recombumin® Elite" (manufactured by Albumedix) in the above GTS buffer to a concentration of 1% by mass. Subsequently, a virus-containing composition was obtained by adding the virus to the virus stabilizer using the same method as for sample 1. Furthermore, the virus-containing composition was divided into four samples and 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.

[0071] <Sample 14> As a virus stabilizer, parainfluenza virus (PI-3), an enveloped RNA virus, is stabilized with TCID50 at a rate of 2 × 10⁻¹⁶. 5.5 A virus-containing composition was obtained using the same method as for Sample 1, except that it was added to a concentration of / mL. The above virus-containing composition was divided into seven samples 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.

[0072] <Sample 15> As a virus stabilizer, TCID50 was used to stabilize reovirus (Reo-3), an RNA virus that does not have an envelope, at a concentration of 2 × 10⁻¹⁶. 6.5A virus-containing composition was obtained using the same method as for Sample 1, except that it was added to a concentration of / mL. The above virus-containing composition was divided into seven samples 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.

[0073] <Sample 16> As a virus stabilizer, TCID50 is used to stabilize adenovirus (Ad5), a DNA virus that does not have an envelope, in a ratio of 2 × 10⁻¹⁶. 8 A virus-containing composition was obtained using the same method as for Sample 1, except that it was added to a concentration of / mL. The above virus-containing composition was divided into seven samples 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.

[0074] <Sample 17> The virus stabilizer is a paramyxovirus (Newcastle disease virus: rNDV-GFP), an oncolytic virus that has been genetically modified with GFP and is an enveloped RNA virus, with FFU levels of 4 × 10⁻¹⁴. 6 A virus-containing composition was obtained using the same method as for Sample 1, except that it was added to contain / mL. The above virus-containing composition was divided into four samples and stored at 4°C for 56 days, at 25°C for 21 days, and at -80°C for 21 days and 56 days, respectively. Table 1 shows a list of Samples 1 to 17. Here, the pH of the isoelectric point of the gelatin hydrolysate contained in the virus stabilizer in Samples 1 to 4, Samples 9 to 12, and Samples 14 to 17 is shown as the value measured by the isoelectric point measurement method using the zeta potential as an indicator, as described above.

[0075] [Table 1]

[0076] [Experiment 1: Investigation of the appropriate weight-average molecular weight of gelatin hydrolysates] For samples 1, 2, 3, 4, and 5, the TCID50 was determined for each sample stored at 4°C for 56 days and at 25°C for 21 days. Based on these TCID50 values, the LRV (Liquid Resistance Value) at 4°C and at 25°C was calculated. Furthermore, for samples 1, 2, 3, 4, and 5, the TCID50 was determined for each sample stored at -80°C for 21 days and at 56 days. Based on these TCID50 values, the LRV (Liquid Resistance Value) at -80°C was calculated. Based on these results, the difference between the LRV (Liquid Resistance Value) of each sample stored at 25°C for 21 days and the LRV (Liquid Resistance Value) of each sample stored at -80°C for 21 days was calculated for samples 1, 2, 3, 4, and 5. The results are shown in Table 2. Furthermore, Table 3 shows the results of calculating the difference between the LRV (Liquid Resistance Value) of the viral titer in each sample stored at 4°C for 56 days and the LRV of the viral titer in each sample stored at -80°C for 56 days for Samples 1, 2, 3, and 4.

[0077] [Table 2]

[0078] [Table 3]

[0079] According to Tables 2 and 3 above, samples 1, 2, 3, and 4 have LRV differences of 1.0 log or less and 1.5 log or less, respectively, and can be evaluated as being able to stably store and transport the virus at 4°C (refrigeration temperature) and 25°C (room temperature). Sample 5, due to gelation, showed an LRV difference exceeding 1.5 log at 25°C. However, evaluation of sample 5 at 4°C for 56 days was not performed due to gelation.

[0080] [Experiment 2: Investigation of the appropriate isoelectric point of gelatin hydrolysates] For samples 1, 2, 3, 4, and 6, the TCID50 was determined for each sample stored at 4°C for 56 days and at 25°C for 21 days. Based on these TCID50 values, the LRV (Liquid Resistance Value) of the viral titer at 4°C and at 25°C was calculated. Furthermore, for samples 1, 2, 3, 4, and 6, the TCID50 was determined for each sample stored at -80°C for 21 days and at 56 days. Based on these TCID50 values, the LRV (Liquid Resistance Value) of the viral titer at -80°C was calculated. Based on these results, the difference between the LRV (Liquid Resistance Value) of the viral titer in each sample stored at 25°C for 21 days and the LRV (Liquid Resistance Value) in each sample stored at -80°C for 21 days was calculated for the virus-containing compositions of samples 1, 2, 3, 4, and 6. The results are shown in Table 4. Furthermore, Table 5 shows the results of calculating the difference between the LRV (Liquid Resistance Value) of the viral titer in each sample stored at 4°C for 56 days and the LRV of the viral titer in each sample stored at -80°C for 56 days for samples 1, 2, 3, 4, and 6.

[0081] [Table 4]

[0082] [Table 5]

[0083] According to Tables 4 and 5 above, samples 1, 2, 3, and 4 can be evaluated as being able to stably store and transport the virus at 4°C (refrigeration temperature) and 25°C (room temperature) because the LRV difference for each sample is 1.0 log or less and 1.5 log or less, respectively. Sample 6 had an LRV difference exceeding 1.5 log at 25°C and exceeding 1.0 log at 4°C.

[0084] [Experiment 3: Investigation of the appropriate concentration of gelatin hydrolysate in the virus stabilizer] For samples 1, 7, 8, 9, 10, 11, and 12, the TCID50 was determined for each sample stored at 4°C for 56 days and at 25°C for 21 days. Based on these TCID50 values, the LRV (Liquid Resistance Value) of the viral titer at 4°C and at 25°C was calculated. Furthermore, for samples 1, 7, 8, 9, 10, 11, and 12, the TCID50 was determined for each sample stored at -80°C for 21 days and for 56 days. Based on these TCID50 values, the LRV (Liquid Resistance Value) of the viral titer at -80°C was calculated. Based on these results, for samples 1, 7, 8, 9, 10, 11, and 12, the difference between the LRV of the viral titer in each sample stored at 25°C for 21 days and the LRV of each sample stored at -80°C for 21 days was calculated. The results are shown in Table 6. Furthermore, Table 7 shows the results of calculating the difference between the LRV (Liquid Resistance Value) of the viral titer in each sample stored at 4°C for 56 days and the LRV of each sample stored at -80°C for 56 days for samples 1, 7, 8, 9, 10, 11, and 12.

[0085] [Table 6]

[0086] [Table 7]

[0087] According to Tables 6 and 7 above, Samples 1, 9, 10, 11, and 12 can be evaluated as being able to stably store and transport the virus at 4°C (refrigeration temperature) and 25°C (room temperature) because the LRV difference for each sample is 1.0 log or less and 1.5 log or less, respectively. For Samples 7 and 8, the LRV difference for each sample was greater than 1.5 log at 25°C and greater than 1.0 log at 4°C.

[0088] [Experiment 4: Comparison with conventional virus stabilizers] For samples 1 and 13, the TCID50 was determined for each sample stored at 4°C for 56 days and at 25°C for 21 days. Based on these TCID50 values, the LRV (Liquid Resistance Value) at 4°C and 25°C was calculated. Furthermore, for samples 1 and 13, the TCID50 was determined for each sample stored at -80°C for 21 days and 56 days. Based on these TCID50 values, the LRV (Liquid Resistance Value) at -80°C was calculated. Based on these, the difference between the LRV (Liquid Resistance Value) of each sample stored at 25°C for 21 days and the LRV (Liquid Resistance Value) of each sample stored at -80°C for 21 days was calculated for samples 1 and 13. The results are shown in Table 8. Furthermore, Table 9 shows the results of calculating the difference between the LRV (Liquid Resistance Value) of each sample stored at 4°C for 56 days and the LRV (Liquid Resistance Value) of each sample stored at -80°C for 56 days for samples 1 and 13.

[0089] [Table 8]

[0090] [Table 9]

[0091] According to Tables 8 and 9 above, Sample 1 can be evaluated as being able to suppress the decrease in viral titer at 4°C (refrigerated temperature) and 25°C (room temperature) compared to Sample 13.

[0092] [Experiment 5: Examination of the effectiveness of virus stabilizers for each type of virus] For samples 1, 14, 15, 16, and 17, the TCID50 was determined for each sample stored at 25°C for 21 days, and the LRV (Liquid Resistance Value) of the viral titer at 25°C was calculated based on the TCID50 value. Furthermore, for samples 1, 14, 15, 16, and 17, the TCID50 was determined for each sample stored at -80°C for 21 days, and the LRV (Liquid Resistance Value) of the viral titer at -80°C was calculated based on the TCID50 value. Based on these, the difference between the LRV of the viral titer in each sample stored at 25°C for 21 days and the LRV of the viral titer in each sample stored at -80°C for 21 days was calculated for samples 1, 14, 15, 16, and 17. For sample 17, FFU (Fever-Factor Unit) was calculated instead of TCID. The results are shown in Table 10.

[0093] Furthermore, for samples 1, 14, 15, and 16, the TCID50 was determined for each sample stored at 4°C for 21 days, 56 days, and 210 days, respectively, and the LRV (Liquid Resistance Value) of the viral titer at 4°C was calculated based on these TCID50 values. For samples 1, 14, 15, and 16, the TCID50 was also determined for each sample stored at -80°C for 21 days, 56 days, and 210 days, respectively, and the LRV (Liquid Resistance Value) of the viral titer at -80°C was calculated based on these TCID50 values. Based on these, the difference between the LRV of the viral titer in each sample stored at 4°C for 21 days, 56 days, and 210 days for samples 1, 14, 15, and 16 and the LRV of the viral titer in each sample stored at -80°C for 21 days, 56 days, and 210 days was calculated. The results are shown in Table 11.

[0094] [Table 10]

[0095] [Table 11]

[0096] According to Tables 10 and 11 above, samples 1, 14, 15, 16, and 17 have LRV differences of 1.0 log or less and 1.5 log or less, respectively, and can be evaluated as being able to stably store and transport the virus at 4°C (refrigeration temperature) and 25°C (room temperature). Furthermore, according to Tables 10 and 11 above, samples 1, 14 to 16, which maintained activity for 3 weeks at 25°C, were all able to maintain activity for up to 30 weeks at 4°C, so it is highly likely that sample 17 can similarly maintain activity for up to 30 weeks at 4°C (refrigeration temperature).

[0097] Considering the results of experiments 1 to 5 described above, each of the examples of samples 1 to 4, samples 9 to 12, and samples 14 to 17 can be evaluated as being able to stably store and transport the virus for 21 days at a temperature of 4°C.

[0098] [Experiment 6: Effects of repeated freeze-thaw cycles on virus stabilizers] Sample 1 was frozen at -80°C and then thawed in ice water. This procedure was repeated three times. The viral titer of Sample 1 was measured after each thawing (first, second, and third freeze-thaw cycles), and the logarithmic reduction value (LRV) was calculated to compare it with the viral titer of Sample 1 before freeze-thawing. The results are shown in Table 12.

[0099] [Table 12]

[0100] According to Table 12 above, the LRV of sample 1 remained near 0 after 1 to 3 freeze-thaw cycles, indicating that the viral titer can be maintained even after freeze-thawing.

[0101] As described above, embodiments and examples of the present invention have been explained, but it is also intended from the outset that the configurations of each of the above embodiments and examples may be combined as appropriate.

[0102] The embodiments and examples disclosed herein should be considered in all respects to be illustrative and not restrictive. The scope of the present invention is indicated by the claims rather than by the foregoing description, and all modifications within the meaning and scope of the claims are intended to be included.

Claims

1. A virus stabilizer comprising a gelatin hydrolysate and an aqueous medium, The virus stabilizer contains 1% by mass or more and 20% by mass or less of the gelatin hydrolysate, The aforementioned gelatin hydrolysate has a weight-average molecular weight of 10,000 or less. The aforementioned gelatin hydrolysate has an isoelectric point pH of 4.0 or higher and 7.0 or lower. The aforementioned gelatin hydrolysate is a hydrolysate of alkali-treated gelatin, The aqueous medium is a virus stabilizer, which is at least one selected from the group consisting of GTS buffer, PBS buffer, Tris buffer, and HEPES buffer.

2. The virus stabilizer according to claim 1, wherein the gelatin hydrolysate has a weight-average molecular weight of 6000 or less.

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

4. The virus stabilizer according to any one of claims 1 to 3, wherein the aqueous medium contains a salt having a buffering effect.

5. The virus stabilizer according to any one of claims 1 to 4, wherein the aqueous medium comprises at least one sugar selected from the group consisting of sucrose, lactose, sorbitol, inositol, trehalose, mannitol, maltitol, xylitol, erythritol, and glycerol.

6. The virus stabilizer according to any one of claims 1 to 5, wherein the aqueous medium contains at least one amino acid selected from the group consisting of methionine, arginine, tryptophan, glutamine, and glutamic acid.

7. A gelatin hydrolysate for use in a virus stabilizer according to any one of claims 1 to 6, The weight-average molecular weight is 10,000 or less, and the pH at the isoelectric point is 4.0 or higher and 7.0 or lower, and Gelatin hydrolysate for use as a virus stabilizer, which is a hydrolyzed product of alkali-treated gelatin.

8. The gelatin hydrolysate for virus stabilization according to claim 7, wherein the gelatin hydrolysate for virus stabilization is a liquid or a powder.

9. A virus-containing composition comprising a virus stabilizer according to any one of claims 1 to 6 and a virus.

10. The virus-containing composition according to claim 9, wherein the virus comprises at least one selected from the group consisting of an enveloped DNA virus, an envelopeless DNA virus, an enveloped RNA virus, and an envelopeless RNA virus.

11. The virus-containing composition according to claim 9 or claim 10, wherein the virus has been genetically modified.

12. The virus-containing composition according to any one of claims 9 to 11, wherein the difference between the logarithmic decrease in viral titer when stored at 25°C for 21 days and the logarithmic decrease in viral titer when stored at -80°C for 21 days is 1.5 log or less.

13. The virus-containing composition according to any one of claims 9 to 12, wherein the difference between the logarithmic decrease in viral titer when stored at 4°C for 210 days and the logarithmic decrease in viral titer when stored at -80°C for 210 days is 1.0 log or less.

14. The virus-containing composition according to any one of claims 9 to 13, wherein the logarithmic decrease in viral titer after three freeze-thaw cycles is 0.3 log or less.

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