Method for producing inactivated influenza vaccine by egg culture method

Abstract

A method for producing an inactivated influenza vaccine by an egg culture method according to the present disclosure includes a pathogen inactivation step for inactivating pathogens mixed into an egg other than influenza viruses.

Description

TECHNICAL FIELD

[0001] The present application relates to a manufacturing method of an inactivated influenza vaccine based on a chicken egg culture method. BACKGROUND

[0002] Influenza viruses belong to the Orthomyxoviridae family classified as "negative-strand single-strand RNA viruses with envelope" in virus taxonomy, and are known to have types A, B, C, D. The difference in types is based on the difference in antigenicity of the structural protein, i.e., M1 protein and nucleoprotein (NP), in the protein that constitutes the virion. In addition, there are differences in pathology, morphology, and genes, and in particular, A and B types differ greatly from C and D types. Types A and B are repeatedly prevalent every year and in many cases cause human influenza.

[0003] Influenza viruses were isolated from pigs in 1931, and human influenza viruses were isolated in 1933. At the same time, since it was known that influenza viruses proliferate in developing chicken eggs, an inactivated whole virus vaccine obtained by inactivating the virus with formaldehyde after culturing, collecting, and purifying the virus by inoculating the virus into developing chicken eggs was used. By chemically modifying the virus gene or virus protein, the virus only loses infectivity, and thus the virus becomes a vaccine antigen in a state of maintaining the particle shape.

[0004] However, the original inactivated whole virus vaccine had high side effects such as fever, and thus a method of removing the lipid membrane component considered to be the cause thereof was devised. For example, a vaccine in which hemagglutinin (HA), which is the main component of the prototype of the current split vaccine, is purified by zonal ultracentrifugation and separated with a surfactant, and the lipid membrane component that causes fever is removed with diethyl ether, is highly safe. The split vaccine has been manufactured since 1964 and until now (non-patent literature 1). Note that it has been reported that in the split vaccine, the nucleic acid and high molecular weight protein of the virus are removed together with the splitting agent, and HA and neuraminidase (NA), which are antigen components, are retained (non-patent literature 2).

[0005] For the manufacture of influenza vaccines, a method of inoculating a target virus strain into hatched chicken eggs to proliferate and purify it is widely used. In Japan, the aforementioned split vaccine containing purified HA (and NA) as the main antigen is circulated. As an example of the manufacture of a seasonal influenza vaccine, A-type strains and B-type strains as seasonal strains are separately cultured in developing chicken eggs to recover and formulate them with the HA component as the main antigen. HA is one of the surface antigens of influenza viruses and participates in the adsorption of viruses to host cells. By inoculating the formulation, antibodies against HA are produced, and they function as defense antibodies against influenza viruses, and thus it is expected to prevent influenza.

[0006] Instead of the conventional egg, a vaccine produced by proliferating a virus in cultured cells is called a cell culture influenza vaccine. As the cells, EB66 cells (derived from duck embryonic stem cells), MDCK cells (derived from canine kidney tubular epithelial cells), Vero cells (derived from African green monkey kidney epithelial cells), and the like are used. The cell culture method has advantages in flexibility and rapidity of vaccine production, but in seasonal influenza vaccines in Japan, an inactivated split vaccine based on the egg culture method is mainstream. Regardless of the production method, since the vaccine is inoculated into animals such as humans, it is necessary to be highly safe, and it is required to inactivate the proliferated virus and purify the main antigen of the vaccine at a high purity. Further, in biological pharmaceuticals such as vaccines, since a raw material derived from a living organism is used, necessary measures to be taken in order to ensure the quality, effectiveness, and safety of pharmaceuticals are stipulated in Japan by "Biological Raw Material Standards" (Non-Patent Document 3: Ordinance of the Ministry of Health, Labour and Welfare No. 37 / February 28, 2018). Since the raw material derived from these living organisms is a biological macromolecule derived from a living organism, there is a risk of infection with a pathogen such as a virus. In the production method of an influenza vaccine based on the egg culture method, a pathogen inactivation or removal process caused by an egg as a raw material is also important.

[0007] For a typical production method of an inactivated split influenza vaccine based on the egg culture method, two strains of type A and two strains of type B of influenza viruses, a total of four production strains, are separately cultured in developing eggs. Then, the allantoic fluid containing the proliferated viruses is recovered, and a vaccine bulk (single bulk) is prepared by processes of ultracentrifugation, sucrose density gradient centrifugation, ether treatment, and formalin inactivation. After the single bulk is prepared for the four production strains, dilution with a buffer is performed to prepare a formulation containing a predetermined amount of HA of each strain of virus and to be formulated.

[0008] In the typical production method of an inactivated split influenza vaccine based on the egg culture method, the inactivation process of the influenza virus is performed as the last process of the production method by formalin treatment or the like. When a pathogen is mixed in an egg as a raw material, the pathogen is usually excluded in the sorting inspection of the developing egg or in the subsequent purification process. However, since this exclusion is not complete, a bulk in which the pathogen cannot be removed before the preparation of the bulk is discarded.

[0009] β-propiolactone (BPL; C3H4O2) is a β-lactone having a ring strain with a molecular weight of 72.06, and is also referred to as 2-oxetanone or β-proprione. BPL easily cleaves between O-H2C to become an alkylating agent that attacks a nucleophilic functional group of a biological macromolecule, and thus, an inactivation effect on influenza viruses is expected. Evaluation of BPL treatment of influenza viruses is disclosed in Non-Patent Document 4 and the like.

[0010] As a method for decomposing host cell nucleic acid accompanying influenza virus or viral antigen thereof produced by cell culture, a method of performing BPL treatment is disclosed in patent documents 1 and 2.

[0011] On the other hand, for the production of inactivated influenza vaccine based on the egg culture method, the introduction of the BPL treatment process has been studied in patent document 3. Patent document 3 discloses that it was found that if formalin treatment is performed after pre-treatment of influenza virus with BPL, the decrease in the natural immunization activation ability of influenza vaccine antigen caused by formalin can be suppressed. In addition, patent document 3 also discloses that it aims to provide an inactivated influenza vaccine (specifically, a whole virus vaccine) that improves the natural immunization activation ability (immunogenicity) of influenza vaccine antigen accompanying formalin treatment, and a method for producing the same.

[0012] Prior Art Documents

[0013] Patent Documents

[0014] Patent document 1: Japanese Patent Application Laid-Open No. 2009-513694.

[0015] Patent document 2: Japanese Patent Application Laid-Open No. 2012-507272.

[0016] Patent document 3: International Publication No. 2021 / 172418.

[0017] Non-Patent Documents

[0018] Non-patent document 1: Yakushi 131 (12), 1723-1731 (2011): https: / / www.jstage.jst.go.jp / article / yakushi / 131 / 12 / 131_12_1723 / _pdf / -char / ja.

[0019] Non-patent document 2: Frontiers in Immunology 2021, vol. 12, Article 711997.

[0020] Non-patent document 3: Ministerial Ordinance No. 37 of the Ministry of Health, Labour and Welfare, enacted on February 28, 2018 "Biological Raw Material Standards".

[0021] Non-patent document 4: Vaccine 2019, vol. 37, pp. 1630-1637. SUMMARY

[0022] PROBLEMS TO BE SOLVED BY THE INVENTION

[0023] When the frequent occurrence of unqualified influenza vaccine bulk solution manufactured using the chicken egg culture method due to the mixing of an egg-transmissible pathogen such as avian reovirus (ARV) and the unbalanced production of a multivalent (e.g., tetravalent) vaccine bulk solution potentially cause an obstacle to product supply, countermeasures against egg-transmissible pathogens are required in the manufacture of influenza vaccines based on the chicken egg culture method. For example, for avian reovirus, a countermeasure of additional inoculation of inactivated reovirus to breeders is taken. However, since these inactivated reovirus are vaccines for suppressing the onset, they cannot prevent the infection of breeders with ARV itself, and when breeders are infected with ARV, ARV is mixed in the eggs. Therefore, in the manufacturing method of inactivated influenza vaccines based on the chicken egg culture method, the elimination of egg-transmissible pathogens is required.

[0024] One aspect of the present application is to establish an inactivation process for pathogens other than influenza viruses that are likely to be mixed in the eggs as a raw material in the manufacturing method of inactivated influenza vaccines based on the chicken egg culture method.

[0025] Means for solving the problem

[0026] The present inventors have conducted intensive studies to solve the above problem. As a result, in the manufacturing method of inactivated influenza vaccines based on the chicken egg culture method, a process capable of inactivating pathogens mixed in the eggs was found. Furthermore, for inactivated influenza vaccines manufactured by a manufacturing method including a process capable of inactivating pathogens mixed in the eggs, it was unexpectedly found that the quality mainly in terms of immunogenicity does not change, and thus the present application was completed.

[0027] The manufacturing method of one aspect of the present application that solves the above problem is a manufacturing method of inactivated influenza vaccines based on the chicken egg culture method, wherein the manufacturing method includes a pathogen inactivation process of inactivating pathogens other than influenza viruses mixed in the eggs.

[0028] Effects of the invention

[0029] According to one aspect of the present application, in the manufacturing method of inactivated influenza vaccines based on the chicken egg culture method, pathogens other than influenza viruses that are likely to be mixed in the eggs as a raw material can be inactivated. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 A flowchart of the manufacturing method of inactivated influenza vaccines based on the chicken egg culture method according to one aspect of the present application is shown. DETAILED DESCRIPTION

[0031] In the present specification, "A to B" means A or more and B or less, unless otherwise specified.

[0032] Method for producing inactivated influenza vaccine

[0033] One embodiment of the method for producing inactivated influenza vaccine based on the chicken egg cultivation method (hereinafter, sometimes referred to as "the present production method") of the present application includes a pathogen inactivation step. One example of the flow of the present production method is shown in Figure 1 .

[0034] (Inactivated influenza vaccine)

[0035] In the present specification, the inactivated influenza vaccine refers to a vaccine containing inactivated influenza virus. The inactivation of influenza virus will be described later.

[0036] (Influenza virus)

[0037] As an example of the influenza virus used in the present production method, A type, B type, C type, or D type, or a subtype thereof, and the like can be given. The influenza virus contained in the inactivated influenza vaccine can be one, or two or more. That is, the inactivated influenza vaccine produced by the present production method can be a monovalent vaccine, or a multivalent vaccine.

[0038] The influenza virus used in the present production method can be a strain isolated from an infected animal or patient, or a recombinant virus strain established in a culture cell by genetic engineering.

[0039] (Chicken egg cultivation method)

[0040] In the present specification, the chicken egg cultivation method refers to the cultivation of an influenza virus strain by inoculating the influenza virus strain into a developing chicken egg. The cultivation is, for example, performed by inoculating the influenza virus strain into a developing chicken egg of about 10 days old, and then culturing at 30 to 37°C for 1 to 7 days.

[0041] (Pathogen inactivation step)

[0042] In the above pathogen inactivation step, a pathogen other than the influenza virus mixed into the chicken egg is inactivated.

[0043] (Pathogen)

[0044] As an example of the pathogen mixed into the chicken egg, avian leukosis virus (ALV), egg drop syndrome-1976 virus (EDSV), avian reovirus (ARV), Mycoplasma gallisepticum (Mg), and the like can be given. In the present specification, ALV, EDSV, ARV, and Mg are sometimes collectively referred to as "ovine infectious pathogen".

[0045] As an example of the pathogen inactivation process, there can be mentioned a process of collecting allantoic fluid containing influenza viruses cultured by the egg culture method (hereinafter, sometimes referred to as "virus-containing allantoic fluid") and inactivating the pathogen contained in the virus-containing allantoic fluid. The virus-containing allantoic fluid can also be subjected to centrifugation such as ultracentrifugation before the pathogen contained in the virus-containing allantoic fluid is inactivated. In addition, the virus-containing allantoic fluid can be subjected to purification or concentration by, for example, sucrose density gradient centrifugation before the pathogen contained in the virus-containing allantoic fluid is inactivated.

[0046] (BPL inactivation process)

[0047] The pathogen inactivation process can be a process of treating the pathogen with β-propiolactone (hereinafter, sometimes simply referred to as "BPL"). Hereinafter, this process is sometimes referred to as a BPL inactivation process. In the BPL inactivation process, for example, the pathogen can be inactivated by adding BPL to the virus-containing allantoic fluid.

[0048] From the viewpoint of the inactivation efficiency of the pathogen and the maintenance of the quality of the influenza vaccine, the final concentration of BPL added to the virus-containing allantoic fluid is preferably 0.05 vol% or more, and more preferably 0.08 vol% or more. In addition, the final concentration of BPL is preferably 0.15 vol% or less, and more preferably 0.1 vol% or less.

[0049] The treatment temperature of the pathogen inactivation reaction in the BPL inactivation process can be room temperature (1°C to 30°C), or can be 3°C or higher and 7°C or lower. In addition, from the viewpoint of the inactivation efficiency of the pathogen and the maintenance of the quality of the influenza vaccine, the treatment time of the pathogen inactivation reaction in the BPL inactivation process is preferably 1 minute or more, more preferably 3 hours or more, further preferably 5 hours or more, and more further preferably 10 hours or more. In addition, the pathogen inactivation reaction time is preferably 44 hours or less, and more preferably 24 hours or less.

[0050] After the virus-containing allantoic fluid is warmed to 34°C to 40°C, the pathogen inactivation reaction in the BPL inactivation process can be completed by maintaining the warmed state for 2 hours to 4 hours to hydrolyze BPL.

[0051] The immunogenicity of the inactivated influenza vaccine produced by the present production method does not change due to the pathogen inactivation reaction in the BPL inactivation process. In the production of an influenza vaccine, if the inactivation process is performed in two stages (pathogen inactivation and influenza virus inactivation), there is usually a concern that the quality of the influenza vaccine such as the immunogenicity will change. However, the present inventors have found that even if the inactivation process is performed in two stages, the quality of the inactivated influenza vaccine represented by the immunogenicity does not change.

[0052] (influenza virus inactivation step)

[0053] The production method can further include an influenza virus inactivation step of inactivating influenza viruses using formaldehyde after the pathogen inactivation step.

[0054] In the influenza virus inactivation step, for example, influenza viruses can be inactivated by adding formalin to the treatment solution after the pathogen inactivation step. In the present specification, formalin refers to an aqueous solution containing 35 to 41 mass% of formaldehyde.

[0055] From the viewpoint of inactivation efficiency of influenza viruses and maintenance of quality of influenza vaccines, the final concentration of formalin added to the urine bladder fluid containing viruses is preferably 0.003 vol% or more, and more preferably 0.005 vol% or more. In addition, the final concentration of formalin is preferably 0.03 vol% or less, and more preferably 0.01 vol% or less.

[0056] The inactivation reaction of influenza viruses can be performed at room temperature (1°C to 30°C), or can be performed at a temperature of 2°C or higher and 6°C or lower. In addition, from the viewpoint of inactivation efficiency of influenza viruses and maintenance of quality of influenza vaccines, the inactivation reaction time of influenza viruses is preferably 7 days or more, and more preferably 14 days. In addition, the inactivation reaction time of influenza viruses is preferably 21 days or less. Further, an inactivation treatment condition of reacting for several days, for example, 3 days at a temperature of 20°C to 30°C can also be selected.

[0057] (degreasing step)

[0058] The production method can further include a degreasing step of treating influenza viruses using an ether after the pathogen inactivation step and before the influenza virus inactivation step. By the degreasing step, an inactivated split vaccine can be produced.

[0059] In the degreasing step, for example, influenza virions can be split and the lipid component of the virions can be removed by adding an ether to the treatment solution after the pathogen inactivation step. The treatment solution after the pathogen inactivation step can be subjected to a filtration treatment such as microfiltration (MF) before the degreasing step.

[0060] As examples of the ether used in the degreasing step, diethyl ether, diisopropyl ether, and the like can be given. The ether used in the degreasing step can be one kind, or two or more kinds.

[0061] The amount of the ether used in the degreasing step can be 10 vol% to 400 vol%, can be 12.5 vol% to 100 vol%, or can be 33 vol% to 50 vol%, based on the total amount of the treatment solution after the pathogen inactivation step.

[0062] In the defatting step, a surfactant can be used in addition to the ether. As the surfactant, for example, polyoxyethylene octylphenyl ether, polysorbate 80, and combinations thereof can be mentioned. The amount of the surfactant used in the defatting step can be 0.002 vol% to 0.3 vol%, can be 0.005 vol% to 0.1 vol%, or can be 0.05 vol% to 0.075 vol%, based on the total amount of the treatment solution after the pathogen inactivation step.

[0063] The treatment temperature of the defatting step can be appropriately changed depending on the type of the influenza virus, the type and concentration of the ether used, and the like, and can be, for example, room temperature (1°C to 30°C), can be 4°C to 25°C, or can be 14°C to 24°C.

[0064] The treatment time of the defatting step can be appropriately changed depending on the type of the influenza virus, the type and concentration of the ether used, the treatment temperature, and the like, and can be, for example, 1 to 2 hours.

[0065] After the defatting step, the ether is removed by centrifugation or the like, and thus the defatting reaction can be completed.

[0066] [Inactivated split vaccine or inactivated whole virus vaccine of influenza virus]

[0067] The inactivated split vaccine or inactivated whole virus vaccine of influenza virus (hereinafter, sometimes referred to as "the present inactivated influenza vaccine") produced by the present production method is also included in one aspect of the present application.

[0068] As examples of the main antigen of the inactivated split vaccine, hemagglutinin (HA) antigen and neuraminidase (NA) antigen can be mentioned. From the viewpoint of high immunogenicity, the antigen of the inactivated split vaccine is preferably the HA antigen.

[0069] The amount of the influenza virus included in the present inactivated influenza vaccine can be 1 to 40 pg / ml per viral strain in terms of the hemagglutinin concentration.

[0070] The present inactivated influenza vaccine can include a pharmaceutically acceptable carrier. As the above carrier, a carrier generally used in vaccine production can be used. Specifically, as the above carrier, saline, buffered saline, dextrose, water, glycerol, isotonic aqueous buffer, and combinations thereof can be mentioned. The vaccine can also be appropriately combined with an emulsifying agent, a preservative (e.g., thiomersal), an isotonic agent, a pH adjusting agent, an inactivating agent (e.g., formalin), and the like.

[0071] To further enhance immunogenicity, the present inactivated influenza vaccine can also contain an adjuvant. As an adjuvant, for example, mention can be made of an aluminum adjuvant or an oil-in-water emulsion adjuvant containing squalene (AS03, MF59, etc.); a ligand of a Toll-like receptor such as CpG and 3-O-deacyl-4'-monophosphoryl lipid A (MPL); a saponin-based adjuvant, a polymer-based adjuvant such as poly-gamma-glutamic acid; a polysaccharide such as chitosan and inulin; and the like.

[0072] The dosage form of the present inactivated influenza vaccine can be, for example, a liquid, a powder (lyophilized powder, dry powder), a capsule, a tablet, a frozen state.

[0073] The administration route of the present inactivated influenza vaccine can be, for example, transdermal administration, sublingual administration, ocular administration, intradermal administration, intramuscular administration, oral administration, enteral administration, nasal administration, intravenous administration, subcutaneous administration, intraperitoneal administration, or administration from the mouth to the lungs. The administration method of the present inactivated influenza vaccine can be, for example, a method of administration using a syringe, a transdermal patch, a microneedle, an implantable sustained-release device, a syringe with a microneedle, a needleless device, or a nebulizer.

[0074] The subject to be administered the present inactivated influenza vaccine can be, for example, a human or a non-human animal, and more specifically, a vertebrate such as a bird or a mammal. As a mammal, mention can be made of an experimental animal such as a mouse, a rat, a rabbit, a guinea pig, a rhesus monkey, a cynomolgus monkey, and a non-human primate; a pet such as a dog and a cat; a livestock such as a pig, a cow, a goat, a sheep, and a horse; or a human.

[0075] The amount and the number of administrations of the present inactivated influenza vaccine can be appropriately selected depending on the degree of symptoms, age, sex, body weight, administration form, and the like.

[0076] [SUMMARY]

[0077] The production method of the present application according to the aspect 1 is a production method of an inactivated influenza vaccine based on a chicken egg culture method, which includes a pathogen inactivation step of inactivating a pathogen other than an influenza virus mixed into a chicken egg.

[0078] The production method of the present application according to the aspect 2 is the production method according to the aspect 1 of the present application, wherein the pathogen can be at least one pathogen selected from the group consisting of avian leukosis virus (ALV), egg drop syndrome-1976 virus (EDSV), avian reovirus (ARV), and Mycoplasma gallisepticum (Mg).

[0079] The production method of the present application according to the aspect 3 is the production method according to the aspect 1 or 2 of the present application, wherein the pathogen inactivation step can be a step of treating the pathogen with β-propiolactone.

[0080] The manufacturing method of the present application of item 4, in the present application of item 3, the final concentration of the β-propiolactone in the pathogen inactivation process can be 0.05 vol% or more and 0.15 vol% or less.

[0081] The manufacturing method of the present application of item 5, in the present application of item 3 or 4, the treatment temperature using the β-propiolactone in the pathogen inactivation process can be 3°C or more and 7°C or less, and the treatment time can be 1 minute or more and 24 hours or less.

[0082] The manufacturing method of the present application of item 6, in any one of the present application of items 3 to 5, the immunogenicity of the inactivated influenza vaccine does not change due to the treatment using the β-propiolactone.

[0083] The inactivated split vaccine or inactivated whole virus vaccine of the influenza virus of the present application of item 7 is manufactured by the manufacturing method of any one of the present application of items 1 to 6.

[0084] The manufacturing method of the present application of item 8, in any one of the present application of items 1 to 6, after the pathogen inactivation process, an influenza virus inactivation process of inactivating the influenza virus using formaldehyde can be further included.

[0085] The manufacturing method of the present application of item 9, in the present application of item 8, after the pathogen inactivation process and before the influenza virus inactivation process, a delipidation process of treating the influenza virus using an ether can be further included.

[0086] The inactivated split vaccine or inactivated whole virus vaccine of the influenza virus of the present application of item 10 is manufactured by the manufacturing method of the present application of item 8.

[0087] The inactivated split vaccine of the influenza virus of the present application of item 11 is manufactured by the manufacturing method of the present application of item 9.

[0088] The following examples show the embodiments of the present application in more detail. Of course, the present application is not limited to the following examples, and various modes are possible in details. Also, the present application is not limited to the above-described embodiments, and various modifications can be made within the scope shown in the claims, and the embodiments obtained by appropriately combining the respective disclosed technical means are also included in the technical scope of the present application. In addition, all the documents described in the present specification are cited as references.

[0089] Example

[0090] 〔Example 1〕 Preparation of inactivated influenza vaccine (with pathogen inactivation process)

[0091] According to Figure 1The preparation of the inactivated influenza vaccine was performed in accordance with the flow of the manufacturing method of the inactivated influenza vaccine. The influenza virus of strain B / Brisbane / 60 / 2008 was inoculated into the allantoic cavity of 11-day-old embryonated chicken eggs, and the allantoic fluid containing the influenza virus was collected after culturing at 34°C for 2 to 3 days. The influenza virus recovered from the allantoic fluid collected by concentration and ultracentrifugation was resuspended using a phosphate buffered sodium chloride solution (PBS, pH 7.4), subjected to sucrose density gradient centrifugation, and the fraction containing the influenza virus was recovered to perform purification. BPL was added to the purified virus solution so that the final concentration became 0.1 vol%, and a pathogen inactivation reaction was performed at 5°C for 22 hours. Then, the pathogen inactivation reaction was terminated by warming at 37°C for 2 hours.

[0092] To the treated solution after the pathogen inactivation reaction, diethyl ether (100 vol%) was added, and degreasing treatment was performed by stirring at room temperature for 1 hour. After the degreasing treatment, the diethyl ether was removed by centrifugal separation, and a virus suspension (hemagglutinin (HA) fraction) was recovered. The recovered virus suspension was subjected to ultrafiltration to remove BPL decomposition products. An aqueous solution containing formaldehyde was added so that the final concentration of formalin became 0.005 vol%. Then, after performing an influenza virus inactivation reaction under refrigeration for 14 days, the target inactivated influenza vaccine was obtained.

[0093] 〔Reference Example 1〕 Preparation of Inactivated Influenza Vaccine (Without Pathogen Inactivation Process)

[0094] The inactivated influenza vaccine was prepared by the same steps as in Example 1, except that the pathogen inactivation reaction using BPL and the ultrafiltration after the degreasing treatment (removal of BPL decomposition products) were not performed.

[0095] The antigens of the inactivated influenza vaccines prepared in Example 1 and Reference Example 1 were HA antigens.

[0096] 〔Evaluation Example 1〕 Test for Removal of Pathogens Mixed into Eggs

[0097] (1) Test substance

[0098] The allantoic fluid after purification using sucrose density gradient centrifugation in Example 1 was used as the test substance in Evaluation Example 1.

[0099] (2) Test sample

[0100] In a glass beaker, 1 part of virus incorporated was added to 9 parts of the test substance, and stirred for 1 minute at room temperature using a stirrer, and divided into two conical tubes made of polypropylene (PP). To one of them, BPL stock solution was added at a final concentration of 0.05 vol% or 0.1 vol%. Then, after gentle inversion mixing for 10 times at room temperature, the sample was transferred to a new conical tube made of PP, and then divided equally into 4 serum tubes made of PP at different sampling time points (0 hours, 5 hours, 10 hours, and 19 hours). After the division, the liquid was mixed at cold darkness (5°C) using a rotary incubator until each sampling time point. After sampling, the container was quickly moved to a constant temperature water bath at 37°C, and reacted for 2 hours at 37°C, and BPL was decomposed.

[0101] (3) Results of removal test of pathogen mixed into egg

[0102] In the manufacture of influenza vaccine, considering the use of the egg cultivation method, from pathogenic viruses possibly derived from a chicken, avian leukosis virus (ALV), egg drop syndrome-1976 virus (EDSV), avian reovirus (ARV), and Mycoplasma gallisepticum (Mg) were selected as examples of infectious pathogenic viruses (Table 1). Among the five, the non-enveloped viruses infected by the egg were ARV (RNA virus) and EDSV (DNA virus).

[0103] Table 1

[0104] To the above test substance, the pathogen of Table 1 was added (incorporated), respectively, and the degree of reduction of each pathogen before and after the BPL treatment process was calculated by the following formula (1), and the inactivation ability of the BPL treatment process for the pathogen was evaluated. Hereinafter, the pathogen of Table 1 is sometimes referred to as "virus or the like".

[0105] LRV = log (titer of virus or the like in the sample before the BPL treatment process / titer of virus or the like in the sample after the BPL treatment process) (1)

[0106] In formula (1), LRV means logarithmic reduction value.

[0107] Evaluation of the inactivation ability of the BPL treatment process with respect to pathogens was performed twice for each of the two conditions of a final BPL concentration of 0.1 vol% and 0.05 vol%, with the BPL treatment time set to 0 hours, 5 hours, 10 hours, and 19 hours. A BPL treatment time of 0 hours indicates that the BPL was added, mixed upside down for 10 times, and then sampled immediately, as the measured time, which corresponds to an operation of less than 1 minute.

[0108] The evaluation results are shown in Table 2. Here, the World Health Organization indicates that in a virus clearance test, LRV4 or more is considered to be a robust and highly reliable effective process (WHO Technical Report, Series No. 924, 2004, Annex 4). However, depending on the virus titer and the limit of quantification in the sample before the BPL treatment process, even if viruses and the like are cleared, the LRV is sometimes less than 4.

[0109] Table 2

[0110] ALV: Avian leukosis virus.

[0111] EDSV: Egg drop syndrome-1976 virus.

[0112] ARV: Avian Reovirus.

[0113] Mg: Mycoplasma gallisepticum.

[0114] PRV: Pseudorabies virus.

[0115] As shown in Table 2, when the final concentration of BPL was 0.1 vol%, for EDSV, ARV, Mg, and PRV, the LRV was 4 or more regardless of the treatment time (0 hours, 5 hours, 10 hours, 19 hours) under the condition of 5°C, and no pathogens were detected. For ALV, depending on the virus titer and the limit of quantification before the BPL treatment, the results were “>3.7” (first time) or “>3.9” (second time), but for ALV, it can also be said that viruses and the like have been cleared. That is, when the final concentration of BPL was 0.1 vol%, regardless of the treatment time (0 hours, 5 hours, 10 hours, 19 hours) under the condition of 5°C, all of the incorporated viruses and the like including ARV were not detected. It was confirmed that all types of pathogens had been cleared.

[0116] In addition, when the final concentration of BPL was 0.05 vol%, no incorporation of viruses or the like was detected for ALV, PRV, and Mg, regardless of the treatment time. On the other hand, for EDSV, live viruses were detected in the samples taken at 0 hour and 5 hours after BPL treatment, but it was confirmed that they had been eliminated after 10 hours and 19 hours. For ARV, live viruses were detected in the samples taken at 0 hour after BPL treatment, but it was confirmed that they had been eliminated after 5 hours, 10 hours, and 19 hours.

[0117] 〔Evaluation Example 2〕 Mouse immunogenicity (HI (hemagglutination inhibition) antibody titer)

[0118] Ten female mice (3 weeks old, ddY system) were used as one group, and each of the inactivated influenza vaccines prepared in Example 1 (with pathogen inactivation process) and each of the inactivated influenza vaccines prepared in Reference Example 1 (without pathogen inactivation process) was administered intraperitoneally at about 0.6 μg of HA (equivalent value) per mouse in 0.5 mL. Blood was collected 21 days after the immunization, and the HI antibody titer in the serum was evaluated. The geometric mean of the HI antibody titer and the 95% confidence interval are shown in Table 3. The HI antibody titer with or without the 0.1 vol% BPL treatment process was evaluated for each of the four influenza virus strains in three batches.

[0119] The results of the F test on the data in Table 3 were that the other strains except Victoria had equal variances, and therefore the Student's t test (two-sided test at a significance level of 5%) was used. For Victoria, the F test result showed that the variances were not equal, and therefore the Welch's t test (two-sided test at a significance level of 5%) was used. The test results were that the HI antibody titers of all strains did not show significant differences with or without the BPL treatment process, and it was not detected that the implementation of the BPL treatment process had an effect on the mouse immunogenicity.

[0120] Table 3

[0121] [a]: The value in ( ) indicates the 95% confidence interval.

[0122] 〔Evaluation Example 3〕 Red blood cell agglutination activity (HA titer)

[0123] The HA titers were determined for each of the inactivated influenza vaccines prepared in Example 1 and Reference Example 1 using chicken red blood cells. The protein concentrations in the stock solutions of each strain were prepared to the same concentration, and were applied to 96-well V-bottom microplates. Clear hemagglutination reactions with chicken red blood cells were observed in all strains. In addition, no difference in reactivity with chicken red blood cells was observed in the stock solutions with or without the BPL treatment process.

[0124] As described above, by adding the pathogen inactivation process using BPL treatment, no difference in HI antibody titer and HA titer was observed, and it was confirmed that the quality of the drug substance was the same and homogeneous with or without the BPL treatment process.

[0125] 〔Evaluation Example 4〕 Comparison of the incidence of failure in the foreign virus negative test in the manufacture of inactivated influenza vaccine caused by the presence or absence of the BPL treatment process

[0126] (1) Foreign virus negative test (chick embryo fibroblast inoculation test)

[0127] It was performed with reference to "Foreign Virus Negative Test Method" (Standards for Biological Products for Animals: Revised on June 30, Heisei 2 (Notice No. 1246)) as a method for investigating the absence of a detectable foreign virus in a live vaccine and a serum-like specimen. During the observation period, when no cytopathic effect (CPE) appeared in the culture cells, it was determined that the test was passed.

[0128] (2) Results before and after the introduction of the BPL treatment process

[0129] The incidence of failure in the foreign virus negative test of the inactivated influenza vaccine prepared in Reference Example 1 was about 4% on average. On the other hand, for the inactivated influenza vaccine prepared in Example 1, no failure was observed in the foreign virus negative test.

[0130] Industrial applicability

[0131] According to the production method of the present application, an inactivated influenza vaccine with improved elimination of egg-infectious pathogens can be provided, and in particular, can be utilized in the medical field.

Claims

1. A manufacturing method for an inactivated influenza vaccine based on an egg culture method, wherein, The manufacturing method includes a pathogen inactivation step, which inactivates pathogens other than influenza viruses that have mixed into the eggs.

2. The manufacturing method as described in claim 1, wherein, The pathogen is at least one pathogen selected from the group consisting of avian leukosis virus (ALV), egg drop syndrome-1976 virus (EDSV), avian reovirus (ARV), and chicken mycoplasma Mg.

3. The manufacturing method as described in claim 1, wherein, The pathogen inactivation process is a process of treating the pathogen with β-propiolactone.

4. The manufacturing method as described in claim 3, wherein, The final concentration of β-propiolactone in the pathogen inactivation process is above 0.05 vol% and below 0.15 vol%.

5. The manufacturing method as described in claim 4, wherein, The pathogen inactivation process using β-propiolactone involves a treatment temperature of 3°C or higher and 7°C or lower, and a treatment time of 1 minute or higher and 24 hours or lower.

6. The manufacturing method as described in claim 3, wherein, The immunogenicity of the inactivated influenza vaccine is not altered by the treatment with the β-propiolactone described above.

7. An inactivated split vaccine or an inactivated whole virus vaccine for influenza virus, wherein, The inactivated split vaccine or inactivated whole virus vaccine of the influenza virus is manufactured by any one of the manufacturing methods according to claims 1 to 6.

8. The manufacturing method according to any one of claims 1 to 6, wherein, Following the pathogen inactivation step, an influenza virus inactivation step is also included, which uses formaldehyde to inactivate the influenza virus.

9. The manufacturing method as described in claim 8, wherein, After the pathogen inactivation step and before the influenza virus inactivation step, a degreasing step is also included, in which the influenza virus is treated with ether.

10. An inactivated split vaccine or an inactivated whole virus vaccine for influenza virus, wherein, The inactivated split vaccine or inactivated whole virus vaccine of the influenza virus is manufactured by the manufacturing method described in claim 8.

11. An inactivated split vaccine against influenza virus, wherein, The inactivated split vaccine of the influenza virus is manufactured by the manufacturing method described in claim 9.

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