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 in eggs, wherein the pathogens are other than influenza viruses.

Description

Manufacturing method for inactivated influenza vaccine using chicken egg culture method

[0001] The present invention relates to a method for producing an inactivated influenza vaccine by a chicken egg culture method.

[0002] Influenza viruses belong to the Orthomyxoviridae family, which is classified as "enveloped, negative-sense, single-stranded RNA viruses," and are known to be of types A, B, C, and D. The differences between types are based on differences in the antigenicity of the structural proteins M1 protein and nucleoprotein (NP) among the proteins that make up the virus particle. In addition to this, there are also pathological, morphological, and genetic differences, with significant differences between types A and B and types C and D in particular. Types A and B are epidemics every year and are often the cause of influenza in humans.

[0003] The influenza virus was isolated from pigs in 1931, and a human influenza virus was isolated in 1933. Around the same time, it was discovered that the influenza virus could grow in embryonated chicken eggs, and so inactivated whole particle vaccines began to be used, in which the virus was inoculated into embryonated chicken eggs, cultured, harvested, purified, and then inactivated with formaldehyde. Chemical modification of the viral genes or viral proteins simply eliminates the infectivity of the virus, so the virus remains in particle form and serves as a vaccine antigen.

[0004] However, the original whole particle inactivated vaccines had a high incidence of side effects, such as fever, and methods were devised to remove the lipid membrane components thought to be the cause. For example, vaccines based on the influenza antigen hemagglutinin (HA), which is the prototype of the current split vaccine, are highly safe, and are purified by zonal ultracentrifugation, separated with a surfactant, and then the lipid membrane components that cause fever are removed with ether. Split vaccines have been produced since 1964 and continue to be produced to the present day (Non-Patent Document 1). It has also been reported that split vaccines have viral nucleic acids and high molecular weight proteins removed along with the splitting agent, while retaining the antigenic components HA and neuraminidase (NA) (Non-Patent Document 2).

[0005] Influenza vaccines are widely produced by inoculating the desired virus strain into embryonated chicken eggs, growing it, and then purifying it. In Japan, the aforementioned split vaccine, which contains purified HA (and NA) as the major antigen, is available. As an example of the production of seasonal influenza vaccines, seasonal strains, type A and type B, are separately cultured in embryonated chicken eggs, and the HA fraction is recovered as the major antigen and formulated. HA is one of the surface antigens of the influenza virus and is involved in the adsorption of the virus to host cells. Inoculation of this formulation is expected to produce antibodies against HA, which act as protective antibodies against the influenza virus and thereby prevent influenza.

[0006] Vaccines produced by growing viruses in cultured cells, instead of traditional chicken eggs, are called cell-cultured influenza vaccines. Cells used include EB66 cells (derived from duck embryonic stem cells), MDCK cells (derived from canine renal tubular epithelial cells), and Vero cells (derived from African green monkey kidney epithelial cells). While the advantages of cell culture methods include flexibility and speed in vaccine production, inactivated split vaccines produced using chicken egg culture methods are the mainstream for seasonal influenza vaccines in Japan. Regardless of the production method, vaccines must be highly safe for humans and other animals, requiring inactivation of the grown virus and highly purified purification of the vaccine's primary antigen. Furthermore, because biopharmaceuticals such as vaccines use biologically derived raw materials, Japan has established the "Standards for Biologically Derived Raw Materials" (Non-Patent Document 3: Ministry of Health, Labour and Welfare Notification No. 37, enacted February 28, 2018) to ensure the quality, efficacy, and safety of pharmaceuticals. Because these biologically derived raw materials are biological macromolecules, they pose a risk of infection with pathogens such as viruses. Even in the production of influenza vaccines using egg culture methods, the process of inactivating or removing pathogens originating from the raw material, chicken eggs, is important.

[0007] In a typical method for producing inactivated split influenza vaccines using egg culture, four production strains, two type A and two type B influenza virus strains, are cultured individually in embryonated chicken eggs. The allantoic fluid containing the grown viruses is then collected and subjected to ultracentrifugation, sucrose density gradient centrifugation, ether treatment, and formalin inactivation to prepare a vaccine stock solution (single stock solution). After the single stock solutions for the four production strains have been prepared, they are diluted with a buffer solution to contain a specified amount of HA from each strain of virus, and formulated.

[0008] In a typical production method for inactivated split influenza vaccines using egg culture, the inactivation process of influenza viruses is carried out as the final step of the production method using formalin treatment or the like. If pathogens are contaminated in the raw material eggs, the pathogens are usually eliminated by selection inspection of the embryonated eggs or in the subsequent purification process. However, this elimination is not complete, and the stock solution from which the pathogens cannot be removed before preparation is discarded.

[0009] β-propiolactone (BPL; C ３ H ４ O ２ ) is a ring-strained β-lactone with a molecular weight of 72.06, and is also known as 2-Oxetanone or Betaprone. BPL can be easily converted into O-H ２ Since BPL cleaves between C and C to become an alkylating agent that attacks nucleophilic functional groups of biopolymers, it is expected to have an inactivating effect on influenza viruses. Evaluation of BPL treatment against influenza viruses is disclosed, for example, in Non-Patent Document 4.

[0010] BPL treatment has been disclosed in patent documents, including U.S. Patent Nos. 5,999,023 and 5,999,032, as a method for degrading host cell nucleic acids associated with influenza viruses or viral antigens produced in cell culture.

[0011] Meanwhile, in the production of inactivated influenza vaccines using chicken egg culture, the introduction of a BPL treatment step is considered in Patent Document 3. Patent Document 3 discloses that it has been found that pre-treating influenza viruses with BPL followed by formalin treatment can suppress the formalin-induced decrease in the innate immune activation ability of influenza vaccine antigens. Patent Document 3 also discloses that its object is to provide an inactivated influenza vaccine (specifically, a whole particle vaccine) and a method for producing the same, in which the innate immune activation ability (immunogenicity) of influenza vaccine antigens is enhanced by formalin treatment.

[0012] Japanese Patent Application Publication No. 2009-513694 Japanese Patent Application Publication No. 2012-507272 International Publication No. 2021 / 172418

[0013] Pharmaceutical Journal 131(12), 1723-1731 (2011): https: / / www.jstage.jst.go.jp / article / yakushi / 131 / 12 / 131_12_1723 / _pdf / -char / jaFrontiers in Immunology 2021, vol.12, Article 711997Ministry of Health, Labour and Welfare Notification No. 37 / Established February 28, 2018 "Standards for Biologically Originated Raw Materials"Vaccine 2019, vol.37, pp.1630-1637

[0014] If incompatibility of influenza vaccine concentrates produced by egg culture methods occurs frequently due to contamination with egg-transmitted pathogens such as avian reovirus (ARV), the production volume balance of polyvalent (e.g., quadrivalent) vaccine concentrates will be disturbed, raising concerns about disruption to product supply. Therefore, measures against egg-transmitted pathogens are necessary in the production of influenza vaccines by egg culture methods. For example, with regard to avian reovirus, a countermeasure is taken in which breeding hens are boosted with inactivated reo vaccines. However, because these inactivated reo vaccines are designed to suppress the onset of disease, they cannot prevent ARV infection in breeding hens, and ARV infection in breeding hens results in ARV contamination of eggs. Therefore, there is a demand for the realization of clearance of egg-transmitted pathogens in the production method of inactivated influenza vaccines by egg culture methods.

[0015] One aspect of the present invention is to establish an inactivation step for pathogens other than influenza viruses that may be contaminated in the raw material chicken eggs in a method for producing an inactivated influenza vaccine using chicken egg culture methods.

[0016] The present inventors have conducted extensive research to solve the above problems. As a result, they have discovered a step in which pathogens contaminating chicken eggs can be inactivated in a method for producing an inactivated influenza vaccine using a chicken egg culture method. They have also unexpectedly found that the quality, including immunogenicity, of an inactivated influenza vaccine produced by a production method including a step in which pathogens contaminating chicken eggs can be inactivated remains unchanged, leading to the completion of the present invention.

[0017] A production method according to one embodiment of the present invention that solves the above-mentioned problems is a method for producing an inactivated influenza vaccine using a chicken egg culture method, and includes a pathogen inactivation step of inactivating pathogens other than influenza viruses that may be present in chicken eggs.

[0018] According to one aspect of the present invention, in a method for producing an inactivated influenza vaccine by a chicken egg culture method, pathogens other than influenza viruses that may be contaminated in chicken eggs, which are the raw material, can be inactivated.

[0019] 1 shows a flow diagram of a method for producing an inactivated influenza vaccine by a chicken egg culture method according to one embodiment of the present invention.

[0020] In this specification, "A to B" means A or more and B or less unless otherwise specified.

[0021] [Method for producing inactivated influenza vaccine] A method for producing an inactivated influenza vaccine by a chicken egg culture method according to one embodiment of the present invention (hereinafter sometimes referred to as "the present production method") includes a pathogen inactivation step. An example of the flow chart of the present production method is shown in Figure 1.

[0022] (Inactivated influenza vaccine) As used herein, an inactivated influenza vaccine refers to a vaccine containing an inactivated influenza virus. The inactivation of influenza viruses is described below.

[0023] (Influenza virus) Examples of influenza viruses used in the present production method include types A, B, C, and D, or subtypes thereof. The inactivated influenza vaccine may contain one type of influenza virus or two or more types of influenza viruses. That is, the inactivated influenza vaccine produced by the present production method may be a monovalent vaccine or a polyvalent vaccine.

[0024] The influenza virus used in this production method may be a strain isolated from an infected animal or patient, or a recombinant virus strain established in cultured cells by genetic engineering.

[0025] (Egg culture method) As used herein, the egg culture method refers to culturing an influenza virus strain by inoculating the influenza virus strain into an embryonated chicken egg. For example, the culture may be performed by inoculating an influenza virus strain into an approximately 10-day-old embryonated chicken egg and then culturing the egg at 30 to 37°C for 1 to 7 days.

[0026] (Pathogen inactivation step) In the pathogen inactivation step, pathogens other than influenza viruses that may be present in the eggs are inactivated.

[0027] (Pathogens) Examples of pathogens that may be contaminated in eggs include avian leukosis virus (ALV), egg-laying drop syndrome-1976 virus (EDSV), avian reovirus (ARV), Mycoplasma gallisepticum (Mg), etc. In this specification, ALV, EDSV, ARV, and Mg may be collectively referred to as "ovo-transmitted pathogens."

[0028] An example of a pathogen inactivation step includes a step of collecting allantoic fluid containing influenza virus cultured by a chicken egg culture method (hereinafter sometimes referred to as "virus-containing allantoic fluid") and then inactivating the pathogens contained in the virus-containing allantoic fluid. Before inactivating the pathogens contained in the virus-containing allantoic fluid, the virus-containing allantoic fluid may be subjected to centrifugation, such as ultracentrifugation. Furthermore, before inactivating the pathogens contained in the virus-containing allantoic fluid, the virus-containing allantoic fluid may be purified or concentrated by sucrose density gradient centrifugation, for example.

[0029] (BPL inactivation step) The pathogen inactivation step may be a step of treating pathogens with beta-propiolactone (hereinafter sometimes abbreviated as "BPL"). Hereinafter, this step may be referred to as a BPL inactivation step. In the BPL inactivation step, for example, pathogens can be inactivated by adding BPL to virus-containing allantoic fluid.

[0030] From the viewpoints of pathogen inactivation efficiency and maintaining 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, more preferably 0.08 vol% or more, and preferably 0.15 vol% or less, more preferably 0.1 vol% or less.

[0031] The treatment temperature for the pathogen inactivation reaction in the BPL inactivation step may be room temperature (1°C to 30°C), or may be 3°C or higher and 7°C or lower. From the standpoint of pathogen inactivation efficiency and maintaining the quality of the influenza vaccine, the treatment time for the pathogen inactivation reaction in the BPL inactivation step is preferably 1 minute or longer, more preferably 3 hours or longer, even more preferably 5 hours or longer, and even more preferably 10 hours or longer. The pathogen inactivation reaction time is preferably 44 hours or shorter, and more preferably 24 hours or shorter.

[0032] The virus-containing allantoic fluid is heated to 34 to 40°C and then maintained at this temperature for 2 to 4 hours to hydrolyze BPL, thereby completing the pathogen inactivation reaction in the BPL inactivation step.

[0033] The pathogen inactivation reaction in the BPL inactivation step does not change the immunogenicity of the inactivated influenza vaccine produced by this production method. When producing an influenza vaccine, if the inactivation step is performed in two stages (pathogen inactivation and influenza virus inactivation), there is usually concern that the quality of the influenza vaccine, such as its immunogenicity, may change. However, the present inventors have found that performing the inactivation step in two stages does not change the quality, including the immunogenicity, of the inactivated influenza vaccine.

[0034] (Influenza Virus Inactivation Step) The present production method may further include, after the pathogen inactivation step, an influenza virus inactivation step in which influenza viruses are inactivated with formaldehyde.

[0035] In the influenza virus inactivation step, influenza viruses can be inactivated, for example, by adding formalin to the treatment solution after the pathogen inactivation step. In this specification, formalin refers to an aqueous solution containing 35 to 41% by mass of formaldehyde.

[0036] From the viewpoints of influenza virus inactivation efficiency and maintaining the quality of the influenza vaccine, the final concentration of formalin added to the virus-containing allantoic fluid is preferably 0.003 vol% or more, more preferably 0.005 vol% or more, and preferably 0.03 vol% or less, more preferably 0.01 vol% or less.

[0037] The influenza virus inactivation reaction may be carried out at room temperature (1°C to 30°C) or at a temperature between 2°C and 6°C. From the viewpoints of the efficiency of influenza virus inactivation and maintaining the quality of the influenza vaccine, the influenza virus inactivation reaction time is preferably 7 days or more, with 14 days being a more preferred embodiment. The influenza virus inactivation reaction time is preferably 21 days or less. Furthermore, inactivation treatment conditions can also be selected such that the reaction is carried out at 20 to 30°C for several days, for example, 3 days.

[0038] (Delipidation step) The present production method may further include a delipidation step of treating influenza viruses with ether after the pathogen inactivation step and before the influenza virus inactivation step. The delipidation step allows the production of an inactivated split vaccine.

[0039] In the delipidation step, for example, by adding ether to the treatment liquid after the pathogen inactivation step, influenza virus particles can be cleaved and lipid components of the particles can be removed. The treatment liquid after the pathogen inactivation step may be subjected to a filtration process such as microfiltration (MF) before the delipidation step.

[0040] Examples of the ether used in the degreasing step include diethyl ether, diisopropyl ether, etc. The number of types of ether used in the degreasing step may be one or more.

[0041] The amount of ether used in the degreasing step may be 10 vol% to 400 vol%, 12.5 vol% to 100 vol%, or 33 vol% to 50 vol%, based on the total amount of the treatment liquid after the pathogen inactivation step.

[0042] In addition to the ether, a surfactant may also be used in the degreasing step. Examples of surfactants include polyoxyethylene octylphenyl ether, polysorbate 80, and combinations thereof. The amount of surfactant used in the degreasing step may be 0.002 vol% to 0.3 vol%, 0.005 vol% to 0.1 vol%, or 0.05 vol% to 0.075 vol%, based on the total amount of the treatment liquid after the pathogen inactivation step.

[0043] The treatment temperature in the degreasing step may be changed as appropriate depending on the type of influenza virus, the type and concentration of the ether used, etc., and may be, for example, room temperature (1°C to 30°C), 4°C to 25°C, or 14°C to 24°C.

[0044] The treatment time for the degreasing step may be appropriately changed depending on the type of influenza virus, the type and concentration of the ether used, the treatment temperature, etc., and may be, for example, 1 to 2 hours.

[0045] After the degreasing step, the degreasing reaction can be terminated by removing the ether by centrifugation or the like.

[0046] [Inactivated split vaccine or inactivated whole particle vaccine of influenza virus] An inactivated split vaccine or inactivated whole particle 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 invention.

[0047] Examples of major antigens in inactivated split vaccines include hemagglutinin (HA) antigen and neuraminidase (NA) antigen. In terms of high immunogenicity, the antigen in inactivated split vaccines is preferably HA antigen.

[0048] The amount of influenza virus contained in the inactivated influenza vaccine may be 1 to 40 μg / ml per virus strain in terms of hemagglutinin concentration.

[0049] The inactivated influenza vaccine may contain a pharmaceutically acceptable carrier. Carriers commonly used in vaccine production can be used as the carrier. Specific examples of the carrier include saline, buffered saline, dextrose, water, glycerol, isotonic aqueous buffer solutions, and combinations thereof. The vaccine may further contain, as appropriate, emulsifiers, preservatives (e.g., thimerosal), tonicity agents, pH adjusters, inactivators (e.g., formalin), and the like.

[0050] To further enhance immunogenicity, the inactivated influenza vaccine may further contain an adjuvant. Examples of adjuvants include aluminum adjuvants or squalene-containing oil-in-water emulsion adjuvants (e.g., AS03, MF59), toll-like receptor ligands such as CpG and 3-O-deacylated-4'-monophosphoryl lipid A (MPL), saponin-based adjuvants, polymer-based adjuvants such as poly-γ-glutamic acid, and polysaccharides such as chitosan and inulin.

[0051] The dosage form of the inactivated influenza vaccine may be, for example, liquid, powder (lyophilized powder, dry powder), capsule, tablet, or frozen.

[0052] The inactivated influenza vaccine may be administered via a route such as transdermal, sublingual, ophthalmic, intradermal, intramuscular, oral, enteral, nasal, intravenous, subcutaneous, or intraperitoneal route, or via inhalation from the mouth to the lungs. The inactivated influenza vaccine may be administered via a syringe, a transdermal patch, a microneedle, an implantable sustained-release device, a syringe equipped with a microneedle, a needleless device, or a spray.

[0053] The inactivated influenza vaccine can be administered to humans and non-human animals, more specifically, vertebrates such as birds and mammals. Mammals include laboratory animals such as mice, rats, rabbits, guinea pigs, rhesus monkeys, cynomolgus monkeys, and non-human primates; pet animals such as dogs and cats; livestock such as pigs, cows, goats, sheep, and horses; and humans.

[0054] The dose and frequency of administration of the present inactivated influenza vaccine can be appropriately selected depending on the severity of symptoms, age, sex, body weight, administration form, etc.

[0055] [Summary] The production method according to aspect 1 of the present invention is a method for producing an inactivated influenza vaccine by a chicken egg culture method, and includes a pathogen inactivation step of inactivating pathogens other than influenza viruses that may be contaminating chicken eggs.

[0056] A production method according to Aspect 2 of the present invention is the production method according to Aspect 1 of the present invention, wherein the pathogen is at least one pathogen selected from the group consisting of avian leukosis virus (ALV), egg-laying drop syndrome-1976 virus (EDSV), avian reovirus (ARV), and Mycoplasma gallisepticum (Mg).

[0057] A manufacturing method according to a third aspect of the present invention may be the same as that according to the first or second aspect of the present invention, wherein the pathogen inactivation step is a step of treating the pathogen with beta-propiolactone.

[0058] A fourth aspect of the present invention relates to the production method of the third aspect of the present invention, wherein the final concentration of the beta-propiolactone in the pathogen inactivation step is 0.05 vol% or more and 0.15 vol% or less.

[0059] A fifth aspect of the present invention relates to the production method of the third or fourth aspect of the present invention, and the treatment temperature with beta-propiolactone in the pathogen inactivation step may be 3°C or higher and 7°C or lower, and the treatment time may be 1 minute or higher and 24 hours or shorter.

[0060] A sixth aspect of the present invention relates to the production method of any one of the third to fifth aspects of the present invention, wherein the immunogenicity of the inactivated influenza vaccine is not altered by the treatment with beta-propiolactone.

[0061] An inactivated split vaccine or inactivated whole particle vaccine of influenza virus according to aspect 7 of the present invention is produced by the production method according to any one of aspects 1 to 6 of the present invention.

[0062] The production method according to Aspect 8 of the present invention is the production method according to any one of Aspects 1 to 6 of the present invention, which may further comprise, after the pathogen inactivation step, an influenza virus inactivation step of inactivating influenza viruses with formaldehyde.

[0063] The production method according to Aspect 9 of the present invention may be the same as Aspect 8 of the present invention, further comprising a delipidation step of treating the influenza virus with ether after the pathogen inactivation step and before the influenza virus inactivation step.

[0064] An inactivated split vaccine or inactivated whole particle vaccine of influenza virus according to Aspect 10 of the present invention is produced by the production method according to Aspect 8 of the present invention.

[0065] An inactivated split influenza virus vaccine according to an eleventh aspect of the present invention is produced by the production method according to the ninth aspect of the present invention.

[0066] The following examples are provided to further explain the embodiments of the present invention. It goes without saying that the present invention is not limited to the following examples, and various modifications are possible in detail. Furthermore, the present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed herein are also included in the technical scope of the present invention. Furthermore, all of the documents described in this specification are incorporated by reference.

[0067] Example 1: Preparation of inactivated influenza vaccine (with pathogen inactivation step) An inactivated influenza vaccine was prepared according to the manufacturing method flow shown in Figure 1. The influenza virus B / Brisbane / 60 / 2008 strain was inoculated into the allantoic cavity of 11-day-old embryonated chicken eggs, and after 2-3 days of incubation at 34°C, allantoic fluid containing influenza virus was collected. The collected allantoic fluid was concentrated and ultracentrifuged to recover influenza virus. The influenza virus was resuspended in phosphate-buffered sodium chloride (PBS, pH 7.4) and subjected to sucrose density gradient centrifugation to recover the influenza virus-containing fraction for purification. BPL was added to the purified virus solution to a final concentration of 0.1 vol%, and a pathogen inactivation reaction was carried out at 5°C for 22 hours. The pathogen inactivation reaction was then completed by heating at 37°C for 2 hours.

[0068] Diethyl ether (100 vol%) was added to the treatment solution after the pathogen inactivation reaction, and the mixture was stirred at room temperature for 1 hour to perform a delipidation treatment. After the delipidation treatment, the diethyl ether was removed by centrifugation, and the virus suspension (hemagglutinin (HA) fraction) was recovered. The recovered virus suspension was ultrafiltered to remove BPL degradation products. A formaldehyde-containing aqueous solution was added to the solution so that the final formalin concentration was 0.005 vol%. The influenza virus inactivation reaction was then carried out under refrigeration for 14 days, and the desired inactivated influenza vaccine was obtained.

[0069] Reference Example 1 Preparation of Inactivated Influenza Vaccine (without Pathogen Inactivation Step) An inactivated influenza vaccine was prepared in the same manner as in Example 1, except that the pathogen inactivation reaction using BPL and ultrafiltration (removal of BPL degradation products) after delipidation were not performed.

[0070] The antigen of the inactivated influenza vaccines prepared in Example 1 and Reference Example 1 is HA antigen.

[0071] Evaluation Example 1 Clearance Test of Pathogens Contaminating Chicken Eggs (1) Test Substance The allantoic fluid purified by sucrose density gradient centrifugation in Example 1 was used as the test substance in Evaluation Example 1.

[0072] (2) Sample: Nine volumes of the test substance and one volume of spike virus were added to a glass beaker and stirred at room temperature using a stirrer for 1 minute. The mixture was then divided into two polypropylene (PP) conical tubes. BPL stock solution was added to one of the tubes at a final concentration of 0.05 vol% or 0.1 vol%. The mixture was then gently inverted 10 times at room temperature and mixed. The mixture was then transferred to a new PP conical tube and aliquoted into four PP cryogenic vials, each representing a different sampling time point (0, 5, 10, and 19 hours). The aliquots were mixed using a rotary incubator in a cool, dark place (5°C) until each sampling time point. After sampling, the container was quickly transferred to a 37°C thermostatic water bath and incubated at 37°C for 2 hours to degrade BPL.

[0073] (3) Results of clearance tests for pathogens contaminating eggs Considering the use of egg culture methods in influenza vaccine production, five types of infectious pathogens were selected as examples from among pathogens that may originate from chickens: avian leukosis virus (ALV), egg-laying drop syndrome-1976 virus (EDSV), avian reovirus (ARV), and Mycoplasma gallisepticum (Mg), in addition to Pseudorabies virus (PRV), which is commonly used in virus clearance tests (Table 1). Of these five types, ARV (RNA virus) and EDSV (DNA virus) are non-enveloped viruses that are transmitted via eggs.

[0074]

[0075] The pathogens in Table 1 were individually added (spiked) to the test substance, and the degree of reduction of each pathogen before and after the BPL treatment step was calculated using the following formula (1), to evaluate the pathogen inactivation ability of the BPL treatment step. Hereinafter, the pathogens in Table 1 may be referred to as "viruses, etc." LRV = log (titer of viruses, etc. in the sample before the BPL treatment step / titer of viruses, etc. in the sample after the BPL treatment step) (1) In formula (1), LRV refers to the logarithmic reduction value.

[0076] The pathogen inactivation ability of the BPL treatment step was evaluated under two conditions: final BPL concentrations of 0.1 vol% and 0.05 vol%, with four treatment times of 0, 5, 10, and 19 hours, each of which was repeated twice. A BPL treatment time of 0 hours indicates that sampling was performed immediately after adding BPL and mixing by inverting 10 times, which corresponds to an actual measurement time of less than 1 minute.

[0077] The evaluation results are shown in Table 2. Here, the WHO recognizes that in virus clearance tests, an LRV of 4 or more represents a robust, reliable, and effective process (WHO Technical Report, Series No. 924, 2004, Annex 4). However, depending on the virus titer and quantitation limit in the sample before the BPL treatment process, the LRV may be less than 4 even if viruses, etc. are cleared.

[0078]

[0079] As shown in Table 2, when the final concentration of BPL was 0.1 vol%, the LRV values ​​for EDSV, ARV, Mg, and PRV were 4 or greater, and no pathogens were detected, regardless of the treatment time at 5°C (0, 5, 10, or 19 hours). For ALV, the virus titer before BPL treatment and the limit of quantification gave results of >3.7 (first run) or >3.9 (second run), indicating that viruses were also cleared from ALV. That is, when the final concentration of BPL was 0.1 vol%, no spike viruses, including ARV, were detected, regardless of the treatment time at 5°C (0, 5, 10, or 19 hours), confirming clearance of all types of pathogens.

[0080] Furthermore, when the final concentration of BPL was 0.05 vol%, spike viruses were not detected for ALV, PRV, and Mg regardless of the treatment time. On the other hand, for EDSV, surviving virus was detected in samples sampled at 0 and 5 hours after BPL treatment, but clearance was confirmed at 10 and 19 hours. For ARV, surviving virus was detected in samples sampled at 0 hour after BPL treatment, but clearance was confirmed at 5, 10, and 19 hours.

[0081] [Evaluation Example 2] Mouse immunogenicity (HI antibody titer) Ten female mice (3 weeks old, ddY strain) were divided into groups, and 0.5 mL of each of the inactivated influenza vaccines prepared in Example 1 (with pathogen inactivation step) and Reference Example 1 (without pathogen inactivation step) was administered intraperitoneally once to each mouse, resulting in approximately 0.6 μg HA (equivalent value). Twenty-one days after immunization, blood was collected, and the HI antibody titer in the serum was evaluated. The geometric mean values ​​and 95% confidence intervals of the HI antibody titers are shown in Table 3. The HI antibody titers with and without the 0.1 vol% BPL treatment step were evaluated for three batches of each of four types of influenza virus strains.

[0082] As a result of the F test in Table 3, all strains except the Victoria strain showed equal variances, so a Student's T-test (two-tailed test with a significance level of 5%) was performed. As for the Victoria strain, the F-test showed unequal variances, so a Welch's T-test (two-tailed test with a significance level of 5%) was performed. As a result of the test, no significant difference was observed in the HI antibody titer between the presence and absence of the BPL treatment step for any of the strains, and no effect of the BPL treatment step on mouse immunogenicity was detected.

[0083]

[0084] Evaluation Example 3: Hemagglutination Activity (HA Titer) The HA titer of each of the inactivated influenza vaccines prepared in Example 1 and Reference Example 1 was measured using chicken red blood cells. The protein concentrations in the stock solutions of each strain were adjusted to be equal and applied to a 96-well V-bottom microplate. A clear hemagglutination reaction with chicken red blood cells was confirmed for each strain. Furthermore, no difference in reactivity with chicken red blood cells was observed between the stock solutions with and without the BPL treatment step.

[0085] As described above, the addition of the pathogen inactivation step by BPL treatment did not result in any differences in HI antibody titers or HA titers, and it was confirmed that the quality of the drug substance was equivalent and homogeneous regardless of whether or not the BPL treatment step was performed.

[0086] [Evaluation Example 4] Comparison of the frequency of failure in the adventitious virus test in the manufacture of inactivated influenza vaccines with and without a BPL treatment process (1) Adventitious virus test (chicken kidney primary culture cell inoculation test) This test was conducted with reference to the "Adventitious Virus Test Method" (Standards for Biological Products for Animals: Revised June 30, 2020 (Notice No. 1246)), which is a method for checking the absence of detectable adventitious viruses in live vaccine and serum samples. If no CPE was observed in the cultured cells during the observation period, the vaccine was considered to have passed the test.

[0087] (2) Results before and after introduction of the BPL treatment process The incidence of non-compliance in the adventitious virus test for the inactivated influenza vaccine prepared in Reference Example 1 was about 4% on average. On the other hand, no non-compliance in the adventitious virus test was observed for the inactivated influenza vaccine prepared in Example 1.

[0088] The production method of the present invention can provide an inactivated influenza vaccine with improved clearance of egg-borne pathogens, and can be used particularly in the medical field.

Claims

1. A method for producing an inactivated influenza vaccine by a chicken egg culture method, comprising: The method includes a pathogen inactivation step of inactivating pathogens other than influenza viruses that may be present in eggs, the pathogen inactivation step is a step of treating the pathogen with beta-propiolactone; The production method further comprises, after the pathogen inactivation step, an influenza virus inactivation step of inactivating influenza viruses with formaldehyde.

2. 2. The method according to claim 1, wherein the pathogen is at least one pathogen selected from the group consisting of avian leukosis virus (ALV), egg-laying drop syndrome-1976 virus (EDSV), avian reovirus (ARV), and Mycoplasma gallisepticum (Mg).

3. The method according to claim 1, wherein the final concentration of beta-propiolactone in the pathogen inactivation step is 0.05 vol% or more and 0.15 vol% or less.

4. The method according to claim 3, wherein the treatment temperature with beta-propiolactone in the pathogen inactivation step is 3°C or higher and 7°C or lower, and the treatment time is 1 minute or higher and 24 hours or lower.

5. 2. The method of claim 1, wherein the treatment with beta-propiolactone does not alter the immunogenicity of the inactivated influenza vaccine.

6. An inactivated split vaccine or inactivated whole particle vaccine of influenza virus, produced by the production method according to any one of claims 1 to 5.

7. The method according to claim 1 , further comprising a delipidation step of treating the influenza virus with ether after the pathogen inactivation step and before the influenza virus inactivation step.

8. An inactivated split influenza virus vaccine produced by the production method of claim 7.