Composition containing four types of attenuated dengue virus

Abstract

There has been a need to find an optimal composition to obtain a stable formulation composition of a tetravalent attenuated vaccine (lyophilized formulation) against dengue virus. The present disclosure provides a lyophilized composition containing four types of attenuated dengue virus, the composition further containing D-sorbitol, glycine, and trehalose hydrate.

Description

Composition containing attenuated live quadrivalent dengue virus

[0001] Related Application This application claims priority to application no. 2024-227582, filed with the Japan Patent Office on 24 December 2024. The priority application in whole is incorporated herein by attribution.

[0002] This invention relates to a composition containing a weakened live quadrivalent dengue virus.

[0003] Dengue virus (DENV) is a mosquito-borne virus belonging to the Flaviviridae family that causes dengue fever, dengue hemorrhagic fever, and dengue shock syndrome in humans. Currently, four serotypes (DENV1 to DENV4), from type 1 to type 4, are involved in human outbreaks.

[0004] Dengue fever has spread to over 120 countries in tropical and subtropical regions, and in recent years, the infection has spread rapidly, particularly due to the concentration of population in urban areas caused by economic development. It is estimated that approximately 100 to 400 million people become ill each year, putting 3.9 billion people, or about 50% of the world's population, at risk. An estimate reported in 2010 indicated that 390 million people were infected annually, and 96 million required hospitalization or other treatment. Every year, 500,000 people develop severe symptoms from dengue fever or dengue hemorrhagic fever and require hospitalization, with an estimated annual mortality rate of approximately 2.5% (12,500 people). According to Non-Patent Literature 1, the population at risk is estimated to increase to approximately 5 to 6.5 billion by 2050 and to approximately 5 to 9 billion by 2080.

[0005] Dengue fever is characterized by severe flu-like symptoms (fever, vomiting, rash, etc.) in addition to intense pain (headache, eye pain, muscle pain, joint pain). Dengue hemorrhagic fever (Grade 1 / 2) and dengue shock (Grade 3 / 4) can cause plasma leakage, bleeding tendencies, and organ damage, and can be fatal if not treated appropriately.

[0006] There is no established effective treatment for dengue virus infection, and preventive measures are limited. Current measures mainly rely on mosquito control, environmental improvements, and the wearing of personal protective equipment, but their effectiveness is limited, and cost-effectiveness remains a challenge. As a more effective infection prevention measure, there is a strong need for the development of a safe and effective dengue vaccine that can be administered to a wide range of age groups. Patent document 1 discloses a quadrivalent attenuated dengue vaccine with excellent efficacy and safety results, and it is hoped that this attenuated quadrivalent dengue vaccine, which exhibits excellent efficacy and safety results whether administered as a single dose or multiple doses, will be launched.

[0007] To ensure that the correct dose of attenuated live quadrivalent dengue vaccine is administered reliably, the dengue virus itself must be protected from inactivation during preparation and transport. Many vaccines are sensitive to temperature changes and can be inactivated by excessive heat or accidental freeze-thaw cycles. Therefore, maintaining a cold chain throughout the entire distribution process is necessary, but this is a major challenge, especially in low- and middle-income countries. Against this backdrop, there is a need to find a formulation that can maintain the stability of attenuated live quadrivalent dengue virus.

[0008] Japanese Patent No. 6910956, JP 2023-52624

[0009] The current and future global distribution and population at risk of dengue, Nature Microbiology;4, 1508-1515(2019)

[0010] Except for Patent Document 2 and others, there is little publicly available information on formulation compositions that can maintain the stability of vaccines (lyophilized formulations) containing attenuated live quadrivalent dengue virus. Therefore, when formulating the vaccine (lyophilized formulation) containing attenuated live quadrivalent dengue virus that the present inventors are developing, it was necessary to find the optimal composition.

[0011] We have found a formulation composition (lyophilized preparation) that can maintain the stability of a vaccine containing attenuated live quadrivalent dengue virus. Specifically, we have demonstrated that a combination of D-sorbitol, glycine, and trehalose hydrate contributes to the stability of the vaccine. We also confirmed that adding L-histidine or albumin (Alb) is effective.

[0012] As a result of diligent research to solve the above problems, the inventors of the present invention have found that D-sorbitol, glycine, and trehalose hydrate are optimal formulations for maintaining the stability of a vaccine (lyophilized formulation) containing attenuated live quadrivalent dengue virus, and that adding L-histidine or albumin is also effective. Sodium hydrogen phosphate hydrate, sodium dihydrogen phosphate hydrate, and sodium chloride are preferred as buffer components.

[0013] Accordingly, the present invention includes the following: [Item 1] A freeze-dried composition comprising a weakened tetravalent dengue virus, comprising D-sorbitol, glycine, and trehalose hydrate. [Item 2] The composition according to Item 1, further comprising L-histidine, wherein, for example, when dissolved in a solvent, the final concentration of L-histidine may be 0.5 to 20 mM, 2.5 to 10 mM, preferably 3.0 to 7.5 mM, more preferably 4.0 to 6.0 mM, and even more preferably about 5 mM. [Item 3] The composition according to Item 1 or 2, further comprising a buffer, wherein the buffer comprises sodium hydrogen phosphate hydrate, sodium dihydrogen phosphate hydrate, and sodium chloride. [Item 4] The composition according to any one of items 1-3, further comprising albumin, wherein, for example, when dissolved in a solvent, the final concentration is 0.025 to 0.6 w / v%, 0.05 to 0.45 w / v%, 0.075 to 0.3 w / v%, preferably 0.1 to 0.2 w / v%, more preferably 0.05 to 0.2 w / v%, and even more preferably about 0.1 w / v%. [Item 5] The composition according to any one of items 1-4, wherein, when dissolved in a solvent, the final concentration of trehalose is 0.25 to 20 w / v%, 1.0 to 15 w / v%, 2.0 to 10 w / v%, preferably 3.0 to 7.0 w / v%, more preferably 4.0 to 6.0 w / v%, and even more preferably about 5 w / v%. [Item 6] The composition according to any one of items 1-5, wherein, when dissolved in a solvent, the final concentration of D-sorbitol may be 0.5 to 10.0 w / v%, 1.0 to 9.5 w / v%, 1.5 to 9.0 w / v%, 2.0 to 8.5 w / v%, 2.5 to 8.0 w / v%, 3.0 to 7.5 w / v%, 3.5 to 7.0 w / v%, 4.0 to 6.5 w / v%, 4.5 to 6.0 w / v%, or 5.0 to 5.5 w / v%, preferably 2.0 to 8.0 w / v%, more preferably 4.0 to 6.0 w / v%, and even more preferably about 5 w / v%.[Item 7] The composition according to any one of items 1-6, wherein, when dissolved in a solvent, the final concentration of glycine is 10-150 mM, 30-120 mM, 50-110 mM, preferably 70-100 mM, more preferably 20-100 mM, and even more preferably about 80 mM. [Item 8] The composition according to any one of items 1-7, wherein, when dissolved in a solvent, the final concentration of trehalose is 5±1 w / v%, and the final concentration of D-sorbitol is 1±0.5 w / v%. [Item 9] The composition according to any one of items 1-8, wherein, when dissolved in a solvent, the final concentration of trehalose is 5±1 w / v%, and the final concentration of D-sorbitol is 5±1 w / v%.

[0014] According to this disclosure, the stability of a vaccine (lyophilized preparation) containing attenuated live quadrivalent dengue virus can be maintained.

[0015] Changes in infectivity titer when stored at 25°C (DENV1) Changes in infectivity titer when stored at 25°C (DENV2) Changes in infectivity titer when stored at 25°C (DENV3) Changes in infectivity titer when stored at 25°C (DENV4) Changes in infectivity titer of lyophilized preparation (DENV1) Changes in infectivity titer of lyophilized preparation (DENV2) Changes in infectivity titer of lyophilized preparation (DENV3) Changes in infectivity titer of lyophilized preparation (DENV4) Stability of final bulk liquid when stored at 25°C (DENV1) Stability of final bulk liquid when stored at 25°C (DENV2) Stability of final bulk liquid when stored at 25°C (DENV3) Final bulk liquid when stored at 25°C Stability (DENV4) Infectivity titer before and after freeze-drying and harsh treatment (DENV1) Infectivity titer before and after freeze-drying and harsh treatment (DENV2) Infectivity titer before and after freeze-drying and harsh treatment (DENV3) Infectivity titer before and after freeze-drying and harsh treatment (DENV4) Changes in infectivity titer over 12 months after the start of storage (DENV1) Changes in infectivity titer over 12 months after the start of storage (DENV2) Changes in infectivity titer over 12 months after the start of storage (DENV3) Changes in infectivity titer over 12 months after the start of storage (DENV4) Infectivity titer after freeze-drying (Composition G) Changes in infectivity titer of long-term stored specimens (Composition G) Infectivity titer before and after harsh treatment (geometric mean) (Composition G)

[0016] The present invention provides a formulation composition that can maintain the stability of a vaccine (lyophilized formulation) containing attenuated live quadrivalent dengue virus. Preferred embodiments of the present invention will be described in detail below, but the invention is not limited to these embodiments.

[0017] The present invention provides a stable formulation composition for lyophilized preparations by adding excipients, stabilizers, or isotonic agents to a weakened tetravalent dengue virus obtained, for example, by the method described in Patent Document 1. Examples of stabilizers include sorbitol and glycine. Histidine or albumin (Alb) may also be included as stabilizers, but according to the present invention, a stable formulation composition can be obtained even without histidine or albumin. The composition of this disclosure differs from that of Patent Document 2 in that it exhibits good stability even without albumin. Examples of excipients for lyophilized preparations include histidine and trehalose.

[0018] In some embodiments, the formulations disclosed herein may further contain buffer components. In these embodiments, examples of buffer components include sodium chloride, sodium hydrogen phosphate hydrate, and sodium dihydrogen phosphate hydrate.

[0019] The freeze-drying of dengue virus may be carried out using a general-purpose freeze-dryer in the following general steps: pre-freezing → vacuum evacuation → primary drying → secondary drying → sealing. The temperatures for each step are exemplified as -50°C to -30°C for pre-freezing, -15°C to 5°C for primary drying, and 20°C to 40°C for secondary drying. The duration for each step is exemplified as 5 to 30 hours, and it generally takes 3 to 5 days to complete all steps.

[0020] The infectivity titer of the lyophilized preparation may be measured by immunohistochemistry. For a preparation to be considered stable, it is preferable that the decrease in infectivity titer before and after lyophilization is as small as possible. The lyophilized composition of this disclosure is preferable because the decrease in infectivity titer is within the specified value (e.g., less than 1 Log) both before and after lyophilization and after storage.

[0021] Stability tests, such as long-term storage tests, thermal stability tests, and accelerated stability tests, may be performed by storing the lyophilized formulation (vial) in a temperature-controlled constant-temperature bath for several days to several months. Vials may also be periodically sampled from the constant-temperature bath and used for various tests.

[0022] The freeze-dried state (cake shape) of the freeze-dried formulation may be confirmed by visual inspection to check for expansion or contraction, or for melting. In addition, the color, height (thickness), gap between the cake and the vial, and whether there are any shape defects may also be checked.

[0023] In one embodiment, the present disclosure relates to a freeze-dried composition comprising a weakened tetravalent dengue virus, comprising D-sorbitol, glycine, and trehalose hydrate.

[0024] The buffer used in the composition of the present disclosure preferably contains sodium hydrogen phosphate hydrate, sodium dihydrogen phosphate hydrate, and sodium chloride. In this case, the pH when the composition of the present disclosure is dissolved is not particularly limited, but the lower limit of the pH may be 4, 4.5, 5, 5.5, 6, 6.3, 6.6, 6.9, 7, or 7.2, and the upper limit of the pH may be 10, 9.5, 9, 8.5, 8, 7.8, 7.6, 7.5, 7.3, or 7.2, and in one embodiment the pH may be 4 to 10, 5 to 9, 6 to 8, 6.8 to 7.8, preferably 6.9 to 7.5, and more preferably about 7.

[0025] In another embodiment, the present disclosure relates to a method for producing a composition comprising a weakened tetravalent dengue virus, comprising the step of obtaining a solution comprising D-sorbitol, glycine, trehalose, and the virus.

[0026] In other embodiments, the disclosure relates to a method for stabilizing a composition comprising attenuated tetravalent dengue virus, comprising the step of obtaining a solution comprising D-sorbitol, glycine, trehalose, and the virus.

[0027] In one embodiment, the process may include a step of obtaining a solution containing D-sorbitol, glycine, trehalose, and a virus solution, followed by a step of freeze-drying the obtained solution.

[0028] In yet another embodiment, the disclosure relates to a kit comprising a freeze-dried composition containing a weakened tetravalent dengue virus, the composition comprising D-sorbitol, glycine, and trehalose hydrate.

[0029] The composition comprising the attenuated tetravalent dengue virus of this disclosure may further contain L-histidine or albumin.

[0030] The compositions comprising attenuated tetravalent dengue virus as disclosed herein include attenuated serotype 1 dengue virus, attenuated serotype 2 dengue virus, attenuated serotype 3 dengue virus, and attenuated serotype 4 dengue virus. For example, serotypes 1 to 4 of the herein were deposited with the ATCC (American Type Culture Collection; 10801 University Boulevard, Manassas, VA 20110 USA) on October 19, 2016, under depositary numbers PTA-123506, PTA-123505, PTA-123507, and PTA-123508, respectively.

[0031] The lyophilized compositions of this disclosure may be used dissolved in a solvent, for example, water. By dissolving the lyophilized compositions of this disclosure in a solvent, they are reconstituted as liquid formulations having a predetermined concentration.

[0032] The freeze-dried composition containing the attenuated tetravalent dengue virus of this disclosure may, for example, contain, at a final bulk liquid concentration of approximately 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20 w / v% or more, or any number in between, of D-sorbitol when dissolved in a solvent. Regarding the concentration of D-sorbitol in the liquid after dissolution of the freeze-dried composition of this disclosure, the lower limit of the concentration may be 0.5 w / v%, 1.0 w / v%, 1.5 w / v%, 2.0 w / v%, 2.5 w / v%, 3.0 w / v%, 3.5 w / v%, 4.0 w / v%, 4.5 w / v%, or 5.0 w / v%, and the upper limit of the concentration may be 10.0 w / v%, 9.5 w / v%, 9.0 w / v%, 8.5 w / v%, 8.0 w / v%, 7.5 w / v%, 7.0 w / v%, 6.5 w / v%, 6.0 w / v%, or 5.5 w / The concentration may be v%, and in one embodiment, the concentration may be 0.5 to 10.0 w / v%, 1.0 to 9.5 w / v%, 1.5 to 9.0 w / v%, 2.0 to 8.5 w / v%, 2.5 to 8.0 w / v%, 3.0 to 7.5 w / v%, 3.5 to 7.0 w / v%, 4.0 to 6.5 w / v%, 4.5 to 6.0 w / v%, or 5.0 to 5.5 w / v%, preferably 2.0 to 8.0 w / v%, more preferably 4.0 to 6.0 w / v%, and even more preferably about 5 w / v%.

[0033] The freeze-dried composition containing the attenuated tetravalent dengue virus of this disclosure may, for example, contain glycine at a final bulk liquid concentration of about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150 mM or more, or any number in between, when dissolved in a solvent. Regarding the concentration of glycine in the liquid after dissolution of the freeze-dried composition of this disclosure, the lower limit of the concentration may be 10 mM, 30 mM, 50 mM, or 70 mM, and the upper limit of the concentration may be 150 mM, 120 mM, 110 mM, or 100 mM. In one embodiment, the concentration may be 10 to 150 mM, 30 to 120 mM, 50 to 110 mM, preferably 70 to 100 mM, more preferably 20 to 100 mM, and even more preferably about 80 mM.

[0034] The freeze-dried composition containing the attenuated tetravalent dengue virus of this disclosure may contain, for example, trehalose in a final bulk liquid concentration of about 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20 w / v% or more, or any number in between, when dissolved in a solvent. Regarding the concentration of trehalose in the liquid after dissolution of the freeze-dried composition of this disclosure, the lower limit of the concentration may be 0.25 w / v%, 1.0 w / v%, 2.0 w / v%, or 3.0 w / v%, and the upper limit of the concentration may be 20 w / v%, 15 w / v%, 10 w / v%, or 7.0 w / v%, and in one embodiment, the concentration may be 0.25 to 20 w / v%, 1.0 to 15 w / v%, 2.0 to 10 w / v%, preferably 3.0 to 7.0 w / v%, more preferably 4.0 to 6.0 w / v%, and even more preferably about 5 w / v%.

[0035] The freeze-dried composition containing the attenuated tetravalent dengue virus of this disclosure may contain, for example, a final bulk liquid concentration of histidine of about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10 mM, 15, 20 mM or more, or any number in between, when dissolved in a solvent. Regarding the concentration of histidine in the liquid after dissolution of the freeze-dried composition of this disclosure, the lower limit of the concentration may be 0.5 mM, 2.5 mM, 3.0 mM, or 4.0 mM, and the upper limit of the concentration may be 20 mM, 10 mM, 7.5 mM, or 6.0 mM. In one embodiment, the concentration may be 0.5 to 20 mM, 2.5 to 10 mM, preferably 3.0 to 7.5 mM, more preferably 4.0 to 6.0 mM, and even more preferably about 5 mM. It is preferable to include histidine at a concentration of less than 30 mM in the liquid after dissolution because the infectivity decreases when the histidine concentration is 30 mM.

[0036] The freeze-dried composition containing the attenuated tetravalent dengue virus of this disclosure may contain, for example, at a final bulk liquid concentration of albumin of about 0.025, 0.05, 0.075, 0.1, 0.125, 0.15, 0.175, 0.2, 0.225, 0.25, 0.275, 0.3, 0.325, 0.35, 0.375, 0.4, 0.425, 0.45, 0.475, 0.5, 0.525, 0.55, 0.575, 0.6 w / v% or more, or any number in between, when dissolved in a solvent. With respect to the concentration of albumin in the liquid after dissolution of the freeze-dried composition of this disclosure, the lower limit of the concentration may be 0.025 w / v%, 0.05 w / v%, 0.075 w / v%, or 0.1 w / v%, and the upper limit of the concentration may be 0.6 w / v%, 0.45 w / v%, 0.3 w / v%, or 0.2 w / v%, and in one embodiment the concentration may be 0.025 to 0.6 w / v%, 0.05 to 0.45 w / v%, 0.075 to 0.3 w / v%, preferably 0.1 to 0.2 w / v%, more preferably 0.05 to 0.2 w / v%, and even more preferably about 0.1 w / v%.

[0037] In one embodiment, when the lyophilized composition of the present disclosure is dissolved in a solvent, for the final concentration, the concentration of trehalose is 5 ± 1%, or D-sorbitol is 5 ± 1%, preferably a combination of those concentrations. In yet another embodiment, when the lyophilized composition of the present disclosure does not contain histidine, when dissolved in a solvent, for the final concentration, the concentration of trehalose is 5 ± 1%, or D-sorbitol is 1 ± 0.5%, preferably a combination of those concentrations.

[0038] In one embodiment, the composition of the present disclosure may be a composition that does not contain L-histidine. Histidine may be used as a buffering agent and a stabilizer. When L-histidine is contained at a high concentration (for example, 30 mM or more at the time of dissolution), the infectious titer may decrease, so it is preferably used at a low concentration. In another embodiment, the composition of the present disclosure may be a composition that does not contain albumin. When the composition of the present disclosure does not contain albumin, animal-derived components can be excluded, so it may have advantages such as reducing the risk of allergy. When albumin is contained, it is preferable for suppressing cake shrinkage.

[0039] The kit of the present disclosure is not particularly limited, but may include a suitable solvent and instructions for use. The kit may be configured to include a container enclosing a lyophilized live attenuated tetravalent dengue virus composition, a solvent for dissolving the composition, and instructions.

[0040] As used herein, "about" means a range of ±10%, preferably ±5%.

[0041] Hereinafter, the present invention will be described in detail with reference to examples, which are used for illustrative purposes only, and the present invention is not limited to these examples.

[0042] [Example 1] Virus solution: A live attenuated dengue virus (DENV1 to DENV4) obtained by the method described in Patent Document 1 was mixed to prepare a tetravalent virus solution. The content of each serotype is shown in Table 1. Dengue virus information (FFU: Focus Forming Unit)

[0043] Equipment: The following equipment was used: • Freeze dryer: Manufactured by Kyowa Vacuum Technology Co., Ltd. • Seam machine: Custom-made • 37°C set CO2 incubator: Manufactured by Thermo, HERACELL 150i or equivalent

[0044] Vero cells: Derived from African green monkey kidney epithelial cells. Purchased from ATCC (ATCC number CCL-81), cultured, and stored. Vero cells obtained by passage from a working cell bank (129th generation) were used.

[0045] Anti-dengue virus monoclonal antibodies: Four types of monoclonal antibodies against each serotype of dengue virus, prepared separately, were used. Details are shown in Table 2. Monoclonal antibody information [a] The calculation was performed assuming an IgG concentration of 1.0 mg / mL when Abs. 280 nm = 1.4.

[0046] The following describes the equipment and reagents used in this example, as well as the substrates for cell culture and immunostaining.

[0047] Equipment: 1) 48-well plate: Costar, 3548 2) 37°C CO2 incubator: Sanyo, Model MCO-19AIC or equivalent 3) 34°C CO2 incubator: ESPEC, BNA-111 or equivalent 4) Microscope: Olympus, Model CK30-F100 5) ELISpot analyzer: ImmunoSpot S5 VERSA Analyzer, CTL

[0048] Reagents: 1) Sodium chloride: Wako, Cat. No. 283-55147 2) Sodium hydrogen phosphate: Wako, Cat. No. 192-02837 3) Sodium dihydrogen phosphate: Wako, Cat. No. 192-02815 4) Sorbitol: Wako, Cat. No. 198-03755 5) Glycine: Nacalai Tesque, Cat. No. 17109-35 6) Histidine: Nacalai Tesque, Cat. No. 18116-92 7) Trehalose: Wako, Cat. No. 206-18455

[0049] Cell culture: 1) MEM: GIBCO, Cat. No. 11095-080 2) Inactivated fetal bovine serum (hereinafter "FBS") 3) PBS (-): In-house prepared 4) Penicillin-Streptomycin, Liquid (hereinafter "PS"): GIBCO, 15140-122 5) 0.25% Trypsin-EDTA (1×), Phenol Red: GIBCO, 25200-072 6) 0.4% Trypan Blue 7) Cell culture medium: 10% FBS, 200 units / 200 μg / mL PS-containing MEM

[0050] Immunostaining method: 1) MEM (Minimum Essential Medium): GIBCO, Cat. No. 11095-080 2) Virus culture medium: 1 × MEM containing 2% FBS 3) Methylcellulose (MC): Wako Pure Chemical Industries, Ltd., 136-02155 4) 2.92% L-glutamine 5) PS: GIBCO, Ltd., 15140-122 6) 5 × Conc. 1 / 15 mol / L PBS (pH 7.5) 7) Blocking buffer, primary antibody diluent: 1 / 15 mol / L PBS-T containing 1% BSA and 0.1% sodium azide 8) Washing PBS-T: 1 / 15 mol / L PBS (pH 7.5) containing 0.05% Tween 20 9) Formaldehyde: Wako Pure Chemical Industries, Ltd., Cat. No. 064-00406 10) Secondary antibody diluent: 1 / 15mol / L PBS-T containing 1% BSA, 0.005% BCP 11) Secondary antibody: Anti-mouse IgG HRP-labeled antibody: Manufactured by Dako, Cat. No. P0447 12) TRITON X-100: Manufactured by SIGMA, Cat. No. T-6878 13) Color former (TMB): Manufactured by MOS, Cat.No.TMBH-1000

[0051] Quadrivalent Bulk Preparation: Quadrivalent dengue virus bulk solutions were prepared as shown in Tables 3 and 4 (compositions G to OG30). The composition shown is for 0.5 mL of solution when dissolved in 0.7 mL of solvent (Japanese Pharmacopoeia Water for Injection). Ingredients Table Composition of each tetravalent bulk liquid

[0052] Lyophilization: This was carried out as follows: 1) 0.7 mL of the prepared tetravalent bulk solution was dispensed into 2 mL vials. 2) FBS was added to the remaining tetravalent bulk solution to a final concentration of 20 v / v%, and the solution was divided into cryotubes and stored at -60°C or below. This was used as the sample before lyophilization. 3) The 2 mL vials after dispensing the tetravalent bulk solution were partially capped and then lyophilized. Lyophilization was carried out using the general steps of pre-freezing → vacuum evacuation → primary drying → secondary drying → sealing. 4) The samples after lyophilization were stored at 5°C or below.

[0053] Cake shape observation: The following items were evaluated and recorded for the prepared lyophilized formulation: 1) Color of the formed cake 2) Height 3) Gap with the vial 4) Presence or absence of shape defects

[0054] Infectivity titer measurement (immunostaining): The lyophilized preparation was dissolved in 0.7 mL of sterile water, and the infectivity titer was measured by immunostaining.

[0055] Preparation of cell sheets: 1) Prepare a cell suspension with a cell density of 1.0 × 10⁶ 5 1) Prepare the cell suspension to a concentration of cells / mL. 2) Seed 300 μL of the cell suspension per well in a 48-well plate (3.0 × 10⁶). 4 (cells / well). 3) Cultured in a CO2 incubator (37°C, 5% CO2) for 3 days.

[0056] Virus Inoculation: 1) The cell plates were observed under a microscope to confirm that there were no abnormalities such as contamination by bacteria and that a monolayer confluent was formed. 2) The virus was diluted with diluent (treated on ice). Dilution ratios: DENV1: 7.0E+04 DENV2: 1.2E+04 DENV3: 2.0E+03 DENV4: 1.2E+05 *The dilution ratio was set to approximately 20 FFU / Well for each serotype. 3) The cell culture medium in the 48-well plate was removed, and any small amount of medium remaining on the plate was aspirated with a Kimwipe or similar. 4) 100 μL of the diluted virus solution from step 2) was added to each well of the 48-well plate. 5) The cells were incubated in a CO2 incubator (34°C, 5% CO2) for 60 minutes (adsorption). Tilting was performed every 15 minutes during this time.

[0057] Addition of layered medium: The preparation method for layered medium is shown below. The reagents were added in the following order, and the solution was stirred after all reagents had been added. Example of layered medium preparation: 1.5% methylcellulose: 100 mL, Inactivated FBS: 2 mL, 2.92% L-glutamine: 1 mL, PS: 1 mL 1) Remove the virus solution and add 200 μL of layered medium to each well of a 48-well plate. 2) Incubate in a CO2 incubator (34°C, 5% CO2) for 2 to 3 days.

[0058] Fixation: 1) Remove the stratified culture medium from the wells and wash three times with washing PBS-T, then gently pat dry with a Kimwipe or similar material. 2) Add 100 μL of formaldehyde solution diluted to 1% with PBS(-) to each well. 3) Allow to stand at room temperature for 30 minutes.

[0059] Blocking: 1) Remove the liquid from the plate and wash three times with washing PBS-T, then gently tap to remove excess water with a Kimwipe or similar. 2) Add 100 μL of TRITON X-100 diluted to 0.1% with PBS(-) to each well. 3) Let stand at room temperature for 5 minutes. 4) Remove the liquid from the wells and wash three times with washing PBS-T, then gently tap to remove excess water with a Kimwipe or similar. 5) Add 300 μL of blocking buffer to each well. 6) Let stand at room temperature for 30 minutes.

[0060] Primary reaction: 1) The primary antibody was diluted to 5 μg / mL with primary antibody diluent. 2) The liquid in the wells was removed, and the remaining small amount of culture medium was absorbed with a Kimwipe or similar. 3) 100 μL of the diluted primary antibody was added to each well. 4) The mixture was allowed to stand at room temperature for 1 hour.

[0061] Secondary reaction: 1) The secondary antibody was diluted to 1 μg / mL with blocking buffer. 2) The liquid in the wells was removed, and the wells were washed three times with washing PBS-T, then gently tapped with Kimwipes to remove excess water. 3) 100 μL of the diluted secondary antibody was added to each well. 4) The wells were allowed to stand at room temperature for 1 hour.

[0062] Color development reaction: 1) Remove the liquid from the wells and wash three times with washing PBS-T, then gently pat dry with a Kimwipe or similar material. 2) Add 100 μL of color developer to each well. 3) Let stand at room temperature, protected from light, for 10 minutes.

[0063] Focus detection: 1) Remove the liquid from the plate and wash it 2-3 times with water, then gently pat it dry with a Kimwipe or similar material. 2) Dry it in a light-shielded place. 3) Use ELISpot to capture images of the plate and count the foci in the wells. 4) Calculate the infectious titer.

[0064] Stability evaluation of lyophilized formulations: The stability of lyophilized formulations was evaluated under severe temperature conditions (37°C ± 2°C, 1 week) and accelerated conditions (25°C ± 2°C, 2 to 18 months). The evaluation items were the change in infectivity titer and the change in cake shape.

[0065] Long-term storage stability evaluation of lyophilized formulations: The stability of lyophilized formulations was evaluated at storage temperatures (2.9±1.5℃, 18 months). The evaluation items were the change in infectious titer and the change in cake shape.

[0066] Humidity test of freeze-dried formulations: The humidity of freeze-dried formulations was measured.

[0067] Test results and changes in infectivity titer during the formulation process (bulk preparation, filling, and freeze-drying): The results of each test were compared with the quality targets in Table 5 to confirm the degree to which the targets were achieved. Quality targets for each formulation process

[0068] Tables 6-9 show the results of the infectivity titer test. The geometric mean ± 2SD of three samples (n=3) is shown as the infectivity titer. For all serotypes, the decrease in infectivity titer before and after the freeze-drying process was small for compositions G and N. On the other hand, composition OH30 (containing 30 mM histidine) showed the largest decrease in infectivity titer among the 10 compositions, with a maximum decrease of 1.13 Log (DENV3). Since the yield of composition OH30 was lower than that of composition OH10 (containing 10 mM histidine), this result suggests that the yield decreases in a histidine concentration-dependent manner. The decrease in infectivity titer before and after 7 days of storage under harsh temperature conditions (37°C ± 2°C) was smallest for composition G among the 10 compositions, followed by composition OG (containing glycine), while the decrease was large for composition OH, with composition OH1 showing the largest decrease. Therefore, it was considered that sorbitol or glycine contributes to thermal stability. Infectivity titer (DENV1) of freeze-dried preparations. *SD calculations for composition G and composition N are not available because n=2. Samples that met the quality targets shown in Table 5 are indicated with *. Infectious titer (DENV2) of lyophilized preparations *Samples that met the quality targets shown in Table 5 are marked with an asterisk (*). Infectivity titer (DENV3) of lyophilized preparations. *Samples that met the quality targets shown in Table 5 are marked with an asterisk (*). Infectious titer (DENV4) of lyophilized preparations. *Samples that met the quality targets shown in Table 5 are indicated with an asterisk (*).

[0069] Evaluation of Cake Shape of Freeze-Dried Formulations: Table 10 shows the humidity content of the freeze-dried formulations. While the significant cake expansion observed in composition G was improved, the formation of a concentrated film and shrinkage were observed, and cake melting occurred after one week of storage at 37°C. Significant shrinkage was also observed during storage at 3°C ​​or 25°C. On the other hand, good cake shape was obtained in compositions N and O, but the bottom of the cake shrank after heat loading. Since the humidity content of compositions G and N exceeded the target value of 1%, it was considered that high humidity was the cause of cake shrinkage and melting. Humidity Content

[0070] Accelerated testing of lyophilized formulations (25°C): Results are shown in Tables 11-14. The sample size was n=1, and each sample was subjected to infectivity titer measurement immediately after sampling. When evaluating the viral stability of each composition at 25°C using infectivity titer as an indicator, composition G was the most stable of the three compositions, showing a decrease of less than 0.5 Log after 2 months of storage (2M), while composition N showed a maximum decrease of 1.0 Log (DENV3), and composition O showed a maximum decrease of 1.7 Log (DENV4). These results indicate that sorbitol contributes to viral stability at 25°C. Changes in infectivity titer of lyophilized formulations stored at 25°C (DENV1) Trends in infectivity titer of lyophilized preparations stored at 25°C (DENV2) Trends in infectivity titer of lyophilized preparations stored at 25°C (DENV3) Trends in infectivity titer of lyophilized preparations stored at 25°C (DENV4)

[0071] Long-term storage stability of lyophilized preparations: The results are shown in Table 15. The number of samples was n=1, the storage temperature was 2.9±1.5℃, and each sample was subjected to infectivity titer measurement immediately after sampling. In this study, only composition G was measured. For DENV1 and DENV2, the decrease in infectivity titer up to 18 months (18M) after the start of storage was less than 0.3 Log, which is the measurement error range of the infectivity titer test. However, DENV3 showed a decrease of 0.64 Log and DENV4 showed a decrease of 0.53 Log, which exceeded the measurement error range. Changes in infectivity titer of samples stored at 3℃.

[0072] [Example 2] Test materials and methods: Prepared tetravalent bulk (frozen) and lyophilized formulations were used. Table 16 shows the composition, and Table 17 shows the list of samples. The time notation (hrs) for each sample name in Table 17 indicates the secondary drying time in the lyophilization process (the same applies hereafter). Composition *The final bulk fluid concentration (viral load per dose) is shown. Sample List

[0073] Infectivity testing of quadrivalent bulk: To confirm that each bulk composition contains the viral loads listed in Table 16, quadrivalent bulk (frozen) was thawed in running water and then subjected to infectivity testing.

[0074] Viral stability evaluation of lyophilized formulations: The viral stability of lyophilized formulations was evaluated using infectivity titer as an indicator, before and after being stored for 7 days under harsh temperature conditions (37°C ± 2°C). The lyophilized formulations were dissolved in 0.7 mL of Milli-Q water and subjected to infectivity titer testing.

[0075] Solubility evaluation of lyophilized formulations: The time required to dissolve the lyophilized formulations in 0.7 mL of Milli-Q water was measured.

[0076] Evaluation of viral stability of lyophilized formulations after dissolution: For lyophilized formulations, viral stability was evaluated using infectivity titer as an indicator before and after dissolution in 0.7 mL of Milli-Q water and storage at 25°C ± 2°C for 24 hours.

[0077] Evaluation of cake shape of freeze-dried formulations: The change in cake shape of freeze-dried formulations was evaluated before and after being held under harsh temperature conditions (37°C ± 2°C) for 7 days.

[0078] Changes in infectivity titer during the formulation process (bulk preparation, filling, and freeze-drying): The results of each test were compared with the quality targets in Table 5 to confirm the degree to which the targets were achieved.

[0079] The results of the infectivity tests described above are shown in Tables 18-21. The geometric mean ± 2SD of the three samples (n=3) is shown as the infectivity titer. Regarding the infectivity titer of the lyophilized formulations, no difference in infectivity titer was observed for any of the compositions due to the secondary drying time. Therefore, the viral stability during the formulation process was evaluated using the infectivity titer of the sample with the highest heat load, a secondary drying time of 20 hours. The decrease in infectivity titer during the formulation process for composition G was 0.67 Log for DENV2, 0.56 Log for DENV3, and 0.54 Log for DENV4. Note that for DENV1, the decrease in infectivity titer during the formulation process was not calculated because the infectivity titer of the sample before lyophilization (freeze-drying) for composition G and composition N+Alb was lower than the estimated value from the measurement results of the preliminary study.

[0080] Under harsh temperature conditions (37°C ± 2°C), the decrease in infectivity titer before and after 7 days of storage was DENV1: 0.28 Log, DENV2: 0.33 Log, DENV3: 0.43 Log, and DENV4: 0.36 Log for composition G, satisfying the WHO standard (less than 1 Log decrease in infectivity titer over 1 week at 37°C). Composition N+Alb, which met the quality objectives similarly to composition G, showed DENV1: 0.57 Log, DENV2: 0.65 Log, DENV3: 0.65 Log, and DENV4: 0.49 Log. While this met the WHO standard, the yield of all serotypes was lower compared to composition G. Based on these results, composition G was determined to be the most suitable composition. DENV1 infectivity titer DENV2 infection titer DENV3 infection titer DENV4 infection titer

[0081] Solubility evaluation of lyophilized formulations: Table 22 shows the dissolution time for each sample. No significant difference in dissolution time was observed for any of the samples. Dissolution time for each sample

[0082] Evaluation of viral stability after thawing of lyophilized formulations: Figures 1-4 show the changes in infectivity titer when samples were stored at 25°C after thawing (samples with a secondary drying time of 10 hours were used). In DENV3, composition N+Alb showed the highest viral stability, followed by composition G, and then composition N, in which order of maintaining high infectivity titer. On the other hand, only in DENV2 did composition G show the best viral stability. In DENV1 and DENV4, the contribution of composition G and composition N to viral stability was equivalent. However, in composition N+Alb, it was suggested that DENV1 alone showed a faster rate of decrease in infectivity titer than the other two compositions. From this, it was shown that albumin is the most effective viral stabilizer in liquid form, and that sorbitol is effective in DENV2.

[0083] Evaluation of the cake shape of the freeze-dried formulation: Regarding humidity, the long-term dried samples of composition G and composition N+Alb were below the target value of 1%, but all samples of composition N and the short-term dried samples of composition G and composition N+Alb were above 1% (Table 23).

[0084] Humidity Evaluation of Freeze-Dried Formulations: Regarding cake shape, composition G showed cracking and shrinkage, and cake melting was confirmed after 24 hours of storage at 37°C. On the other hand, while cracking was observed in the cakes of composition N and composition N+Alb, no change in cake shape was observed before and after heat loading. The reason why composition G melted under heat loading is presumed to be that the high concentration of amorphous sorbitol caused the sorbitol to react with bound water and melt under heat loading. However, since this formulation is stored under refrigeration and can be controlled to avoid exposure to harsh heat loading conditions, composition G was judged to be the most appropriate from the standpoint of viral stability and humidity. Humidity

[0085] [Example 3] The effects of adding albumin to compositions G and N were investigated in detail. Similar investigations were also conducted on proline and furialazine as potential stabilizers to replace albumin.

[0086] Test materials: Table 24 shows the composition of each additive in the lyophilized preparation used as the test substance in this example. Concentration of each additive in 1 mL of the final bulk liquid.

[0087] Quality evaluation of freeze-dried formulations: Observation of cake shape: The cake shape of the freeze-dried formulations was observed for each composition. The color, depth, degree of shrinkage, and presence of cracks or foreign matter were observed and recorded visually.

[0088] Infectivity testing: Each preparation was dissolved in 0.7 mL of purified water, and an infectivity test (immunostaining method) was performed.

[0089] The stability of the lyophilized formulations was evaluated under severe temperature conditions (37°C ± 2°C, 7 days). The evaluation items were observation of the cake shape and the change in infectivity titer. Each formulation was dissolved in 0.7 mL of purified water, and an infectivity titer test (immunostaining method) was performed.

[0090] Stability Evaluation of the Final Bulk Solution: The stability of the final bulk solution before freeze-drying was evaluated under accelerated temperature conditions (25°C ± 2°C, 27 hours). The evaluation item was the change in infectivity titer. Infectivity titer testing (immunostaining method) was performed.

[0091] Regarding the formulation of the dengue vaccine, the quality targets shown in Table 5 were established based on the test results. The degree to which the targets were achieved was confirmed by comparing each test result with Table 5.

[0092] Observation of cake shape: After freeze-drying, good cake formation was observed for all compositions except composition 9, which contains proline. On the other hand, composition 9, which contains proline, was unable to form a cake, and a foamy structure was observed. In the stress test, cake melt was observed in the composition group based on composition G (compositions 1-3) and composition 9, which contains proline. On the other hand, although cake melt was not observed in compositions 4-8 and 10, cake shrinkage was observed. However, for compositions 5-8, it was found that the degree of shrinkage tended to be reduced in all compositions by the addition of albumin.

[0093] Infectivity Titer Testing of Lyophilized Formulations: The progression of infectivity titers of lyophilized formulations is shown in Figures 5-8 below. The geometric mean ± 2SD of the values ​​measured for 3 samples (n=3) per composition is shown as the infectivity titer. Note that BT is an abbreviation for "Back Titration" and refers to tetravalent virus solution (without additives).

[0094] Table 25 shows the decrease in infectivity titer during the freeze-drying process and before and after the stress test. Measurement results that met the quality targets (decrease in infectivity titer during the freeze-drying process: less than 0.5 Log10 FFU / mL, decrease before and after the stress test: less than 1 Log10 FFU / mL) are indicated with an asterisk (*). During the freeze-drying process, no composition met the target for all serotypes (DENV1-4). On the other hand, the decrease in infectivity titer before and after the stress test was less than 1 Log10 FFU / mL for all compositions, thus achieving the quality target regarding the decrease in infectivity titer during the stress test. (Decline infectivity titer during freeze-drying process and before and after the stress test) Products that met the quality targets (decrease in the freeze-drying process: less than 0.5 Log10 FFU / mL, decrease before and after the harsh test: less than 1 Log10 FFU / mL) are marked with an asterisk (*).

[0095] Stability Evaluation of Final Bulk Solution Figures 9-12 show the changes in infectivity titer when the final bulk solution before freeze-drying was stored at 25°C for 27 hours. When samples were subjected to infectivity titer testing at the start of storage, 5 hours after the start of storage, and 27 hours after the start of storage, it was confirmed that the infectivity titer decreased by approximately 1 log in three compositions: composition G (composition 1), N (composition 4), and 1.5% Sor + Pro300 + 5% Tre (composition 9). On the other hand, compositions containing albumin or flialadine showed high stability across all serotypes.

[0096] [Example 4] Anticipating a decrease in infectivity during the freeze-drying process, an excess amount of virus was added to the final bulk to achieve the target infectivity of 1.0 × 10⁶. 5 We conducted studies to aim for achieving FFU / dose. Considering that the filling volume per vial is 0.5 mL, in order to achieve the target infectivity titer, the infectivity titer of the lyophilized preparation must be 2.0 × 10⁶. 5 The FFU / mL level must be above this. In this example, freeze-drying tests and harsh tests were conducted under conditions of excessive virus load, and the cake shape and infectivity titer tests were performed on the samples subjected to each test to evaluate the effect of excessive virus load.

[0097] Test materials, equipment, and reagents: The reagents used in this example are listed below. Reagents: 1) Sodium chloride: Wako, Cat. No. 195-01663 2) Disodium hydrogen phosphate hydrate: Wako, Cat. No. 198-02834 3) Sodium dihydrogen phosphate: Wako, Cat. No. 192-02815 4) Glycine: Nacalai Tesque, Cat. No. 17109-35 5) Histidine: Nacalai Tesque, Cat. No. 18116-92 6) Trehalose: Wako, Cat. No. 206-18455 7) Sorbitol: Wako, Cat. No. 198-03755 8) Sorbitol syrup: Merck, Cat. No. 1.02994.9050 9) Recombinant human serum albumin: In-house manufactured

[0098] Virus: The infectivity titers for each bulk price are shown below. Infectivity titers for each bulk price

[0099] Container and closure system: 1) Glass vial: Vial 2mL VIST (Yamato Special Glass Co., Ltd.) 2) Rubber stopper: F-1451 CP19G HS02 (Nipro Corporation)

[0100] Equipment: 1) Freeze dryer: LyoStarII (manufactured by FTS Systems)

[0101] Test Method Additive Preparation: The composition of the additives in this example is shown in Table 27 below. The numbers in the table indicate the concentration of each additive in the final bulk liquid. Each additive was measured and mixed to achieve the concentrations shown in the table. Additive composition per 1 mL of bulk liquid

[0102] Preparation of final bulk solution: 1) The infectivity titer of each serotype in the final bulk solution before lyophilization of virus (+) samples is 8.0 × 10⁻⁶. 6 A tetravalent virus solution was prepared by mixing the unit-valence bulk solutions of DENV1 to DENV4 to achieve an FFU / mL ratio. The tetravalent virus solution was added to the pre-prepared additive solutions of each composition and mixed until homogeneous. The prepared bulk solutions were stored on ice. 2) For virus-negative samples, the same amount of 10 mMPB as the tetravalent virus solution added during the preparation of virus-positive samples was added to each additive solution.

[0103] Filling and Loading: Using a continuous dispenser and pipetteman, 0.35 mL each of virus(+) and virus(-) bulk solution were filled into vials and partially capped. The vials were arranged on a tray attached to the freeze-dryer according to a predetermined layout diagram and freeze-dried.

[0104] Sample storage: After freeze-drying, the samples were stored at 5°C or below.

[0105] Quality evaluation of freeze-dried samples: Observation of cake shape: The cake shape of the freeze-dried formulation was observed for each composition. The color, depth, degree of shrinkage, and presence of cracks or foreign matter were observed and recorded visually.

[0106] Infectivity testing: Infectivity testing was performed on samples after observation of the cake shape. The lyophilized preparation was dissolved in 0.7 mL of solvent (water for injection or purified water) and subjected to infectivity testing (immunostaining).

[0107] Severity Test: After observing the cake shape, the stability of the samples under severe temperature conditions (37°C ± 2°C, 7 days) was evaluated. The evaluation items were the change in cake shape and infectivity titer after the severity test. The formulations after the severity test were dissolved in 0.7 mL of solvent (water for injection or purified water) and subjected to infectivity testing (immunostaining method).

[0108] Long-term storage stability study: After observing the cake shape, the stability of samples (compositions N, N+0.1%Alb, 1.5%Sor+5%Tre, and 1.5%Sor+5%Tre+0.1%Alb) at actual storage temperatures (5°C ± 3°C) was evaluated. The evaluation items were the cake shape after storage and the change in infectivity titer. The lyophilized preparation was dissolved in 0.7 mL of solvent (water for injection or purified water) and subjected to an infectivity titer test (immunostaining method).

[0109] Observation of cake shape in test results: After freeze-drying, good cake shape was obtained for all compositions 1-7. However, after the harsh test, significant melting was observed in compositions 1-3, which are based on composition G. No change in cake shape was observed before and after the harsh test for compositions 4-7.

[0110] Infectivity Test: Infectivity titers before and after freeze-drying and after harsh testing are shown in Tables 28-31 and Figures 13-16. The quality target was "infectivity titer after freeze-drying of 2.0 × 10⁻⁶". 5 We confirmed that the two conditions of "FFU / mL or higher" and "decrease in infectivity titer after exposure within 1 Log for all compositions and serotypes" were achieved for all compositions and serotypes. In particular, it became clear that compositions 1-3, based on composition G, tended to show higher infectivity titers. Infectivity titers (DENV1) before and after freeze-drying and exposure. (Unit: FFU / mL) Infectivity titer (DENV2) before and after freeze-drying and after harsh treatment (Unit: FFU / mL) Infectivity titer (DENV3) before and after freeze-drying and after harsh treatment (Unit: FFU / mL) Infectious titer (DENV4) before and after freeze-drying and after harsh treatment (Unit: FFU / mL)

[0111] Long-term storage stability test: Figures 17-20 show the results of the long-term storage stability test conducted for compositions N, N+0.1%Alb, 1.5%Sor+5%Tre, and 1.5%Sor+5%Tre+0.1%Alb. Although a slight decrease in infectivity titer was observed from 6 to 12 months after the start of storage, the decrease in infectivity titer was confirmed to be less than 0.5 Log for all compositions and all serotypes. Furthermore, no difference in trends was observed among the four compositions tested.

[0112] Based on the above results, the target infectious titer of the final formulation is (2.0 × 10⁻⁶). 5 8.0 × 10¹⁰ is an overdose relative to FFU / mL. 6 By preparing the final bulk solution to achieve an FFU / mL ratio, it was confirmed that the target infectious titer could be achieved for all compositions.

[0113] [Example 5] Regarding a vaccine containing attenuated live quadrivalent dengue virus (lyophilized formulation / composition G), the purpose of the study was to obtain results such as the potency of the formulation, storage stability at 5℃±3℃ (actual storage conditions) and 37℃±2℃ (severe conditions), and stability after dissolution.

[0114] Test Materials and Methods Test materials: Dengue Tetravalent Vaccine for Injection (small product / composition G) * *To investigate the effect of filling time on cake shape and infectivity, this product was classified into three categories: Beginning, Middle, and End, in order of filling time, and used for evaluation.

[0115] Test Method: Observation of Cake Shape: The shape of the cake was observed. The color, depth, degree of shrinkage, and presence of cracks or foreign matter were observed and recorded with the naked eye.

[0116] Titer testing: Infectivity testing was performed on samples after observation of the cake shape. The lyophilized preparation was dissolved in 0.7 mL of purified water and subjected to infectivity testing (immunostaining).

[0117] Long-term storage stability evaluation under actual storage conditions: The stability during long-term storage under actual storage conditions (5°C ± 3°C) was evaluated. The evaluation items were the changes in cake shape and infectious titer after sampling, and the sampling times of the specimens were 1, 3, 6, 12, 18, 24, and 36 months. The formulations after long-term storage were dissolved in 0.7 mL of purified water and subjected to an infectious titer test (immunostaining method).

[0118] Storage stability evaluation under harsh conditions: The storage stability under temperature harsh conditions (37°C ± 2°C, 7 days) was evaluated. The evaluation items were the changes in cake shape and infectious titer after the harsh test. The formulations after the harsh test were dissolved in 0.7 mL of purified water and subjected to an infectious titer test (immunostaining method).

[0119] Observation of the test result cake shape: Although some cracks and adhered filling liquid on the side were observed in the cake shape, the cake shape was generally good. Also, when comparing the lyophilized products at the Beginning, Middle, and End, no remarkable differences in the cake shape were found, so it was considered that the influence of the filling time on the cake shape was negligible.

[0120] Potency test: The infectious titers obtained for the formulations after freeze-drying are shown in Table 32 and Figure 21. Infectious titer after freeze-drying (Unit: Log10 FFU / mL)

[0121] For DENV1 - 4, when the infectious titers were measured respectively, although only DENV3 did not reach the target of 5 Log 10 FFU / dose (0.5 mL), it was confirmed that all serotypes met the shipment specification of 3 Log 10 FFU / dose (0.5 mL).

[0122] Long-term storage stability evaluation under actual storage conditions Observation of cake shape of long-term stored samples: When comparing the cake shape at 1, 3, 6, 12, 18, 24, and 36 months after the start of storage, no significant difference in cake shape was observed up to 12 months, but shrinkage of the cake was observed from 18 months after the start of storage. At the start of storage, the cake height was approximately 3.5-4.0 mm, while at 36 months after the start of storage, it was confirmed that it had shrunk to approximately 2.0-3.5 mm. Furthermore, when comparing the cake shape at each time point for Beginning, Middle, and End, the degree of shrinkage of the freeze-dried End product appeared to be greater than that of Beginning and Middle after 18 months after the start of storage, but at 36 months after the start of storage, it was confirmed that the order of shrinkage was End > Beginning > Middle. No significant effect was observed on the degree of maintenance of cake shape after long-term storage due to differences in filling time.

[0123] Infectivity titer testing of long-term stored specimens: Table 33 and Figure 22 show the infectious titers obtained for long-term stored specimens. Changes in infectious titers of long-term stored specimens. (Unit: Log 10 FFU / mL)

[0124] Infectivity titers of the preparations were measured at 1, 3, 6, 12, 18, 24, and 36 months after the start of storage. The infectivity titers remained almost constant until 24 months after the start of storage. A slight decrease was observed at 36 months after the start of storage, but the decrease from the start of storage was less than 1 Log10 FFU / mL for all serotypes.

[0125] Evaluation of storage stability under harsh conditions: Observation of cake shape of harsh-condition samples: When comparing the cake shape before and after the harsh test, melting of the cake was observed in the freeze-dried product after harsh conditions. The exact same trend was observed when comparing the first and second tests.

[0126] Infectivity titer testing of stress-treated specimens: The infectious titers obtained for specimens before and after stress testing are shown in Table 34 and Figure 23. Infectivity titers (geometric mean) before and after stress testing. (Unit: Log 10 FFU / mL)

[0127] When comparing infectivity titers before and after the harsh test, DENV1, 2, and 4 showed a decrease in infectivity titer of 0.2 to 0.3 log. 10 While the decrease in infectious titer was small, around FFU / mL, the decrease in DENV3 was 0.9 log. 10 The FFU / mL level was somewhat high. On the other hand, the quality target of "a decrease in infectivity titer of 1.0 Log before and after the harsh test" was met. 10 We confirmed that the "less than FFU / mL" threshold was achieved across all serotypes.

[0128] Compositions 5% trehalose, 5% D-sorbitol, glycine, and L-histidine (composition G) and 5% trehalose, 1% sorbitol, and glycine (composition N) + albumin all met quality targets of a formulation yield of 10% or more and a decrease in infectivity titer of less than 1 log before and after the thermal stability test (37°C ± 2°C, 1 week). Composition G, in particular, showed excellent thermal stability. The humidity content met the quality target of less than 1%. Composition G also showed excellent stability in long-term storage tests at 5°C ± 3°C (actual storage conditions). Furthermore, high stability and good freeze-dried cake formation were confirmed with 5% trehalose + 1.5% sorbitol + albumin.

[0129] This invention can be used as a vaccine composition using the dengue virus.

Claims

1. A freeze-dried composition containing a weakened tetravalent dengue virus, comprising D-sorbitol, glycine, and trehalose hydrate.

2. The composition according to claim 1, further comprising L-histidine.

3. The composition according to claim 1 or 2, further comprising a buffer, wherein the buffer comprises sodium hydrogen phosphate hydrate, sodium dihydrogen phosphate hydrate, and sodium chloride.

4. The composition according to claim 3, further comprising albumin.

5. The composition according to claim 1, wherein, when dissolved in a solvent, the final concentration is 5±1% trehalose and 1±0.5% D-sorbitol.

6. The composition according to claim 2, wherein, when dissolved in a solvent, the final concentration is 5±1% trehalose and 5±1% D-sorbitol.

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