TW201839130A - Japanese Encephalitis Vaccine And Method For Production Thereof - Google Patents

Abstract

The present invention relates to a method for production of Japanese encephalitis vaccine. The method includes inoculating cells for vaccine production on a cell culture carrier, culturing the cells in a tidal culture system for 3 to 10 days, inoculating the Japanese encephalitis virus (JEV) into the cells, culturing the inoculated cells for 3 to 10 days, harvesting the virus, and purifying and inactivating the virus to obtain inactivated JEV vaccine. The present invention also relates to an inactivated JEV vaccine prepared by the method above.

Description

Japanese encephalitis vaccine and preparation method thereof

The present invention relates to a Japanese encephalitis vaccine and a preparation method thereof, in particular, a Japanese encephalitis death vaccine having a stable antigen, and a preparation method capable of increasing the stability of the Japanese encephalitis death vaccine antigen.

Japanese encephalitis is a kind of endemic epidemic disease in Taiwan in summer. The peak is usually in June and July. The epidemic areas are almost all over Taiwan. Young children under the age of 9 are common infections. Due to the poor prognosis of Japanese encephalitis, it has a great impact on individuals, families and society, as well as the burden of economic and medical care. Therefore, how to reduce the incidence of Japanese encephalitis cases is an important public health issue. At present, there is no effective anti-Japanese encephalitis virus drug available for treatment, only supportive defamation. The most effective and only way to fight Japanese encephalitis virus infection is to vaccinate Japanese encephalitis vaccine.

The earliest Japanese encephalitis vaccine preparation method was to inoculate a virulent strain of Japanese encephalitis virus into a rat brain for virus proliferation, and then remove the virus for inactivation to prepare a Japanese encephalitis inactivated whole virus vaccine. However, the biggest problem with the use of such inactivated virus vaccines is that they do not provide long-term immunity, and the number of additional vaccine injections must be increased, which causes cost burden and inconvenient injection, and increasing the number of vaccine injections also has an opportunity to trigger an allergic reaction. In addition, the Japanese encephalitis vaccine prepared from the rat brain has safety concerns. About 20% of the Japanese encephalitis vaccine prepared by the rat brain will have redness, pain and other reactions at the inoculation site, and about 10% of the people have moderate Systemic side effects such as fever, chills, headache, rash, muscle pain and other symptoms. What's more, in Japan, a 15-year-old teenager developed a suspected acute disseminated encephalomyelitis (ADEM) after being vaccinated with a Japanese encephalitis vaccine prepared from rat brain. So the Japanese government announced in 2005 that it would stop the use of the non-activated Japanese encephalitis vaccine prepared by the rat brain for routine vaccination, and also stop the production of the vaccine, and instead use cell culture to produce the Japanese encephalitis vaccine. . In line with international trends, Taiwan's Disease Control Agency also announced that Taiwan will switch to cell culture in 2017 to replace the Japanese encephalitis vaccine.

The Japanese encephalitis vaccine produced by the cell culture method has higher safety than the rat brain preparation. However, the Japanese encephalitis vaccine produced by the cell culture method has a problem of preservation stability. Gelatin is added as a stabilizer to increase the stability of the antigen in the vaccine, but gelatin may also induce an allergic reaction in the subject, thereby causing other safety problems. Therefore, the development of a Japanese encephalitis vaccine with a stable antigen and a preparation method thereof are urgently and extremely important.

In the first part of the present invention, a method for preparing a Japanese encephalitis vaccine comprises the following steps: (1) inoculating a cell for producing a vaccine on a cell culture carrier in a tidal culture system at 34~ The medium-culture medium and the serum-free medium are cultured at 37 ° C and 5 to 10% CO ₂ in a volume-free medium, and the volume ratio of the serum-free medium is 1:5 to 1:20. 1000~2000 ml/min, the residence time of the serum-free medium is 0-60 seconds after the liquid level rises completely, and the residence time of the serum-free medium is 0-60 seconds after the liquid level is completely lowered, and the adjusted CO ₂ concentration maintains the pH value. Between 7.0 and 7.4, the glucose content is maintained above 1 g/L, and the cells are inoculated for 1 to 12 hours. (2) The cells continue to be cultured for 3 to 10 days at 34 to 37 ° C and 0 to 10% CO ₂ . The serum-free medium has a liquid surface lifting speed of 1000-2000 ml/min, and the residence time of the serum-free medium is 0-60 seconds after the liquid level rises completely, and the residence time of the serum-free medium is 0-60 seconds after the liquid level is completely lowered. The adjusted CO ₂ concentration maintains the pH between 7.0 and 7.4 and the glucose content remains above 1 g/L; 3) Inoculate Japanese encephalitis virus to the cells, and the multiplicity of infection (MOI) is 1x10 ^-2 ~ 1x10 ^-6 at 34~37 °C, 5~10% CO ₂ , The serum medium is cultured, and the liquid level of the serum-free medium is 1000-2000 ml/min, and the residence time of the serum-free medium is 0-60 seconds, and the residence time of the serum-free medium is completely decreased. 0~60 seconds, the adjusted CO ₂ concentration keeps the pH between 6.8 and 7.2, and the virus is inoculated for 1~12 hours, and the glucose content is maintained above 1 g/L. (4) Japanese encephalitis virus after inoculation and The cells are further cultured in a culture environment of 34-37 ° C and 0-10% CO ₂ , and the liquid-free lifting rate of the serum-free medium is 1000-2000 ml/min, and the residence time of the serum-free medium is completely increased. 0~60 seconds, the residence time of the serum-free medium is 10~60 seconds after the liquid level is completely lowered, the adjusted CO ₂ concentration is maintained between pH 6.8 and 7.2, and the glucose content is maintained above 1 g/L; 5) The virus solution is harvested once every 5~8 days after virus inoculation, and purified and inactivated to obtain Encephalitis virus vaccine deactivation; wherein after deactivation of the Japanese encephalitis virus vaccines stored for 4 months at 2 ~ 8 ° C, viral proteolytic 5% or less.

In the second part of the present invention, a Japanese encephalitis inactivated virus vaccine prepared by the above method is provided.

In the first part of the present invention, a method for preparing a Japanese encephalitis vaccine comprises the following steps: (1) inoculating a cell for producing a vaccine on a cell culture carrier in a tidal culture system at 34~ The medium-culture medium and the serum-free medium are cultured at 37 ° C and 5 to 10% CO ₂ in a volume-free medium, and the volume ratio of the serum-free medium is 1:5 to 1:20. 1000~2000 ml/min, the residence time of the serum-free medium is 0-60 seconds after the liquid level rises completely, and the residence time of the serum-free medium is 0-60 seconds after the liquid surface is completely lowered, and the cells are inoculated for 1 to 12 hours, and The CO ₂ concentration is adjusted according to the pH value of the medium, and the CO ₂ concentration is about 8-10%. The pH of the medium is maintained between 7.0 and 7.4 during the cell inoculation, and the glucose content is maintained above 1 g/L; (2) The cells are further cultured at 34~37 ° C, 0~10% CO ₂ for 3-10 days, and the serum-free medium liquid surface lifting speed is 1000-2000 ml/min, and the serum-free medium liquid level rises completely after the residence time. 0 to 60 seconds, the serum-free medium liquid level drops completely after the residence time is 0 to 60 seconds, and according to The pH of the medium is adjusted to a CO ₂ concentration, and the CO ₂ concentration is about 0 to 8%. The pH of the medium is maintained between 7.0 and 7.4 during the culture period, and the glucose content is maintained at 1 g/L or more; (3) Japan The encephalitis virus is inoculated onto the cell, and the multiplicity of infection (MOI) is 1×10 ^-2 ~ 1x10 ^-6 , and cultured in the serum-free medium at 34 to 37 ° C and 5 to 10% CO ₂ . The liquid-free medium has a liquid surface lifting speed of 1000-2000 ml/min, and the residence time of the serum-free medium is 0-60 seconds after the liquid level rises completely, and the residence time of the serum-free medium is 0-60 seconds after the liquid level is completely lowered. The virus is inoculated for 1 to 12 hours, and the CO ₂ concentration is adjusted according to the pH value of the medium, the CO ₂ concentration is 8 to 10%, and the pH of the medium is maintained between 6.8 and 7.2 during the virus inoculation, and the glucose content is maintained at 1 g/L or more; (4) The Japanese encephalitis virus and cells after inoculation continue to be cultured in a culture environment of 34 to 37 ° C and 0 to 10% CO ₂ , and the liquid level of the serum-free medium is 1000~ 2000 ml/min, the serum-free medium liquid level rises completely after the residence time is 0-60 seconds, the serum-free medium solution After completion of the residence time decreased from 0 to 60 seconds, and adjusting the concentration of CO ₂ depending on the pH of the medium, CO ₂ concentration of 0 to 8%, the pH during the culture is maintained between 6.8 to 7.2, glucose was maintained at 1 g/L or more; and (5) virus virus is harvested once every 5-8 days after virus inoculation, and purified and non-activated to obtain a Japanese encephalitis deactivated virus vaccine; wherein the Japanese encephalitis deactivates the virus vaccine After 4 months of storage at 2-8 ° C, the viral protein cleavage rate was 5% or less.

In certain embodiments, the cell for making the vaccine is an African green monkey kidney cell (VERO cell).

In certain embodiments, the serum-free medium is cultured as VP-SFM.

In certain embodiments, the volume ratio of the cell culture carrier to the serum-free medium is between about 1:5 and 1:20. In some embodiments, the cell culture carrier and the serum-free medium preferably have a volume of about 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, and 1:11. , 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, or 1:20.

In certain embodiments, the cells in step (1) are inoculated for about 1 to 12 hours. In certain embodiments, the cell seeding in step (1) is preferably about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours.

In some embodiments, the serum-free medium in the step (1) has a liquid level rise and fall speed of about 1000 to 2000 ml/min, which is equivalent to about 1000 to 2000 mm/sec. In some embodiments, the serum-free medium liquid level rise and fall speed in the step (1) is preferably about 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 ml/ Min.

In some embodiments, the serum-free medium is raised to just cover all of the cell culture carrier, and the cell culture carrier is completely immersed in the serum-free medium, but is not suspended in the solution. The serum-free medium is used to avoid damage to the cells caused by the shear force generated by suspending the cell culture carrier in the serum-free medium.

In certain embodiments, the pH of the serum-free medium is maintained by adjusting the concentration of CO ₂ in the culture environment to avoid adjusting the pH of the medium to the cells using hydrochloric acid (HCl) and/or sodium hydroxide (NaOH). cause some damages.

In some embodiments, the serum-free medium in the step (1) has a liquid level rise and a residence time of about 0 to 60 seconds. In some embodiments, the retention time of the serum-free medium in the step (1) is preferably about 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50. , 55, or 60 seconds.

In some embodiments, the serum-free medium in the step (1) has a complete liquid level drop and a residence time of about 0 to 60 seconds. In some embodiments, the pH of the serum-free medium in the step (1) is preferably about 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50. , 55, or 60 seconds.

In certain embodiments, the pH of the serum-free medium in step (1) is maintained between about 7.0 and 7.4. In certain embodiments, the pH of the serum-free medium in step (1) is preferably maintained between about 7.0, 7.1, 7.2, 7.3, or 7.4.

In certain embodiments, the concentration of CO ₂ in step (1) is from about 8 to 10%. In some embodiments, the concentration of CO ₂ in the step (1) is preferably about 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3. 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10%.

In certain embodiments, the glucose content in step (1) is maintained above about 1 g/L. In certain embodiments, the glucose content in step (1) is preferably maintained at about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 g/L or more.

In some embodiments, the serum-free medium in step (2) is replaced once in the cell culture for about 3 to 4 days and before the virus is challenged.

In some embodiments, the serum-free medium at the step (2) has a liquid level rise and fall speed of about 1000 to 2000 ml/min, which is equivalent to about 1000 to 2000 mm/sec. In some embodiments, the serum-free medium liquid level rise and fall speed in the step (2) is preferably about 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 ml/ Min.

In some embodiments, the serum-free medium in the step (2) has a liquid level rise and a residence time of about 0 to 60 seconds. In some embodiments, the retention time of the serum-free medium in the step (2) is preferably about 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50. , 55, or 60 seconds.

In some embodiments, the serum-free medium in the step (2) has a complete liquid level drop after a complete residence time of about 0 to 60 seconds. In some embodiments, the pH of the serum-free medium in the step (2) is preferably about 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50. , 55, or 60 seconds.

In certain embodiments, the pH of the serum-free medium in step (2) is maintained between about 7.0 and 7.4. In certain embodiments, the pH of the serum-free medium in step (2) is preferably maintained between about 7.0, 7.1, 7.2, 7.3, or 7.4.

In certain embodiments, the concentration of CO ₂ in the step (2) is from about 0 to 8%. In some embodiments, the concentration of CO ₂ in the step (2) is preferably about 0, 0.5, 1.0, 1.5, _2.0 , 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5. 7.0, 7.5, or 8%.

In certain embodiments, the glucose content in step (2) is maintained above 1 g/L. In certain embodiments, the glucose content in step (2) is preferably maintained at about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 g/L or more.

In certain embodiments, the viral infection dose (MOI) in step (3) is from about 1 x 10 ^-2 to 1 x 10 ^-6 . In some embodiments, the viral infection dose (MOI) in step (3) is about 1 x 10 ^-2 , 1 x 10 ^-3 , 1 x 10 ^-4 , 1 x 10 ^-5 , or 1 x 10 ^-6 .

In some embodiments, the serum-free medium in the step (3) has a liquid level rise and fall speed of about 1000 to 2000 ml/min, which is equivalent to about 1000 to 2000 mm/sec. In some embodiments, the serum-free medium liquid level rise and fall speed in the step (3) is preferably about 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 ml/ Min.

In some embodiments, the serum-free medium in the step (3) has a liquid surface rise completely after a residence time of about 0 to 60 seconds. In some embodiments, the retention time of the serum-free medium in the step (3) is preferably about 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50. , 55, or 60 seconds.

In some embodiments, the serum-free medium in the step (3) has a complete liquid level drop after a complete residence time of about 0 to 60 seconds. In some embodiments, the pH of the serum-free medium in the step (3) is preferably about 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50. , 55, or 60 seconds.

In certain embodiments, the pH of the serum-free medium in step (3) is maintained between about 6.8 and 7.2. In certain embodiments, the pH of the serum-free medium in step (3) is preferably maintained between about 6.8, 6.9, 7.0, 7.1, or 7.2.

In certain embodiments, the concentration of CO ₂ in the step (3) is about 8 to 10%. In some embodiments, the concentration of CO ₂ in the step (3) is preferably about 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3. 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10%.

In certain embodiments, the glucose content in step (3) is maintained above 1 g/L. In some embodiments, the glucose content in step (3) is preferably maintained at about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 g/L or more.

In some embodiments, the serum-free medium liquid level rise and fall speed in the step (4) is about 1000 to 2000 ml/min, which is equivalent to about 1000 to 2000 mm/sec. In some embodiments, the serum-free medium liquid level rise and fall speed in the step (4) is preferably about 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 ml/ Min.

In some embodiments, the serum-free medium in the step (4) has a complete liquid level rise time of about 0 to 60 seconds. In some embodiments, the retention time of the serum-free medium in the step (4) is preferably about 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50. , 55, or 60 seconds.

In some embodiments, the serum-free medium in the step (4) has a complete liquid level drop after a complete residence time of about 0 to 60 seconds. In some embodiments, the residence time of the serum-free medium in the step (4) is preferably about 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50. , 55, or 60 seconds.

In certain embodiments, the pH of the serum-free medium in step (4) is maintained between about 6.8 and 7.2. In certain embodiments, the pH of the serum-free medium in step (4) is preferably maintained between about 6.8, 6.9, 7.0, 7.1, or 7.2.

In certain embodiments, the concentration of CO ₂ in step (4) is from about 0 to 8%. In some embodiments, the CO ₂ concentration in the step (4) is preferably about 0, 0.5, 1.0, 1.5, _2.0 , 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5. 7.0, 7.5, or 8%.

In certain embodiments, the glucose content in step (4) is maintained above 1 g/L. In certain embodiments, the glucose content in step (4) is preferably maintained at about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 g/L or more.

In certain embodiments, the viral solution is harvested once every 5 to 8 days after inoculation of the virus in step (5). In some embodiments, the viral solution is preferably harvested once every 5, 6, 7, or 8 days after inoculation of the virus in step (5).

In some embodiments, the inactivated treatment in the step (5) includes, but is not limited to, an inactivator treatment, a heat treatment, and the like, which are inactivated by the present invention. The inactivating agent includes, but is not limited to, formaldehyde, paraformaldehyde, beta-propionolactone (BPL), binary ethyleneimine (BEI), or the present invention. Inactivated agents, etc. In a preferred embodiment, the inactivating agent is 2-bromoethylamine (BEI).

In part, the present invention provides a Japanese encephalitis inactivated virus vaccine prepared by the above method.

In one embodiment, the vaccine comprises a pharmaceutically acceptable carrier.

Wherein the pharmaceutically acceptable carrier comprises one or more agents selected from the group consisting of: solvents, emulsifiers, suspending agents, decomposers, binding agents. , excipient, stabilizing agent, chelating agent, diluent, gelling agent, preservative, lubricant, interfacial activity Surfactant, adjuvant, and other carriers similar or suitable for use in the present invention.

Wherein the adjuvant includes, but is not limited to, an oil adjuvant (eg, mineral oil, vegetable oil, animal oil, Freund's complete adjuvant, Freund's incomplete adjuvant, etc.), water adjuvant (eg, aluminum hydroxide), double Phase oil adjuvant (eg, water-in-oil-in-water dosage form, w/o/w), bio-type adjuvant (eg, CpG oligonucleotide, bacterial toxoid). Wherein the dual phase oily adjuvant comprises a surfactant and an oil phase material; the surfactant comprises one or more of the following selected groups: sorbitol fatty acid ester; sorbitol fatty acid Ester and ethylene oxide or propylene oxide concentrate; mannitol fatty acid ester; mannitol fatty acid ester and ethylene oxide or propylene oxide concentrate; mannitol fat Acid esters with the following selected hydrophilic groups: carboxylic acid, amine, amide, alcohol, polyester, ether, oxygen Oxide conjugate; anhydrous mannitol fatty acid ester; anhydrous mannitol fatty acid ester with the following selected hydrophilic groups: carboxylic acid, amine, decylamine, alcohol, polyester polyol, ether a conjugate of an oxy group; a saccharose fatty acid ester; a sucrose fatty acid ester with an ethylene oxide or propylene oxide concentrate; a glycerin fatty acid ester; a glycerin fatty acid ester with ethylene oxide or propylene oxide Concentrate; fatty acid and ethylene oxide or propylene oxide concentrate; fatty alcohol and epoxy Dioxane or propylene oxide concentrate; and glycerophospholipids (glycerophospholipid). The oil phase material includes one or more of the following selected groups: mineral oil, vegetable oil, and animal oil.

In certain embodiments, the vaccine of the present invention may further comprise one or more pathogenic antigens, including but not limited to: Coxsackie virus, Echovirus, poliovirus (Poliovirus), Enterovirus, Hepatitis B virus, Mycobacterium bovis, Corynebacterium diphtheriae, Clostridium tetani, Bordetella pertussis, Haemophilus influenza, varicella-zoster virus, Measles virus, Mumps virus, Rubella virus, Rotavirus and Influenza virus.

As used herein, the term "tidal culture system" refers to a cell culture system comprising a cell culture vessel and a medium containment vessel for loading the cell culture carrier and housing the vessel with the medium. The medium is allowed to circulate between the cell culture container and the medium container. When a part of the medium flows from the medium containing container to the cell culture container, causing the medium level in the cell culture container to rise; conversely, when a part of the medium present in the cell culture container flows to the medium containing container, The medium level in the cell culture vessel is lowered. In a preferred embodiment, the cell culture vessel is a serum bottle having an opening at the bottom thereof and connected to the medium containing container in a line. In some preferred embodiments, the medium-receiving container can be, but is not limited to, a plastic bag, a glass bottle, a culture bottle, a serum bottle, and the like. In certain preferred embodiments, the air in the tidal culture system is pumped to control the direction of media flow. In some preferred embodiments, the direction of flow of the medium is controlled by the height difference between the cell culture vessel and the medium containment vessel.

As used herein, the articles "a", "an" and "sai" are meant to mean one or more than one (ie, at least one). For example, "a component" means one element or more than one element.

The term "about", "about" or "nearly" as used herein essentially means that the stated value or range is within 20%, preferably within 10%, and more preferably within 5%. The amount of digitization provided herein is an approximation and is intended to be derived if the terms "about", "about" or "nearly" are not used.

All of the technical and scientific terms described in this specification, unless otherwise defined, are intended to be common to those of ordinary skill in the art.

The present invention is exemplified by the following examples, but the present invention is not limited by the following examples.

Embodiment 1 Preparation of Japanese encephalitis inactivated virus vaccine

African green monkey kidney cells (VERO cells) were inoculated onto a cell culture vector in a tidal culture system, cultured in VP-SFM serum-free medium (GIBCOâ, Thermo Fisher Scientific Inc., Waltham, MA, USA), vector and medium. The volume ratio is 1:10. Cell inoculation was carried out in an incubator at 34~37 ° C and about 10% CO ₂ . The liquid level of the culture medium in the tidal culture system was 1800 mm/sec. When the medium level rose to just cover all the cell culture carriers. That is, the rise is complete, the residence time is about 20 seconds after the liquid level rises completely, and the residence time is about 5 seconds after the liquid level is completely lowered. The cells are inoculated for 1 to 12 hours under the conditions, and adjusted according to the pH of the medium. The CO ₂ concentration and the CO ₂ concentration were 8 to 10%, and the pH of the medium was maintained between 7.0 and 7.4 during the cell inoculation, and the glucose content was maintained at 2 g/L or more.

After the cell inoculation is completed, the cells are cultured for 3 to 10 days in an incubator at 34 to 37 ° C and 0 to 10% CO ₂ , and the liquid level of the medium in the tidal culture system is 1800 mm/sec. When it rises to cover all the cell culture carriers, the rise is complete, the residence time is 10 seconds after the liquid level rises completely, and the residence time is 30 seconds after the liquid level is completely lowered, and the CO ₂ concentration is adjusted according to the pH value of the medium. The CO ₂ concentration is 0 to 8%, and the pH of the medium is maintained between 7.0 and 7.4 during the culture period, and the glucose content is maintained at 2 g/L or more. Further, in the cell culture stage, the dissolved oxygen amount of the medium is about 50% or more of the dissolved oxygen amount of the fresh unused medium. VP-SFM serum-free medium was replaced at 3 to 5 days in cell culture and before virus challenge.

After 3 to 10 days of VERO cell culture, the Japanese encephalitis virus Beijing strain (a virulent strain obtained from the Ministry of Health and Welfare Disease Control Agency, Taiwan) was used as a vaccine strain for virus inoculation. The virus infection dose (MOI) was 1x10 ^-2 ~ 1x10 ^-6 . The cells were inoculated in an incubator at 34~37 °C and 5~10% CO ₂ . The liquid level of the medium in the tidal culture system was 1800 mm/sec. When the medium level rose to just cover all the cell culture carriers. When the temperature rises completely, the residence time is 20 seconds after the liquid level rises completely, and the residence time is 5 seconds after the liquid level is completely lowered. The virus is inoculated for 1 to 12 hours under this condition, and the CO is adjusted according to the pH value of the medium. ₂ concentration, CO ₂ concentration is 8~10%, the pH of the medium is maintained between 6.8 and 7.2 during virus inoculation, and the glucose content is maintained above 2 g/L.

After virus inoculation, the virus culture was continued in an incubator at 34~37 °C and 0~10% CO ₂ . The liquid level of the medium in the tidal culture system was 1800 mm/sec. When the medium level rose to just right. When all cell culture carriers were covered, the rise was complete. The residence time was 10 seconds after the liquid level was completely increased, and the residence time was 30 seconds after the liquid level was completely lowered. The CO ₂ concentration was adjusted according to the pH of the medium, CO _{2 .} The concentration was 0-8%, the pH was maintained between 6.8 and 7.2 during virus culture, and the glucose content was maintained above 2 g/L. Further, in the virus culture stage, the dissolved oxygen amount of the medium is about 40% or more of the dissolved oxygen amount of the fresh unused medium. After the virus is inoculated, the virus solution is harvested once every 5 to 8 days, and a new medium is added. The viral power price is determined, and the viral power price can reach 1 x 10 ^8.6 TCID ₅₀ /ml, and 10 L of virus liquid can be harvested in each batch of medium.

Then, after the virus solution is harvested, it is concentrated/dialyzed with a 100 kDa filter cassette, the host cell DNA is removed by benzoonase, and the sepharoase 6 FF column is subjected to column chromatography, and then the commercially available JEV E is used. The antigenic antibody was subjected to virus detection (Dot Blot) on each fraction of the column chromatography to determine the fraction of the virus. The fraction of the virus was collected and concentrated in a 100 kDa cassette, and 2-bromoethylamine was used. Binary ethyleneimine (BEI) deactivates the virus solution and finally sterilizes it with a 0.22 μm filter. The prepared inactivated virus purification solution can be directly used as an inactivated vaccine, or can be formulated as an inactivated vaccine containing an adjuvant by using an Al(OH) ₃ adjuvant.

Embodiment 2 Japanese encephalitis inactivated virus vaccine stability test

A Japanese encephalitis inactivated virus vaccine obtained by using a serum-free medium in a spinner flask culture, and a Japanese encephalitis inactivated virus vaccine prepared by the above Example 1, respectively, were stored at 4 ° C and stored at different times. The bicinchoninic acid (BCA) assay was used to quantify the content and yield of antigenic proteins in the Japanese encephalitis inactivated virus vaccine in vaccine samples. Japanese encephalitis inactivated virus vaccine obtained by spinner flask culture, and Japanese encephalitis inactivated virus vaccine prepared by the above Example 1, the difference between the two is that the former Japanese encephalitis virus is produced in a spinner flask culture, and the latter The Japanese encephalitis virus is produced by the tidal culture system of the above-mentioned first embodiment, and the rest such as VERO cells, medium (VP-SFM serum-free medium), virus, viral infection dose (MOI), culture conditions (34~ 37 ° C, 0 ~ 10% CO ₂ ), follow-up process, etc. are the same.

The yield analysis of the antigen protein in the vaccine showed that the yield of the Japanese encephalitis inactivated virus vaccine prepared and stored in the same process was different except that the Japanese encephalitis virus was cultured in a different manner. The Japanese encephalitis virus vaccine prepared by rotary bottle culture has a virus protein yield of 1.84 mg/L per liter of virus protein, and is calculated by containing 15 μg of viral protein per dose of Japanese encephalitis vaccine. The rate is about 123 doses per liter of viral fluid. The Japanese encephalitis inactivated virus vaccine prepared in the above Example 1 has a viral protein yield of 5.6 mg/L per liter of virus protein, and is calculated by containing 15 μg of viral protein per dose of Japanese encephalitis vaccine. The yield of the Japanese encephalitis vaccine prepared by the method of the present invention is about 373 doses per liter of virus solution, which is three times the yield of the Japanese encephalitis virus vaccine prepared by spinner flask culture. From the above results, it is known that the Japanese encephalitis vaccine prepared by the method of the present invention produces a much higher dose per unit volume than the conventional bottle culture.

The stability analysis of the antigen protein in the vaccine showed that the viral proteins of the Japanese encephalitis inactivated virus vaccine prepared and stored in the same process were different in stability except for the manner in which the Japanese encephalitis virus was cultured. The Japanese encephalitis virus vaccine prepared by spinner flask culture, after about 4 months of cold storage, has been lysed from 300 μg/ml to 230 μg/ml (the viral protein cleavage rate is 23.3%). On the other hand, the Japanese encephalitis inactivated virus vaccine prepared in the above Example 1 was maintained at 280 μg/ml in the vaccine after about 4 months of cold storage, and there was almost no lysis (viral proteolytic rate). 1.75%).

It can be seen from the above results that the Japanese encephalitis inactivated virus vaccine prepared by the method of the present invention has high stability and the yield is significantly higher than that of the Japanese encephalitis virus vaccine prepared by spinner culture, and the virus protein in the vaccine is of good quality. Not easy to crack.

Embodiment 3 Immunogenicity test of Japanese encephalitis inactivated virus vaccine

Mouse immunization program

Thirty healthy BALB/c mice aged 6 to 8 weeks were used as test animals. The serum of all mice showed a negative state of Japanese encephalitis virus antibody before the test. The 30 mice were randomly divided into 6 groups, 5 in each group. The Japanese encephalitis virus vaccine was administered according to the following groups, and immunized by intraperitoneal injection. The injection volume was 200 ml/d/time, respectively. The first immunization and the second immunization were carried out on the 0th day and the 14th day, and blood was collected on the 0th day, the 14th day, and the 28th day of the test, and the serum sample was isolated. The test group was given the following vaccines each time as follows: Group 1: Guoguang Japanese encephalitis vaccine (inactivated Japanese encephalitis vaccine prepared from rat brain) (positive control group); Group 2: 15 μg inactivated by BEI roller bottle culture Japanese encephalitis virus + 50 μg Al (OH) ₃ adjuvant; group 3: 3 μg BEI to inactivate roller bottle culture Japanese encephalitis virus + 50 μg Al (OH) ₃ adjuvant; group 4 : 15 μg of Japanese encephalitis virus + 50 μg Al(OH) ₃ adjuvant in tidal mode with BEI inactivation; Group 5: 3 μg of tidal culture of Japanese encephalitis virus + 50 μg Al(OH) inactivated by BEI ₃ adjuvant; Group 6: phosphate buffered saline (PBS) (negative control group).

2. Neutralizing antibody test:

The Japanese encephalitis virus neutralizing antibody titer was determined by the BHK-21 cell 50% Plaque Reduction Neutralization Test (PRNT ₅₀ ) (Russell, 1967). A single layer of BHK-21 cells was prepared in a 24-well plate and placed in a 37 ° C carbon dioxide incubator. Serum samples for testing were inactivated for 30 minutes prior to the 56 ° C water bath. Test sera, positive control sera, and negative control sera were diluted 1:10 and used after a 2-fold serial dilution. The Japanese encephalitis virus strain was diluted with a virus dilution (viral amount: 600-700 PFU/ml), uniformly mixed with an equal amount (130 μl) of the inactivated serum sample, and reacted at 25 ° C for 90 minutes. 50 μl of the virus serum mixture was further infected with BHK-21 cells, and cultured at 37 ° C for 60 minutes in a 5% CO ₂ incubator to adsorb the virus. Then, a medium containing 1% methylcellulose was added to each well, and cultured at 35 ° C in a 5% CO ₂ incubator for 3 to 7 days. Finally, the methylcellulose medium was washed away with a 0.9% NaCl solution and stained with Naphthol blue-black solution to calculate the number of spots. Neutralization titers are defined as the highest serum dilution factor reduced by 50% of the number of spots.

The results of the neutralizing antibody test are shown in Fig. 1, and the results show that the Japanese encephalitis vaccine (Group 4, Group 5) prepared by the method of the present invention can cause higher neutralizing antibodies. The neutralizing antibody titer caused by applying 15 μg of Japanese encephalitis vaccine (Group 2) produced by spinner flask culture was neutralized with the neutralizing antibody caused by Shikatsu Shikoku Japanese encephalitis vaccine (Group 1). The price is equivalent. From the above results, the immunogenicity of the Japanese encephalitis inactivated virus vaccine prepared by the method of the present invention is significantly higher than that of the Japanese encephalitis virus vaccine prepared by spinner culture, and the rat brain which is higher than the current use. A commercially available Japanese encephalitis vaccine was prepared.

Figure 1 shows the results of neutralization antibody titer analysis of blood samples collected from mice on the 28th day after the second immunization (after 2 weeks of immunization) after immunization with different Japanese encephalitis virus inactivated vaccines.

Claims (13)

A method for preparing a Japanese encephalitis vaccine comprises the following steps: (1) inoculating a cell for producing a vaccine on a cell culture carrier in a tidal culture system at 34 to 37 ° C, 5 to 10% CO ₂ , the medium is cultured in a serum-free medium, the volume ratio of the cell culture carrier to the serum-free medium is 1:5 to 1:20, and the liquid-free lifting rate of the serum-free medium is 1000-2000 ml/min, The retention time of the serum medium is 0-60 seconds after the liquid level rises completely. The residence time of the serum-free medium is 0-60 seconds, and the adjusted CO ₂ concentration keeps the pH between 7.0 and 7.4. Maintaining at 1 g/L or more, performing cell inoculation for 1 to 12 hours; (2) The cells are further cultured at 34 to 37 ° C, 0 to 10% CO ₂ for 3 to 10 days, and the serum-free medium is raised and lowered at a liquid level. For 1000~2000 ml/min, the residence time of the serum-free medium is 0-60 seconds after the liquid level rises completely, and the residence time of the serum-free medium is 0-60 seconds after the liquid level is completely lowered, and the adjusted CO ₂ concentration makes the pH value Maintaining between 7.0 and 7.4, the glucose content is maintained above 1 g/L; (3) Inoculation of Japanese encephalitis virus to The virus has a multiplicity of infection (MOI) of 1×10 ⁻² to 1×10 ^-6 , and is cultured in the serum-free medium at 34 to 37° C. and 5 to 10% CO ₂ , and the serum-free medium is cultured. The liquid surface lifting speed is 1000~2000 ml/min, and the residence time of the serum-free medium is 0-60 seconds after the liquid level rises completely, and the residence time of the serum-free medium is 0-60 seconds after the liquid level is completely lowered, and the adjusted CO ₂ The concentration is maintained at a pH between 6.8 and 7.2, and the virus is inoculated for 1 to 12 hours, and the glucose content is maintained above 1 g/L. (4) The Japanese encephalitis virus and cells after inoculation continue at 34 to 37 ° C. The medium is cultured in a culture environment of 0 to 10% CO ₂ , and the liquid level of the serum-free medium is 1000-2000 ml/min, and the serum-free medium has a complete liquid level and the residence time is 0 to 60 seconds. The serum-free medium is used. The residence time after the liquid level is completely lowered is 0~60 seconds, the adjusted CO ₂ concentration is maintained at pH 6.8~7.2, the glucose content is maintained above 1 g/L; and (5) every 5~ after virus inoculation The virus solution was harvested once in 8 days, and purified and non-activated to obtain Japanese encephalitis deactivated virus vaccine. Wherein after activation of the Japanese encephalitis virus vaccine to be stored for 4 months at 2 ~ 8 ° C, viral proteolytic 5% or less. The method of claim 1, wherein the cell for producing the vaccine is African green monkey kidney cells (VERO cells). The method of claim 1, wherein the serum-free medium is cultured as VP-SFM. The method of claim 1, wherein the serum-free medium is raised to just cover all of the cell culture carrier, and the whole cell culture carrier is completely immersed in the serum-free medium, but Not suspended in the serum-free medium. The method of claim 1, wherein the serum-free medium in the step (1) has a liquid surface rise completely after a residence time of 20 seconds, and the serum-free medium has a complete liquid level and a residence time of 5 seconds; wherein The liquid-free medium in the step (2) has a liquid surface rise completely after a residence time of 10 seconds, and the serum-free medium has a complete liquid surface drop time of 30 seconds; wherein the serum-free medium liquid level in the step (3) The residence time after the completion of the rise is 20 seconds, the residence time of the serum-free medium is completely decreased after the liquid level is 5 seconds; and the residence time of the serum-free medium in the step (4) is 10 seconds, which is no The residence time of the serum medium was reduced to 30 seconds after completion. The method of claim 1, wherein the amount of dissolved oxygen in the serum-free medium in the step (2) is 50% or more of the dissolved oxygen amount of the fresh unused serum-free medium. The method of claim 1, wherein the amount of dissolved oxygen in the serum-free medium in the step (4) is 40% or more of the dissolved oxygen amount of the fresh unused serum-free medium. The method of claim 1, wherein the serum-free medium in the step (2) is replaced once in the cell culture for 3 to 5 days and before the virus is challenged. The method of claim 1, wherein the inactivation treatment in the step (5) is one of an inactivation treatment and a heat treatment. The method of claim 7, wherein the inactivating agent comprises formaldehyde, paraformaldehyde, beta-propionolactone (BPL), and 2-ethylethylamine (binary ethyleneimine) , BEI) one of them. A Japanese encephalitis inactivated virus vaccine prepared by the method of claim 1 of the patent application. The vaccine of claim 12, further comprising a pharmaceutically acceptable carrier. The vaccine according to claim 12, further comprising one or more pathogenic antigens selected from the group consisting of: Coxsackievirus, Echovirus, and poliovirus (Poliovirus), Enterovirus, Hepatitis B virus, Mycobacterium bovis, Corynebacterium diphtheriae, Clostridium tetani, Bordetella pertussis, Haemophilus influenza, varicella-zoster virus, Measles virus, Mumps virus, Rubella virus, Rotavirus, And Influenza virus.