WO2015059714A1 - Mode d'urgence dans un véhicule hybride - Google Patents

Mode d'urgence dans un véhicule hybride Download PDF

Info

Publication number
WO2015059714A1
WO2015059714A1 PCT/IN2014/000585 IN2014000585W WO2015059714A1 WO 2015059714 A1 WO2015059714 A1 WO 2015059714A1 IN 2014000585 W IN2014000585 W IN 2014000585W WO 2015059714 A1 WO2015059714 A1 WO 2015059714A1
Authority
WO
WIPO (PCT)
Prior art keywords
virus
bulk
purified
formalin
japanese encephalitis
Prior art date
Application number
PCT/IN2014/000585
Other languages
English (en)
Other versions
WO2015059714A8 (fr
Inventor
Krishna Mohan Vadrevu
Venkatesan RAMASAMY
Prasanna Kumar DUVVURU
Original Assignee
Bharat Biotech International Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Bharat Biotech International Limited filed Critical Bharat Biotech International Limited
Priority to CN201480050520.1A priority Critical patent/CN105744952A/zh
Priority to AU2014338520A priority patent/AU2014338520B2/en
Priority to RU2016114285A priority patent/RU2706693C2/ru
Publication of WO2015059714A1 publication Critical patent/WO2015059714A1/fr
Publication of WO2015059714A8 publication Critical patent/WO2015059714A8/fr
Priority to PH12016500500A priority patent/PH12016500500A1/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5252—Virus inactivated (killed)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
    • C12N2770/24134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
    • C12N2770/24151—Methods of production or purification of viral material
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention pertains to the field of viral vaccine compositions. Particularly, the present invention relates to Japanese encephalitis vaccine composition and processes or methods of manufacture of vaccine composition against Japanese encephalitis infections. The present invention relates to processes for production and purification of virus bulk and inactivation techniques of the said Japanese encephalitis virus bulk thereof.
  • JEV Japanese encephalitis virus
  • flavivirus family and genus flavivirus It is a small enveloped virus (50nm) containing a 10.9 kb single-strand, negative sense RNA genome.
  • Viron RNA codes for 11 proteins include four structural and seven non structural Proteins.
  • the surface envelope protein (E protein) serves as the cell receptor binding Protein and fusion protein for virus attachment and entry into the host. So antibodies against E protein neutralize the virus and play an important role in protection.
  • the surface structural E protein is an important component of the vaccine so it is very important to stabilize this protein molecule with suitable stabilizer for vaccine efficacy.
  • the bioprocess of Japanese encephalitis involves either growing in roller bottle flasks, or cell factory units. Production of industrial amounts of the Japanese encephalitis vaccine through roller bottle cell culture, or through cell factories needs higher amounts of time, with comparatively lesser yields, and also occupying large amount of space, which makes it difficult to control and regulate experimental parameters at a regular and constant basis in each cell factories.
  • the production of virus bulk therefore inherently lacks uniformity of operation.
  • the present invention overcomes these difficulties through its new bio-process technique wherein the time required to yield high amounts of industrial Japanese encephalitis bulk is reduced to great extents.
  • Commercial production of the vaccine bulk is enabled within much lesser time as compared to earlier culture techniques.
  • Vero cells are first grown in a disposable bioreactor followed by infection with JEV, wherein multiple viral harvests is accomplished as a novel feature in this invention.
  • Production of the virus is followed by simultaneous inactivation and stabilization methods particularly applicable to Japanese encephalitis vaccine antigen.
  • Stabilization of .Japanese encephalitis vaccine i general require maintaining the vaccine vials or the bulk at prescribed temperature ranges at 2°-8°C for stabilization for larger periods of time. This requires added infrastructure facility to transport and store the vaccine thereby increasing cost of the vaccine.
  • the vaccine so obtained from the virus bulk according to the methods disclosed in this invention do not require any special refrigeration facilities mentioned above and are stable at ambient temperature conditions i.e. at room temperatures (25°C).
  • the stabilities obtained by the present invention are comparable to the stabilities observed in case of Japanese encephalitis vaccines stored at refrigeration temperatures (2°- 8°C). The potency and the immunogenecity of the viral bulk and the vaccine to confer sufficient immunization to the subjects also are retained.
  • One object of the present invention is to provide a stable inactivated vaccine composition that is capable to prevent as well as provide treatment from Japanese encephalitis infections caused by Indian olar strain of the Japanese encephalitis virus.
  • Another object of the invention is to provide for a method of adaptation of the Kolar strain of the Japanese encephalitis virus in Vero Cells.
  • Yet another object of the invention is to provide for a suitable method of commercial production of purified inactivated Japanese encephalitis bulk at specified experimental parameters, and techniques which enables multiple harvesting of Japanese encephalitis bulk at much higher yields using a disposable bioreactor.
  • One more object of the invention is to provide for novel purification techniques using specific matrix column during the downstream processing of the Japanese encephalitis virus bulk at a commercial scale.
  • Yet another object of the invention is to provide a simultaneous method of inactivation and stabilization of the purified Japanese encephalitis virus bulk.
  • various methods of culture of Vero cells in a disposable bioreactor is disclosed. Satisfactory levels of growth of Vero cells at various experimental parameters involving proper media composition and requirement of change in media is optimized, to obtain satisfactory levels of cell count within a desired amount of time.
  • Vero cells are infected with the live Japanese encephalitis virus, Indian Kolar strain, and accordingly the embodiment describes, a method of adaptation of Japanese encephalitis virus to grow in Vero cells in a disposable bioreactor.
  • the adaptation of Kolar strain of Japanese encephalitis virus in Vero cells is scaled up from a disposable bioreactor to next subsequent sizes of the disposable bioreactor to generate production batch size yield of purified JEV bulk.
  • the disposable bioreactors have been named as DB-1 to DB-100 to DB- 500, depending upon cell capacity.
  • a further embodiment of the invention provides alternative methods of downstream purification of the live JEV bulk using various novel chromatographic techniques, which results in obtaining high amounts of purified JEV bulk.
  • One more embodiment of the invention discloses novel methods of simultaneous inactivation and stabilization of the purified JEV bulk.
  • the invention also provides experimental data of stability of the purified inactivated JEV bulk at 25 °C - 37°C, ready for vaccine formulation.
  • a further embodiment of the invention provides a Japanese encephalitis vaccine composition comprising a vaccine antigen, wherein the antigen is Kolar strain of Japanese encephalitis virus.
  • DMEM Dulbecco's Modified Eagle Medium
  • the present invention discloses a novel Indian strain of the Japanese encephalitis virus JEV821564-XY which is a Kolar strain of the Japanese encephalitis virus.
  • This Kolar strain JEV821564 of the Japanese encephalitis virus (hereafter mentioned as 'Kolar strain') was transferred to Bharat Biotech International Limited through a Material Transfer Agreement from National Institute of Virology, Pune. At present there is no vaccine composition against the Indian strain (Kolar) of the Japanese encephalitis virus.
  • this particular strain of the Japanese encephalitis virus is grown in a method wherein the strain is adapted in Vero cells for its culture. Virus culture is generally grown in liquid media. Cell culture in Vero cells of any biological product includes very high number of roller bottles or cell factories. Virus culture in Cell-Factories takes longer durations of time. Cell Factories occupy higher amounts of space, and it is also difficult to monitor all the experimental parameters during virus culture in cell factories individually. The produce of Japanese encephalitis through conventional cell factories is minimal.
  • the present invention discloses a novel method of manufacture of this Kolar strain of the Japanese encephalitis virus in a disposable bioreactor.
  • Japanese encephalitis virus is adapted to be grown in Vero cells, and this particular strain of the virus is adapted to propagate in Vero cells supplied with specific media.
  • the same Vero cells are capable to give multiple harvests of the virus bulk, within single infection.
  • This particular technique of multiple virus harvest of Japanese encephalitis virus (JEV) presented in this invention is unique and distinct, although propagation of JE virus in Vero cells is already known.
  • obtaining desired amount of cell growth (of Vero Cells) in a new apparatus such as the one used in this invention which is a disposable bioreactor, and scaled up from smaller capacity disposable bioreactors to higher capacity production size batches of disposable bioreactors is not provided earlier.
  • the conventionally used method of propagation of the Japanese encephalitis virus in Vero cells are generally grown in Cell Factories or roller bottles in which the Vero cells are utilized in a layer formation for only once.
  • the Vero cell line is utilized by infecting the Japanese encephalitis virus, and the cell line is exhausted after only one infection by the virus seed bank.
  • the Vero cells are grown in such unique and novel technique and the experimental parameters are set in such manner that, a single infection of the seed Japanese encephalitis virus is capable of producing at least thrice to five time harvest of the virus bulk from the same.
  • Vero cell line culture involving the disposable bioreactor. At the same time, this reduces the number of infections as well as the amount of space and more quantity of cell factories required to infect and grow the same viral cell culture quantity. Additionally, multiple harvests from the cell culture, also automatically adds on the amount of yields, both in terms of quantity and quality of the viral harvest to very high amounts as compared to the processes involved in doing and attaining similar yields with Cell Factories.
  • the present invention is directed to stabilize the purified JEV bulk both during inactivation process and during storage of virus bulk.
  • the composition of each stabilizer ranges from 0.5 to 1% w/v.
  • the chemical stabilizers which are added to viral antigen are D- sorbitol and glycine.
  • the viral antigen with suitable stabilizer was stored in the sufficient physiologically acceptable phosphate buffer saline to maintain pH at from 7.0 to about 7.4.
  • Example 1 Cell Culture and adaptation of olar strain of JE virus to Vero Cells.
  • Initial cell seeding of Vero cells are taken in a T- 175 flask comprising of Vero Cells.
  • Vero cell culture is done in T-175 flasks and grown in suitable media.
  • Cell growth obtained from 1 T-175 flask is transferred to 5 T-175 flasks after 4-5 days. This is further transferred to 1 cell-factory-10 (CF10) after 4-5 days again, and then again transferred to 5 Cell Factory-40 (CF-40).
  • CF-40 is used presently for commercial production of JEV bulk.
  • One batch of JEV bulk is produced involving at least 30 CF-40s in a period of one month for production of one commercial batch of JEV bulk.
  • the present invention teaches a particular method of propagation of the said Kolar strain of JE in Vero cells, useful for commercial scale-up of the bulk production and is capable to produce JEV bulk upto approximately 140 CF-40s within the same period of time i.e. approximately one month.
  • Such a high yield of the JEV bulk within the same period of time is a big advantage to increase the production capacity commercially.
  • a minimum requirement of a walk-in incubator room which can accommodate at least 3-4 people for operation and carrying out the experiments is needed as the culture facility.
  • the incubator room shall retain a constant prescribed temperature at 37°C throughout the batch.
  • the requirement of having run an incubator room constantly for a period of 5-6 months which would provide output of 200 cell factory-40s (200 CF40s) will definitely increase the production costs.
  • for running approximately upto 150 CF-40s at a time within a period of one month will require a huge and an extra large sized incubator room maintained constantly at 37°C to provide sufficient space for at least 20 people to operate and control the experimental parameters.
  • the present invention discloses a novel method of manufacture of JEV bulk in a disposable bioreactor, and not in cell factories (CF40) wherein all the technical specifications in the disposable bioreactor can be regulated with required amount of accuracy and precision required for cell culture and virus culture which cannot be attained in the case of cell factories.
  • the present invention replaces the use of CF40s in JEV bulk production, with such suitable disposable bioreactor.
  • the given disposable bioreactor provides the necessary channels, probes and sensors through which it is possible to regulate the cell culture and virus culture process automatically. Hence, control of various experimental parameters like temperature, pH, dissolved oxygen, agitation and media supply are easily done in the given disposable bioreactor with the required accuracy and precision. Additionally, adaptation of the Kolar strain of the JE virus into Vero cells in a bioreactor is capable of production of JEV bulk to a quantity of at least 40 CF40s to 200 CF40s in one batch of produce at the same time as required in case of 30 CF40s in one batch.
  • the yields obtained are also higher both in case of equivalent corresponding production capacity and output of 30 CF40s and obviously very very high in case of corresponding production capacity and produce that would have been obtained for up to 200 CF40s in 5-6 months. Also, the amount of loss is significantly minimized to considerable extents for use of bioreactors instead of CF40s. It is very evident that there must have been or it is bound to experience loss in the batch produce for a period of 5-6 months in case of production involving approximately 200 Cell Factories. This possibility is entirely omitted because of use of bioreactor for JEV bulk production according to the methods disclosed in this invention.
  • the present invention of adapting the particular Kolar strain of the JE virus in Vero cells grown in the given bioreactor increases the yield of the virus production both in cases of comparable amount of produce as in 30 CF40s and/or 200 CF40s.
  • one further advantage lies in the fact that, contamination risks are minimized to great extents, in case of disposable bioreactors as disclosed in the present invention. Since all the experimental parameters are controlled through electronic instructions, manual operation is minimal in case of JE bulk production in the disposable bioreactor.
  • One further advantage of the disposable bioreactor is that it can produce the consistent cells thereby resulting in consistent yields, whereas in case of cell factories, it is not possible to maintain consistent yields to such similar extents to that of disposable bioreactors due to manual handling for regulating the experimental parameters.
  • the total available surface area for cell growth in 30CF40s is 763200 cm 2 which might be having the capacity of ⁇ 75000-90000 x 10 6 cells
  • the disposable bioreactor used in this invention has total surface area available for cell growth ranging from 26000 cm 2 to 5000000 cm 2 which supports the required amount of cells to grow and further availability of Vero cells as the substrate for the virus so that it can infect more and more number of Vero Cells, to suffice the required amounts of increased bulk production of JE in the bioreactor.
  • the total cell count capacity ranges from approximately 7800 x 10 6 to 1500000 x 10 6 cells. Increase in number of availability of cells signifies increase in available substrate of the virus to infect more and more number of cells, and hence, larger amount of JE bulk productions can be achieved.
  • Table 1.1 Comparison between cell factories and disposable bioreactor (s).
  • Cell culture is a widely practiced technique in biotechnological processes. However, while a given cell is capable to grow in more than one given medium, their characteristics may alter when the medium is changed. It is also crucial to note that, respective to specific cell line, the time and duration of cell growth also varies. Cell growth depends upon a few important parameters such as, medium, growth factors, concentration of glucose, oxygen availability, and the amount of initial seeding and multiplicity of infection (MOI). Furthermore, growths of cells are also prone to irregular pH changes, which require constant monitoring at different phases of cell culture. Hence, it is already established that growing of cells is specific to the purpose and the surrounding environmental, and physico- chemical factors.
  • Vero cell line in the mentioned bioreactor instead of cell factories unlike the earlier practice is a separate hurdle that has been overcome in the present invention. Additionally, growing the Vero cell line in the bioreactor may need frequent media changes at specific and appropriate time intervals for optimum cell growth. Changing in media also requires particular calculated cost-effective measures to corroborate maximum cell growth. Control of temperature fluctuations in the bioreactor also is an important issue to support good cell growth. At the same time, very high amount of cell growth may result into poor yields, whereas in case of very or low cell growth below the desired levels also results into failure of the experiments. Therefore, it is desirable to enhance and hone all the above mentioned parameters in a new environment (i.e.
  • Adaptation of a specific strain of a virus to a particular cell substrate is again a challenge.
  • adaptation of JE virus on Vero cells is known, yet it is always strain specific.
  • Each individual strain of the virus involves specific conditions to grow and infect Vero cells in a given manner which ensures high growth and quick multiplication of the virus.
  • the invention as presented in this application refers to adaptation of Kolar strain of the Japanese encephalitis virus in Vero Cells.
  • Characterization of a laboratory adapted strain of JE virus may exhibit different characteristics from those of its earlier isolated strains in terms of rapid growth in Vero cells, viral titer and their resultant potency.
  • Virus characterization and genetic stability can differ the yield, and the necessary processes required for commercial production of the JE bulk.
  • this present invention discloses, adaptation of a new strain of Japanese encephalitis virus which is Kolar strain of JE virus isolated and endemic to the Indian subcontinent in Vero cells in a specific disposable bioreactor as mentioned in the above examples, which is capable of high yield of JE bulk with high potency sufficient to elicit protective immune response in the given subjects against Japanese encephalitis virus infections.
  • the examples as presented in this invention discloses the unique method of adaptation of the novel Kolar strain of the Japanese encephalitis virus in Vero cells, and its multiple harvests obtained from the initial cell line.
  • Trypsinisation is a process by which the adhered cells are made to detach from the monolayer so that they may be transferred into the next level called as Cell expansion. Such trypsinisation is a critical process and requires skill to execute it, as well as trypsinisation at each particular intermediate step is also time taking.
  • Initial experiments performed with initial cell seeding of 26000 cells/cm 2 in the disposable bioreactor of initial size volume referred as DB meant that despite using DB100 or DB500, trypsinisation will still be required several times prior to initially seed the cells in the disposable bioreactor. Therefore we need to minimize the cell seeding in the disposable bioreactor since it is directly proportional to the large scale bioreactor seeding.
  • DB-100 seeding will require the 10 CF-40 grown cells (26000xl0 6 cells) where as for DB-500 will require the 50 CF-40 cells (130000xl0 6 cells). It is highly difficult to handle these many CF to trypsinise for collecting the cells from it. With the modified or minimized 3000 cells/cm 2 seeding DB-100 requires the 1 CF-40 grown cells (3000xl0 6 cells) where as for DB-500 will require the 6 CF-40 cells (15000xl0 6 cells).
  • Table 1.2 Scale up from Disposable Bioreactor (DB) to DB-100 and DB-500 and multiple virus harvest values. Viral Harvest Data are all mentioned in plaque forming units.
  • the viral harvests were clarified by microfiltration or depth filters.
  • the clarified harvests were concentrated by using tangential flow filtration 300 kDa cassette and the concentrated harvest further subjected for column purification.
  • VHl showed titer value 10 6 ⁇ 88 PFUs and VH2 showed titer value of 10 7 02 PFUs.
  • VH3 and VH4 virus titers showed 10 6'49 PFUs and 10 5 84 PFUs respectively.
  • Experiment II - Objective Scale up from Disposable Bioreactor to Commercial Batch size Disposable Bioreactor 100.
  • Viral harvest 1 has been collected using serum free medium on the 4th day following the infection and VH2 and VH3 were collected with 3 day intervals respectively starting from the day of infection.
  • the viral harvests were clarified by microfiltration or depth filters.
  • the clarified harvests were concentrated by using tangential flow filtration 300 kDa cassette and the concentrated harvest further subjected for column purification.
  • Example 2 Purification of JE bulk using celufine sulfate ester matrix and Inactivation of the JE bulk.
  • the purification of the JE bulk produced in the disposable bioreactor is carried out using celufine sulfate column based on affinity chromatography.
  • Celufine sulphate column purification comprises a chromatographic technique wherein the said celufine sulphate is a column matrix made of cellulose support matrix with the presence of an activated group sulphate ester.
  • the total harvest volume is first concentrated with 300 kDa membrane from 300 liter to 50 liter to obtain the retentate containing live JEV. The permeate is discarded.
  • the retentate contains the JE virus.
  • the retentate is subjected to column purification using the said celufine sulphate matrix.
  • the concentrated harvest volume of 50 liter is passed through the celufine sulphate column and the flowthrough is discarded since it contains unwanted protein and other genetic material.
  • the virus which is bound to the column will be eluted out by using elution buffer containing high amount of salts.
  • the elute is quantified through optical density values measured giving a sharp peak, which signifies the presence of purified live Japanese encephalitis virus.
  • the virus collected in the elution is diafiltered to remove the salts, certain unwanted proteins and other genetic material by buffer exchange against phosphate buffered saline repeatedly for at least 5-6 times.
  • the retentate is collected and passed through 0.45 ⁇ PVDF (polyvinylidene fluoride) membrane filtration.
  • the live JE bulk is inactivated with formalin at the ratio between 1 :1500 v/v to 1 :2500 v/v for 7 days at 22°C. along with the stabilizer mixture containing 0.1-1% sorbitol, and 0.1-1% Glycine added during the inactivation process only.
  • Stabilizers at specific concentration of 0.5% w/v glycine and 1% w/v sorbitol are added to the live JE virus along with inactivating agent either formalin or BPL.
  • the stabilizers are added to prevent degradation of the virus during the inactivation incubation period of 7 days only, so mentioned above due to addition of the inactivating agent formalin or betapropio lactone (BPL).
  • the JE bulk is further diafiltered through buffer exchange at least for 5-6 times against phosphate buffered saline to remove the formalin from the purified inactivated JE bulk.
  • the diafiltered JE bulk is sterile filtered by using 0.22 ⁇ PVDF (polyvinylidene fluoride) membrane filtration.
  • the inactivated JE bulk is stored for stability studies at 5°C+ 3°C for at least 24 months to 36 months.
  • the purification and inactivation steps of Japanese encephalitis (Kolar strain) bulk according to one of the methods disclosed in this invention can be summarized below:
  • step (b) concentration of the harvest volume to at least 4 times with 300kDa polyethersulfone (PESU) membrane cassette to obtain the concentrated retentate;
  • step (c) column purification of the concentrated harvest retentate of step (b) through celufine sulphate matrix column, wherein the said matrix consists of a cellulose support matrix with the presence of an activated group sulphate ester to collect the elute containing the desired virus, high amount of salts cellular proteins and nucleic acid material;
  • step (d) diafiltration and buffer exchange using 300kDa PESU membrane cassette against phosphate buffered saline of the elute of step (c) for at least 5-6 times to collect the retentate to remove the undesirable high amount of salts cellular proteins and nucleic acid material from the virus of interest to collect the purified concentrated virus bulk;
  • step (e) subjecting the purified concentrated virus bulk of step (d) to 0.45 ⁇ PVDF (polyvinylidene fluoride) membrane filtration to eliminate any contaminants in the process;
  • PVDF polyvinylidene fluoride
  • step (f) simultaneous stabilization and inactivation of the purified virus of step (e) with formalin at a ratio between 1 :1500 v/v to 1 : 2500 v/v (i.e. to 2500 volume of live JE, 1 volume of formalin is treated) along with stabilizers, wherein the said stabilizers consists of glycine and sorbitol for a period of 7 days at 22°C or with beta-propio lactone at a ratio between 1 :1500 v/v to 1 : 2500 v/v (i.e. to 2500 volume of live JE, 1 volume of formalin is treated) along with stabilizers, wherein the said stabilizers consists of glycine and sorbitol for a period of 7 days at 5°C +3°C;
  • step (g) diafiltration and buffer exchange of the inactivated purified virus bulk of step (f) using 300kDa PESU membrane cassette against phosphate buffered saline for at least 5-6 times to remove formalin only in case of formalin inactivation;
  • step (h) 0.22 ⁇ PVDF (polyvinylidene fluoride) membrane filtration of the purified inactivated virus bulk of step (g) to obtain the final and stabilized inactivated JEV bulk at 5°C+ 3°C for at least 24 months to 36 months free from any contaminants suitable for formulation of the Japanese encephalitis vaccine.
  • PVDF polyvinylidene fluoride
  • Table 2.1 Purified inactivated JEV bulk and number of doses in 1 month using 30 CF40 through celufine sulphate matrix purification. The total virus bulk obtained for one batch of DB100 in one month is mentioned below. Accordingly the number of doses of JE vaccine that may be made available with 7 ⁇ of inactivated JE per dose is also calculated and provided below:
  • Table 2.2 Purified inactivated JEV bulk and number of doses in 1 month using DB- 100 through celufine sulphate matrix purification.
  • the total virus bulk obtained for one batch of DB-500 in one month is mentioned below. Accordingly the number of doses of JE vaccine that may be made available with 7 of inactivated JE per dose is also calculated and provided below:
  • Table 2.3 Purified inactivated JEV bulk and number of doses in 1 month using DB-500 through celufine sulphate matrix purification.
  • the total JE bulk produced in one commercial batch in the disposable bioreactor DB-100 is equivalent to the amount of JE bulk that would have been produced in at least 48 CF40s, for the same period of time or even lesser (38 days).
  • the amount of JE bulk produced is equivalent to the amount of JE bulk that would have been produced in at least 147 CF40s for the same period of time (39 days).
  • Example 3 Purification of JE virus using agarose based matrix column and inactivation of the JE virus.
  • Capto Core-700 Purification of JE virus using an agarose based column Capto Core-700 is another novel aspect of this invention.
  • the number of doses that may be manufactured in one batch size of DB-100 and DB-500 are even higher in comparison with celufine sulphate purification.
  • Capto core 700 column consists of a highly cross-linked agarose matrix with a functional active ligand octylamine, responsible for both hydrophobic interactions and is also positively charged in order to interact strongly with most of the impurities over a wide range of pH and salt concentrations.
  • the advantage is concentration of viral harvest is not required.
  • Capto Core matrix The principle involved in the Capto Core matrix is size exclusion together with affinity chromatographic techniques both acting simltaneously.
  • the column purification is accomplished by passing the entire harvest volume through Capto Core matrix.
  • the proteins having a molecular weight below 700 kDa and other genetic material present in the harvest will be bind to the Capto Core matrix.
  • the flow through is collected comprising the live JE virus because viruses will not bind to the Capto Core matrix. This is followed by concentration through a 300kDa poly ethersulf one (PESU) membranes cassette. The permeate is discarded, and the retentate comprises the live JE virus at this stage.
  • PESU poly ethersulf one
  • the concentrated live JE virus present in the retentate is chemically inactivated through treatment with inactivating agent formalin or betapropiolactone (BPL) at a ratio between 1 :1500 v/v to 1 : 2500 v/v (i.e. to 2500 volume of live JE, 1 volume of formalin is treated) for a period of 7 days at 22°C along with the stabilizer mixture containing 0.1-1% sorbitol, and 0.1-1% Glycine added during the inactivation process only.
  • Stabilizers at specific concentration of 0.5% w/v glycine and 1% w/v sorbitol are added to the live JE virus along with inactivating agent either formalin or BPL.
  • the stabilizers are added to prevent degradation of the virus during the inactivation incubation period of 7 days only, so mentioned above due to addition of the inactivating agent formalin or BPL.
  • the JE bulk is diafiltered and concentrated by buffer exchange against phosphate buffered saline in a repeated manner for at least 5-6 times to remove formalin. In case of BPL inactivation diafiltration is not required. BPL will be neutralized by incubating the bulk at 37°C for 3 hours.
  • the diafiltered JE bulk is sterile filtered by using 0.22 ⁇ PVDF (polyvinylidene fluoride) membrane filtration.
  • the inactivated Japanese encephalitis virus bulk is further stored at 5°C +3°C for at least 24 months to 36 months.
  • step (c) concentration and diafiltration of the flowthrough of step (b) using 300kDa polyethersulfone (PESU) membranes cassette to obtain the purified concentrated vims bulk;
  • PESU polyethersulfone
  • step (d) subjecting the purified concentrated virus bulk of step (c) to 0.45 ⁇ PVDF (polyvinylidene fluoride) membrane filtration to eliminate any contaminants in the process;
  • PVDF polyvinylidene fluoride
  • step (e) simultaneous stabilization and inactivation of the purified virus of step (d) with formalin at a ratio between 1 :1500 v/v to 1 : 2500 v/v (i.e. to 2500 volume of live JE, 1 volume of formalin is treated) along with stabilizers, wherein the said stabilizers consists of glycine and sorbitol for a period of 7 days at 22°C or with beta-propio lactone at a ratio between 1 :1500 v/v to 1 : 2500 v/v (i.e.
  • step (e) diafiltration and buffer exchange of the inactivated purified virus bulk of step (e) using 300kDa PESU membrane cassette against phosphate buffered saline for at least 5-6 times to remove formalin only in case of formalin inactivation;
  • step (g) 0.22 ⁇ PVDF (polyvinylidene fluoride) membrane filtration of the purified inactivated virus bulk of step (f) to obtain the final and stabilized inactivated JEV bulk at 5°C+ 3°C for at least 24 months to 36 months free from any contaminants suitable for formulation of the Japanese encephalitis vaccine.
  • PVDF polyvinylidene fluoride
  • Table 3.1 Purified inactivated JEV bulk and number of doses in 1 month using 30 CF40 through Capto Core-700 matrix purification. Volume Protein
  • Table 3.2 Purified inactivated JEV bulk and number of doses in 1 month using DB-100 through Capto Core-700 purification.
  • Table 3.3 Purified inactivated JEV bulk and number of doses in 1 month using DB-500 through Captocore purification.
  • Table 3.4 Comparison of purified JEV bulk obtained through celufine sulfate matrix and Capto Core 700 matrix.
  • Example 4 Stability studies of inactivated JEV bulk.
  • the dialyzed inactivated JEV bulk was stored with suitable proportion of with or without chemical stabilizer(s) at 5 ⁇ 3°C.
  • the antigen was drawn at different interval and tested by plaque reduction neutralization (PRNT50).
  • PRNT50 plaque reduction neutralization
  • the stability of antigen was performed with a viral neutralization titer of mouse antiserum as per IP.2007; WHO TRS No.771.
  • the virus neutralization of the antiserum was determined by the 50% plaque reduction method.
  • the neutralization titer against the virus was expressed as the reciprocal of the serum dilution that showed 50% plaque reduction compared to the plaque number of the control wells without live viral antigen.
  • Table 4.1 Stability of the purified inactivated JEV bulk for 36 months.

Abstract

L'invention concerne des méthodes nouvelles et des techniques expérimentales appropriées qui maximisent le rendement d'antigènes, et stabilisent également la charge virale de l'encéphalite japonaise inactivée, purifiée, dérivée d'une cellule Vero. L'invention concerne également d'autres méthodes de culture cellulaire et de culture virale pour la fabrication commerciale de formulations vaccinales. La charge virale inactivée stabilisée peut être stockée longtemps à 2°C-8°C jusqu'à formulation du vaccin.
PCT/IN2014/000585 2013-09-14 2014-09-08 Mode d'urgence dans un véhicule hybride WO2015059714A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
CN201480050520.1A CN105744952A (zh) 2013-09-14 2014-09-08 病毒疫苗及其制备方法
AU2014338520A AU2014338520B2 (en) 2013-09-14 2014-09-08 A viral vaccine and methods of manufacture thereof
RU2016114285A RU2706693C2 (ru) 2013-09-14 2014-09-08 Вирусная вакцина и способы ее производства
PH12016500500A PH12016500500A1 (en) 2013-09-14 2016-03-14 A viral vaccine and methods of manufacture thereof

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
IN4336/CHE/2013 2013-09-14
IN4135/CHE/2013 2013-09-14
IN4135CH2013 IN2013CH04135A (fr) 2013-09-14 2014-09-08

Publications (2)

Publication Number Publication Date
WO2015059714A1 true WO2015059714A1 (fr) 2015-04-30
WO2015059714A8 WO2015059714A8 (fr) 2015-06-18

Family

ID=52997726

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IN2014/000585 WO2015059714A1 (fr) 2013-09-14 2014-09-08 Mode d'urgence dans un véhicule hybride

Country Status (6)

Country Link
CN (1) CN105744952A (fr)
AU (1) AU2014338520B2 (fr)
IN (1) IN2013CH04135A (fr)
PH (1) PH12016500500A1 (fr)
RU (1) RU2706693C2 (fr)
WO (1) WO2015059714A1 (fr)

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3581646A1 (fr) * 2018-06-15 2019-12-18 Themis Bioscience GmbH Système chromatographique et de fabrication intégré pour la production de virus
US11008547B2 (en) 2014-03-25 2021-05-18 Terumo Bct, Inc. Passive replacement of media
US11104874B2 (en) 2016-06-07 2021-08-31 Terumo Bct, Inc. Coating a bioreactor
US11162880B2 (en) 2015-11-09 2021-11-02 University Of Notre Dame Du Lac Particle size purification method and devices
US11478541B2 (en) 2017-11-03 2022-10-25 Takeda Vaccines, Inc. Method for inactivating Zika virus and for determining the completeness of inactivation
US11608486B2 (en) 2015-07-02 2023-03-21 Terumo Bct, Inc. Cell growth with mechanical stimuli
US11629332B2 (en) 2017-03-31 2023-04-18 Terumo Bct, Inc. Cell expansion
US11685883B2 (en) 2016-06-07 2023-06-27 Terumo Bct, Inc. Methods and systems for coating a cell growth surface
US11965175B2 (en) 2016-05-25 2024-04-23 Terumo Bct, Inc. Cell expansion
US11975062B2 (en) 2017-11-30 2024-05-07 Takeda Vaccines, Inc. Zika vaccines and immunogenic compositions, and methods of using the same

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111394321A (zh) * 2020-03-30 2020-07-10 无锡加莱克色谱科技有限公司 病毒颗粒纯化用超大孔径层析介质及其应用方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999011762A1 (fr) * 1997-08-28 1999-03-11 Cheil Jedang Corporation Virus de l'encephalite japonaise attenue, adapte a des cellules vero et vaccin contre l'encephalite japonaise
WO2012073257A2 (fr) * 2010-11-30 2012-06-07 Bharat Biotech International Limited Formulations vaccinales pour la prophylaxie et le traitement d'infections par le virus chandipura chez les mammifères
CN102100910B (zh) * 2009-12-21 2013-01-23 北京清大天一科技有限公司 一种生产病毒疫苗的方法

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010137036A2 (fr) * 2009-05-25 2010-12-02 Panacea Biotec Ltd Nouveau vaccin contre l'encéphalite japonaise et procédé de fabrication correspondant
WO2013130001A1 (fr) * 2012-02-29 2013-09-06 Ge Healthcare Bio-Sciences Ab Procédé pour l'élimination des endotoxines

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999011762A1 (fr) * 1997-08-28 1999-03-11 Cheil Jedang Corporation Virus de l'encephalite japonaise attenue, adapte a des cellules vero et vaccin contre l'encephalite japonaise
CN102100910B (zh) * 2009-12-21 2013-01-23 北京清大天一科技有限公司 一种生产病毒疫苗的方法
WO2012073257A2 (fr) * 2010-11-30 2012-06-07 Bharat Biotech International Limited Formulations vaccinales pour la prophylaxie et le traitement d'infections par le virus chandipura chez les mammifères

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
1064/DEL/2009 (RAJESH JAIN, 25 May 2009 (2009-05-25), pages 10 *
HONDA K ET AL.: "Development of Vero cell -derived inactivated Japanese encephalitis vaccine", BIOLOGICALS, vol. 30, no. 4, December 2002 (2002-12-01), pages 303 - 14, XP002599708, DOI: doi:10.1006/BIOL.2002.0345 *
KOMIYA T ET AL.: "Long-term stability of Vero cell -derived inactivated Japanese encephalitis vaccine prepared using serum-free medium", VACCINE, vol. 26, 2008, pages 3680 - 3689 *
SOLOMON T. ET AL.: "Current use and development of vaccines for Japanese encephalitis", EXPERT OPIN. BIOL. THER., vol. 8, no. 1, 2008, pages 95 - 106, XP009173515, DOI: doi:10.1517/14712598.8.1.95 *
THOMAS SJ ET AL.: "New Japanese encephalitis vaccines: alternatives to production in mouse brain", EXPERT REV. VACCINES, vol. 10, no. 3, 2011, pages 355 - 364 *
WEEKLY EPIDEMIOLOGICAL RECORD NO. 34/35, vol. 81, 25 August 2006 (2006-08-25), pages 325 - 340 *

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11008547B2 (en) 2014-03-25 2021-05-18 Terumo Bct, Inc. Passive replacement of media
US11795432B2 (en) 2014-03-25 2023-10-24 Terumo Bct, Inc. Passive replacement of media
US11608486B2 (en) 2015-07-02 2023-03-21 Terumo Bct, Inc. Cell growth with mechanical stimuli
US11162880B2 (en) 2015-11-09 2021-11-02 University Of Notre Dame Du Lac Particle size purification method and devices
US11965175B2 (en) 2016-05-25 2024-04-23 Terumo Bct, Inc. Cell expansion
US11634677B2 (en) 2016-06-07 2023-04-25 Terumo Bct, Inc. Coating a bioreactor in a cell expansion system
US11685883B2 (en) 2016-06-07 2023-06-27 Terumo Bct, Inc. Methods and systems for coating a cell growth surface
US11104874B2 (en) 2016-06-07 2021-08-31 Terumo Bct, Inc. Coating a bioreactor
US11629332B2 (en) 2017-03-31 2023-04-18 Terumo Bct, Inc. Cell expansion
US11702634B2 (en) 2017-03-31 2023-07-18 Terumo Bct, Inc. Expanding cells in a bioreactor
US11478541B2 (en) 2017-11-03 2022-10-25 Takeda Vaccines, Inc. Method for inactivating Zika virus and for determining the completeness of inactivation
US11648304B2 (en) 2017-11-03 2023-05-16 Takeda Vaccines, Inc. Zika vaccines and immunogenic compositions, and methods of using the same
US11730802B2 (en) 2017-11-03 2023-08-22 Takeda Vaccines, Inc. Zika vaccines and immunogenic compositions, and methods of using the same
US11964008B2 (en) 2017-11-03 2024-04-23 Takeda Vaccines, Inc. Method for inactivating zika virus and for determining the completeness of inactivation
US11975062B2 (en) 2017-11-30 2024-05-07 Takeda Vaccines, Inc. Zika vaccines and immunogenic compositions, and methods of using the same
EP3581646A1 (fr) * 2018-06-15 2019-12-18 Themis Bioscience GmbH Système chromatographique et de fabrication intégré pour la production de virus
WO2019238919A1 (fr) * 2018-06-15 2019-12-19 Themis Bioscience Gmbh Système intégré de fabrication et de chromatographie pour la production de virus

Also Published As

Publication number Publication date
RU2706693C2 (ru) 2019-11-20
PH12016500500A1 (en) 2016-06-13
IN2013CH04135A (fr) 2015-08-14
AU2014338520B2 (en) 2019-12-05
WO2015059714A8 (fr) 2015-06-18
RU2016114285A (ru) 2017-10-19
AU2014338520A1 (en) 2016-03-31
RU2016114285A3 (fr) 2018-07-16
CN105744952A (zh) 2016-07-06

Similar Documents

Publication Publication Date Title
AU2014338520B2 (en) A viral vaccine and methods of manufacture thereof
EP1427815B1 (fr) Procedes pour la production industrielle de vaccins
EP1427817B1 (fr) Multiplication de virus dans une culture cellulaire
DE69837211T2 (de) Ein an vero-zellen angepasstes abgeschwächtes japanische enzephalitis virus und ein impfstoff gegen japanische enzephalitis
US6149917A (en) Industrial production process for a vaccine against Japanese encephalitis virus and vaccine produced
ES2653197T3 (es) Procedimiento para producir antígeno ortomixoviral y vacunas
DE19612966A1 (de) Tierische Zellen und Verfahren zur Vermehrung von Influenzaviren
EA023382B1 (ru) Способ получения высококонцентрированного препарата иммуноглобулина для подкожного применения
CN101352570B (zh) 二倍体细胞狂犬疫苗及纯化狂犬疫苗的制备方法
US20080193478A1 (en) Inactivated Poliomyelitis Vaccine Derived From Sabin Strain Of Polio Virus
CN107043784A (zh) 一种慢病毒载体的制备方法
EP2075005B1 (fr) Vaccin de salk (ipv ) et contre la diphtérie, la coqueluche et le tétanos (dpt)
CN101352569B (zh) 二倍体细胞乙型脑炎疫苗及纯化乙型脑炎疫苗的制备方法
CN1248471A (zh) Vero细胞乙型脑炎灭活疫苗及其制备方法
EP2456855B1 (fr) Vidange et réalimentation d'un bioréacteur à microsupport
CN106497850B (zh) 一种纯化猪肺炎支原体的方法
KR20120027381A (ko) 일본뇌염 백신 및 그것의 제조 방법
CN105296438A (zh) 一种制备森林脑炎灭活疫苗的生产方法
CN115141273A (zh) 一种猫杯状病毒的单克隆抗体及其应用
CN102614508B (zh) 一种人用流感病毒裂解疫苗的病毒裂解灭活方法
CN1513553B (zh) Vero细胞森林脑炎灭活疫苗
CN114306587B (zh) 一种低血清乙型脑炎灭活疫苗的制备方法及乙型脑炎灭活疫苗
KR20100017593A (ko) 바이러스의 증식을 위한 ２-단계 온도 프로파일
RU2683508C1 (ru) Штамм вируса для изготовления вакцинных препаратов против геморрагической лихорадки с почечным синдромом (варианты)
RU2314125C1 (ru) Способ получения инактивированной вакцины против вируса гепатита а

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 14856071

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: IDP00201602154

Country of ref document: ID

ENP Entry into the national phase

Ref document number: 2014338520

Country of ref document: AU

Date of ref document: 20140908

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 2016114285

Country of ref document: RU

Kind code of ref document: A

122 Ep: pct application non-entry in european phase

Ref document number: 14856071

Country of ref document: EP

Kind code of ref document: A1