EP4626574A1 - Production et fabrication de vaccin à flavivirus à grande échelle - Google Patents

Production et fabrication de vaccin à flavivirus à grande échelle

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Publication number
EP4626574A1
EP4626574A1 EP23829266.8A EP23829266A EP4626574A1 EP 4626574 A1 EP4626574 A1 EP 4626574A1 EP 23829266 A EP23829266 A EP 23829266A EP 4626574 A1 EP4626574 A1 EP 4626574A1
Authority
EP
European Patent Office
Prior art keywords
harvest
bulk drug
drug substance
harvesting
cells
Prior art date
Legal status (The legal status 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 status listed.)
Pending
Application number
EP23829266.8A
Other languages
German (de)
English (en)
Inventor
Joseph David Santangelo
Jill A. Livengood
Yockann LEE
Weiwen LUO
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Takeda Vaccines Inc
Original Assignee
Takeda Vaccines Inc
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
Priority claimed from PCT/US2023/061230 external-priority patent/WO2023147337A2/fr
Application filed by Takeda Vaccines Inc filed Critical Takeda Vaccines Inc
Publication of EP4626574A1 publication Critical patent/EP4626574A1/fr
Pending legal-status Critical Current

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Classifications

    • 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
    • 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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/36—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving ionic interaction, e.g. ion-exchange, ion-pair, ion-suppression or ion-exclusion
    • B01D15/361—Ion-exchange
    • B01D15/363—Anion-exchange
    • 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)
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2311/00—Details relating to membrane separation process operations and control
    • B01D2311/04—Specific process operations in the feed stream; Feed pretreatment
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2311/00—Details relating to membrane separation process operations and control
    • B01D2311/26—Further operations combined with membrane separation processes
    • B01D2311/2688—Biological processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2315/00—Details relating to the membrane module operation
    • B01D2315/10—Cross-flow filtration
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2315/00—Details relating to the membrane module operation
    • B01D2315/16—Diafiltration
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
    • B01D61/14—Ultrafiltration; Microfiltration
    • B01D61/145—Ultrafiltration
    • 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

Definitions

  • the present invention provides a method for large-scale flaviviral vaccine production and manufacture comprising the following sequential steps (i) providing cells in growth media, (ii) infecting the cells of step (i) with infection media comprising flavivirus, (iii) harvesting to obtain a harvest, (iv) processing the harvest of step (iii) to obtain a processed harvest, and (v) purifying the processed harvest of step (iv), wherein step (v) comprises at least one chromatography step.
  • the infection media can optionally comprise flavivirus at a low MOI.
  • the present invention provides a method for large-scale flaviviral vaccine production and manufacture comprising the following sequential steps (i) providing cells in growth media, (ii) infecting the cells of step (i) with infection media comprising flavivirus (optionally at a low MOI), (iii) harvesting to obtain a harvest, (iv) processing the harvest of step (iii) to obtain a processed harvest, and (v) purifying the processed harvest of step (iv), wherein step (v) comprises at least one chromatography step.
  • the method of infection in step (ii) is static, and wherein the cells are infected in monolayer.
  • the present invention provides a method for large-scale flaviviral vaccine production and manufacture comprising the following sequential steps (i) providing cells in growth media, (ii) infecting the cells of step (i) with infection media comprising flavivirus (optionally at a low MOI), (iii) harvesting to obtain a harvest, wherein harvesting comprises a first harvesting step and at least one further harvesting step, and wherein at least 12 to 30 hours before the first harvesting step, a media change is carried out, (iv) processing the harvest of step (iii) to obtain a processed harvest, and (v) purifying the processed harvest of step (iv), wherein step (v) comprises at least one chromatography step.
  • the present invention provides a method for large-scale flaviviral vaccine production and manufacture comprising the following sequential steps (i) providing cells in growth media, (ii) infecting the cells of step (i) with infection media comprising flavivirus optionally at a low MOI), (iii) harvesting to obtain a harvest, wherein harvesting comprises a first harvesting step and at least one further harvesting step, and wherein at least 12 to 30 hours before the first harvesting step, a media change is carried out, (iv) processing the harvest of step (iii) to obtain a processed harvest, and (v) purifying the processed harvest of step (iv), wherein step (v) comprises at least one chromatography step.
  • the method of infection in step (ii) is static, and wherein the cells are infected in monolayer.
  • the present invention provides a method for large-scale flaviviral vaccine production and manufacture comprising the following sequential steps (i) providing cells in growth media, (ii) infecting the cells of step (i) with infection media comprising flavivirus (optionally at a low MOI), (iii) harvesting to obtain a harvest, (iv) processing the harvest of step (iii) to obtain a processed harvest, and (v) purifying the processed harvest of step (iv), wherein step (v) comprises at least one chromatography step, and wherein step (v) comprises the sequential steps of (v-a) ion exchange chromatography to obtain a purified harvest, and
  • a method of large-scale flaviviral vaccine production and manufacture comprises, a harvesting step to obtain a harvest, obtaining a bulk drug substance from the harvest, processing the bulk drug substance comprising a step of filtering the bulk drug substance followed by a step of freezing the bulk drug substance to obtain a frozen bulk drug substance.
  • processing the bulk drug substance does not involve a freezing step before the filtration step.
  • the harvest does not undergo a freezing step until the step of processing the bulk drug substance.
  • a method of large-scale flaviviral vaccine production and manufacture comprises the following sequential steps:
  • step (ii) infecting the cells of step (i) with media comprising a flavivirus (optionally at a low MOI),
  • step (v) purifying the processed harvest of step (iv) comprising at least one chromatography step to obtain a purified harvest
  • step (ii) infecting the cells of step (i) with media comprising a flavivirus (optionally at a low MOI),
  • step (iv) processing the harvest of step (iii) to obtain a processed harvest
  • step (vi) processing the drug substance obtained in step (v) comprising at least one flushing step of the ultrafiltration membrane with a flushing buffer to obtain a bulk drug substance, wherein a second harvesting step is conducted, and the above steps are repeated to obtain a second bulk drug substance,
  • total surface area of production culture is achievable in the magnitude of 35, 000 cm 2 or more, such as, for example, 50, 000 cm 2 to 1 , 000, 000 cm 2 , 100, 000 cm 2 to 1 , 000, 000 cm 2 , 50, 000 cm 2 to 450, 000 cm 2 , 100, 000 cm 2 to 450, 000 cm 2 .
  • the present invention also provides a method for large-scale flaviviral vaccine production and manufacture comprising formulation of a drug product.
  • the drug product preferably comprises a tetravalent dengue viral vaccine comprising live, attenuated dengue-2 virus serotype, a dengue 2/1 chimera, a dengue 2/3 chimera and a dengue 2/4 chimera.
  • TDV- 1 and TDV-2 are manufactured in the same way.
  • TDV-3 and TDV-4 are manufactured in the same way as TDV-1 and TDV-2 but differ in the scale of manufacturing and/or the processing of the bulk drug substance. In one embodiment, TDV-4 is manufactured at a larger scale than TDV-1 , TDV-2, and TDV-3. In one embodiment, TDV-3 and TDV-4 are manufactured at a larger scale than TDV-1 and TDV-2.
  • Figure 4 Graph of viral titers obtained after infection with low MOI of 0.001 (Group 1) and high MOI of 0.05 (Group 2). Individual viral titers for days 5, 6 and 9 are shown (bars left to right).
  • Figure 5 Graph showing viral yield (grams) dependence on pH fluctuation - lower the fluctuation higher the viral yield.
  • the Corning® CellSTACK® culture chambers that can be used are 1- stack (CF1) with 636 cm 2 cell growth area, 2-stack (CF2) with 1 ,272 cm 2 cell growth area, 5- stack (CF5) with 3,180 cm 2 cell growth area, 10-stack (CF10) with 6,360 cm 2 cell growth area, 40-stack (CF40) with 25,440 cm 2 cell growth area.
  • the recommended medium volume required for each of these vessels is 130 to 200 mL for CF1 , 260 to 400 mL for CF2, 650 to 1 ,000 mL for CF5, 1 ,300 to 2,000 mL for CF10, and 5,200 to 8,000 mL for 40-stack.
  • the method of the present invention specifically concerns large- scale production and manufacture of viral vaccines, the scientific, and economical considerations for which are entirely different from a small-scale process. Accordingly, the present invention preferably excludes small-scale processes of viral vaccine production.
  • production culture refers to a pure culture of the desired strain, i.e., primary strain
  • inoculum refers to a seed culture to be inoculated to a medium for proliferation, which is then considered a production culture.
  • methods used to develop seed stocks, or the methods used for seed culture in general are not applicable or irrelevant and cannot be used for large-scale production and manufacture of viral vaccines.
  • replication rate refers to the factor by which the flaviviral population grows.
  • attenuated means that replication rate of the virus has been compromised.
  • the method of the present invention can be used for large-scale production and manufacture of flaviviruses.
  • the genus flavivirus comprises enveloped positive-stranded RNA viruses such as West Nile (WN) virus, Japanese Encephalitis virus (JEV), Zika virus, Dengue fever virus, yellow fever virus (YF), Kyasanur Forest disease virus, Murray Valley encephalitis virus, St.
  • WN West Nile
  • JEV Japanese Encephalitis virus
  • YF yellow fever virus
  • Kyasanur Forest disease virus Murray Valley encephalitis virus
  • the genus flavivirus contains highly pathogenic and potentially haemorrhagic fever viruses, such as yellow fever virus and dengue virus, Zika virus, encephalitic viruses, such as Japanese encephalitis virus, Murray Valley encephalitis virus and West Nile virus, and several less pathogenic viruses.
  • the flavivirus genome comprises in 5' to 3' direction: a 5'-noncoding region (5'-NCR), a capsid protein (C) encoding region, a pre-membrane protein (prM) encoding region, an envelope protein (E) encoding region, a region encoding non-structural proteins (NS1 , NS2A, NS2B, NS3, NS4A, NS4B, NS5) and a 3' noncoding region (3'-NCR).
  • the flaviviral structural proteins are C, prM and E, and the non-structural proteins are NS1 to NS5.
  • the structural and non-structural proteins are translated as a single polyprotein and processed by cellular and flaviviral proteases.
  • Preferred flavivirus vaccines for manufacture according to the present invention are zika and dengue vaccines.
  • the method of the present invention can be used forthe large- scale production and manufacture of dengue viruses, i.e., each of the dengue serotypes.
  • Dengue virus as used herein is a single stranded, positive sense RNA virus of the family flaviviridae.
  • the family flaviviridae includes three genera, flavivirus, hepacivirus and pestivirus.
  • the term "dengue serotype” refers to a species of dengue virus which is defined by its cell surface antigens and therefore can be distinguished from other dengue serotypes by serological methods known in the art.
  • dengue serotype 1 dengue serotype 1
  • DEV-2 dengue serotype 2
  • DEV- 3 dengue serotype 3
  • DEV-4 dengue serotype 4
  • the live, attenuated dengue virus strains can e.g., be (i) a chimeric dengue serotype 2/1 strain (TDV-1), (ii) a dengue serotype 2 strain (TDV-2), (iii) a chimeric dengue serotype 2/3 strain (TDV-3), and (iv) a chimeric dengue serotype 2/4 strain (TDV-4).
  • TDV-1 , TDV-2, TDV-3 and TDV-4 together form a tetravalent dengue virus composition termed “TDV” or, “TAK-003”, a dengue vaccine marketed under the tradename “Qdenga”.
  • the tetravalent dengure virus composition comprises: live attenuated dengue virus serotype 1 , live attenuated dengue virus serotype 2, live attenuated dengue virus serotype 3, and live attenuated dengue virus serotype 4; wherein the dengue serotype 1 is a chimeric dengue serotype 2/1 strain, the dengue serotype 2 is a non-chimeric dengue serotype 2 strain, the dengue serotype 3 is a chimeric dengue serotype 2/3 strain and the dengue serotype 4 is a chimeric dengue serotype 2/4 strain; wherein the dengue serotype 2 strain is derived from the wild type virus strain DEN-2 16681 and differs in at least three nucleotides from the wild type as follows: a) 5'-noncoding region (NCR)-57 (nt-57) b) NS1-53 Gly-to-Asp (n
  • a “chimeric virus” or “chimeric strain” or “chimeric virus strain” in general comprises parts from at least two different viruses.
  • a chimeric virus can comprise the prM and E proteins of dengue virus and the other proteins from another flavivirus.
  • the chimeric virus can comprise the prM and E proteins of dengue virus and the other proteins from another flavivirus such as yellow fever virus, Zika virus, West Nile virus, Japanese encephalitis virus, St. Louis encephalitis virus and tick-borne encephalitis virus, the chimeric virus can comprise the prM and E proteins of dengue virus and the other proteins from yellow fever virus strain YF-17D.
  • Such chimeric dengue viruses are e.g. described in WO 01/060847 A2, WO 2014/150939 A2 and WO 2017/179017 A1.
  • the live, attenuated dengue-2 virus serotype or DENV-2 or TDV-2 is in the form of DENV-2 16681 derived DEN-2 PDK-53 variant with a triple mutation at NS1-53, at 5’NCR-57 and at NS3-250 such that the amino acid position 250 of the NS3 protein contains a valine residue, and wherein the chimeras have said DEN-2 PDK-53 genome as viral backbone and one or more structural protein genes encoding capsid, premembrane/membrane or envelope of said DEN-2 PDK-53 genome or combinations thereof replaced with one or more corresponding structural protein genes from DEN-1 , DEN-3 or DEN- 4.
  • the live, attenuated dengue-2 virus serotype or DENV-2 or TDV-2 is represented by a polynucleotide of SEQ ID NO: 3, or a polypeptide of SEQ ID NO: 4; the dengue 2/1 chimera or DENV-2/1 chimera, or TDV-1 having nonstructural proteins from a modified live, attenuated dengue-2 virus serotype and structural proteins from a dengue-1 virus serotype, represented by a polynucleotide of SEQ ID NO: 1 , or a polypeptide of SEQ ID NO: 2; the dengue 2/3 chimera or DENV-2/3 chimera, or TDV-3 having nonstructural proteins from a modified live, attenuated dengue-2 virus serotype and structural proteins from a dengue-3 virus serotype, represented by a polynucleotide of SEQ ID NO: 5, or a polypeptide of SEQ ID NO: 6; and the dengue 2/4 chimera or DENV-2/4
  • RNA virus as comprised in TDV, can be characterized by its RNA sequence. It is, alternatively or in addition, also common practice to characterize the genome of RNA viruses by the corresponding DNA sequences and corresponding DNA sequences are readily understood to reflect the RNA genome in the virus. Therefore, reference to “t” or “thymine” in the entire disclosure is to be understood as reference to “u” or “uracil”, respectively, if the genomic RNA virus sequence is meant.
  • the method for large-scale flaviviral vaccine production and manufacture preferably comprises a step of providing cells in growth media.
  • the flaviviral vaccine is a tetravalent dengue vaccine including all four live, attenuated dengue serotypes including dengue serotype 1 such as dengue 2/1 chimera or TDV-1 represented by SEQ ID NO: 1 and/or SEQ ID NO:2 , dengue serotype 2 or TDV-2 represented by SEQ ID NO: 3 and/or SEQ ID NO:4, dengue serotype 3 such as dengue serotype 2/3 or TDV-3 represented by SEQ ID NO: 5 and/or SEQ ID NO:6, dengue serotype 4 such as dengue serotype 2/4 or TDV-4 represented by SEQ ID NO: 7 and/or SEQ ID NO:8.
  • the methods described in this section are performed for each of the four serotypes separately.
  • This step further involves expansion of cells to sufficient numbers in growth media.
  • cell lines can be used including Madin-Darby Canine Kidney cells, monkey cell lines pMK, Vero, and human cell lines HEK 293, MRC 5, Per.C6, PMK, LLCMK2, BHK and WI-38. All of these cell lines are well characterized with a proven safety profile. Furthermore, these cell lines are suitable to support replication of the attenuated vaccine strains. Each of these cell lines represents a separate embodiment of the invention.
  • a cell line (such as selected from Madin-Darby Canine Kidney cells, monkey cell lines pMK, Vero, and human cell lines HEK 293, MRC 5, Per.C6, PMK, LLCMK2, BHK, and WI-38) can be used to develop two cell banks, a master cell bank (MCB) and a working cell bank (WCB).
  • MCB master cell bank
  • WCB working cell bank
  • master cell bank refers to a culture of cells (e.g., fully characterized cells) that have been grown from a single clone and stored under cryopreservation conditions. The cells from the MCB are used to develop a working cell bank.
  • working cell bank refers to a culture of cells (e.g., fully characterized cells) that has been grown from a single vial of the MCB, or from two pooled vials of the master cell bank and stored under cryopreservation conditions. The cells from the WCB are later used in a production cell culture.
  • the method of the present invention also includes the use of growth media and additive formulations for the expansion of the cells, which may or may not be the same as the media in which the WCB and MCB are developed.
  • the growth media used for the expansion of cells comprises one or more of growth media, glutamine or GlutaMAX TM, and serum. In some embodiments, the media is serum-free.
  • growth media refers to a medium for culturing cells containing nutrients that maintain cell viability and support proliferation.
  • the growth media may contain any of the following nutrients in appropriate amounts and combination: salt(s), buffer(s), amino acids, glucose or other sugar(s), antibiotics, and other components such as peptide growth factors, etc.
  • “Growth media” are known in the art and may be classified as natural or artificial cell culture media. Examples of cell culture media that can be used for the method of the present invention include Minimum Essential Medium (MEM), Dulbecco's Modified Eagle' s Medium (DMEM), DMEM/F-12, and Roswell Park Memorial Institute Medium (RPMI).
  • MEM Minimum Essential Medium
  • DMEM Dulbecco's Modified Eagle' s Medium
  • DMEM/F-12 Roswell Park Memorial Institute Medium
  • custom cell culture media or commercially available cell culture media such as Dulbecco's Modified Eagle Medium, Minimum Essential Medium, RPMI medium, HA or HAT medium, or other media available from other commercial sources can be used.
  • the growth media can include one or more antibiotics (e.g., actinomycin D, ampicillin, carbenicillin, cefotaxime, fosmidomycin, gentamycin, kanamycin, neomycin, penicillin, penicillin streptomycin, polymyxin B, streptomycin, tetracycline, or any other suitable antibiotic or any combination of two or more thereof).
  • the growth media can include one or more salts (e.g., balanced salts, calcium chloride, sodium chloride, potassium chloride, magnesium chloride, etc.).
  • the growth media can also include one or more buffers (e.g., HEPES or other suitable buffer).
  • the growth media can also include differentiation factors. Growth or differentiation factors (e.g., WNT-family proteins, BMP- family proteins, IGF-family proteins, etc.) can be added individually or in combination, e.g., as a differentiation cocktail including different factors that bring about differentiation toward a particular lineage. It is understood that each individual possibility can be combined with another possibility to arrive at a combination of features and that each possibility, and each combination represents a separate embodiment of the invention.
  • the concentration of glucose can be, for example, 3.12 g/L, 3.22 g/L, 3.32 g/L, 3.42 g/L, 3.52 g/L, 3.62 g/L, 3.72 g/L, 3.82 g/L, or 3.92 g/L.
  • concentration of glucose can be, for example, 3.12 g/L, 3.22 g/L, 3.32 g/L, 3.42 g/L, 3.52 g/L, 3.62 g/L, 3.72 g/L, 3.82 g/L, or 3.92 g/L.
  • the concentration of glutamine or GlutaMAX TM can be between 3.5 mM to
  • “Serum” as used herein refers to mammalian serum and can be selected from human serum, equine serum, bovine serum, or sheep serum. Serum is widely used as a supplement of the cell growth media for adherent mammalian cell culture as it provides a wide variety of macromolecular proteins, low molecular weight nutrients, carrier proteins for water-insoluble components, and other compounds necessary for in vitro growth of cells, such as hormones and attachment factors. The term “serum” further encompasses a solution based on serum containing a buffer. The addition of substances like salts, buffers, sugars, a chelating agents, preservatives and protease inhibitors to the serum used in the present invention can be included.
  • cell culture media include serum (e.g., fetal bovine serum, bovine calf serum, equine serum, porcine serum, or other serum).
  • cell culture media are serum-free.
  • cell culture media include human platelet lysate (hPL).
  • the media is serum-free. If serum is added, the concentration of serum can be in the range 1 to 10%, for example between 1 to 2%, 2 to 3%, 3 to 4%, 4 to 5%, 5 to 6%, 6 to 7%, 7 to 8%, 8 to 9%, or 9 to 10%.
  • the concentration of serum can be, for example, 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%.
  • the concentration of FBS in the media can be in the range 1 to 10%, for example between 1 to 2%, 2 to 3%, 3 to 4%, 4 to 5%, 5 to 6%, 6 to 7%, 7 to 8%, 8 to 9%, or 9 to 10%.
  • the concentration of equine serum in the media can be in the range 1 to 10%, for example between 1 to 2%, 2 to 3%, 3 to 4%, 4 to 5%, 5 to 6%, 6 to 7%, 7 to 8%, 8 to 9%, or 9 to 10%.
  • the concentration of sheep serum in the media can be in the range 1 to 10%, for example between 1 to 2%, 2 to 3%, 3 to 4%, 4 to 5%, 5 to 6%, 6 to 7%, 7 to 8%, 8 to 9%, or 9 to 10%.
  • the concentration of porcine serum in the media can be in the range 1 to 10%, for example between 1 to 2%, 2 to 3%, 3 to 4%, 4 to 5%, 5 to 6%, 6 to 7%, 7 to 8%, 8 to 9%, or 9 to 10%.
  • concentration of porcine serum in the media can be in the range 1 to 10%, for example between 1 to 2%, 2 to 3%, 3 to 4%, 4 to 5%, 5 to 6%, 6 to 7%, 7 to 8%, 8 to 9%, or 9 to 10%.
  • the invention includes preparing the growth media in any of the two ways described above. Further, the invention includes using one way of preparing the growth media for one flavivirus and another way for another flavi virus . Further, if a flavivirus with different serotypes is being produced, the invention includes producing one serotype in growth media prepared in one way and another serotype in growth media prepared in another way. For example, for the purpose of the invention, if dengue serotypes are being separately produced, serotype 4 may be produced with a filtered growth media while serotype 1 may be produced with aseptic mixing and vice versa. Each possibility represents a separate embodiment of the invention. When filtration is used, preferably a filter less than 1 pm is used, more preferably a filter less than 0.5 pm is used.
  • Another optional additive that can be included is a dissociation reagent for adherent cell passaging to dislodge the cells from the surface.
  • the term “dissociation reagent” as used herein refers to a solution or fluid which is contacted with anchorage-dependent cells and causes the cells to dissociate (loosen their cell attachment and may become detached) from the surface to which they adhere.
  • the proteolytic enzymes used in the present invention can be recombinant e.g., commercially available TrypLE Select or of mammalian origin e.g., trypsin of porcine origin.
  • the growth media can also comprise a combination of chelators and proteolytic enzymes. Each possibility represents a separate embodiment of the invention.
  • the targeted volume of the dissociation reagent is determined by the surface area of the culture vessel.
  • the volume to surface area ratio of the dissociation reagent can be between 0.01 to 0.02 mL/cm 2 , or 0.01 1 to 0.016 mL/cm 2 .
  • the volume to surface area ratio of proteolytic enzymes can be between 0.01 to 0.02 mL/cm 2 , or 0.01 1 to 0.016 mL/cm2.
  • the volume to surface area ratio of pepsin can be between 0.01 to 0.02 mL/cm 2 , or 0.01 1 to 0.016 mL/cm2.
  • the duration of treatment with a proteolytic enzyme can be up to 100 minutes, up to 90 minutes, up to 80 minutes, up to 70 minutes, up to 60 minutes, up to 50 minutes, up to 40 minutes, up to 30 minutes, up to 20 minutes.
  • the duration of treatment with TrypLE select can be up to 100 minutes, up to 90 minutes, up to 80 minutes, up to 70 minutes, up to 60 minutes, up to 50 minutes, up to 40 minutes, up to 30 minutes, up to 20 minutes.
  • the duration of treatment with pepsin can be up to 100 minutes, up to 90 minutes, up to 80 minutes, up to 70 minutes, up to 60 minutes, up to 50 minutes, up to 40 minutes, up to 30 minutes, up to 20 minutes.
  • DPBS phosphate-buffered saline
  • the cells are washed with the growth media that was used for cell expansion.
  • the targeted volume of the growth media used per wash is determined by the surface area of the culture vessel.
  • the volume to surface area ratio of the wash with the growth media can be between 0.04 to 0.06 ml_/cm 2 or 0.046 to 0.053 mL/cm2.
  • Another parameter is the maximum process duration for passaging, i.e., the time period starting from when the cells are out of the incubator, passaged and are on hold with growth media.
  • the maximum process duration for passaging can be up to 20 hours, preferably less than 20 hours, or less than 15 hours or less than 10 hours such as, for example, 5 hours, 6 hours, 7 hours, 8 hours or 9 hours.
  • Another parameter is the maximum cell split ratio, which can be set up to 1 :8 irrespective of the culture vessel used.
  • This parameter largely depends on the cell density and confluency up to which the cells are expanded before infection.
  • the minimum confluency of cells before infection is between 80 to 90%, preferably more than 80%.
  • the cell seeding density is in the range 1.5 x 10 4 to 3 x 10 4 cells/cm 2 , such as, for example, 1.75 x 10 4 or 2 x 10 4 cells/cm 2 and the cell density before infection is the range of 9 x 10 4 to 2 x 10 5 cells/cm 2 .
  • the duration of the cell thawing process can be within the range of 3 to 5 minutes, the incubation CO 2 percentage can be less than 6%, the dry period of the cells can be less than two hours, the maximum process duration for passaging can be up to 20 hours, and the maximum cell split ratio can be up to 1 :8.
  • the method for large-scale flaviviral vaccine production and manufacture comprises the sequential steps of (i) providing cells in growth media, (ii) infecting the cells of step (i) with media comprising a flavivirus (optionally at a low MOI).
  • the cells can be Vero cells.
  • the flavivirus can be a dengue virus.
  • the vaccine can be a live, attenuated viral vaccine.
  • the flaviviral vaccine is a tetravalent dengue vaccine including all four live, attenuated dengue serotypes including dengue serotype 1 such as dengue 2/1 chimera or TDV-1 represented by SEQ ID NO: 1 and/or SEQ ID NO:2 , dengue serotype 2 or TDV-2 represented by SEQ ID NO: 3 and/or SEQ ID NO:4, dengue serotype 3 such as dengue serotype 2/3 or TDV-3 represented by SEQ ID NO: 5 and/or SEQ ID NO:6, dengue serotype 4 such as dengue serotype 2/4 or TDV-4 represented by SEQ ID NO: 7 and/or SEQ ID NO:8.
  • dengue serotype 1 such as dengue 2/1 chimera or TDV-1 represented by SEQ ID NO: 1 and/or SEQ ID NO:2
  • dengue serotype 2 or TDV-2 represented by SEQ ID NO: 3 and/or SEQ ID NO:4
  • dengue serotype 3 such as dengue serotype 2
  • the methods described in this section are performed for each of the four serotypes separately.
  • the cells are infected with media comprising the working virus seed (WVS) comprising flavivirus.
  • WVS working virus seed
  • This media is called the infection media.
  • the infection media may or may not be the same as the growth media.
  • the objective of this step is to ensure that the virus infects the cells and replicates to a sufficient potency for harvesting and purification. Furthermore, when an attenuated virus is used, the additional objective is that the virus retains its attenuating loci.
  • the WCB is used for the manufacture of the master and working virus seeds, MVS and VWS, respectively.
  • MVS master virus seeds
  • production vessels refers to “culture vessels” that are inoculated with infection media comprising the VWS for the purpose of large-scale production and manufacture of flaviviral vaccines.
  • the term “culture vessel” may referto any container in which cells may be cultured.
  • Culture vessels include, but are not limited to, tissue culture flasks, 96 well plates, culture dishes, culture slides, and rotating wall vessels.
  • tissue culture flasks that can be used are Corning® CellSTACK® culture chambers or NuncTM EasyFill TM Cell FactoryTM systems.
  • the Corning® CellSTACK® culture chambers that can be used alone or in combination are 1 -stack (CF1) with 636 cm 2 cell growth area, 2-stack (CF2) with 1 ,272 cm 2 cell growth area, 5-stack (CF5) with 3,180 cm 2 cell growth area, 10-stack (CF10) with 6,360 cm 2 cell growth area, 40-stack (CF40) with 25,440 cm 2 cell growth area.
  • the method of the present invention also includes the use of infection media comprising a VWS comprising a flavivirus for infection of cells.
  • the present invention includes the use of infection media comprising a flavivirus for infection of cells.
  • the infection media can be the same as the growth media used for the expansion of cells or it can be different. In some embodiments, the media is serum-free.
  • the infection media can comprise one or more of growth media, and additionally, glutamine or GlutaMAX TM, and a non-ionic surfactant.
  • non-ionic surfactant means a surfactant that contains neither positively nor negatively charged functional groups. In contrast to anionic and cationic surfactants, non-ionic surfactants do not ionize in solution.
  • Non-ionic surfactants may be selected from block copolymers, sorbitan esters, ethoxylated or propoxylated sorbitan esters, alkyl-polyglycosides (APG), alkoxylated mono- or di-alkylamines, fatty acid monoethanolamides (FAMA), fatty acid diethanolamides (FADA), ethoxylated fatty acid monoethanolamides (EFAM), propoxylated fatty acid monoethanolamides (PFAM), polyhydroxy alkyl fatty acid amides, or N-acyl N-alkyl derivatives of glucosamine (glucamides, GA, or fatty acid glucamide, FAGA), and combinations thereof.
  • APG alkyl-polyglycosides
  • FAMA
  • the non-ionic surfactant can be a high molecular weight non-ionic surfactant. “High molecular weight” means a molecular weight of 1500 or more.
  • the non-ionic surfactant can be a non-ionic triblock copolymer.
  • the surfactant can be a non-ionic, hydrophilic, polyoxyethylene-polyoxypropylene block copolymer (or EO-PO block copolymer).
  • the EO-PO block copolymers can include blocks of polyethylene oxide (-CH2CH2O-designated EO) and polypropylene oxide (-CH2CHCH3O- designated PO).
  • the PO block can be flanked by two EO blocks in a EOx-POy-Eox arrangement.
  • the PO component is hydrophilic and the EO component is hydrophobic
  • the overall hydrophilicity, molecular weight and the surfactant properties of the copolymer can be adjusted by varying x and y in the EOx-POy-Eox block structure.
  • the EO-PO block copolymers will self-assemble into micelles with a PO core and a corona of hydrophilic EO groups.
  • the non-ionic surfactant can be a poloxamer.
  • Poloxamers are non-ionic triblock copolymers composed of a central hydrophobic chain of poly(propyleneoxide) flanked by two hydrophilic chains of polyethylene oxide). The length of the polymer blocks can be customized, leading to different poloxamers with slightly different properties.
  • the non-ionic surfactant can be Pluronic F127 (poloxamer 407), Pluronic F123 (poloxamer 403), Pluronic F-68 (poloxamer 188), Pluronic P123, Pluronic P85, other polyethylene oxide-polypropylene oxide (EO-PO) block copolymers of greater than 3,000- 4,000 MW or combinations thereof.
  • “Poloxamer 407” as used herein is a hydrophilic non-ionic surfactant which consists of a triblock copolymer consisting of a central propylene glycol block with about 56 repeat units and two flanking hydrophilic polyethylene glycol blocks each comprising about 101 repeat units.
  • Polyxamer 407 is also known by its trade names Pluronic F127 and Synperonic PE/F127.
  • Polyxamer 188 as used herein (P188) is a non-ionic linear copolymer having an average molecular weight of 8400 Daltons and is also referred to as Pluronic F68, FLOCOR and RheothRx.
  • Pluronic P123 is a symmetric triblock copolymer comprising polyethylene oxide) (PEG) and polypropylene oxide) (PPG) in an alternating linear fashion, PEO-PPO-PEO.
  • Pluronic P85 is a difunctional block copolymer surfactant terminating in primary hydroxyl groups.
  • the preparation of the infection media can include adding glutamine or GlutaMAX TM and non-ionic surfactant, resulting in specific concentrations of glucose, glutamine or GlutaMAX TM and non-ionic surfactant in the infection media.
  • concentration of glucose in the infection media can be between 3 g/L and 4 g/L of glucose, 3.1 to 3.2 g/L, 3.2 to 3.3 g/L, 3.3 to 3.4 g/L, 3.4 to 3.5 g/L, 3.5 to 3.6 g/L, 3.6 to 3.7 g/L, 3.7 to 3.8 g/L, 3.8 to 3.9 g/L, or 3.9 to 4.0 g/L.
  • the concentration of glucose can be, for example, 3.1 1 g/L, 3.22 g/L, 3.33 g/L, 3.44 g/L, 3.55 g/L, 3.66 g/L, 3.77 g/L, 3.88 g/L, or 3.99 g/L.
  • the concentration of glutamine or GlutaMAX TM in the infection media can be between 3.5 mM to 4.5 mM, 3.5 mM to 3.6 mM, 3.6 mM to 3.7 mM, 3.7 mM to 3.8 mM. 3.8 mM to 3.9 mM, 4.0 mM to 4.1 mM, 4.1 to 4.2 mM, 4.2 to 4.3 mM, 4.3 to 4.4 mM, or 4.4 to 4.5 mM.
  • the concentration of glutamine or GlutaMAX TM can be, for example 3.5 mM, 3.6 mM, 3.7 mM, 3.8 mM, 3.9 mM, 4.0 mM, 4.1 mM, 4.2 mM, 4.3 mM, 4.4 mM, or 4.5 mM. Each of these possibilities represents a separate embodiment of the invention.
  • the addition of non-ionic surfactant at specific concentrations enhances the viral production compared to infection media without the addition of non-ionic surfactant.
  • the concentration of the non-ionic surfactant can be between 0.05% to 2.0% (w/v) in the medium.
  • concentration of poloxamer 407 and/or F127 can be between 0.05% to 2.0% (w/v) in the medium.
  • the concentration of P85 can be between 0.05% to 2.0% (w/v) in the medium.
  • the infection media with and without a pH indicator can be used.
  • the pH indicator for the infection media can be phenol red. If no pH indicator is used, e.g., the media does not contain phenol red, visual microbial assessment may be implemented.
  • infection media preparation strategy is also a factor to control microbial growth.
  • the way the infection media is prepared can affect the growth or potency of the virus.
  • the infection media can be prepared by either “aseptically mixing” and homogenizing all components or mixing the components and then “filtering” them depending on the flavivirus that is being produced.
  • the invention includes preparing the infection media in any of the two ways described above. Further, the invention includes using one way of preparing the infection media for one flavivirus and another way for another flavivirus. Further, if a virus with different serotypes is being used, the invention includes producing one serotype in infection media prepared in one way and another serotype in infection media prepared in another way.
  • the cells provided in growth media are infected with media comprising a WVS of the flavivirus.
  • media comprising a WVS of the flavivirus.
  • each flavivirus and each serotype of each flavivirus has its own WVS from which a separate production culture for large-scale production and manufacture of the flaviviral vaccine is initiated.
  • Infection is a complex biological process which involves various operating parameters that will impact the infection efficiency/harvest potency, namely viruses attaching, entering and infecting the cells.
  • the method of the invention includes specific infection parameters.
  • MOI multiplicity of infection
  • P(n) is the probability that a target cell will get infected by n infectious virus particles.
  • the probability that a target cell will get infected by a virus particle is 1 - P(0).
  • infection efficiency or “transduction efficiency” or “virus potency” or “viral titers” are used interchangeably and refer to the ability of a flaviviral particle to bind to, penetrate and deliver its genome to the cytoplasm of a host cell, thereby allowing expression of structural proteins in a host cell.
  • the infection efficiency can be measured using analytical techniques known in the art such as an immunofocus assay (“IFA assay”).
  • IFA assay is a well-known analytical technique used in the art. The principle of the assay is based on classical virus plaque assays where serial dilutions of virus are plated on monolayers of adherent cells from a suitable host.
  • an overlay medium containing thickening agents is added to prevent diffusion of virions. Therefore, progeny virions can only infect cells adjacent to the original infected cell. This results in a roughly circular focus of infection for each infectious unit of virus.
  • Immunofocus assays differ from the classical plaque assay in that foci of infection are detected by immunostaining instead of visual observation of cytopathic effect.
  • the term “viral titre” refers to the number of infectious viral particles, or “transducing units,” that result in the infection of a target cell. Viral titer in the context of the invention can be measured using the IFA assay discussed herein above.
  • Viral titers determined by an IFA assay result in plaque forming units (PFU).
  • the immune focus assay can be carried out as described in detail in section 2.5 of Brewoo et al. (Vaccine. 2012 February 14; 30(8): 1513-1520. doi:10.1016/j. vaccine.201 1.11.072)
  • any other analytical method can be used to measure the viral titer, for example, viral titer can be measured by a functional assay, such as an assay described in Xiao et al., Exp. Neurobiol. 144:113-124, 1997, or Fisher et al., J. Virol. 70:520-532, 1996, the disclosures of both of which are incorporated herein by reference.
  • High viral titers refers to a viral titre greater than 7.0 logw PFU/mL, preferably greater than 7.5 log PFU/mL.
  • an infection media comprising a flavivirus at a low MOI can be used for large-scale flaviviral vaccine production and manufacture. Even more surprisingly, it has been found that a low MOI can be used for large-scale attenuated flaviviral vaccine production and manufacture.
  • the flavivirus can be a dengue virus.
  • a low MOI can be used for large-scale flaviviral vaccine production and manufacture providing higher average viral titres as compared to the same method comprising the same process steps but using a high MOI.
  • a low MOI can be used for large-scale attenuated flaviviral vaccine production and manufacture providing higher average viral titers as compared to the same method comprising the same process steps but using a high MOI.
  • high MOI refers to MOI greater than 0.008.
  • the flavivirus can be a dengue virus.
  • Average viral titers refers to average viral titers of at least two harvesting steps.
  • “Low MOI” as used herein refers to MOI of 0.008 or less, such as less than 0.008, or 0.005 or less, such as less than 0.005, preferably from 0.0001 to 0.008, or from 0.0001 to 0.005 such as, from 0.001 to 0.005, e.g., 0.0011 , 0.0012, 0.0013, 0.0014, 0.0015, 0.002, 0.0021 , 0.0022, 0.0023, 0.0024, 0.0025, 0.003, 0.0031 , 0.0032, 0.0033, 0.0034, 0.0035, 0.004, 0.0041 , 0.0042, 0.0043, 0.0044, 0.0045, or 0.005.
  • a low MOI within the range of 0.008 or less can be used. Accordingly, for the purpose of the present invention, a low MOI within the range of 0.005 or less can be used.
  • the infection of each of the serotypes can be carried out within the low MOI range. It is also possible that one serotype is infected with media comprising a flavivirus at a certain low MOI within the range of 0.008 or less and 0.005 or less, and another serotype is infected with media comprising a flavivirus at another low MOI within the range of 0.008 or less and 0.005 and less.
  • Each possibility represents a separate embodiment of the invention.
  • a low MOI can be used for large-scale flaviviral vaccine production and manufacture providing high viral titres.
  • a low MOI can be used for large-scale attenuated flaviviral vaccine production and manufacture with high viral titers.
  • the flavivirus can be a dengue virus.
  • a low MOI can be used for large-scale flaviviral vaccine production and manufacture providing higher average viral titres as compared to the same method comprising the same process steps but using a high MOI.
  • a low MOI can be used for large-scale attenuated flaviviral vaccine production and manufacture providing higher average viral titers as compared to the same method comprising the same process steps but using a high MOI.
  • high MOI refers to MOI greater than 0.008.
  • the flavivirus can be a dengue virus.
  • Average viral titers refers to average viral titers of at least two harvesting steps.
  • vero cells in more than ten CF-10 flasks are infected with dengue serotype 1 such as dengue 2/1 chimera or TDV-1 represented by SEQ ID NO:1 and/or SEQ ID NO: 2 at a low MOI.
  • dengue serotype 1 such as dengue 2/1 chimera or TDV-1 represented by SEQ ID NO:1 and/or SEQ ID NO: 2 at a low MOI.
  • vero cells in more than ten CF-10 flasks are infected with dengue serotype 2 or TDV-2 represented by SEQ ID NO:3 and/or SEQ ID NO: 4 at a low MOI.
  • vero cells in more than ten CF-10 flasks or CF-40 flasks are infected with dengue serotype 3 such as dengue 3/1 chimera or TDV-3 represented by SEQ ID NO:5 and/or SEQ ID NO: 6 at a low MOI.
  • dengue serotype 3 such as dengue 3/1 chimera or TDV-3 represented by SEQ ID NO:5 and/or SEQ ID NO: 6 at a low MOI.
  • vero cells in more than ten CF-40 flasks are infected with dengue serotype 4 such as dengue 2/4 chimera or TDV-4 represented by SEQ ID NO:7 and/or SEQ ID NO: 8 at a low MOI.
  • dengue serotype 4 such as dengue 2/4 chimera or TDV-4 represented by SEQ ID NO:7 and/or SEQ ID NO: 8 at a low MOI.
  • the method for large-scale flaviviral vaccine production and manufacture comprises the sequential steps of (i) providing cells in growth media, and (ii) infecting the cells of step (i) with media comprising a flavivirus at a low MOI.
  • the growth media of step (i) does not comprise a non-ionic surfactant.
  • the cells can be Vero cells.
  • the flavivirus can be a dengue virus.
  • the vaccine can be a live, attenuated viral vaccine.
  • the large-scale flaviviral vaccine production and manufacture with high viral titers involves the use of a cell line that comprises abundant flaviviral receptors.
  • the large-scale attenuated flaviviral vaccine production and manufacture with high viral titers involves the use of a cell line that comprises abundant flaviviral receptors.
  • the cells can be Vero cells.
  • the flavivirus can be a dengue virus and the flaviviral receptors can be dengue receptors.
  • “Flaviviral receptors” as used herein refers to cell surface receptors.
  • a wide range of cell surface receptors has been implicated in flavivirus entry into different cells types such as avp3 integrins C-type lectin receptors (CLR), phosphatidylserine receptors TIM (T-cell immunoglobulin and mucin domain) and TYRO3, AXL and MER (TAM).
  • a cell line “abundant” in flaviviral receptors as used herein refers to a cell line that has some degree of overlap of flaviviral receptors with the flaviviral receptors in a mosquito cell line such as C636, for example, at least one or two common receptors.
  • “Dengue virus vaccine” as used herein means either a monovalent vaccine, i.e., one of the four dengue serotypes, divalent vaccine, i.e., two of the four serotypes, trivalent vaccine, i.e., three of the four serotypes, or tetravalent vaccine, i.e., all four serotypes.
  • the method for large-scale flaviviral vaccine production and manufacture comprises the sequential steps of (i) providing cells that comprise abundant flaviviral receptors in growth media, and (ii) infecting the cells of step (i) with media comprising a flavivirus at a low MOI.
  • the growth media of step (i) does not comprise a non-ionic surfactant.
  • the cells can be Vero cells.
  • the flavivirus can be a dengue virus.
  • the vaccine can be a live, attenuated viral vaccine
  • Another infection parameter is the volume of infection media per surface area of the production vessel.
  • the volume of infection media per surface area of the production vessel is such that it covers the monolayer of the cells within the tissue culture vessel. However, whether the infection media covers the cell monolayer or not can depend on the physical method of infection, i.e., either static or rocking.
  • a rocking/shaking infection method should be used to ensure infection efficiency and virus potency. “Rocking/shaking as used herein refers to either manually rocking the tissue culture vessels or using a shaking platform.
  • the term “static” infection means that the tissue culture vessel is not moving during the infection process and thus, the infection media is not mechanically dispersed throughout the cell monolayer.
  • the term “rocking” infection means that the tissue culture vessel is kept on a platform that gently moves along a horizontal axis or the tissue culture vessel is manually moved so that the infection media is mechanically dispersed throughout the monolayer.
  • the flavivirus can be a dengue virus.
  • Another infection parameter within the meaning of the method of infection is infecting the cells in suspension or in monolayer.
  • the term “suspension” refers to cells that are dispersed in media.
  • the cells can be adherent cells that have been dissociated from the culture vessel using a dissociation reagent.
  • the cells should be infected immediately after using the dissociation agent (when they are still in suspension) to ensure infection efficiency.
  • the cells can be Vero cells.
  • the flavivirus can be a dengue virus.
  • the cells can be Vero cells.
  • the flavivirus can be a dengue virus.
  • the cells can be Vero cells.
  • the flavivirus can be a dengue virus.
  • the term “monolayer” refers to a layer of cells in which no, or substantially no cell is growing on top of another, but all are growing side by side and are often touching each other on the same growth surface.
  • the growth media of step (i) does not comprise a non-ionic surfactant.
  • the cells can be Vero cells.
  • the flavivirus can be a dengue virus.
  • the vaccine can be a live, attenuated viral vaccine.
  • the pH throughout the steps mentioned above is maintained in a range from 7.6 to 8.1 .
  • the flaviviral vaccine is a tetravalent dengue vaccine including all four live, attenuated dengue serotypes including dengue serotype 1 such as dengue 2/1 chimera or TDV-1 represented by SEQ ID NO: 1 and/or SEQ ID NO:2 , dengue serotype 2 orTDV-2 represented by SEQ ID NO: 3 and/or SEQ ID NO:4, dengue serotype 3 such as dengue serotype 2/3 or TDV-3 represented by SEQ ID NO: 5 and/or SEQ ID NO:6, dengue serotype 4 such as dengue serotype 2/4 or TDV-4 represented by SEQ ID NO: 7 and/or SEQ ID NO:8.
  • dengue serotype 1 such as dengue 2/1 chimera or TDV-1 represented by SEQ ID NO: 1 and/or SEQ ID NO:2
  • dengue serotype 2 orTDV-2 represented by SEQ ID NO: 3 and/or SEQ ID NO:4
  • dengue serotype 3 such as dengue serotype 2
  • harvest refers to the composition obtained from the action of harvesting, e.g., from collecting the supernatant.
  • Harvest or “crude harvest” are used interchangeably and refer to any intermediate composition from the time of collecting the supernatant up until the first processing step.
  • processing the harvest refers to one or more operational steps conducted on the obtained harvest.
  • processing the harvest may comprise one or all of the steps including clarification, stabilization, freezing, thawing and pooling.
  • processing the harvest refers to clarification and stabilization in that order.
  • processed harvest refers to clarified and stabilized harvest in that order.
  • the filters used in the clarification of the harvest can be heterogenous double layer of polyethersulfone with pore sizes of 0.2 pm and 0.45 pm.
  • several heterogenous double layer filters can be used such as Sartoclean (Cellulose Acetate) 0.8 +0.65 pm, Sartopure (Polypropylene) 0.65 pm, Sartoclean (Glassfiber Fleeces) 0.8 + 0.65 pm, Sartopore 2 (Polyethersulfone) 0.8 + 0.45 pm, Sartopore 2 (Polyethersulfone) 0.45 + 0.2 pm, or Sartopore 2 XLG (Polyethersulfone) 0.8 + 0.2 pm.
  • Sartoclean Cellulose Acetate
  • Sartopure Polypropylene
  • Sartoclean Glassfiber Fleeces
  • the composition obtained is termed “clarified harvest”, which refers to a harvest that has been subjected to the processing step of clarification.
  • the buffered excipient composition comprisies a non-ionic surfactant, a sugar and a protein in phosphate buffer saline (PBS).
  • PBS phosphate buffer saline
  • the buffered excipient composition is also termed stabilization buffer.
  • the terms buffered excipient composition and stabilization buffer are used interchangeably.
  • sucrose as used herein also includes sugar alcohols such as mannitol, sorbitol, arabitol, erythritol, maltitol, xylitol, glycitol, glycol, polyglycitol, polyethylene glycol, polypropylene glycol, and glycerol.
  • sugar alcohols such as mannitol, sorbitol, arabitol, erythritol, maltitol, xylitol, glycitol, glycol, polyglycitol, polyethylene glycol, polypropylene glycol, and glycerol.
  • the sugar used for the stabilization buffer can be a non-reducing sugar.
  • Non-reducing sugars do not contain an aldehyde or ketone group which is capable of being oxidized.
  • examples of non-reducing sugars include sucrose, trehalose or its hydrates such as trehalose di hydrate.
  • the concentration of the sugar in the stabilization buffer can be 20% to 60% (w/v), 25% to 55% (w/v), or 35% to 50%, or 45% (w/v). Preferably, 35% to 50% (w/v).
  • the concentration of non-reducing sugar in the stabilization buffer can be 20% to 60% (w/v), 25% to 55% (w/v), or 35% to 50%, or 45% (w/v).
  • the surfactant in the stabilization buffer can be a non-ionic surfactant.
  • the concentration of the non-ionic surfactant in the stabilization buffer can be preferably between 0.25% to 5 %. For example, 1 .5%, 1 .75%, 2%, 2.25%, 2.5%, 2.75% or 3%.
  • the concentration of poloxamer 407 and/or F127 can be preferably between 0.25% to 5 %.
  • 1.5%, 1.75%, 2%, 2.25%, 2.5%, 2.75% or 3% Each possibility represents a separate embodiment of the invention.
  • the concentration of poloxamer 403 and/or F123 can be preferably between 0.25% to 5 %. For example, 1.5%, 1.75%, 2%, 2.25%, 2.5%, 2.75% or 3%.
  • the concentration of P123 can be preferably between 0.25% to 5 %.
  • the concentration of P85 can be preferably between 0.25% to 5 %.
  • the protein which may be present in the stabilization buffer can be any protein which is essentially inert and does not react with the virus. In particular, the protein does not affect the structure or infectivity of the virus.
  • the protein can be a structural protein or a serum protein.
  • the protein can be selected from an albumin, collagen, hydrolyzed collagen, gelatin and hydrolyzed gelatin.
  • the albumins are a family of globular non-glycosylated proteins which are inter alia present in the blood of a vertebrate. They are water-soluble, moderately soluble in concentrated salt solutions, and experience heat denaturation. Suitable albumins for use in the method of the present invention include mammalian serum albumins such as human serum albumin and bovine serum albumin or lactalbumin. Serum albumin is one of the most common proteins in vertebrate blood and has multiple functions. Human serum albumin is not glycosylated and has a single free thiol group. The human serum albumin may be recombinant human serum albumin, or it may be human serum albumin purified from human serum.
  • human serum albumin purified from human serum is used.
  • gelatin refers to a heterogeneous mixture of water-soluble proteins of high average molecular weight. Gelatin is not found in nature but derived from collagen by hydrolytic action. Gelatin is obtained by boiling skin, tendon, bones, ligaments in water. Gelatin is colorless or slightly yellowish, transparent, sheets, flakes or coarse powder which absorbs in a range between about 5 times and about 10 times its weight of water to form a gel in solutions.
  • the concentration of the protein in the stabilization buffer can be 0.1% to 0.5% (w/v), 0.2% to 0.4% (w/v), 0.15% to 0.3% (w/v) or 0.3% (w/v), preferably 0.2% to 0.4% (w/v).
  • the concentration of the albumin in the stabilization buffer can be 0.1 % to 0.5% (w/v), 0.2% to 0.4% (w/v), 0.15% to 0.3% (w/v) or 0.3% (w/v), preferably 0.2% to 0.4% (w/v).
  • the concentration of human serum albumin or human serum albumin purified from human serum in the stabilization buffer can be 0.1 % to 0.5% (w/v), 0.2% to 0.4% (w/v), 0.15% to 0.3% (w/v) or 0.3% (w/v), preferably 0.2% to 0.4% (w/v).
  • the concentration of collagen in the stabilization buffer can be 0.1 % to 0.5% (w/v), 0.2% to 0.4% (w/v), 0.15% to 0.3% (w/v) or 0.3% (w/v), preferably 0.2% to 0.4% (w/v).
  • the concentration of gelatin in the stabilization buffer can be 0.1 % to 0.5% (w/v), 0.2% to 0.4% (w/v), 0.15% to 0.3% (w/v) or 0.3% (w/v), preferably 0.2% to 0.4% (w/v).
  • the non-ionic surfactant in the stabilized harvest can be between 0.01 % to 0.4%.
  • 0.1 %, 0.125%, 0.14%, 0.16%. 0.18%, or 0.2% can be between 0.01 % to 0.4%.
  • concentration of poloxamer 407 and/or F127 can be between 0.01% to 0.4%.
  • concentration of poloxamer 403 and/or F123 can be between 0.01 % to 0.4%.
  • the concentration of P123 can be between 0.01 % to 0.4%.
  • the concentration of P85 can be between 0.01 % to 0.4%.
  • the concentration of P85 can be between 0.01 % to 0.4%.
  • the concentration of the albumin in the stabilized harvest can be 0.007% to 0.04% (w/v), 0.01 % to 0.03% (w/v), 0.01 % to 0.075% (w/v) or 0.02% (w/v).
  • the concentration of human serum albumin or human serum albumin purified from human serum in the stabilized harvest can be 0.007% to 0.04% (w/v), 0.01 % to 0.03% (w/v), 0.01 % to 0.075% (w/v) or 0.02% (w/v).
  • the concentration of collagen in the stabilized harvest can be 0.007% to 0.04% (w/v), 0.01 % to 0.03% (w/v), 0.01 % to 0.075% (w/v) or 0.02% (w/v).
  • the concentration of gelatin in the stabilized harvest can be 0.007% to 0.04% (w/v), 0.01 % to 0.03% (w/v), 0.01% to 0.075% (w/v) or 0.02% (w/v).
  • composition termed “stabilized harvest”, refers to a crude harvest that has been subjected to the processing step of stabilization, or a crude harvest that has been subjected to the processing steps of clarification and stabilization in any order .
  • clarified, and stabilized harvest refers to a crude harvest that has been clarified and stabilized in that order.
  • stabilized and clarified harvest refers to a crude harvest that has been stabilized and clarified in that order.
  • the method for large-scale viral vaccine production and manufacture comprises the sequential steps of (i) providing cells in growth media, (ii) infecting the cells of step (i) with media comprising a flavivirus (optionally at a low MOI), iii) harvesting to obtain a harvest and (iv) processing the harvest of step (iii) comprising stabilization of the harvest.
  • the cells can be Vero cells.
  • the flavivirus can be a dengue virus.
  • the vaccine can be a live, attenuated viral vaccine.
  • the method for large-scale viral vaccine production and manufacture comprises the sequential steps of (i) providing cells in growth media, (ii) infecting the cells of step (i) with media comprising a flavivirus at a low MOI, (iii) harvesting to obtain a harvest and (iv) processing the harvest of step (iii) comprising clarification and stabilization of the harvest.
  • the cells can be Vero cells.
  • the flavivirus can be a dengue virus.
  • the vaccine can be a live, attenuated viral vaccine.
  • Freezing is another way of processing and can be used to store and freeze a composition at any time during the processing at or below -30 °C.
  • Thawing is anotherway of processing and refers to adapting the frozen composition to room temperature.
  • Pooling is anotherway of processing and comprises pooling one or more compositions at any time during the processing in order to carry out further processing steps on the pooled composition or purifying steps on the pooled processed harvest.
  • each “harvest” or “crude harvest” may be frozen immediately after the harvesting step followed by thawing and then may be subjected to one or several processing steps or purifying steps on the processed harvest.
  • a first harvest obtained from the first harvesting step may be immediately frozen after the harvesting step followed by thawing and pooling together with at least one further harvest obtained from at least one further harvesting step and then subjected to one or more further processing steps or purifying steps.
  • a first harvest obtained from the first harvesting step can be immediately clarified and then immediately frozen followed by thawing and pooling with at least one further clarified harvest obtained from at least one further harvesting step (that may or may not have been frozen and thawed) and then subjected to one or more further processing steps or purifying steps.
  • a first harvest obtained from the first harvesting step is immediately clarified and stabilized on the same day.
  • “Immediately on the same day” or “on the same day” as used herein throughout the disclosure refers to a time interval ranging from a few minutes to a few hours but less than 20 hours preferably less than 10 hours, or less than 12 hours such as 1 hour or less, 2 hours or less, 3, hours or less, 4 hours or less or ,5 hours or less. 6 hours or less, 7 hours or less, 8 hours or less, 9 hours or less.
  • a first harvest obtained from the first harvesting step is immediately clarified, stabilized and purified with at least one chromatography step on the same day.
  • a chromatography step is preferably necessarily included and may be an ion exchange chromatography, (/.e., either a cation exchange chromatography, or an anion exchange chromatography).
  • Commonly used anion exchange functional groups are Q-resin, a quaternary amine, and DEAE resin (diethylaminoethane).
  • Commonly used cation exchange groups are CM, a quaternary amine, S, a methyl sulfonate, and SP, a sulphonyl group.
  • the anion or cation exchange chromatography step can be performed with all common commercially available anion or cation exchange resins or membranes.
  • Typical strong anion exchange groups that can be used for the purpose of the invention comprise functional groups such as: quaternary aminoethyl (QAE) moieties, primary amine (PA), quaternary ammonium (Q) moieties and trimethylammoniumethyl (TMAE) groups.
  • Resins having quaternary aminoethyl (QAE) moieties include, e.g., Toyopearl QAE (available from Tosoh Bioscience, Germany), Selectacel QAE (a quaternary aminoethyl derivative of cellulose, available from Polysciences Inc., Pennsylvania USA) and others.
  • the anion exchange chromatography step can be performed with an anion exchange chromatography membrane having quaternary ammonium groups.
  • the membrane base material can be selected from stabilized reinforced cellulose and polyethersulfone.
  • the membrane base material can be stabilized reinforced cellulose and the functional group is a quaternary ammonium group.
  • the anion exchange chromatography step may or may not involve the use of a monolithic support.
  • the nominal pore size of the membrane can be 0.5 pm to 5 pm or greater than 3 pm.
  • the membrane area can be 20 to 50 cm 2 , 25 to 45 cm 2 , 30 to 40 cm 2 such as, for example, 32 cm 2 , 34 cm 2 , 36 cm 2 , or 38 cm 2 .
  • the flow rate can be 1 to 50 ml/min, or 10 to 40 ml/min depending on the column used.
  • host cell DNA is removed from a sample comprising viral particles and host cell DNA.
  • the term “removing host cell DNA” means that the content of host cell DNA after the method of the invention has been performed is lower than the content of host cell DNA before the method of the invention is performed.
  • the method of the present invention results in an at least tenfold reduction of the host cell DNA content.
  • the method of the present invention results in a reduction of the host cell DNA content by at least 12-fold or 15-fold, more preferably by at least 18-fold or 20-fold and most preferably by at least 22-fold.
  • Methods to determine the host cell DNA content in a biological sample obtained from a host cell include quantitative PCR using primers which specifically bind to the host cell DNA, but not to the viral nucleic acids.
  • Kits for determining host cell DNA content are commercially available for example from ThermoFisher.
  • the Picogreen® dye is used to determine the host cell DNA content.
  • feed composition refers to the solution that is introduced to the chromatography column or the ultrafiltration membrane.
  • the feed composition is directly related to how the harvest is processed before the chromatography step for example by clarification, or stabilization, or clarification and stabilization.
  • directly performing a chromatography step after the clarification, stabilization or clarification and stabilization of the harvest does not lead to substantial loss of viral titer as compared to when the same feed composition is first concentrated in a membrane filtration step such as a TFF step.
  • high viral titers can be obtained when a large-scale flaviviral vaccine production and manufacture comprises the following steps sequentially: a chromatography step directly on the clarified and stabilized harvest, and followed by ultrafiltration.
  • the flavivirus can be a dengue virus.
  • the vaccine can be a live, attenuated viral vaccine.
  • the harvesting step, the clarification of the harvest, the stabilization of the harvest, the purification of the clarified and stabilized harvest with a chromatography step followed by ultrafiltration are all performed on the same day.
  • high viral titers can be obtained when a large-scale flaviviral vaccine production and manufacture comprises the following steps sequentially: an ion exchange chromatography step directly on the clarified and stabilized harvest, and followed by ultrafiltration.
  • the flavivirus can be a dengue virus.
  • the vaccine can be a live, attenuated viral vaccine.
  • the harvesting step, the clarification of the harvest, the stabilization of the harvest, the purification of the clarified and stabilized harvest with ion exchange chromatography followed by ultrafiltration are all performed on the same day.
  • high viral titers can be obtained when a large-scale flaviviral vaccine production and manufacture comprises the following steps sequentially: an ion exchange chromatography step directly on the clarified and stabilized harvest, and followed by tangential flow filtration.
  • the flavivirus can be a dengue virus.
  • the vaccine can be a live, attenuated viral vaccine.
  • the harvesting step, the clarification of the harvest, the stabilization of the harvest, the purification of the clarified and stabilized harvest with ion exchange chromatography followed by tangential flow filtration are all performed on the same day.
  • high viral titers can be obtained when a large-scale flaviviral vaccine production and manufacture comprises the following steps sequentially: an anion exchange chromatography step directly on the clarified and stabilized harvest, and followed by ultrafiltration.
  • the flavivirus can be a dengue virus.
  • the vaccine can be a live, attenuated viral vaccine.
  • the harvesting step, the clarification of the harvest, the stabilization of the harvest, the purification of the clarified and stabilized harvest with anion exchange chromatography followed by ultrafiltration are all performed on the same day.
  • high viral titers can be obtained when a large-scale flaviviral vaccine production and manufacture comprises the following steps sequentially: an anion exchange chromatography step directly on the clarified and stabilized harvest, and followed by tangential flow filtration.
  • the flavivirus can be a dengue virus.
  • the vaccine can be a live, attenuated viral vaccine.
  • the harvesting step, the clarification of the harvest, the stabilization of the harvest, the purification of the clarified and stabilized harvest with anion exchange chromatography followed by tangential flow filtration are all performed on the same day.
  • high viral titers can be obtained when a large-scale flaviviral vaccine production and manufacture comprises the following steps sequentially: a cation exchange chromatography step directly on the clarified and stabilized harvest, and followed by ultrafiltration.
  • the flavivirus can be a dengue virus.
  • the vaccine can be a live, attenuated viral vaccine.
  • the harvesting step, the clarification of the harvest, the stabilization of the harvest, the purification of the clarified and stabilized harvest with cation exchange chromatography followed by ultrafiltration are all performed on the same day.
  • high viral titers can be obtained when a large-scale flaviviral vaccine production and manufacture comprises the following steps sequentially: a cation exchange chromatography step directly on the clarified and stabilized harvest, and followed by tangential flow filtration.
  • the flavivirus can be a dengue virus.
  • the vaccine can be a live, attenuated viral vaccine.
  • the harvesting step, the clarification of the harvest, the stabilization of the harvest, the purification of the clarified and stabilized harvest with cation exchange chromatography followed by tangential flow filtration are all performed on the same day.
  • high viral titers can be obtained when a large-scale flaviviral vaccine production and manufacture comprises the following steps sequentially: (i) providing cells in growth media, (ii) infecting the cells of step (i) with media comprising a flavivirus (optionally at a low MOI), (iii) harvesting to obtain a harvest, (iv) processing the harvest of step (iii) comprising clarification and stabilization of the harvest (v) purifying the processed harvest of step (iv) by the sequential steps of (v-a) ion exchange chromatography directly on the processed harvest, and (v-b) tangential flow filtration on the purified harvest obtained in step (v-a).
  • high viral titers can be obtained when a large-scale flaviviral vaccine production and manufacture comprises the following steps sequentially: (i) providing cells in growth media, (ii) infecting the cells of step (i) with media comprising a flavivirus at a low MOI, (iii) harvesting to obtain a harvest, (iv) processing the harvest of step (iii) comprising clarification and stabilization of the harvest (v) purifying the processed harvest of step (iv) by the sequential steps of (v-a) anion exchange chromatography directly on the processed harvest and (v-b) tangential flow filtration on the purified harvest obtained in step (v-a).
  • the cells can be Vero cells.
  • the flavivirus can be a dengue virus.
  • the vaccine can be a live, attenuated viral vaccine.
  • steps (iii), (iv) and (v) are all performed on the same day.
  • high viral titers can be obtained when a large-scale flaviviral vaccine production and manufacture comprises the following steps sequentially: (i) providing cells in growth media, (ii) static infection of the cells of step (i) in monolayer with media comprising a virus at a low MOI, (iii) harvesting to obtain a harvest,
  • step (iv) processing the harvest of step (iii) comprising clarification and stabilization of the harvest
  • high viral titers can be obtained when a large-scale flaviviral vaccine production and manufacture comprises the following steps sequentially: (i) providing cells in growth media, (ii) static infection of the cells of step (i) in monolayer with media comprising a virus at a low MOI, (iii) harvesting to obtain a harvest,
  • step (iv) processing the harvest of step (iii) comprising clarification and stabilization of the harvest
  • purified harvest refers to a processed harvest that has been subjected to at least one chromatography steps.
  • the term “clarified and purified harvest” refers to a crude harvest that has been subjected to a clarification step and one or more chromatography steps in that order.
  • the term “clarified, stabilized and purified harvest” refers to a crude harvest that has been subjected to a clarification step, a stabilization step and one or more chromatography steps in that order.
  • the term stabilized, clarified and purified harvest refers to a crude harvest that has been subjected to a clarification step, one or more chromatography steps and a stabilization step in that order.
  • a low MOI can be used for large-scale flaviviral vaccine production and manufacture providing higher average viral titres as compared to the same method comprising the same process steps but using a high MOI.
  • a low MOI can be used for large-scale attenuated flaviviral vaccine production and manufacture providing higher average viral titers as compared to the same method comprising the same process steps but using a high MOI.
  • high MOI refers to MOI greater than 0.008.
  • the flavivirus can be a dengue virus.
  • Average viral titers refers to average viral titers of at least two harvesting steps.
  • a method for large-scale flaviviral vaccine production and manufacture comprising the following sequential steps:
  • step (ii) infecting the cells of step (i) with infection media comprising flavivirus
  • step (v) purifying the processed harvest of step (iv), wherein step (v) comprises at least one chromatography step, wherein the pH throughout steps (iii) to (v) is maintained at a range from 7.6 to 8.1 , and wherein the difference between
  • the method results in higher viral yield as compared to the same method with the same process steps but allowing a difference between (a) and (b) to be greater than 0.4 units.
  • the higher viral yield pertains to the viral yield that is higher by at least 1 mg/cm2, such as 1 .5 mg/cm2.
  • a method for large-scale Flaviviral vaccine production and manufacture comprising the following sequential steps:
  • step (iv) processing the harvest of step (iii) to obtain a processed harvest, (v) purifying the processed harvest of step (iv), wherein step (v) comprises at least one chromatography step, wherein the pH throughout steps (iii) to (v) is maintained at a range from 7.6 to 8.1 , and wherein the difference between
  • the method results in higher viral yield as compared to the same method with the same process steps but allowing a difference between (a) and (b) to be greater than 0.3 units.
  • the higher viral yield pertains to the viral yield that is higher by at least 1 mg/cm2, such as 1 .5 mg/cm2.
  • step (ii) infecting the cells of step (i) with infection media comprising flavivirus at a low MOI
  • step (iv) processing the harvest of step (iii) to obtain a processed harvest
  • “low MOI” pertains to less than 0.008.
  • the method results in higher viral yield as compared to the same method with the same process steps but allowing a difference between (a) and (b) to be greater than 0.4 units.
  • the higher viral yield pertains to the viral yield that is higher by at least 1 mg/cm2, such as 1.5 mg/cm2.
  • a method for large-scale Flaviviral vaccine production and manufacture comprising the following sequential steps:
  • step (iv) processing the harvest of step (iii) to obtain a processed harvest, (v) purifying the processed harvest of step (iv), wherein step (v) comprises at least one chromatography step, wherein the pH throughout steps (iii) to (v) is maintained at a range from 7.6 to 8.1 , and wherein the difference between
  • “low MOI” pertains to less than 0.008.
  • the step of purifying the harvest can comprise a step of ultrafiltration to obtain a drug substance.
  • the feed composition i.e., the input of the ultrafiltration step is either a clarified and purified harvest, a clarified, stabilized, and purified harvest, a stabilized, clarified and purified harvest, a stabilized and purified harvest, or only a purified harvest.
  • the ultrafiltration step can be a direct flow filtration (DFF) or tangential flow filtration step (TFF).
  • DFF direct flow filtration
  • TFF tangential flow filtration step
  • the method for large-scale viral vaccine production and manufacture comprises the sequential steps of (i) providing cells in growth media, (ii) infecting the cells of step (i) with media comprising a flavivirus (optionally at a low MOI), (iii) harvesting to obtain a harvest and (iv) processing the harvest of step (iii) to obtain a processed harvest and (v) purifying the processed harvest of step (iv) comprising at least one chromatography step and an ultrafiltration step.
  • steps (iii), and (iv) and (v) are all performed on the same day.
  • the method for large-scale viral vaccine production and manufacture comprises the sequential steps of (i) providing cells in growth media, (ii) infecting the cells of step (i) with media comprising a flavivirus (optionally at a low MOI), (iii) harvesting to obtain a harvest and (iv) processing the harvest of step (iii) to obtain a processed harvest and (v) purifying the processed harvest of step (iv) comprising at least one chromatography step and a tangential flow filtration step.
  • the cells can be Vero cells.
  • the flavivirus can be a dengue virus.
  • the vaccine can be a live, attenuated viral vaccine.
  • steps (iii), (iv) and (v) are all performed on the same day.
  • Direct flow filtration is applied when all the fluid to be filtered is driven, due to a supply pressure, in a direction perpendicular to a filtering surface. Contaminants are captured within the filtration media or build up on the surface, causing the differential pressure across the filter to rise as it blocks over the duration of the filtration process. The filtrate exits the filter on the downstream side. Once a certain differential pressure has been reached, after which the fluid flow rate decreases and/or the filter reaches its terminal differential pressure, filtration is stopped, and the filter is either discarded or may sometimes be regenerated for re-use.
  • Tangential Flow Filtration is a process where the feed stream flows parallel to the membrane face.
  • the composition obtained can be subjected to one or more tangential flow filtration (TFF) steps, one TFF step and one DFF step, or one TFF step. This step serves to concentrate the sample to ensure there is sufficient potency and to remove small molecular weight impurities.
  • TFF tangential flow filtration
  • a cross-linked cellulose-based polymer filter with a cut-off size of 100 kDa can be used in the TFF step.
  • Typical TFF membranes used for this step include molecular weight cut-off (MWCO) sizes of 100 kDa (Hydrosart) and 300 kDa polyethersulfone (PES).
  • MWCO molecular weight cut-off
  • PES polyethersulfone
  • TWO basic filter configurations are generally used for TFF: cartridge filters and cassette filters.
  • cartridge filters (often called hollow fiber filters)
  • the membrane forms a set of parallel hollow fibers.
  • the feed stream passes through the lumen of the fibers and the permeate is collected from outside the fibers.
  • cassette filters several flat sheets of membrane are held apart from each other and from the cassette housing by support screens. The feed stream passes into the space between two sheets and permeate is collected from the opposite side of the sheets.
  • both a cartridge or cassette filter can be used.
  • a cartridge filter can be used.
  • the solution containing the viruses for flaviviral vaccine manufacture is in the retentate.
  • the retentate obtained is also the drug substance.
  • the feed volume can e.g. be concentrated two- to ten-folds, three- to eight-fold, four- to six-fold or fivefold.
  • a drug substance is obtained.
  • the feed can be a clarified and purified harvest.
  • the feed can be a clarified, stabilized, and purified harvest.
  • the feed can be a stabilized, clarified and purified harvest.
  • the feed can be a stabilized and purified harvest.
  • the feed can be a purified harvest.
  • the pH throughout the steps mentioned above is maintained in a range from 7.6 to 8.1 .
  • a method for large-scale Flaviviral vaccine production and manufacture comprising the following sequential steps:
  • step (iv) processing the harvest of step (iii) to obtain a processed harvest
  • the method results in higher viral yield as compared to the same method with the same process steps but allowing a difference between (a) and (b) to be greater than 0.4 units.
  • the higher viral yield pertains to the viral yield that is higher by at least 1 mg/cm2, such as 1 .5 mg/cm2.
  • step (ii) infecting the cells of step (i) with infection media comprising flavivirus
  • step (iv) processing the harvest of step (iii) to obtain a processed harvest, (v) purifying the processed harvest of step (iv), wherein step (v) comprises the following sequential steps:
  • step (v-b) ultrafiltration to obtain a drug substance preferably, tangential flow filtration, wherein the pH throughout steps (iii) to (v) is maintained at a range from 7.6 to 8.1 , and wherein the difference between
  • the method results in higher viral yield as compared to the same method with the same process steps but allowing a difference between (a) and (b) to be greater than 0.3 units.
  • the higher viral yield pertains to the viral yield that is higher by at least 1 mg/cm2, such as 1 .5 mg/cm2.
  • a method for large-scale Flaviviral vaccine production and manufacture comprising the following sequential steps:
  • step (ii) infecting the cells of step (i) with infection media comprising flavivirus at a low MOI
  • step (iv) processing the harvest of step (iii) to obtain a processed harvest
  • step (v) purifying the processed harvest of step (iv), wherein step (v) comprises the following sequential steps:
  • step (v-b) ultrafiltration to obtain a drug substance preferably, tangential flow filtration, wherein the pH throughout steps (iii) to (v) is maintained at a range from 7.6 to 8.1 , and wherein the difference between
  • “low MOI” pertains to less than 0.008.
  • the method results in higher viral yield as compared to the same method with the same process steps but allowing a difference between (a) and (b) to be greater than 0.4 units.
  • the higher viral yield pertains to the viral yield that is higher by at least 1 mg/cm2, such as 1.5 mg/cm2.
  • step (ii) infecting the cells of step (i) with infection media comprising flavivirus at a low MOI
  • step (iv) processing the harvest of step (iii) to obtain a processed harvest
  • step (v-b) ultrafiltration to obtain a drug substance preferably, tangential flow filtration, wherein the pH throughout steps (iii) to (v) is maintained at a range from 7.6 to 8.1 , and wherein the difference between
  • an embodiment of the method for large-scale flaviviral vaccine production and manufacture of flaviviral vaccines includes a step of processing the harvest and a step of purifying the processed harvest comprising at least one chromatography step followed by an ultrafiltration step.
  • the purification step comprises an ultrafiltration step after the chromatography step.
  • a drug substance is obtained. If a divalent, trivalent, or tetravalent final drug product is intended, the drug substance at this step still comprises each individual monovalent drug substance.
  • each of the individual method steps and/or components of ingredients described herein above are contemplated alone or in combination with each other. Thus, each possibility described herein above alone and in combination with each other represents a separate embodiment of the invention. Processing the drug substance to obtain a bulk drug substance
  • the method for large-scale flaviviral vaccine production and manufacture comprises the sequential steps of (i) providing cells in growth media, (ii) infecting the cells of step (i) with media comprising a flavivirus (optionally at a low MOI), (Hi) harvesting to obtain a harvest and (iv) processing the harvest of step (iii) to obtain a processed harvest and (v) purifying the processed harvest of step (iv) comprising at least one chromatography step to obtain a purified harvest (v) an ultrafiltration step to obtain a drug substance (vi) processing the drug substance obtained in step (v) comprising at least one flushing step of the ultrafiltration membrane with a flushing buffer to obtain a composition comprising the buffer flush and drug substance which is the bulk drug substance.
  • the ultrafirltration is tangential flow filtration.
  • the chromatography step is anion exchange chromatography.
  • the cells can be Vero cells.
  • the flaviviral vaccine is a tetravalent dengue vaccine including all four live, attenuated dengue serotypes including dengue serotype 1 such as dengue 2/1 chimera or TDV-1 represented by SEQ ID NO: 1 and/or SEQ ID NO:2 , dengue serotype 2 or TDV-2 represented by SEQ ID NO: 3 and/or SEQ ID NO:4, dengue serotype 3 such as dengue serotype 2/3 or TDV-3 represented by SEQ ID NO: 5 and/or SEQ ID NO:6, dengue serotype 4 such as dengue serotype 2/4 or TDV-4 represented by SEQ ID NO: 7 and/or SEQ ID NO:8.
  • the methods described in this section are performed for each of the four serotypes separately.
  • the TFF step for the purpose of the invention may or may not be followed by diafiltration.
  • Diafiltration is a fractionation process that washes smaller molecules through a membrane and leaves larger molecules in the retentate without ultimately changing concentration. It can be used to remove salts or exchange buffers.
  • the method of the invention includes a step of flushing the filter membrane with buffer one, two, three, four or five times to obtain a composition comprising the buffer flush and drug substance, which is the bulk drug substance.
  • the harvesting step, the clarification of the harvest, the stabilization of the harvest, the purification of the clarified and stabilized harvest with a chromatography step and tangential flow filtration followed by the step of flushing are all performed on the same day.
  • the TFF step for the purpose of the invention may or may not be followed by diafiltration.
  • Diafiltration is a fractionation process that washes smaller molecules through a membrane and leaves larger molecules in the retentate without ultimately changing concentration. It can be used to remove salts or exchange buffers.
  • the method of the invention includes a step of flushing the filter membrane with buffer one, two, three, four or five times to obtain a buffer flush
  • the harvesting step, the clarification of the harvest, the stabilization of the harvest, the purification of the clarified and stabilized harvest with a chromatography step and tangential flow filtration followed by the step of flushing are all performed on the same day.
  • the composition comprising the drug substance and the buffer flush is the bulk drug substance.
  • a bulk drug substance is a composition comprising the drug substance (TFF retentate) and at least two buffer flushes of the TFF membrane.
  • a bulk drug substance is a composition comprising the drug substance (TFF retentate) and at least three buffer flushes of the TFF membrane.
  • a bulk drug substance is a composition comprising the drug substance (TFF retentate) and at least four buffer flushes of the TFF membrane.
  • a bulk drug substance is a composition comprising the drug substance (TFF retentate) and at least two buffer flushes of the TFF membrane.
  • a bulk drug substance is a composition comprising the drug substance (TFF retentate) and at least three buffer flushes of the TFF membrane.
  • a bulk drug substance is a composition comprising the drug substance (TFF retentate) and at least four buffer flushes of the TFF membrane.
  • the flushing buffer comprises the same buffered excipient composition/stabilization buffer added at the step of processing the harvest, more specifically at the step of stabilization of the harvest except that the excipients in the flushing buffer are at a lower concentration as compared to the buffered excipient composition/stabilization buffer.
  • the flushing buffer can comprise the same excipients as that of the stabilization buffer added at step (ii) of the method including sugar, non-ionic surfactant, and protein.
  • the flushing buffer comprises the same excipients as that of the stabilization buffer added at step (ii) of the method including sugar, non-ionic surfactant, and protein except that the excipients in the flushing buffer are at a lower concentration as compared to the excipients in the stabilization buffer.
  • the flushing buffer can comprise the same excipients as that of the stabilized or purified harvest including sugar, non-ionic surfactant, and protein.
  • the flushing buffer comprises the same excipients as that of the stabilized or purified harvest including sugar, non-ionic surfactant, and protein except that the excipients in the flushing buffer are at a higher concentration as compared to the excipients in the stabilized or purified harvest.
  • the flushing buffer can comprise the same excipients as that of the drug substance including sugar, non-ionic surfactant, and protein.
  • the flushing buffer comprises the same excipients as that of the drug substance including sugar, non-ionic surfactant, and protein except that the sugar in the flushing buffer is at a higher concentration as compared to the sugar in the drug substance.
  • an embodiment of the method for large-scale production and manufacture of flaviviral vaccine includes further step (vi) of processing the drug substance comprising at least one flushing step of the ultrafiltration membrane with a flushing buffer to obtain a composition comprising the buffer flush and the drug substance, which is the bulk drug substance, wherein the flushing buffer comprises a sugar, a surfactant, and a protein.
  • steps (iii), (iv), (v), and (vi) are all performed on the same day.
  • the method for large-scale production and manufacture of flaviviral vaccine includes further step (vi) of processing the drug substance comprising at least one flushing step of the ultrafiltration membrane with a flushing buffer to obtain a buffer flush, wherein the flushing buffer comprises a sugar, a surfactant, and a protein.
  • steps (iii), (iv), (v), and (vi) are all performed on the same day.
  • the drug substance and the bulk drug substance comprise the same excipients as that of the stabilization buffer added at step (ii) of the method including sugar, non-ionic surfactant, and protein.
  • the concentration of the excipients including sugar, non-ionic surfactant, and protein in the drug substance and bulk drug substance is the same.
  • the concentration of the excipients in the bulk drug substance from the buffered excipient composition/stabilization buffer added at step (ii) is two to five times higher as compared to the concentration of these excipients in the stabilized harvest or the purified harvest. In a preferred embodiment, the concentration of these excipients in the bulk drug substance is five times higher as compared to the concentration of these excipients in the stabilized harvest or the purified harvest.
  • the pH throughout the steps mentioned above is maintained in a range from 7.6 to 8.1 .
  • a method for large-scale Flaviviral vaccine production and manufacture comprising the following sequential steps:
  • step (iv) processing the harvest of step (iii) to obtain a processed harvest
  • step (vi) processing the drug substance obtained from step (v-b) to obtain a bulk drug substance, wherein the pH throughout steps (iii) to (vi) is maintained at a range from 7.6 to 8.1 , and wherein the difference between
  • the method results in higher viral yield as compared to the same method with the same process steps but allowing a difference between (a) and (b) to be greater than 0.4 units.
  • the higher viral yield pertains to the viral yield that is higher by at least 1 mg/cm2, such as 1 .5 mg/cm2.
  • step (ii) infecting the cells of step (i) with infection media comprising flavivirus, (iii) harvesting to obtain a harvest,
  • step (iv) processing the harvest of step (iii) to obtain a processed harvest
  • step (vi) processing the drug substance obtained from step (v-b) to obtain a bulk drug substance, wherein the pH throughout steps (iii) to (vi) is maintained at a range from 7.6 to 8.1 , and wherein the difference between
  • a method for large-scale Flaviviral vaccine production and manufacture comprising the following sequential steps:
  • step (iv) processing the harvest of step (iii) to obtain a processed harvest
  • step (v) purifying the processed harvest of step (iv), wherein step (vi) comprises the following sequential steps:
  • step (vi) processing the drug substance obtained from step (v-b) to obtain a bulk drug substance, wherein the pH throughout steps (iii) to (v) is maintained at a range from 7.6 to 8.1 , and wherein the difference between
  • the maximum pH occurring at any time point throughout steps (iii) to (vi), is no greater than 0.4 units.
  • “low MOI” pertains to less than 0.008.
  • the method results in higher viral yield as compared to the same method with the same process steps but allowing a difference between (a) and (b) to be greater than 0.4 units.
  • the higher viral yield pertains to the viral yield that is higher by at least 1 mg/cm2, such as 1.5 mg/cm2.
  • step (ii) infecting the cells of step (i) with infection media comprising flavivirus at a low MOI
  • step (iv) processing the harvest of step (iii) to obtain a processed harvest
  • step (vi) processing the drug substance obtained from step (v-b) to obtain a bulk drug substance, wherein the pH throughout steps (iii) to (v) is maintained at a range from 7.6 to 8.1 , and wherein the difference between
  • “low MOI” pertains to less than 0.008.
  • the method results in higher viral yield as compared to the same method with the same process steps but allowing a difference between (a) and (b) to be greater than 0.3 units.
  • the higher viral yield pertains to the viral yield that is higher by at least 1 mg/cm2, such as 1 .5 mg/cm2.
  • an embodiment of the method for large-scale production and manufacture of flaviviral vaccines includes a step of processing the drug substance comprising mixing the drug substance with a buffer flush to obtain a bulk drug substance.
  • the flushing step results in a composition that comprises the buffer flush and the drug substance, which is the bulk drug substance. If a divalent, trivalent, or tetravalent final drug product is intended, the bulk drug substance at this step still comprises each individual monovalent bulk drug substance.
  • each of the individual method steps and/or components or ingredients described herein above are contemplated alone or in combination with each other. Thus, each possibility described herein above alone and in combination with each other represents a separate embodiment of the invention.
  • the ultrafiltration is tangential flow filtration.
  • the chromatography step is anion exchange chromatography.
  • the cells can be Vero cells.
  • the flaviviral vaccine is a tetravalent dengue vaccine including all four live, attenuated dengue serotypes including dengue serotype 1 such as dengue 2/1 chimera or TDV-1 represented by SEQ ID NO: 1 and/or SEQ ID NO:2 , dengue serotype 2 or TDV-2 represented by SEQ ID NO: 3 and/or SEQ ID NO:4, dengue serotype 3 such as dengue serotype 2/3 orTDV-3 represented by SEQ ID NO: 5 and/or SEQ ID NO:6, dengue serotype 4 such as dengue serotype 2/4 or TDV-4 represented by SEQ ID NO: 7 and/or SEQ ID NO:8.
  • the methods described in this section are performed for each of the four serotypes separately.
  • Processing the bulk drug substance may comprise a depth filtration step. This filtration step can be carried out in the same manner as the step of clarification of the harvest.
  • depth filtration uses a porous filtration medium to separate particles and solids from a liquid.
  • the filter media which may be used in depth filtration can be cellulose acetate, polypropylene, cellulose acetate protected by glassfiber fleece and polyethersulfone.
  • the filter used in depth filtration can have a pore size of 0.1 pm to 1 pm, 0.2 pm to 0.8 pm, or 0.2 pm to 0.45 pm.
  • Freezing is another way of processing the bulk drug substance and can be used to store and freeze the bulk drug substance preferably below -30 °C.
  • Thawing is another way of processing the bulk drug substance and refers to adapting the frozen bulk drug substance to room temperature.
  • Pooling is another way of processing the bulk drug substance and comprises pooling two or more bulk drug substances.
  • a first harvest obtained from the first harvesting step is clarified and stabilized on the same day.
  • the clarified and stabilized harvest is also purified on the same day with at least one chromatography step, preferably an anion exchange chromatography.
  • the purified harvest is also subjected to an ultrafiltration step on the same day, preferably a tangential flow filtration to obtain a first drug substance.
  • the first drug substance is also mixed on the same day with a flushing buffer to obtain a composition comprising the buffer flush and the first drug substance, which is the first bulk drug substance.
  • a second harvesting step is conducted, and the above steps are repeated to obtain a second bulk drug substance.
  • up to ten harvesting steps are conducted, and the above steps are repeated to obtain up to ten bulk drug substances, wherein the interval between each harvesting step is 20-30 hours.
  • the above steps are conducted in the same way for each of the four live, attenuated dengue serotypes including including dengue serotype 1 such as dengue 2/1 chimera or TDV-1 represented by SEQ ID NO: 1 and/or SEQ ID NO:2 , dengue serotype 2 or TDV-2 represented by SEQ ID NO: 3 and/or SEQ ID NO:4, dengue serotype 3 such as dengue serotype 2/3 or TDV-3 represented by SEQ ID NO: 5 and/or SEQ ID NO:6, dengue serotype 4 such as dengue serotype 2/4 or TDV-4 represented by SEQ ID NO: 7 and/or SEQ ID NO:8.
  • dengue serotype 1 such as dengue 2/1 chimera or TDV-1 represented by SEQ ID NO: 1 and/or SEQ ID NO:2
  • dengue serotype 2 or TDV-2 represented by SEQ ID NO: 3 and/or SEQ ID NO:4
  • dengue serotype 3 such as dengue serotype 2/3 or TDV-3
  • each bulk drug substance obtained at the end of each day of harvest wherein the interval between each harvesting step is 20-30 hours.
  • six bulk drug substances are obtained from six harvesting steps, each bulk drug substance obtained at the end of each day of harvest, wherein the interval between each harvesting step is 20-30 hours.
  • for dengue serotype 3 six bulk drug substances are obtained from six harvesting steps, each bulk drug substance obtained at the end of each day of harvest, wherein the interval between each harvesting step is 20-30 hours.
  • for dengue serotype 4 six bulk drug substances are obtained from six harvesting steps, each bulk drug substance obtained at the end of each day of harvest, wherein the interval between each harvesting step is 20-30 hours.
  • each bulk drug substance can be frozen on the same day or subjected to a step of depth filtration on the same day and then frozen on the same day.
  • each bulk drug substance of dengue serotype 1 is immediately frozen on the same day.
  • each of the frozen bulk drug substances of dengue serotype 1 are thawed, pooled, and then subjected to a step of depth filtration.
  • six bulk drug substances of dengue serotype 1 are thawed, pooled, and then subjected to a step of depth filtration.
  • each bulk drug substance of dengue serotype 2 is immediately frozen on the same day.
  • each of the frozen bulk drug substances of dengue serotype 2 are thawed, pooled, and then subjected to a step of depth filtration.
  • six bulk drug substances of dengue serotype 2 are thawed, pooled, and then subjected to a step of depth filtration.
  • each bulk drug substance of dengue serotype 3 is immediately subjected to a step of depth filtration and then frozen on the same day.
  • six bulk drug substances of dengue serotype 3 are subjected to a step of depth filtration individually and then frozen on the same day individually.
  • each bulk drug substance of dengue serotype 4 is immediately subjected to a step of depth filtration and then frozen on the same day.
  • six bulk drug substances of dengue serotype 4 are subjected to a step of depth filtration individually and then frozen on the same day individually.
  • the method of the invention includes the following steps for dengue serotype 1 such as dengue 2/1 chimera or TDV-1 represented by SEQ ID NO: 1 and/or SEQ ID NO:2, (i) providing cells in growth media as explained in the earlier section, (ii) infection of cells with media comprising dengue serotype 1 (optionally at a low MOI as described in the earlier section), (iii) a first harvest obtained from the first harvesting step (iv) processing the first harvest comprising clarification and stabilization of the harvest on the same day (v) clarified and stabilized harvest is purified on the same day comprising (v-a) at least one chromatography step, preferably an anion exchange chromatography and (v-b) purified harvest is also subjected to an ultrafiltration step on the same day, preferably a tangential flow filtration to obtain a first drug substance (vi) the first drug substance is also processed on the same say, preferably mixed on the same day with a flushing buffer to obtain a
  • the method includes step (vii-b) of thawing the six bulk drug substances, and (vii-c) pooling the six bulk drug substances, and (vii-d) subject the pooled six bulk drug substances to a depth filtration step, and (vii-e) freezing the filtered bulk drug substances.
  • the method of the invention includes the following steps for dengue serotype 2 or TDV-2 represented by SEQ ID NO: 3 and/or SEQ ID NO:4, (i) providing cells in growth media as explained in the earlier section, (ii) infection of cells with media comprising dengue serotype 2 (optionally at a low MOI as described in the earlier section), (iii) a first harvest obtained from the first harvesting step (iv) processing the first harvest comprising clarification and stabilization of the harvest on the same day (v) clarified and stabilized harvest is purified on the same day comprising (v-a) at least one chromatography step, preferably an anion exchange chromatography and (v-b) purified harvest is also subjected to an ultrafiltration step on the same day, preferably a tangential flow filtration to obtain a first drug substance (vi) the first drug substance is also processed on the same say, preferably mixed on the same day with a flushing buffer to obtain a composition comprising the buffer flush and the first
  • the method includes step (vii-b) of thawing the six bulk drug substances, and (vii-c) pooling the six bulk drug substances, and (vii-d) subject the pooled six bulk drug substances to a depth filtration step, and (vii-e) freezing the filtered bulk drug substances.
  • the method of the invention includes the following steps for dengue serotype 3 or TDV-3 represented by SEQ ID NO: 5 and/or SEQ ID NO:6, (i) providing cells in growth media as explained in the earlier section, (ii) infection of cells with media comprising dengue serotype 3 at a low MOI as described in the earlier section, (iii) a first harvest obtained from the first harvesting step (iv) processing the first harvest comprising clarification and stabilization of the harvest on the same day (v) clarified and stabilized harvest is purified on the same day comprising (v-a) at least one chromatography step, preferably an anion exchange chromatography and (v-b) purified harvest is also subjected to an ultrafiltration step on the same day, preferably a tangential flow filtration to obtain a first drug substance (vi) the first drug substance is also processed on the same day, preferably mixed on the same day with a flushing buffer to obtain a composition comprising the buffer flush and the first drug substance,
  • the method of the invention includes the following steps for dengue serotype 4 or TDV-4 represented by SEQ ID NO: 7 and/or SEQ ID NO:8, (i) providing cells in growth media as explained in the earlier section, (ii) infection of cells with media comprising dengue serotype 4 at a low MOI as described in the earlier section, (iii) a first harvest obtained from the first harvesting step (iv) processing the first harvest comprising clarification and stabilization of the harvest on the same day (v) clarified and stabilized harvest is purified on the same day comprising (v-a) at least one chromatography step, preferably an anion exchange chromatography and (v-b) purified harvest is also subjected to an ultrafiltration step on the same day, preferably a tangential flow filtration to obtain a first drug substance (vi) the first drug substance is also processed on the same day preferably mixed on the same day with a flushing buffer to obtain a composition comprising the buffer flush and the first drug substance, which is
  • an embodiment of the method for large-scale production and manufacture of flaviviral vaccines includes a step of processing the bulk drug substance comprising a depth filtration step. If a divalent, trivalent, or tetravalent final drug product is intended, the bulk drug substance at this step still comprises each individual monovalent bulk drug substance.
  • a divalent, trivalent, or tetravalent final drug product is intended, the bulk drug substance at this step still comprises each individual monovalent bulk drug substance.
  • a method of large-scale flaviviral vaccine production and manufacture comprises, a harvesting step to obtain a harvest, obtaining a bulk drug substance from the harvest, processing the bulk drug substance comprising a step of filtering the bulk drug substance followed by a step of freezing the bulk drug substance to obtain a frozen bulk drug substance.
  • a method of large-scale flaviviral vaccine production and manufacture comprises, a harvesting step to obtain a harvest, obtaining a bulk drug substance from the harvest, processing the bulk drug substance comprising a step of filtering the bulk drug substance followed by a step of freezing the bulk drug substance to obtain a frozen bulk drug substance.
  • the harvest does not undergo a freezing step until the step of processing the bulk drug substance.
  • a method of large-scale flaviviral vaccine production and manufacture comprises, a harvesting step to obtain a harvest, obtaining a bulk drug substance from the harvest, processing the bulk drug substance comprising a step of filtering the bulk drug substance followed by a step of freezing the bulk drug substance to obtain a frozen bulk drug substance.
  • processing the bulk drug substance does not involve a freezing step before the filtration step.
  • the harvest does not undergo a freezing step until the step of processing the bulk drug substance.
  • a method of large-scale flaviviral vaccine production and manufacture comprises, a harvesting step to obtain a harvest, ultrafiltration such as tangential flow filtration to obtain a drug substance, and at least one flushing step of the ultrafiltration membrane with a flushing buffer to obtain a composition comprising a buffer flush and drug substance, which is the bulk drug substance, processing the bulk drug substance comprising a step of filtering the bulk drug substance followed by a step of freezing the bulk drug substance to obtain a frozen bulk drug substance.
  • a method of large-scale flaviviral vaccine production and manufacture comprises, a harvesting step to obtain a harvest, processing the harvest to obtain a processed harvest, ultrafiltration such as tangential flow filtration to obtain a drug substance, and at least one flushing step of the ultrafiltration membrane with a flushing buffer to obtain a composition comprising a buffer flush and drug substance, which is the bulk drug substance, processing the bulk drug substance comprising a step of filtering the bulk drug substance followed by a step of freezing the bulk drug substance to obtain a frozen bulk drug substance.
  • processing the bulk drug substance does not involve a freezing step before the filtration step.
  • the harvest does not undergo a freezing step until the step of processing the bulk drug substance.
  • a method of large-scale flaviviral vaccine production and manufacture comprises, a harvesting step to obtain a harvest, processing the harvest to obtain a processed harvest, purification comprising at least one chromatography step such as an anion exchange chromatography to obtain a purified harvest, ultrafiltration such as tangential flow filtration to obtain a drug substance, and at least one flushing step of the ultrafiltration membrane with a flushing buffer to obtain a composition comprising a buffer flush and drug substance, which is the bulk drug substance, processing the bulk drug substance comprising a step of filtering the bulk drug substance followed by a step of freezing the bulk drug substance to obtain a frozen bulk drug substance.
  • processing the bulk drug substance does not involve a freezing step before the filtration step.
  • a method of large-scale flaviviral vaccine production and manufacture comprises, a harvesting step to obtain a harvest, processing the harvest comprising clarification and stabilization of the harvest to obtain a clarified and stabilized harvest, purification of the clarified and stabilized harvest comprising at least one chromatography step such as an anion exchange chromatography to obtain a purified harvest, ultrafiltration such as tangential flow filtration on the purified harvest to obtain a drug substance, and at least one flushing step of the ultrafiltration membrane with a flushing buffer to obtain a composition comprising a buffer flush and drug substance, which is the bulk drug substance, processing the bulk drug substance comprising a step of filtering the bulk drug substance followed by a step of freezing the bulk drug substance to obtain a frozen bulk drug substance.
  • processing the bulk drug substance does not involve a freezing step before the filtration step.
  • the harvest does not undergo a freezing step until
  • a method of large-scale flaviviral vaccine production and manufacture comprises, providing cells in growth media, infecting cells with media comprising a flavivirus. a harvesting step to obtain a harvest, obtaining a bulk drug substance from the harvest, processing the bulk drug substance comprising a step of filtering the bulk drug substance followed by a step of freezing the bulk drug substance to obtain a frozen bulk drug substance.
  • the harvest does not undergo a freezing step until the step of processing the bulk drug substance.
  • infecting the cells with media comprising a flavivirus optionally comprises infecting the cells at an MOI of 0.1 or less.
  • a method of large-scale flaviviral vaccine production and manufacture comprises, providing cells in growth media, infecting cells with media comprising a flavivirus, a harvesting step to obtain a harvest, obtaining a bulk drug substance from the harvest, processing the bulk drug substance comprising a step of filtering the bulk drug substance followed by a step of freezing the bulk drug substance to obtain a frozen bulk drug substance.
  • processing the bulk drug substance does not involve a freezing step before the filtration step.
  • infecting the cells with media comprising a flavivirus optionally comprises infecting the cells at an MOI of 0.1 or less.
  • a method of large-scale flaviviral vaccine production and manufacture comprises, providing cells in growth media, infecting cells with media comprising a flavivirus. a harvesting step to obtain a harvest, obtaining a bulk drug substance from the harvest, processing the bulk drug substance comprising a step of filtering the bulk drug substance followed by a step of freezing the bulk drug substance to obtain a frozen bulk drug substance.
  • processing the bulk drug substance does not involve a freezing step before the filtration step.
  • the harvest does not undergo a freezing step until the step of processing the bulk drug substance.
  • infecting the cells with media comprising a flavivirus optionally comprises infecting the cells at an MOI of 0.1 or less .
  • a method of large-scale flaviviral vaccine production and manufacture comprises, providing cells in growth media, infecting cells with media comprising a flavivirus. a harvesting step to obtain a harvest, processing the harvest to obtain a processed harvest, ultrafiltration such as tangential flow filtration to obtain a drug substance, and at least one flushing step of the ultrafiltration membrane with a flushing buffer to obtain a composition comprising a buffer flush and drug substance, which is the bulk drug substance, processing the bulk drug substance comprising a step of filtering the bulk drug substance followed by a step of freezing the bulk drug substance to obtain a frozen bulk drug substance.
  • processing the bulk drug substance does not involve a freezing step before the filtration step.
  • the harvest does not undergo a freezing step until the step of processing the bulk drug substance.
  • infecting the cells with media comprising a flavivirus optionally comprises infecting the cells at an MOI of 0.1 or less.
  • a method of large-scale flaviviral vaccine production and manufacture comprises, providing cells in growth media, infecting cells with media comprising a flavivirus. a harvesting step to obtain a harvest, processing the harvest to obtain a processed harvest, purification comprising at least one chromatography step such as an anion exchange chromatography to obtain a purified harvest, ultrafiltration such as tangential flow filtration to obtain a drug substance, and at least one flushing step of the ultrafiltration membrane with a flushing buffer to obtain a composition comprising a buffer flush and drug substance, which is the bulk drug substance, processing the bulk drug substance comprising a step of filtering the bulk drug substance followed by a step of freezing the bulk drug substance to obtain a frozen bulk drug substance.
  • processing the bulk drug substance does not involve a freezing step before the filtration step.
  • the harvest does not undergo a freezing step until the step of processing the bulk drug substance.
  • infecting the cells with media comprising a flavivirus optionally comprises infecting the cells at an MOI of 0.1 or less.
  • a method of large-scale flaviviral vaccine production and manufacture comprises, providing cells in growth media, infecting cells with media comprising a flavivirus. a harvesting step to obtain a harvest, processing the harvest comprising clarification and stabilization of the harvest to obtain a clarified and stabilized harvest, purification of the clarified and stabilized harvest comprising at least one chromatography step such as an anion exchange chromatography to obtain a purified harvest, ultrafiltration such as tangential flow filtration on the purified harvest to obtain a drug substance, and at least one flushing step of the ultrafiltration membrane with a flushing buffer to obtain a composition comprising a buffer flush and drug substance, which is the bulk drug substance, processing the bulk drug substance comprising a step of filtering the bulk drug substance followed by a step of freezing the bulk drug substance to obtain a frozen bulk drug substance.
  • chromatography step such as an anion exchange chromatography
  • ultrafiltration such as tangential flow filtration on the purified harvest to obtain a drug substance
  • processing the bulk drug substance does not involve a freezing step before the filtration step.
  • the harvest does not undergo a freezing step until the step of processing the bulk drug substance.
  • infecting the cells with media comprising a flavivirus optionally comprises infecting the cells at an MOI of 0.1 or less.
  • the cells are vero cells.
  • a method of large-scale flaviviral vaccine production and manufacture as described above in this section provides high viral titers.
  • a method of large-scale flaviviral vaccine production and manufacture as described above in this section provides higher viral titers as compared to a method differing only in that processing the bulk drug substance comprises freezing the bulk drug substance before filtering the bulk drug substance.
  • the step of filtering comprises agitating the bulk drug substance.
  • Agitating herein means for example rocking the bulk drug substance during filtering.
  • the weight of the bulk drug substance is at least 100g such as 100 to 1000g such as 2000g or 3000g.
  • the flavivirus is a live, attenuated dengue virus selected from a group consisting of dengue serotype 1 (DENV-1) such as a dengue 2/1 chimera, dengue serotype 2 (DENV-2), dengue serotype 3 (DENV-3) such as a dengue 2/3 chimera and dengue serotype 4 (DENV-4) such as a dengue 2/4 chimera.
  • DEV-1 dengue serotype 1
  • DEV-2 dengue serotype 2
  • DEV-3 dengue serotype 3
  • DEV-4 dengue serotype 4
  • each of the at least two harvests do not undergo a freezing step until the step of processing the bulk drug substance.
  • a method of large-scale flaviviral vaccine production and manufacture comprises, at least two harvesting steps to obtain at least two harvests, obtaining at least two bulk drug substances from the at least two harvests, processing the at least two bulk drug substances comprising a step of filtering each of the at least two bulk drug substances followed by a step of freezing each of the at least two bulk drug substances to obtain at least two frozen bulk drug substances.
  • processing the bulk drug substance does not involve a freezing step before the filtration step.
  • a method of large-scale flaviviral vaccine production and manufacture comprises, at least two harvesting steps to obtain at least two harvests, and performing the following steps for each of the at least two harvests o ultrafiltration such as tangential flow filtration to obtain a drug substance, and o at least one flushing step of the ultrafiltration membrane with a flushing buffer to obtain a composition comprising a buffer flush and drug substance, which is the bulk drug substance, to obtain at least two drug substances and at least two bulk drug substances, processing the at least two bulk drug substances comprising a step of filtering each of the at least two bulk drug substances followed by a step of freezing each of the at least two bulk drug substances to obtain at least two frozen bulk drug substances,
  • processing the bulk drug substance does not involve a freezing step before the filtration step.
  • each of the at least two harvests do not undergo a freezing step until the step of processing the bulk drug substance.
  • a method of large-scale flaviviral vaccine production and manufacture comprises, at least two harvesting steps to obtain at least two harvests, and performing the following steps for each of the at least two harvests o processing the harvest to obtain a processed harvest, o purification comprising at least one chromatography step such as an anion exchange chromatography to obtain a purified harvest, o ultrafiltration such as tangential flow filtration to obtain a drug substance, and o at least one flushing step of the ultrafiltration membrane with a flushing buffer to obtain a composition comprising a buffer flush and drug substance, which is the bulk drug substance, to obtain at least two processed harvests, at least two purified harvests, at least two drug substances and at least two bulk drug substances, processing the at least two bulk drug substances comprising a step of filtering each of the at least two bulk drug substances followed by a step of freezing each of the at least two bulk drug substances to obtain at least two frozen bulk drug substances.
  • processing the bulk drug substance does not involve a freezing step
  • a method of large-scale flaviviral vaccine production and manufacture comprises, at least two harvesting steps to obtain at least two harvests, and performing the following steps for each of the at least two harvests o processing the harvest comprising clarification and stabilization of the harvest to obtain a clarified and stabilized harvest, o purification of the clarified and stabilized harvest comprising at least one chromatography step such as an anion exchange chromatography to obtain a purified harvest, o ultrafiltration such as tangential flow filtration to obtain a drug substance, and o at least one flushing step of the ultrafiltration membrane with a flushing buffer to obtain a composition comprising a buffer flush and drug substance, which is the bulk drug substance, to obtain at least two clarified and stabilized harvests, at least two purified harvests, at least two drug substances and at least two bulk drug substances, processing the at least two bulk drug substances comprising a step of filtering each of the at least two bulk drug substances followed by a step of freezing each of the at least two bulk drug substances to
  • processing the bulk drug substance does not involve a freezing step before the filtration step.
  • each of the at least two harvests do not undergo a freezing step until the step of processing the bulk drug substance.
  • infecting the cells with media comprising a flavivirus optionally comprises infecting the cells at an MOI of 0.1 or less.
  • the step of filtering comprises agitating the bulk drug substance for at least 1 minute such as 5 minutes, for example from 5 to 15 minutes. Agitating herein means for example rocking the bulk drug substance during filtering.
  • a method of large-scale flaviviral vaccine production and manufacture comprises the following sequential steps:
  • step (vi) processing the drug substance obtained in step (v) comprising at least one flushing step of the ultrafiltration membrane with a flushing buffer to obtain a bulk drug substance, wherein a second harvesting step is conducted, and the above steps are repeated to obtain a second bulk drug substance,
  • steps (iii) to (vii) are conducted on the same day, wherein processing the bulk drug substances comprises a step of filtering the bulk drug substance before the step of freezing the bulk drug substance.
  • step (iv) processing the first harvest obtained in step (iii) comprising clarification and stabilization of the harvest to obtain a first clarified and stabilized harvest
  • At least one flushing step of the ultrafiltration membrane with a flushing buffer to obtain a composition comprising a buffer flush and drug substance, which is the first bulk drug substance, preferably up to ten harvesting steps are conducted, and the above steps are repeated to obtain up to ten bulk drug substances, wherein the interval between each harvesting step is 20-30 hours,
  • step (vii-b) freezing the filtered bulk drug substance obtained from step (vii-a) to obtain a frozen bulk drug substance, wherein all of steps (iii) to (vi) are performed on the same day, preferably wherein steps (vii-a) and (vii-b) are conducted on the same day as step (vi).
  • the frozen bulk drug substances are thawed and pooled.
  • the low MOI is less than 0.005 or preferably 0.008 or less.
  • the method provides for high viral titres.
  • large-scale comprises production cultures of surface area 35,000 cm 2 or more, 50,000 cm 2 or more, 100,000 cm 2 or more.
  • the flavivirus is a live, attenuated dengue virus selected from a group consisting of dengue serotype 1 (DENV-1) such as a dengue 2/1 chimera, dengue serotype 2 (DENV-2), dengue serotype 3 (DENV-3) such as a dengue 2/3 chimera and dengue serotype 4 (DENV-4) such as a dengue 2/4 chimera.
  • DEV-1 dengue serotype 1
  • DEV-2 dengue serotype 2
  • DEV-3 dengue serotype 3
  • DEV-4 dengue serotype 4
  • the flavivirus is a live, attenuated dengue virus selected from a group consisting of TDV-1 represented by SEQ ID NO: 1 and/or 2, TDV-2 represented by SEQ ID NO: 3 and/or 4, TDV-3 represented by SEQ ID NO: 5 and/or 6, and TDV-4 represented by SEQ ID NO: 7 and/or 8.
  • the MOI is from 0.0001 to 0.008, from 0.0001 to 0.005, from 0.001 to 0.005.
  • Processing the bulk drug substance comprises a filtration step according to the above embodiments.
  • this filtration step is a depth filtration step.
  • this filtration step can be carried out in the same manner as the filtration step in the step of clarification of the harvest.
  • Depth filtration uses a porous filtration medium to separate particles and solids from a liquid.
  • the filter media which may be used in depth filtration can be cellulose acetate, polypropylene, cellulose acetate protected by glass fiber fleece and polyethersulfone.
  • the filter used in depth filtration can have a pore size of 0.1 pm to 1 pm, 0.2 pm to 0.8 pm, or 0.2 pm to 0.45 pm.
  • the filtration step comprises using a heterogenous double layer filter. In some embodiments, the filtration step comprises using a heterogenous double layer filter of 0.1 pm to 1 pm such as 0.45 pm and 0.1 pm to 1 pm such as 0.2 pm. In some embodiments, the filtration step comprises using a heterogenous double layer filter of 0.45 pm and 0.2 m. In some embodiments, the filtration step comprises using a heterogenous double layer of polyethersulfone. In some embodiments, the filtration step comprises using a heterogenous double layer of polyethersulfone with pore sizes of 0.2 pm and 0.45 pm.
  • heterogenous double layer filters can be used such as Sartoclean (Cellulose Acetate) 0.8 +0.65 pm, Sartopure (Polypropylene) 0.65 pm, Sartoclean (Glassfiber Fleeces) 0.8 + 0.65 pm, Sartopore 2 (Polyethersulfone) 0.8 + 0.45 pm, Sartopore 2 (Polyethersulfone) 0.45 + 0.2 pm, or Sartopore 2 XLG (Polyethersulfone) 0.8 + 0.2 pm.
  • Sartoclean Cellulose Acetate
  • Sartopure Polypropylene
  • Sartoclean Glassfiber Fleeces
  • Sartopore 2 Polyethersulfone
  • Sartopore 2 Polyethersulfone
  • XLG Polyethersulfone
  • the bulk drug substance after this step comprises the same excipients as present in the bulk drug substance before the processing step.
  • the method of the invention also provides means and ways to freeze and store the bulk drug substance.
  • the bulk drug substance can be frozen and stored at any time.
  • One such time point is after the filtration of the BDS.
  • the bulk drug substance can be frozen and stored.
  • the objective of the freezing step is to ensure the viral potency in order for the material to be used for drug product formulation.
  • any of the commercially available bags can be used to freeze and store the bulk drug substance.
  • the bulk drug substance is frozen and stored after the filtration of the BDS as discussed in the previous section, e.g., for TDV-3 and TDV-4.
  • the bulk drug substance can be immediately frozen and stored after obtaining the bulk drug substance from each harvesting step, thereafter, the bulk drug substances from each harvesting step are thawed, pooled and filtered as discussed above and then frozen and stored e.g., for TDV-1 and TDV-2.
  • the bags are used to freeze the bulk drug substance and store them until they are thawed for use in the formulation of the drug product.
  • the bags can be selected based on certain parameters such as size, manufacturer, material of construction, and storage temperature range, thaw conditions, ability to maintain sterile conditions, ease of transportation and use in previous processes and clinical studies.
  • the supported temperature range for the bags can e.g. be within the range -84 °C to
  • the freezing options supported by the bags used in the method of the invention can be ultra-low freezer, blast freezer, plate freezing and thawing.
  • Ultra-low freezer or “ULTs” as used herein refers to a freezer in operating within the -50°C to -80°C range, ULTs are used to store a variety of analytes and products, from biospecimen samples to enzymes and drugs.
  • Blast freezer as used herein refers to a specialized freezer that allows a product to be frozen rapidly over a short period of time. The temperature of a blast freezer can vary from -10 °C to -120 °C.
  • Platinum freezing as used herein is a fast-freezing method, once the product is in direct contact with two cold metallic surfaces.
  • the thawing options supported by the bags used in certain embodiments of the method of the invention can be water bath, plasma thawing bath, shaker incubator, plate freezethawing.
  • the term "water bath” refers to a bath containing a heating medium having at least 50% of water by volume. The maximum heating temperature of the water bath can be controlled by selecting the composition of the heating medium.
  • “Plasma thawing” as used herein refers to a method of thawing using an equipment designed for rapid and uniform thawing of fresh frozen plasma (FFP) bags at 37°C.
  • the device is made of a stainless- steel chamber and equipped with digital temperature controller and a water circulating pump.
  • the considerations for handling and transportation of the bags used in the present invention can be rigidity of the bottles and hard casting that minimizes damage, EVOH gas barrier with EVA film layer, ULDPE film.
  • the material compatibility and sterility characteristics of the bags include free of animal components, gamma irradiated, and USP standards.
  • EVOH barrier refers to an environmentally friendly substance that is used as a barrier plastic. Unlike PVDC, EVOH does not contain chlorine, dioxins, metals, or other elements that may cause endocrinological disorders.
  • GUILPED as used herein refers to ultra-low-density polyethylene.
  • suitable bags are commercially available bags that can be used to freeze the bulk drug substance including Nalgene PETG/PFA bottles (50 mL-2 L), Sartorius-Stedim Celsius-Pak/ FFT/FFTp, 2D bags (30 mL-16 L) or Pall Allegro 2D Biocontainers (125 mL-50 L).
  • the large-scale production and manufacture of flaviviral vaccines comprises processing the bulk drug substance such as freezing and storing the bulk drug substance. If a divalent, trivalent, or tetravalent final drug product is intended, the bulk drug substance at this step still comprises each individual monovalent bulk drug substance.
  • the bulk drug substance such as freezing and storing the bulk drug substance.
  • the bulk drug substance at this step still comprises each individual monovalent bulk drug substance.
  • the next step is (ix) formulation of the bulk drug product.
  • step (ix-b) Mixing the thawed bulk drug substance of step (ix-a) and a formulation buffer to obtain a bulk drug product.
  • the flaviviral vaccine is a tetravalent dengue vaccine including all four live, attenuated dengue serotypes including dengue serotype 1 such as dengue 2/1 chimera or TDV-1 represented by SEQ ID NO: 1 and/or SEQ ID NO:2 , dengue serotype 2 or TDV-2 represented by SEQ ID NO: 3 and/or SEQ ID NO:4, dengue serotype 3 such as dengue serotype 2/3 or TDV-3 represented by SEQ ID NO: 5 and/or SEQ ID NO:6, dengue serotype 4 such as dengue serotype 2/4 or TDV-4 represented by SEQ ID NO: 7 and/or SEQ ID NO:8.
  • dengue serotype 1 such as dengue 2/1 chimera or TDV-1 represented by SEQ ID NO: 1 and/or SEQ ID NO:2
  • dengue serotype 2 or TDV-2 represented by SEQ ID NO: 3 and/or SEQ ID NO:4
  • dengue serotype 3 such as dengue serotype 2
  • the formulation buffer comprises two buffers, the first excipient buffer (FEB) and a second excipient buffer (SEB).
  • FEB first excipient buffer
  • SEB second excipient buffer
  • the components of each of FEB and SEB may be the same or different. However, quantitatively, the components of each of FEB and SEB are different.
  • excipient refers to a substance added to a liquid pharmaceutical composition in addition to the biological active agent. This can include substances used for the purpose of enhancing stabilization of the active agent, salts, carbohydrates (such as e.g., sugars), surfactants, proteins, bulking agents, fillers, or agents that in combination with the active agent can confer a therapeutic enhancement of the composition.
  • excipients may refer to salts, carbohydrates, non-ionic surfactants and albumins.
  • Excipients may also refer to buffers such as e.g., phosphate buffer.
  • the FEB and SEB buffers can e.g.
  • the FEB and SEB buffer can also comprise water for injection (WFI) as needed.
  • the salts used in the buffer can be anhydrous or hydrates.
  • the salts can be either one, two, three, or four sodium salts, or one, two, three, or four potassium salts, or one, two, three, or four magnesium salts.
  • the sodium salt can be sodium fluoride, or sodium chloride, or sodium bromide, or sodium iodide, or sodium sulphate, or disodium salts such as disodium phosphate, or sodium dihydrogen phosphate or disodium hydrogen phosphate or disodium hydrogen phosphate, dihydrate, or sodium bicarbonate or sodium carbonate.
  • the potassium salt can be potassium fluoride, or potassium chloride, or potassium iodide, or potassium sulphate, or potassium dihydrogen phosphate or potassium dihydrogen sulphate or potassium bicarbonate or potassium carbonate.
  • the magnesium salt can be magnesium fluoride, or magnesium chloride, or magnesium iodide, or magnesium sulphate, or magnesium dihydrogen phosphate or magnesium bicarbonate or magnesium carbonate.
  • Each of the above salts can e.g., be used within the range of 0.9 mM to 130 mM in the SEB buffer.
  • the SEB buffer can comprise sodium chloride in the range 100 to 130 mM, 1 10 to 130 mM, 120 to 130 mM and other three salts within the range of 0.9 to 6 mM.
  • the SEB buffer can comprise potassium chloride in the range 100 to 130 mM, 1 10 to 130 mM, 120 to 130 mM and other three salts within the range of 0.9 to 6 mM.
  • the SEB and SEB buffer can comprise magnesium chloride in the range 100 to 130 mM, 1 10 to 130 mM, 120 to 130 mM and other three salts within the range of 0.9 to 6 mM.
  • the SEB buffer can comprise sodium fluoride in the range 100 to 130 mM, 1 10 to 130 mM, 120 to 130 mM and other three salts within the range of 0.9 to 6 mM.
  • the SEB buffer can comprise potassium fluoride in the range 100 to 130 mM, 1 10 to 130 mM, 120 to 130 mM and other three salts within the range of 0.9 to 6 mM.
  • the SEB buffer can comprise sodium carbonate in the range 100 to 130 mM, 1 10 to 130 mM, 120 to 130 mM and other three salts within the range of 0.9 to 6 mM.
  • the SEB buffer can comprise potassium carbonate in the range 100 to 130 mM, 1 10 to 130 mM, 120 to 130 mM and other three salts within the range of 0.9 to 6 mM.
  • the SEB buffer can comprise sodium chloride in the range 100 to 130 mM, 1 10 to 130 mM, 120 to 130 mM, and potassium chloride in the range 1 mM to 2 mM, 1 mM to 1.5 mM and two other salts within the range of 0.9 to 6 mM.
  • the SEB buffer can comprise sodium chloride in the range 100 to 130 mM, 1 10 to 130 mM, 120 to 130 mM, and magnesium chloride in the range 1 mM to 2 mM, 1 mM to 1 .5 mM and two other salts within the range of 0.9 to 6 mM.
  • the SEB buffer can comprise potassium chloride in the range 100 to 130 mM, 110 to 130 mM, 120 to 130 mM, and magnesium chloride in the range 1 mM to 2 mM, 1 mM to 1 .5 mM and two other salts within the range of 0.9 to 6 mM.
  • the SEB buffer can comprise sodium fluoride in the range 100 to 130 mM, 1 10 to 130 mM, 120 to 130 mM, and potassium chloride in the range 1 mM to 2 mM, 1 mM to 1 .5 mM and two other salts within the range of 0.9 to 6 mM.
  • the SEB buffer can comprise sodium fluoride in the range 100 to 130 mM, 110 to 130 mM, 120 to 130 mM, and potassium carbonate in the range 1 mM to 2 mM, 1 mM to 1 .5 mM and two other salts within the range of 0.9 to 6 mM.
  • the SEB buffer can comprise sodium fluoride in the range 100 to 130 mM, 1 10 to 130 mM, 120 to 130 mM, and magnesium chloride in the range 1 mM to 2 mM, 1 mM to 1.5 mM and two other salts within the range of 0.9 to 6 mM.
  • the SEB buffer can comprise sodium iodide in the range 100 to 130 mM, 110 to 130 mM, 120 to 130 mM, and potassium carbonate in the range 1 mM to 2 mM, 1 mM to 1 .5 mM and two other salts within the range of 0.9 to 6 mM.
  • the SEB buffer can comprise sodium chloride in the range 100 to 130 mM, 1 10 to 130 mM, 120 to 130 mM, and potassium chloride in the range 1 mM to 2 mM, 1 mM to 1.5 mM, potassium dihydrogen phosphate within the range 0.9 to 1.2 mM, 0.9 to 1.0 mM and one other salt within the range of 4 to 6 mM, 5 to 6 mM.
  • the SEB buffer can comprise sodium chloride in the range 100 to 130 mM, 1 10 to 130 mM, 120 to 130 mM, and potassium phosphate in the range 1 mM to 2 mM, 1 mM to 1 .5 mM, potassium dihydrogen sulphate within the range 0.9 to 1 .2 mM, 0.9 to 1 .0 mM and one other salt within the range of 4 to 6 mM, 5 to 6 mM.
  • the SEB buffer can comprise sodium chloride in the range 100 to 130 mM, 1 10 to 130 mM, 120 to 130 mM, and potassium phosphate in the range 1 mM to 2 mM, 1 mM to 1 .5 mM, sodium dihydrogen sulphate within the range 0.9 to 1 .2 mM, 0.9 to 1.0 mM and one other salt within the range of 4 to 6 mM, 5 to 6 mM.
  • the SEB buffer can comprise potassium chloride in the range 100 to 130 mM, 1 10 to 130 mM, 120 to 130 mM, and magnesium chloride in the range 1 mM to 2 mM, 1 mM to 1.5 mM, sodium dihydrogen phosphate within the range 0.9 to 1.2 mM, 0.9 to 1.0 mM and magnesium carbonate within the range of 4 to 6 mM, 5 to 6 mM.
  • the FEB buffer can comprise sodium chloride in the range 20 to 50 mM, 30 to 50 mM, 40 to 50 mM and other three salts within the range of 3 to 25 mM.
  • the FEB buffer can comprise potassium chloride in the range 20 to 50 mM, 30 to 50 mM, 40 to 50 mM and other three salts within the range of 3 to 25 mM.
  • the FEB buffer can comprise magnesium chloride in the range 20 to 50 mM, 30 to 50 mM, 40 to 50 mM and other three salts within the range of 3 to 25 mM.
  • the FEB buffer can comprise sodium fluoride in the range 20 to 50 mM, 30 to 50 mM, 40 to 50 mM and other three salts within the range of 3 to 25 mM.
  • the FEB buffer can comprise potassium fluoride in the range 20 to 50 mM, 30 to 50 mM, 40 to 50 mM and other three salts within the range of 3 to 25 mM.
  • the FEB buffer can comprise sodium carbonate in the range 20 to 50 mM, 30 to 50 mM, 40 to 50 mM and other three salts within the range of 3 to 25 mM.
  • the FEB buffer can comprise potassium carbonate in the range 20 to 50 mM, 30 to 50 mM, 40 to 50 mM and other three salts within the range of 3 to 25 mM.
  • the FEB buffer can comprise sodium chloride in the range range 20 to 50 mM, 30 to 50 mM, 40 to 50 mM, and potassium chloride in the range 4 to 6 mM, 5 to 6 mM and two other salts within the range of 3 to 25 mM.
  • the FEB buffer can comprise sodium chloride in the range range 20 to 50 mM, 30 to 50 mM, 40 to 50 mM, and magnesium chloride in the range 4 to 6 mM, 5 to 6 mM and two other salts within the range of 3 to 25 mM.
  • the FEB buffer can comprise sodium fluoride in the range 20 to 50 mM, 30 to 50 mM, 40 to 50 mM, and magnesium chloride in the range 4 to 6 mM, 5 to 6 mM and two other salts within the range of 3 to 25 mM.
  • the FEB buffer can comprise sodium iodide in the range 20 to 50 mM, 30 to 50 mM, 40 to 50 mM, and potassium carbonate in the range 4 to 6 mM, 5 to 6 mM and two other salts within the range of 3 to 25 mM.
  • the FEB buffer can comprise sodium chloride in the range range range 20 to 50 mM, 30 to 50 mM, 40 to 50 mM, and potassium phosphate in the range 4 to 6 mM, 5 to 6 mM, potassium dihydrogen sulphate within the range 3 to 4 mM, 3.2 to 4 mM and one other salt within the range of 18 to 22 mM.
  • the FEB buffer can comprise potassium chloride in the range 20 to 50 mM, 30 to 50 mM, 40 to 50 mM, and magnesium chloride in the range 4 to 6 mM, 5 to 6 mM, sodium dihydrogen phosphate within the range 3 to 4 mM, 3.2 to 4 mM and disodium hydrogen phosphate dihydrate within the range of 18 to 22 mM, 19 to 22 mM, 20 to 22 mM.
  • the FEB buffer can comprise potassium chloride in the range 20 to 50 mM, 30 to 50 mM, 40 to 50 mM, and magnesium chloride in the range 4 to 6 mM, 5 to 6 mM, sodium dihydrogen phosphate within the range 3 to 4 mM, 3.2 to 4 mM and magnesium carbonate within the range of 18 to 22 mM, 19 to 22 mM, 20 to 22 mM.
  • the sugar in the FEB and SEB buffer can be monosaccharides, (e.g. glucose, galactose, ribose, mannose, rhamnose, talose, xylose, or allose arabinose.), disaccharides (e.g. trehalose, sucrose, maltose, isomaltose, cellibiose, gentiobiose, laminaribose, xylobiose, mannobiose, lactose, or fructose), trisaccharides (e.g.
  • monosaccharides e.g. glucose, galactose, ribose, mannose, rhamnose, talose, xylose, or allose arabinose.
  • disaccharides e.g. trehalose, sucrose, maltose, isomaltose, cellibiose, gentiobiose, laminaribose
  • sugar polymers e.g. dextran, xanthan, pullulan, cyclodextrins, amylose, amylopectin, starch, cello-oligosaccharides, cellulose, maltooligosaccharides, glycogen, chitosan, or chitin).
  • the sugar in the FEB and SEB buffer can be sugar alcohols such as mannitol, sorbitol, arabitol, erythritol, maltitol, xylitol, glycitol, glycol, polyglycitol, polyethylene glycol, polypropylene glycol, and glycerol.
  • the sugar in the FEB and SEB buffer can be a nonreducing sugar.
  • the sugar in the FEB and SEB buffer can be sucrose, trehalose or its hydrates such as trehalose dihydrate.
  • Each of the above sugars can be used within the range of 120 to 160 g/L in the SEB buffer and within the range of 180 to 220 g/L in the FEB buffer.
  • sucrose can be within the range of 120 to 160 g/L, 130 to 160 g/L, 140 to 150 g/L in the SEB buffer and 180 to 220 g/L, 190 to 210 g/L, 200 to 210 g/L in the FEB buffer.
  • trehalose can be within the range of 120 to 160 g/L, 130 to 160 g/L, 140 to 150 g/L in the SEB buffer and 180 to 220 g/L, 190 to 210 g/L, 200 to 210 g/L in the FEB buffer.
  • the non-ionic surfactant in the FEB and SEB buffer can be a high molecular weight non-ionic surfactant.
  • the non-ionic surfactant can be a non-ionic triblock copolymer.
  • the surfactant can be a non-ionic, hydrophilic, polyoxyethylene-polyoxypropylene block copolymer (or EO-PO block copolymer).
  • the EO-PO block copolymers can include blocks of polyethylene oxide (-CH2CH2O-designated EO) and polypropylene oxide (-CH2CHCH3O- designated PO).
  • the PO block can be flanked by two EO blocks in a EOx-POy-Eox arrangement.
  • the PO component is hydrophilic and the EO component is hydrophobic
  • the overall hydrophilicity, molecular weight and the surfactant properties of the copolymer can be adjusted by varying x and y in the EOx-POy-Eox block structure.
  • the EO-PO block copolymers will self-assemble into micelles with a PO core and a corona of hydrophilic EO groups.
  • the non-ionic surfactant in the FEB and SEB buffer can be a poloxamer.
  • Poloxamers are non-ionic triblock copolymers composed of a central hydrophobic chain of poly(propyleneoxide) flanked by two hydrophilic chains of polyethylene oxide). The length of the polymer blocks can be customized, leading to different poloxamers with slightly different properties.
  • the non-ionic surfactant in the FEB and SEB buffer can be Pluronic F127 (poloxamer 407), Pluronic F68 (poloxamer 403), Pluronic P123, Pluronic P85, other polyethylene oxide-polypropylene oxide (EO-PO) block copolymers of greater than 3,000- 4,000 MW or combinations thereof.
  • Pluronic F127 polyxamer 407
  • Pluronic F68 polyoxamer 403
  • Pluronic P123 Pluronic P85
  • EO-PO polyethylene oxide-polypropylene oxide
  • the concentration of the non-ionic surfactant in the FEB and SEB buffer can be the same.
  • the concentration of the non-ionic surfactant in the FEB and SEB buffer each can be with the range 2.0 to 12 g/L, 4 to 12 g/L, 6 to 12 g/L, 8 to 12 g/L, or 10 to 12 g/L.
  • the concentration of a high molecular weight non-ionic surfactant in the FEB and SEB buffer each can be with the range 2.0 to 12 g/L, 4 to 12 g/L, 6 to 12 g/L, 8 to 12 g/L, or 10 to 12 g/L.
  • the concentration of a poloxamer in the FEB and SEB buffer each can be with the range 2.0 to 12 g/L, 4 to 12 g/L, 6 to 12 g/L, 8 to 12 g/L, or 10 to 12 g/L.
  • the concentration of Pluronic F127 (poloxamer 407) in the FEB and SEB buffer each can be with the range 2.0 to 12 g/L, 4 to 12 g/L, 6 to 12 g/L, 8 to 12 g/L, or 10 to 12 g/L.
  • the concentration of a Pluronic P85 in the FEB and SEB buffer each can be with the range 2.0 to 12 g/L, 4 to 12 g/L, 6 to 12 g/L, 8 to 12 g/L, or 10 to 12 g/L.
  • the protein in the FEB and SEB buffer can be any protein which is essentially inert and does not react with the virus.
  • the protein does not affect the structure or infectivity of the virus.
  • the protein can be a structural protein or a serum protein.
  • the protein can be selected from an albumin, human serum albumin, collagen, hydrolyzed collagen, gelatin and hydrolyzed gelatin.
  • the concentration of the protein in the FEB and SEB buffer can be the same.
  • the concentration of protein in the FEB and SEB buffer each can be within the range 0.5 to 1.5 g/L, 0.7 to 1.2 g/L, 0.8 to 1 .0 g/L.
  • the concentration of protein in the FEB and SEB buffer each can be within the range 0.5 to 1.5 g/L, 0.7 to 1.2 g/L, 0.8 to 1.0 g/L.
  • the concentration of albumin in the FEB and SEB buffer each can be within the range 0.5 to 1.5 g/L, 0.7 to 1 .2 g/L, 0.8 to 1.0 g/L.
  • the concentration of human serum albumin in the FEB and SEB buffer each can be within the range 0.5 to 1.5 g/L, 0.7 to 1.2 g/L, 0.8 to 1.0 g/L.
  • the concentration of collagen in the FEB and SEB buffer each can be within the range 0.5 to 1.5 g/L, 0.7 to 1 .2 g/L, 0.8 to 1.0 g/L.
  • the concentration of gelatin in the FEB and SEB buffer each can be within the range 0.5 to 1 .5 g/L, 0.7 to 1.2 g/L, 0.8 to 1 .0 g/L.
  • one embodiment of the large-scale production and manufacture of flaviviral vaccines comprises formulation of a bulk drug product. If a divalent, trivalent, or tetravalent final drug product is intended, the drug product obtained at this stage comprises a divalent, trivalent or tetravalent bulk drug product.
  • the individual method steps and/or components of ingredients described herein above are contemplated alone or in combination with each other. Thus, each possibility described herein above alone and in combination with each other represents a separate embodiment of the invention.
  • the formulated and mixed bulk drug product material can be filtered through a sterile filtration assembly.
  • the pore size of the filter can be up to a maximum of 0.2 pm.
  • the specific types and characteristics of the filters that can be used have been discussed previously under clarification of the harvest and can also be used in this step.
  • sterile filtration of the bulk drug product can be conducted via a peristaltic pump characterized by up to 260 rpm and a maximum inlet pressure of 1 .0 to 1 .2 bar.
  • the filtered drug product can be collected in a separate vessel that is held at a temperature within the range 2°C to 8°C.
  • the sterile filtered bulk drug product can be mixed at approximately 100 to 150 rpm for at least 10 minutes and then further sampled for sterility.
  • the temperature of the isolator filling line is maintained at 10°C to 25°C, more preferably between 15°C to 25°C.
  • the method of the invention utilizes vials that can be used to fill the filtered drug product.
  • the vials can be either plastic or glass vials.
  • the vials are glass vials.
  • any of the USP Type I, II or III glass vials can be used.
  • USP Type I glass vials are used.
  • Glass refers to any suitable glass.
  • neutral glass is a borosilicate glass containing significant amounts of boric oxide, aluminum oxide, alkali and/or alkaline earth oxides. It has a high hydrolytic resistance and a high thermal shock resistance.
  • Soda-lime-silica glass is a silica glass containing alkali metal oxides, mainly sodium oxide and alkaline earth oxides, mainly calcium oxide. It has only a moderate hydrolytic resistance.
  • glass containers are classified as:
  • Type II glass containers which are usually of soda-lime- silica glass with high hydrolytic resistance resulting from suitable treatment of the surface. They are suitable for most acidic and neutral, aqueous preparations whether or not for parenteral use,
  • Type III glass containers which are usually of soda- lime-silica glass with only moderate hydrolytic resistance. They are generally suitable for non-aqueous preparations for parenteral use, for powders for parenteral use (except for freeze-dried preparations) and for preparations not for parenteral use.
  • the vials used in the present invention are made of borosilicate glass.
  • the vials used in the present invention have high hydrolytic resistance and a high thermal shock resistance.
  • the vials used are USP Type I glass vials.
  • the USP Type I glass vials which can be used in the present invention can be either 2R or 4R tubular glass vials with a capacity of 2 mL or 4 mL, respectively.
  • a USP Type I 2R vial or a USP Type 1 2R vial can be used to fill the filtered bulk drug product.
  • a number of steps can be carried including but not limited to one or more of washing of the vials, sterilization of the vials, or de-pyrogenation of the vials.
  • the method can use pre-sterilized glass supplied directly to the filling station as a cost- and space-saving alternative to operating a washing and sterilizing line.
  • Pre-sterilized glass vials are washed and depyrogenated by the supplier, double bagged and then gamma irradiated to sterilize before being shipped to the end user for use.
  • the vials Once the vials have been processed, they are reading for filling. At this time, the sterile filtered bulk is aseptically connected to the single use filling manifold. Prior to filling, the bulk can be mixed at approximately 100 to 150 rpm, 1 10 to 120 rpm, 130 to 140 rpm to 150 rpm for a minimum of 20 to 30 minutes, 15 to 20 minutes, or 15 minutes. In a preferred embodiment, mixing is carried out at 140 to 150 rpm, for about 15 minutes.
  • the mixing and filling continue in parallel with each other.
  • the mixing continues for 15 minutes until approximately 60 to 80% of the vials are filled, for example, 70 to 80%, 75 to 80% of the vials are filled.
  • the mixing is switched off.
  • the mixing is not continued in parallel with the further filling.
  • at least 60 to 80% of the vials for example, 70 to 80%, 75 to 80% vials are filled in parallel with the mixing of bulk drug product.
  • the filling range is one of the important parameters of this step in certain embodiments of the method of the invention.
  • the critical vial fill volume can be set according to the circumstances and the required use. For example, critical vial fill volume can be set at 0.5 to 0.8 mL, 0.6 to 0.7 mL, 0.65 to 0.66 mL, 0.651 , 0.652, 0.653, 0.654, or 0.655 mL.
  • a skilled person knows how to set a critical vial fill volume by manually setting the pump seeds for the filling needles in the isolator filling line at the initation of the fill run and then maintaining the volume by automatic feedback control.
  • additional checks are incorporated to ensure that the content remains within the critical fill volume. These checks can be for example weight checks.
  • the method of the invention may or may not include weight checks to confirm that the volume is within the critical fill volume. If weight checks are employed, a standard deviation of ⁇ 5 to 7% may be considered as fulfilling the requirements of the critical fill volume.
  • one embodiment of the method of the invention involves stoppering of the vial.
  • the filling machine may partially stopper the vials.
  • the partial stoppering is accomplished before transferring the vial into the freeze-drying units.
  • the stoppers may or may not undergo a step of sterilization before being transferred to the filler.
  • the transfer is carried out aseptically.
  • a step of “proper stoppering” inspection may or may not be implemented.
  • the minimum product yield is confirmed to be at least 90%, 95% or 97% good vials out of total number of filled vials.
  • the total time in solution post formulation is defined as the time that begins with the end of transfer of the last lot per serotype and ends at beginning of lyophilization cycle.
  • the post-TIS may be within the range of 10-30 hours, more preferably 12-29 hours. It is possible that the post-TIS for one serotype is different as compared to the post-TIS for another serotype.
  • the post-TIS for TDV-4 is the longest
  • the post-TIS for TDV 2 is the shortest or the same as TDV-1 and TDV-3
  • the post-TIS for TDV-1 and TDV-3 is the same.
  • the post-TIS for TDV-4 is within the range of 15-30 hours, 16- 30 hours, or 16-29 hours
  • the post-TIS for TDV 2 is within the rage 10-24 hours, or 12-24 hours
  • the post-TIS for TDV-1 and TDV-3 is 1 1-25 hours, or 12-25 hours.
  • the large-scale production and manufacture of flaviviral vaccines may comprise filling and loading the bulk drug product in vials.
  • each of the individual method steps and/or components described herein above are contemplated alone or in combination with each other.
  • each possibility described herein above alone and in combination with each other represents a separate embodiment of the invention.
  • the drug product can be lyophilized, such as in commercial scale freeze-dryers. Lyophilization involves manipulating the temperature and pressure of the solution so that the phase of the solution can move directly from the frozen state to the gaseous state without moving through the liquid phase/state. This is achieved by cooling the solution and lowering the pressure to below the triple point of water (the temperature and pressure at which water can exist in equilibrium in the liquid, solid, and gaseous states - which is 0.01 °C). This allows for the removal of the solvent from the product without subjecting the product to intense heat.
  • Lyophilization involves manipulating the temperature and pressure of the solution so that the phase of the solution can move directly from the frozen state to the gaseous state without moving through the liquid phase/state. This is achieved by cooling the solution and lowering the pressure to below the triple point of water (the temperature and pressure at which water can exist in equilibrium in the liquid, solid, and gaseous states - which is 0.01 °C). This allows for the removal of the solvent from the product without subjecting the product to intense heat.
  • the lyophilization process cycle for freeze-drying liquid is e.g. comprised of five primary phases: equilibration, freezing, primary drying, secondary drying, and stoppering, each controlled by three basic process parameters: temperature, pressure, and time.
  • one embodiment of the method of the invention comprises a freeze-drying method for preparing a dry composition, the method comprising the steps of (i) providing an aqueous composition, (ii) freezing the aqueous composition to form a frozen composition, (iii) subjecting the frozen composition to a primary drying step in an apparatus with a shelf temperature, which ranges from above the Tg of the composition to not more than 15 °C above the Tg and at a pressure below 0.3 mbar to form a primary dried product, and (iv) subjecting the primary dried product to a secondary drying step at a temperature above the temperature of the primary drying step to form a secondary dried product.
  • the primary drying step (iii) is carried out in an apparatus with a shelf temperature of from -33 °C to -10 °C, or from -33 °C to -20 °C, or from -31 °C to -23 °C, or from 29 °C to -25 °C, such as about -27 °C.
  • the primary drying step (c) can be carried out in an apparatus with a shelf temperature above the collapse temperature of the composition (Tc).
  • the primary drying step (iii) is carried out for a period of from 100 hours to 10 hours, or from 60 hours to 30 hours, or from 50 hours to 40 hours, such as around 48 hours.
  • the primary drying step (iii) can be carried out at a pressure of less than 0.1 mbar, or less than 0.05 mbar, or less than 0.025 mbar, and optionally above about 0.01 mbar.
  • the secondary drying step (iv) can be carried out at a temperature of at least 20 °C, or at least 25 °C.
  • the large-scale production and manufacture of flaviviral vaccines may comprise lyophilization of the drug product.
  • each of the individual method steps and/or components described herein above are contemplated alone or in combination with each other.
  • each possibility described herein above alone and in combination with each other represents a separate embodiment of the invention.
  • the vials can be capped.
  • the chamber pressure can be backfilled to -0.125 to -0.075 bar (partial vacuum) for stoppering using dry sterile nitrogen gas (N2).
  • Vials can be stoppered with >6.5 N/cm2 pressure for 60 seconds and the shelf temperature is then decreased to 3°C to 7°C until unloaded into a Grade A environment.
  • the vials can be sealed, such as with aluminium/plastic, e.g. while in the sterile isolator.
  • Every vial is visually inspected either manually or through a semi-automated process for potential defects of product, glass, crimping, stoppering or others including but not limited to cake appearance, cap defects, glass defects, and visible particles.
  • the drug product is frozen for storage.
  • Each drug product batch can e.g, be transferred to an additional storage location at 2°C to 8°C before being frozen, or the product batch can be transferred directly to a dedicated blast freezer, for example with temperature -15°C to -35°C, for blast freezing which can last e.g., at least 36 hours blast freezer.
  • the batch can e.g. remain in the blast freezer for at least 36 hours.
  • drug product batches can be transferred to long-term storage, for example at -15°C to -25°C.
  • Example 1 - Low MOI range shows that high viral titers can be obtained in large scale flaviviral vaccine production and manufacture when cells in monolayer are infected with infection media comprising a flavivirus at a low MOI (less than 0.008, or 0.005 or less) irrespective of the volume of infection media used.
  • Vero WCB is thawed in 37°C water bath. The thawing should not exceed 5 minutes. Post-thawed cells are then transferred into appropriate tissue culture vessels. Vero cells were grown in tissue culture vessels comprising growth media.
  • DGM Dulbecco growth media
  • DMEM Dulbecco's Modified Eagle's Medium
  • FBS fetal bovine serum
  • the growth media comprised DMEM + 4.0 mM glutamine + 3.52 g/L glucose + 10% fetal bovine serum (FBS).
  • Vero cells were cultured at 37°C, 5% CO 2 , with operating ranges of 36°C to 39°C and 4% to 6% respectively.
  • Vero cells were grown to at least 90% confluency on the surface of the culture vessel prior to TDV infection. When the cells are confluent, the cells are dislodged from the surface by TrypLE Select within 10 to 30 minutes. When the desired cell density had reached, infection and virus production was performed using the infection media.
  • the infection media comprises DMEM + 4.0 mM glutamine + 3.88 g/L glucose + 0.1 % (w/v) of F127. The volume of infection media was between 0.014 to 0.114 mL/cm 2 .
  • Vero cell line for the manufacture of viral vaccines is widely accepted by regulatory authorities.
  • the continuous mammalian cell line is well characterized with a proven safety profile as it has been used in several approved vaccines.
  • the Vero cell line was shown to support replication of the attenuated dengue vaccine strains, therefore, was used for the manufacture of the master and working virus seeds (MVS) and (WVS) as well as for production of monovalent drug substances.
  • a recombinant dissociation trypsin enzyme TrypLE Select was used for dissociation of the cells from the surface of culture vessels. When all the cells are dislodged from the surfaces of the culture vessels, DGM is added to neutralize the activity of TrypLE Select.
  • Example 5.2 Infection of Vero cells and Harvest [00467]After the Vero cells have expanded to sufficient numbers in TDV production vessels, the cells are subsequently infected with monovalent dengue WVS.
  • the infection media used was DMEM without phenol red.
  • the final concentrations of glucose, glutamine and F127 in the infection media were 3.88 g/L, 4.0 mM and 0.1 % (w/v), respectively.
  • DMP was prepared by aseptically mixing the sterile liquid components including DMEM, F127 and glutamine, the mixed liquid components was further filtered using Sartopore 2 0.45 + 0.2 pm filter.
  • Vero cells are grown to at least 80% confluency on the surface of the culture vessel prior to TDV infection. Vero cells were required to be minimally 75% confluent to attain a cell density of approximately 1.0 x 10 5 cells/cm 2 . When the desired cell density has been reached, infection and virus production is performed using DMP.
  • Monovalent TDV working virus seed was used for the infection of the Vero cells.
  • Vero cells with at least 85% confluency in vessels were infected in monolayer with WVS at MOI 0.001 and the infection volume per cm 2 was 0.057 mL/cm 2 .
  • the method of infection was static, i.e., no rocking mechanism was used for the vessels during the infection process.
  • the infection and virus production parameters; temperature and CO 2 set points were kept at 38°C and 4%, respectively.
  • the duration of the infection was set at 70 minutes.
  • Each harvesting step is conducted by collecting the supernantant from the chambers. 28 hours before the first harvesting step, a media change was carried out. After each harvesting step, fresh DMP was added to the cells. The daily harvest volume was up to approximately 12.8 L per daily harvest and up to 76.8 L total harvest volume. All daily harvests achieved greater than 7.0 log 10 PFU/mL with I FA assay.
  • the turbidity of harvests ranged between 1 to 9 nephelometric turbidity units (NTUs).
  • NTUs nephelometric turbidity units
  • a decrease of turbidity (below 1 NTU) is observed. This indicates that the cell debris from the harvest are effectively removed in this clarification step.
  • the average filter backpressure ranged between 0.1 to 0.3 bar. The robustness of the clarification step to reduce the turbidity to the low levels, with relatively low operating backpressure, ensured that the clarification process operated consistently and away from the process boundary.
  • the stabilization buffer was a stock solution of 3xFTA to be added to the harvest to yield a final concentration of 0.2* FTA (0.2% F127, 3% trehalose dihydrate, 0.02% HSA in PBS).
  • a small volume of 3xFTA was used (which was calculated based on one-14th of the total volume of clarified harvest) to yield a concentration of 0.2xFTA.
  • the primary objective of this step is removal of the host cell DNA (HC DNA) while ensuring maximal virus recovery.
  • AEX column operating in a flow through mode is used in this step. It was found that the stabilized harvest as described above can be directly used as feed material for the AEX column. No buffer exchange or buffer concentration step was needed. Sartobind Q mini column was used in this step that efficiently removed HC DNA with minimal viral loss.
  • the DBC of Sartobind Q 7 mL membrane ranged from 2.15 to 3.69 mg of DNA per 1 mL of Membrane bed volume (MV).
  • [OO481]AEX step was sufficient to reduce the amount of host cell DNA to values below the specification of ⁇ 50 ng/mL.
  • Example 5.7 Processing the drug substance to obtain a bulk drug substance [00485] Three flushing steps of the TFF membrane were conducted to obtain a bulk drug substance.
  • step 8 comprises filtration of the bulk drug substance, and both the filtration of the bulk drug substance and the final freeze of step 8 occur on the same day, and wherein steps 1 to 7 do not involve a freezing step.
  • a vial of Vero WCB is thawed in 37°C water bath.
  • the thawing duration is controlled within 8 minutes.
  • Post-thawed cells are then transferred into the vessels already containing DMEM comprising 4.0 g/L of glucose + 5% FBS + 4.5 mM glutamine and cultured at 38°C, 4% CO2, respectively. Phenol red was excluded from the media and visual microscopic check was employed. The media used was mixed together and filtered to maintain microbial control prior to use.
  • the vessels used for cell expansion in this example include 4 x CF40 chambers. This corresponds to a surface are of 101 ,760 cm 2 . When the cells are confluent, the cells are dislodged from the surface by TrypLE Select within 20 minutes. During the cell passaging process, allowable cells “dry” duration and maximum duration for cell passaging are defined up to 30 minutes and 5.0 hours respectively.
  • a recombinant dissociation trypsin enzyme TrypLE Select was used for dissociation of the cells from the surface of culture vessels was used as in Example 5.1. However, in this example, prior to adding TrypLE Select, the cells are washed with DPBS to remove residual FBS. When all the cells are dislodged from the surfaces of the culture vessels, DGM is added to neutralize the activity of TrypLE Select.
  • the infection media was DMEM without phenol red.
  • the final concentrations of glucose, glutamine and F68 in the infection media were 3 g/L , 4.5 mM and 0.2% (w/v), respectively.
  • DMP was filtered using Sartopore 2 0.45 + 0.2 pm filter with aseptically mixing the liquid components.
  • Vero cells are grown to at least 90% confluency on the surface of the culture vessel prior to infection.
  • Vero cells were required to be minimally 90% confluent to attain a cell density of approximately 2 x 105 cells/cm 2 .
  • infection and virus production is performed using DMP.
  • Monovalent TDV working virus seed was used for the infection of the Vero cells. Vero cells with at least 90% confluency were infected in monolayer with WVS at MOI 0.0014 and the infection volume per cm 2 was 0.014 mL/cm 2 . Manual rocking of CF10s during the infection process was performed. In this example, a DPBS washing step was conducted prior to infection, as part of a series of dilution steps. The infection and virus production parameters; temperature and CO 2 set points were kept at 37°C and 5%, respectively. The duration of the infection was set at 90 minutes.
  • Harvesting step included collecting the supernatant from the chambers. 12 hours before the first harvesting step, a media change was carried out. After each harvesting step, fresh DMP was added to the cells. The daily harvest volume was up to approximately 12.8 L per daily harvest and up to 38.4 L total harvest volume. All daily harvests achieved greater than 7.0 Iog10 PFU/mL with IFA assay.
  • the stabilization buffer was a stock solution of 3*FTA to be added to the harvest to yield a final concentration of 0.2* FTA (0.2% F123, 3% trehalose dihydrate, 0.02% HSA in PBS). A smaller volume of 3*FTA was used (which was calculated based on one-14th of the total volume of clarified harvest) to yield a concentration of 0.2*FTA.
  • the flushing buffer was 1xFTA. However, to compensate for the trehalose dihydrate passage through the TFF membrane, the buffer flush composition comprised of 27% trehalose dihydrate concentration, to yield a 15% trehalose dihydrate concentration in the monovalent bulk drug substance.
  • Sartopore 2 filter was also adopted for the filtration of the bulk drug substance. This is a hydrophilic polyethersulfone membrane with pore size 0.45 + 0.2 pm.
  • the filter size of 525.0 cm2 was used when concentrated individual harvests are pooled prior to filtration, and 87.6 cm2 when each daily harvest is filtered separately.
  • the closest membrane area sizes available from the filter manufacturer are 0.1 m 2 and 150 cm 2 respectively.
  • the bulk drug substances are frozen and stored. While for TDV-1 and TDV-2, the bulk drug substance of each harvesting day was frozen and thawed on the day of obtaining the last bulk drug substance from the last harvesting step and then all bulk drug substances were pooled and then subjected to a filtration step followed by final freezing and storing.
  • the objective of the freezing step is to ensure the drug substance potency and other quality attributes are maintained in order for the material to be used for drug product formulation.
  • the formulation buffer comprised two buffers, the first excipient buffer (FEB) and a second excipient buffer (SEB). Qualitatively, the components of each of FEB and SEB are the same. However, quantitatively, the components of each of FEB and SEB are different.
  • Freezing rate Rate (°C/time) 0.5°C/min (95-105 min to reach - 45°C ⁇ 2°C)
  • step 6.14 After Unloading and Capping (step 6.14) and Visual Inspection (step 6.15), the final drug product is frozen in step 6.16 and finally in step 6.17 - labelled and packaged.
  • Group A pH was maintained within 7.6 to 8.1 but allowed to fluctuate by 0.5 units
  • Group B pH was maintained within 7.6 to 8.1 but allowed to fluctuate by less than 0.5 units (0.3 units in this group).
  • the yield of the virus can be improved significantly by not only maintaining the pH throughout the process steps at a given pH range (here 7.6 to 8.1) but also by ensuring that the pH is not allowed to fluctuate by more than 0.3 units.
  • Example 8 Straight through process improves viral titers
  • dengue virus was manufactured according to the invention in two different ways as provided in the table below (only the difference is provided in detail, the other steps are conducted according to the previous examples).
  • Table 11 Summary of Process A and B [00539]Tables 12 and below shows the results of titers achieved by Process A and B.
  • Table 12 provides a fair comparison between Process A and B while the titer values for Process B are taken post-thaw, the titer values of Process A are taken before the pooled freezing step (i.e., before the second freezing step in step 9 of Table 11 , above). This means that the titer values represented in Table 12 below are taken after freezing the samples only once so that the only difference between the samples is that while in Process B the samples were filtered before freezing, the samples in Process A were filtered after freezing.
  • Item 1 A method for large-scale flaviviral vaccine production and manufacture comprising the following sequential steps:

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Abstract

La présente invention concerne des procédés de production et de fabrication de vaccin à flavivirus à grande échelle. Les procédés de l'invention sont spécifiquement envisagés pour la production et la fabrication à grande échelle de vaccins à partir de flavivirus vivants atténués tels que des vaccins à partir de virus de la dengue vivants, atténués. En outre, les procédés de l'invention concernent la formulation de produits vaccinaux viraux vivants, atténués, monovalents, divalents, trivalents, ou tétravalents.
EP23829266.8A 2022-11-29 2023-11-29 Production et fabrication de vaccin à flavivirus à grande échelle Pending EP4626574A1 (fr)

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US202263385309P 2022-11-29 2022-11-29
US202263385274P 2022-11-29 2022-11-29
PCT/US2023/061230 WO2023147337A2 (fr) 2022-01-25 2023-01-25 Production et fabrication de vaccin à flavivirus à grande échelle
PCT/US2023/061238 WO2023147342A2 (fr) 2022-01-25 2023-01-25 Production et fabrication de vaccin flaviviral à grande échelle
PCT/US2023/081551 WO2024118740A1 (fr) 2022-11-29 2023-11-29 Production et fabrication de vaccin à flavivirus à grande échelle

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Publication number Priority date Publication date Assignee Title
CA2282790C (fr) 1997-02-28 2011-04-19 St. Louis University Vaccins chimeres a base de flavivirus
CA2941182A1 (fr) 2000-02-16 2001-08-23 The Government Of The United States Of America As Represented By The Secretary, Department Of Health And Human Services Flavivirus chimeriques avirulents et immunogenes
AU2003239932A1 (en) 2002-05-31 2003-12-19 Acambis, Inc. Tetravalent dengue vaccines
EP1924280A4 (fr) 2005-08-10 2008-12-10 Acambis Inc Vaccination contre l'infection par le virus de la dengue
FR2903605A1 (fr) 2006-07-12 2008-01-18 Sanofi Pasteur Sa Methode d'immunisation contre les quatres serotypes de la dengue
FR2906724B1 (fr) 2006-10-04 2009-03-20 Sanofi Pasteur Sa Methode d'immunisation contre les 4 serotypes de la dengue.
FR2909286B1 (fr) 2006-12-01 2012-06-08 Sanofi Pasteur Methode d'immunisation contre les 4 serotypes de la dengue
CN105219734B (zh) * 2008-12-05 2020-03-03 武田疫苗公司 用于诱导病毒生长的组合物、方法和用途
CA3177572C (fr) 2013-03-15 2026-02-03 The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Compositions et procedes pour constructions chimeriques du virus de la dengue dans des vaccins
EP3442571A1 (fr) 2016-04-13 2019-02-20 Takeda Vaccines, Inc. Compositions et procédés de vaccination contre le virus de la dengue chez des enfants et de jeunes adultes
CA3079151A1 (fr) * 2017-10-16 2019-04-25 Serum Institute Of India Private Limited Compositions stables de vaccin comprenant, entre autres, un flavivirus recombinant vivant attenue et procede pour sa preparation
EP4110381A1 (fr) * 2020-02-27 2023-01-04 Takeda Vaccines, Inc. Procédé d'élimination d'adn de cellule hôte à partir d'une préparation virale
WO2023147342A2 (fr) * 2022-01-25 2023-08-03 Takeda Vaccines, Inc. Production et fabrication de vaccin flaviviral à grande échelle

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