WO2025219920A1 - Procédés de production d'un virus enveloppé - Google Patents

Procédés de production d'un virus enveloppé

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Publication number
WO2025219920A1
WO2025219920A1 PCT/IB2025/054027 IB2025054027W WO2025219920A1 WO 2025219920 A1 WO2025219920 A1 WO 2025219920A1 IB 2025054027 W IB2025054027 W IB 2025054027W WO 2025219920 A1 WO2025219920 A1 WO 2025219920A1
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WIPO (PCT)
Prior art keywords
cells
cell
cell culture
vvd
suspension
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PCT/IB2025/054027
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English (en)
Inventor
Maximillian KLIMPEL
Holger Laux
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CSL Behring LLC
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CSL Behring LLC
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Publication of WO2025219920A1 publication Critical patent/WO2025219920A1/fr
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    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • 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
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16051—Methods of production or purification of viral material

Definitions

  • the present disclosure relates generally to the manufacturing of gene therapy products, and specifically to methods of producing an enveloped virus in a suspension perfusion cell culture comprising filtering the cell culture fluid through a filter at a harvest rate of at least 2 vessel volumes per day (VVD) to produce a filtered cell culture fluid comprising the enveloped virus, wherein the filter retains cells from the suspension cell line.
  • VVD vessel volumes per day
  • Retroviruses e.g., lentiviruses (LVs) are one of the most studied viral vectors for gene therapy. Retroviruses in general are RNA-based viruses which integrate their genetic information into the target cell chromosomes permanently. The advantages of retroviruses include long-term transgene expression in target cells, a low immunogenic potential, and the ability to transduce into dividing and non-dividing cells.
  • LVs lentiviruses
  • HIV-1 human immunodeficiency virus 1
  • VSV-G protein from Vesicular stomatitis Indiana virus
  • lentiviruses To produce lentiviruses, cells such as human embryonic kidney cells HEK 293T are transfected with 3-4 plasmids. These include the transfer plasmid with the gene of interest and several packaging plasmids encoding, vesicular stomatitis virus G protein (VSV-G), and essential viral proteins responsible for gene integration or self-assembly. These plasmids can be transiently transfected into the cells, or a producer cell line is created with stable integration of the plasmids with inducible promoters, in which lentivirus production can be induced.
  • VSV-G vesicular stomatitis virus G protein
  • the lentivirus is harvested from the producer cells and subsequently purified and concentrated in the downstream process.
  • the resultant lentivirus can be used to modify patient cells, such as hemopoietic stem cells, for clinical benefit.
  • the inventors sought to produce a method of producing enveloped viruses, e.g., for gene therapy, at commercial scale and suitable for regulatory requirements.
  • the upstream process for producing an enveloped virus in a suspension cell culture comprises culturing cells in the cell culture and harvesting the enveloped virus at the completion of the culture period.
  • the inventors determined that during production enveloped viruses have a low half-life when retained in cell culture at 37 °C. It was also hypothesized that VSV-G degrades when the virus is exposed to 37 °C in chemically defined medium, causing lower infectious titers. To address this problem, the inventors identified that they could perform a perfusion harvest over the culture period to increase the virus recovery due to the shorter average time of the enveloped virus in the bioreactor.
  • the inventors found that by retaining the cells in a filter and recirculating the retained cells back into the cell culture to produce further enveloped virus, the infectious virus recovery was further increased. Unexpectedly, increasing the perfusion harvest rates further increased yields and increased cell specific virus yields.
  • the findings by the inventors provide methods of producing enveloped viruses.
  • the present disclosure provides a method of producing an enveloped virus in a suspension perfusion cell culture, the method comprising:
  • the method further comprises recirculating cell culture fluid through the filter to return the retained suspension cell line cells into the suspension cell culture.
  • the present disclosure further provides a method of producing an enveloped virus in a suspension perfusion cell culture, the method comprising:
  • the harvest rate is greater than about 1 VVD.
  • the harvest rate is between about 1 and 10 VVD.
  • the harvest rate is about 1 VVD, or about 2 VVD, or about 3 VVD, or about 4 VVD, or about 5 VVD, or about 6 VVD, or about 7 VVD, or about 8 VVD, or about 9 VVD, or about 10 VVD.
  • the harvest rate is 2 VVD. In another example, the harvest rate is 3 VVD.
  • the filter is an acoustic standing wave.
  • Acoustic standing waves suitable for use in the present disclosure will be apparent to the skilled person and/or described herein.
  • the method comprises recirculating the retained suspension cell line cells into the suspension cell culture at a recirculation rate greater than the harvest rate.
  • the recirculation rate is about 1.5 times, or about 2 times, or about 2.5 times, or about 3 times the harvest rate. In one example, the recirculation rate is about 1.5 times the harvest rate. In another example, the recirculation rate is 2 times the harvest rate. In a further example, the recirculation rate is 2.5 times the harvest rate. In one example, the recirculation rate is about 3 times the harvest rate. In one example, the recirculation rate is about 4 times the harvest rate. In one example, the recirculation rate is about 5 times the harvest rate.
  • the recirculation rate is about 6 times the harvest rate. In one example, the recirculation rate is about 7 times the harvest rate. In one example, the recirculation rate is about 8 times the harvest rate. In one example, the recirculation rate is about 9 times the harvest rate. In one example, the recirculation rate is about 10 times the harvest rate.
  • the suspension cell line is initially cultured in a cell culture medium that suppresses production of the enveloped virus and allows expansion of the suspension cell line.
  • the disclosure provides a method of producing an enveloped virus in a suspension cell culture, the method comprising culturing a suspension cell line expressing a tetracycline-suppressible gene expression system in a cell culture medium.
  • tetracycline-suppressible gene expression system is also known as a Tet-Off expression system.
  • the suspension cell line is initially cultured in a cell culture medium comprising a sufficient amount of tetracycline or a derivative thereof to suppress production of the enveloped virus and allow expansion of the suspension cell line.
  • the sufficient amount of tetracycline or a derivative thereof in the cell culture medium is at least 0.1 ng/mL of cell culture medium. In one example, the sufficient amount of tetracycline or a derivative thereof in the cell culture medium is between about 0.1 ng/mL and about 10,000 ng/mL of cell culture medium.
  • the sufficient amount of tetracycline or a derivative thereof in the cell culture medium is between about 0.1 ng/mL and about 1,000 ng/mL of cell culture medium. In one example, the sufficient amount of tetracycline or a derivative thereof in the cell culture medium is between about 0.1 ng/mL and about 100 ng/mL of cell culture medium. In one example, the sufficient amount of tetracycline or a derivative thereof in the cell culture medium is between about 0.1 ng/mL and about 10 ng/mL of cell culture medium.
  • the sufficient amount of tetracycline or a derivative thereof in the cell culture medium is about 0.1 ng/mL, or about 0.2 ng/mL, or about 0.3 ng/mL, or about 0.4 ng/mL, or about 0.5 ng/mL, or about 0.6 ng/mL, or about 0.7 ng/mL, or about 0.8 ng/mL, or about 0.9 ng/mL, or about 1 ng/mL of cell culture medium.
  • the method comprises reducing the concentration of tetracycline or a derivative thereof in the cell culture medium such that production of the enveloped virus is induced.
  • the concentration of tetracycline or derivative thereof in the cell culture medium is reduced to a concentration of 0.5 ng/mL or less of cell culture medium. In one example, the concentration of tetracycline or derivative thereof in the cell culture medium is reduced to a concentration of 0.5 ng/mL to 0.001 ng/mL of cell culture medium.
  • the concentration of tetracycline or derivative thereof in the cell culture medium is reduced to a concentration of about 0.5 ng/mL, or about 0.45 ng/mL, or about 0.4 ng/mL, or about 0.35 ng/mL, or about 0.3 ng/mL, or about 0.25 ng/mL, or about 0.2 ng/mL, or about 0.1 ng/mL of cell culture medium.
  • the tetracycline derivative is selected from the group consisting of minocycline, doxycycline, demeclocycline, oxy tetracycline, and tigecy cline. In one example, the tetracycline derivative is doxycycline.
  • the harvest rate is about 1 VVD from day 0 to about day 5 following induction of enveloped virus production.
  • the harvest rate is about 2 VVD from about day 5 following induction of enveloped virus production.
  • the harvest rate is about 3 VVD from about day 6 following induction of enveloped virus production.
  • the recirculation rate is between about 2 VVD and 10 VVD.
  • the recirculation rate is about 2 VVD, or about 4 VVD, or about 6 VVD. In one example, the recirculation rate is 6 VVD.
  • the harvest rate is 3 VVD and the recirculation rate is 6 VVD. In another example, the harvest rate is 2 VVD and the recirculation rate is 4 VVD.
  • the harvest rate is about 1 VVD from day 0 to about day 5, about 2 VVD from about day 5 and about 3VDD from about day 6 to the end of culture;
  • the recirculation rate is about 2 VVD from day 0 to about day 5, about 4 VVD from about day 5 and about 6VDD from about day 6 to the end of culture.
  • the method further comprises performing a suspension cell line cell bleed.
  • the suspension cell line cell bleed is performed at a rate of 0.1 VVD to 0.5 VVD.
  • the suspension cell line cell bleed is performed at a rate of 0.1 VVD.
  • the suspension cell line cell bleed is performed at a rate of 0.2 VVD.
  • the suspension cell line cell bleed is performed at a rate of 0.3 VVD.
  • the suspension cell line cell bleed is performed at a rate of 0.4 VVD.
  • the suspension cell line cell bleed is performed at a rate of 0.5 VVD.
  • the method involving a harvest rate of greater than 1 VVD results in an increase in yield per reactor volume of at least 1-fold, at least 2-fold, at least 3 -fold, at least 4-fold, or at least 5-fold compared to a method involving a harvest rate of about 1 VVD.
  • the suspension cell line cell bleed is performed to maintain a target cell viability of at least 70%. In one example, the suspension cell line cell bleed is performed to maintain a target cell viability of at least 75%. In one example, the suspension cell line cell bleed is performed to maintain a target cell viability of at least 80%. For example, the suspension cell line cell bleed is performed to maintain a target cell viability of at least 85%, or at least 90%, or at least 95%. In one example, the suspension cell line cell bleed is performed to maintain a target cell viability of about 80%, or about 81%, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%. In another example, the suspension cell line cell bleed is performed to maintain a target cell viability of about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%.
  • the suspension cell line cell bleed is performed to maintain a target cell density of at least about 1 x 10 7 cells/mL of cell culture fluid.
  • the suspension cell line cell bleed is performed to maintain a target cell density of between about 1 x 10 7 cells/mL and 5 x 10 7 cells/mL of cell culture fluid.
  • the suspension cell line cell bleed is performed to maintain a target cell density of about 1 x 10 7 cells/mL of cell culture fluid.
  • the suspension cell line cell bleed is performed to maintain a target cell density of about 2 x 10 7 cells/mL of cell culture fluid.
  • the suspension cell line cell bleed is performed to maintain a target cell density of about 3 x 10 7 cells/mL of cell culture fluid.
  • the suspension cell line cell bleed is performed to maintain a target cell density of about 4 x 10 7 cells/mL of cell culture fluid. In one example, the suspension cell line cell bleed is performed to maintain a target cell density of about 5 x 10 7 cells/mL of cell culture fluid.
  • the suspension cell line is a stable producer cell line, i.e., cells having stably incorporated therein the genetic material required to produce the lentivirus. Such cells are distinguished from cells having the genetic elements transiently incorporated therein.
  • the suspension cell line is initially seeded in the cell culture medium at a density of between about 1 x 10 5 cells/mL and 1 x 10 10 cells/mL of cell culture medium.
  • the suspension cell line is initially seeded in the cell culture medium at a density of between about 1 x 10 5 cells/mL and 1 x IO 10 cells/mL, or 1 x 10 5 cells/mL and 1 x 10 7 cells/mL, or about 0.1 x 10 6 cells/mL and 1 x 10 8 cells/mL, or about 0.5 x 10 6 cells/mL and 1 x 10 7 cells/mL, or about 0.5 x 10 6 cells/mL and 5 x 10 6 cells/mL, or about 0.5 x 10 6 cells/mL and 2.5 x 10 6 cells/mL of cell culture medium.
  • the suspension cell line is initially seeded in the cell culture medium at a density of 1 x 10 5 cells/mL and 1 x 10 7 cells/mL In one example, the suspension cell line is initially seeded in the cell culture medium at a density of 0.5 x 10 6 cells/mL to 5.0 x 10 6 cells/mL. In one example, the suspension cell line is initially seeded in the cell culture medium at a density of between 0.8 x 10 6 cells/mL and 1.2 x 10 6 . In one example, the suspension cell line is initially seeded in the cell culture medium at a density of between 1 x 10 6 cells/mL and 2.5 x 10 6 .
  • the suspension cell line is initially seeded in the cell culture medium at a density of between 1.5 x 10 6 cells/mL and 2 x 10 6 . In one example, the suspension cell line is initially seeded in the cell culture medium at a density of about 1 x 10 5 cells/mL, or about 2 x 10 5 cells/mL, or about 3 x 10 5 cells/mL, or about 4 x 10 5 cells/mL, or about 5 x 10 5 cells/mL, or about 6 x 10 5 cells/mL, or about 7 x 10 5 cells/mL, or about 8 x 10 5 cells/mL, or about 9 x 10 5 cells/mL, or about 10 x 10 5 cells/mL of cell culture medium.
  • the suspension cell line is initially seeded in the cell culture medium at a density of about 1 x 10 6 cells/mL, or about 2 x 10 6 cells/mL, or about 3 x 10 6 cells/mL, or about 4 x 10 6 cells/mL, or about 5 x 10 6 cells/mL, or about 6 x 10 6 cells/mL, or about 7 x 10 6 cells/mL, or about 8 x 10 6 cells/mL, or about 9 x 10 6 cells/mL, or about 10 x 10 6 cells/mL of cell culture medium.
  • the suspension cell line is initially seeded in the cell culture medium at a density of about 0.5 x 10 6 cells/mL.
  • the suspension cell line is initially seeded in the cell culture medium at a density of about 1 x 10 6 cells/mL. In one example, the suspension cell line is initially seeded in the cell culture medium at a density of about 1.5 x 10 6 cells/mL. In one example, the suspension cell line is initially seeded in the cell culture medium at a density of about 1.8 x 10 6 cells/mL. In one example, the suspension cell line is initially seeded in the cell culture medium at a density of about 2 x 10 6 cells/mL. In one example, the suspension cell line is initially seeded in the cell culture medium at a density of 2.5 x 10 6 cells/mL.
  • the suspension cell line is initially seeded in the cell culture medium at a density of 3.0 x 10 6 cells/mL. In one example, the suspension cell line is initially seeded in the cell culture medium at a density of 3.5 x 10 6 cells/mL. In one example, the suspension cell line is initially seeded in the cell culture medium at a density of 4.0 x 10 6 cells/mL. In one example, the suspension cell line is initially seeded in the cell culture medium at a density of 4.5 x 10 6 cells/mL. In one example, the suspension cell line is initially seeded in the cell culture medium at a density of 5.0 x 10 6 cells/mL.
  • the suspension cell line is initially seeded in the cell culture medium at a density of about 1 x 10 7 cells/mL, or about 2 x 10 7 cells/mL, or about 3 x 10 7 cells/mL, or about 4 x 10 7 cells/mL, or about 5 x 10 7 cells/mL, or about 6 x 10 7 cells/mL, or about 7 x 10 7 cells/mL, or about 8 x 10 7 cells/mL, or about 9 x 10 7 cells/mL, or about 10 x 10 7 cells/mL of cell culture medium.
  • the suspension cell line is initially seeded in the cell culture medium at a density of about 1 x 10 8 cells/mL, or about 2 x 10 8 cells/mL, or about 3 x 10 8 cells/mL, or about 4 x 10 8 cells/mL, or about 5 x 10 8 cells/mL, or about 6 x 10 8 cells/mL, or about 7 x 10 8 cells/mL, or about 8 x 10 8 cells/mL, or about 9 x 10 8 cells/mL, or about 10 x 10 8 cells/mL of cell culture medium.
  • the suspension cell line is initially seeded in the cell culture medium at a density of about 1 x 10 9 cells/mL, or about 2 x 10 9 cells/mL, or about 3 x 10 9 cells/mL, or about 4 x 10 9 cells/mL, or about 5 x 10 9 cells/mL, or about 6 x 10 9 cells/mL, or about 7 x 10 9 cells/mL, or about 8 x 10 9 cells/mL, or about 9 x 10 9 cells/mL, or about 10 x 10 9 cells/mL of cell culture medium.
  • the suspension cell line is grown to a viable cell density of between about 1 x 10 5 cells/mL to about 1 x 10 10 cells/mL of cell culture medium.
  • the suspension cell line is grown to a viable cell density of between about 1 x
  • the suspension cell line is grown to a viable cell density of between about
  • the suspension cell line is grown to a viable cell density of between about 6 x 10 6 cells/mL to about 1 x 10 7 of cell culture medium. In another example, the suspension cell line is grown to a viable cell density of between about 0.5 x 10 6 cells/mL to 5.0 x 10 6 cells/mL. In one example, the suspension cell line is grown to a viable cell density of between about 1 x
  • the suspension cell line is grown to a viable cell density of between about 1.5 x 10 6 cells/mL and 2 x 10 6 . In one example, the suspension cell line is grown to a viable cell density of about 1 x 10 5 cells/mL, or about
  • the suspension cell line is grown to a viable cell density of about 1 x 10 6 cells/mL, or about 2 x 10 6 cells/mL, or about 3 x 10 6 cells/mL, or about 4 x 10 6 cells/mL, or about 5 x 10 6 cells/mL, or about 6 x 10 6 cells/mL, or about 7 x 10 6 cells/mL, or about 8 x 10 6 cells/mL, or about 9 x 10 6 cells/mL, or about 10 x 10 6 cells/mL of cell culture medium.
  • the suspension cell line is grown to a viable cell density of about 0.5 x 10 6 cells/mL.
  • the suspension cell line is grown to a viable cell density of about 1 x 10 6 cells/mL. In one example, the suspension cell line is grown to a viable cell density of about 1.5 x 10 6 cells/mL. In one example, the suspension cell line is grown to a viable cell density of about 1.8 x 10 6 cells/mL. In one example, the suspension cell line is grown to a viable cell density of about 2 x 10 6 cells/mL. In one example, the suspension cell line is grown to a viable cell density of about 2.5 x 10 6 cells/mL. In one example, the suspension cell line is grown to a viable cell density of about 3.0 x 10 6 cells/mL.
  • the suspension cell line is grown to a viable cell density of about 3.5 x 10 6 cells/mL. In one example, the suspension cell line is grown to a viable cell density of about 4.0 x 10 6 cells/mL. In one example, the suspension cell line is grown to a viable cell density of about 4.5 x 10 6 cells/mL. In one example, the suspension cell line is grown to a viable cell density of about 5.0 x 10 6 cells/mL.
  • the suspension cell line is grown to a viable cell density of about 1 x 10 7 cells/mL, or about 2 x 10 7 cells/mL, or about 3 x 10 7 cells/mL, or about 4 x 10 7 cells/mL, or about 5 x 10 7 cells/mL, or about 6 x 10 7 cells/mL, or about 7 x 10 7 cells/mL, or about 8 x 10 7 cells/mL, or about 9 x 10 7 cells/mL, or about 10 x 10 7 cells/mL of cell culture medium.
  • the suspension cell line is grown to a viable cell density of about 1 x 10 8 cells/mL, or about 2 x 10 8 cells/mL, or about 3 x 10 8 cells/mL, or about 4 x 10 8 cells/mL, or about 5 x 10 8 cells/mL, or about 6 x 10 8 cells/mL, or about 7 x 10 8 cells/mL, or about 8 x 10 8 cells/mL, or about 9 x 10 8 cells/mL, or about 10 x 10 8 cells/mL of cell culture medium.
  • the suspension cell line is grown to a viable cell density of about 1 x 10 9 cells/mL, or about 2 x 10 9 cells/mL, or about 3 x 10 9 cells/mL, or about 4 x 10 9 cells/mL, or about 5 x 10 9 cells/mL, or about 6 x 10 9 cells/mL, or about 7 x 10 9 cells/mL, or about 8 x 10 9 cells/mL, or about 9 x 10 9 cells/mL, or about 10 x 10 9 cells/mL of cell culture medium.
  • the method results in a viral infectious titer yield of at least 1 x 10 5 transducing units (TU) /mL.
  • the method results in a viral infectious titer yield of between about 1 x 10 5 TU /mL and 1 x 10 10 TU/mL.
  • the method results in a viral infectious titer yield of about 1 x 10 5 TU/mL, or about 1.25 x 10 5 TU/mL, or about 1.5 x 10 5 TU/mL, or at least 1.75 x 10 5 TU/mL, or at least 2 x 10 5 TU/mL, or at least 2.5 x 10 5 TU/mL, or at least 3 x 10 5 TU/mL, or about 3.5 x 10 5 TU/mL, or about 4 x 10 5 TU/mL, or about 5 x 10 5 TU/mL.
  • the method results in a viral infectious titer yield of about 6 x 10 5 TU/mL, or about 7 x 10 5 TU/mL, or at least 8 x 10 5 TU/mL, or at least 9 x 10 5 TU/mL, or at least 10 x 10 5 TU/mL.
  • the method results in a viral infectious titer yield of about 1 x 10 6 TU/mL, or about 1.5 x 10 6 TU/mL, or about 2 x 10 6 TU/mL, or about 5 x 10 6 TU/mL, or about 7 x 10 6 TU/mL, or about 10 x 10 6 TU/mL.
  • the method results in a viral infectious titer yield of about 1 x 10 7 TU/mL, or about 5 x 10 7 TU/mL, or about 10 x 10 8 TU/mL, or about 5 x 10 8 TU/mL, or about 10 x 10 8 TU/mL, or about 5 x 10 9 TU/mL, or about 1 x 10 10 TU/mL.
  • the method results in a viral infectious titer yield of at least 1 x
  • the method results in a viral infectious titer yield of at least 1 x 10 6 TU/mL of culture medium at day 3 of culture.
  • the method results in a viral infectious titer yield of about 1.5 x 10 6 TU/mL, or about 2 x 10 6 TU/mL, or about 5 x 10 6 TU/mL, or about 7 x
  • the method results in a viral infectious titer yield of at least 1 x 10 7 TU/mL of culture medium at day 3 of culture.
  • the method results in a viral infectious titer yield of about 1.1 x 10 7 TU/mL, or about 1.2 x 10 7 TU/mL, or about 1.3 x 10 7 TU/mL, or about 1.4 x 10 7 TU/mL, or about 1.5 x 10 7 TU/mL of culture medium at day 3 of culture.
  • the method results in a viral infectious titer yield of at least 1.5 x 10 7 TU/mL of culture medium at day 3 of culture.
  • the method results in a viral infectious titer yield of about 1.6 x 10 7 TU/mL, or about 1.7 x 10 7 TU/mL, or about 1.8 x 10 7 TU/mL, or about 1.9 x 10 7 TU/mL of culture medium at day 3 of culture.
  • the method results in a viral infectious titer yield of at least 2 x 10 7 TU/mL of culture medium at day 3 of culture.
  • the method results in a viral infectious titer yield of about 2.1 x 10 7 TU/mL, or about 2.2 x 10 7 TU/mL, or about 2.3 x 10 7 TU/mL, or about 2.4 x 10 7 TU/mL, or about 2.5 x 10 7 TU/mL, or about 2.6 x 10 7 TU/mL, or about 2.7 x 10 7 TU/mL, or about 2.8 x 10 7 TU/mL, or about 2.9 x 10 7 TU/mL of culture medium at day 3 of culture.
  • the method results in a viral infectious titer yield of at least 1 x
  • the method results in a viral infectious titer yield of at least 1 x 10 6 TU/mL of culture medium at day 5 of culture.
  • the method results in a viral infectious titer yield of about 1.5 x 10 6 TU/mL, or about 2 x 10 6 TU/mL, or about 5 x 10 6 TU/mL, or about 7 x
  • the method results in a viral infectious titer yield of at least 1 x 10 7 TU/mL of culture medium at day 5 of culture.
  • the method results in a viral infectious titer yield of about 1.1 x 10 7 TU/mL, or about 1.2 x 10 7 TU/mL, or about 1.3 x 10 7 TU/mL, or about 1.4 x 10 7 TU/mL, or about 1.5 x 10 7 TU/mL of culture medium at day 5 of culture.
  • the method results in a viral infectious titer yield of at least 1.5 x 10 7 TU/mL of culture medium at day 5 of culture.
  • the method results in a viral infectious titer yield of about 1.6 x 10 7 TU/mL, or about 1.7 x 10 7 TU/mL, or about 1.8 x 10 7 TU/mL, or about 1.9 x 10 7 TU/mL of culture medium at day 5 of culture.
  • the method results in a viral infectious titer yield of at least 2 x 10 7 TU/mL of culture medium at day 5 of culture.
  • the method results in a viral infectious titer yield of about 2.1 x 10 7 TU/mL, or about 2.2 x 10 7 TU/mL, or about 2.3 x 10 7 TU/mL, or about 2.4 x 10 7 TU/mL, or about 2.5 x 10 7 TU/mL, or about 2.6 x 10 7 TU/mL, or about 2.7 x 10 7 TU/mL, or about 2.8 x 10 7 TU/mL, or about 2.9 x 10 7 TU/mL of culture medium at day 5 of culture.
  • the method results in a viral infectious titer yield of at least 1 x
  • the method results in a viral infectious titer yield of at least 1 x 10 6 TU/mL of culture medium at day 10 of culture.
  • the method results in a viral infectious titer yield of about 1.5 x 10 6 TU/mL, or about 2 x 10 6 TU/mL, or about 5 x 10 6 TU/mL, or about 7 x
  • the method results in a viral infectious titer yield of at least 1 x 10 7 TU/mL of culture medium at day 10 of culture.
  • the method results in a viral infectious titer yield of about 1.1 x 10 7 TU/mL, or about 1.2 x 10 7 TU/mL, or about 1.3 x 10 7 TU/mL, or about 1.4 x 10 7 TU/mL, or about 1.5 x 10 7 TU/mL of culture medium at day 10 of culture.
  • the method results in a viral infectious titer yield of at least 1.5 x 10 7 TU/mL of culture medium at day 10 of culture.
  • the method results in a viral infectious titer yield of about 1.6 x 10 7 TU/mL, or about 1.7 x 10 7 TU/mL, or about 1.8 x 10 7 TU/mL, or about 1.9 x 10 7 TU/mL of culture medium at day 10 of culture.
  • the method results in a viral infectious titer yield of at least 2 x 10 7 TU/mL of culture medium at day 10 of culture.
  • the method results in a viral infectious titer yield of about 2.1 x 10 7 TU/mL, or about 2.2 x 10 7 TU/mL, or about 2.3 x 10 7 TU/mL, or about 2.4 x 10 7 TU/mL, or about 2.5 x 10 7 TU/mL, or about
  • the method results in a viral infectious titer yield of at least 1 x
  • the method results in a viral infectious titer yield of at least 1 x 10 6 TU/mL of culture medium at day 15 of culture.
  • the method results in a viral infectious titer yield of about 1.5 x 10 6 TU/mL, or about 2 x 10 6 TU/mL, or about 5 x 10 6 TU/mL, or about 7 x
  • the method results in a viral infectious titer yield of at least 1 x 10 7 TU/mL of culture medium at day 15 of culture.
  • the method results in a viral infectious titer yield of about 1.1 x 10 7 TU/mL, or about 1.2 x 10 7 TU/mL, or about 1.3 x 10 7 TU/mL, or about 1.4 x 10 7 TU/mL, or about 1.5 x 10 7 TU/mL of culture medium at day 15 of culture.
  • the method results in a viral infectious titer yield of at least 1.5 x 10 7 TU/mL of culture medium at day 15 of culture.
  • the method results in a viral infectious titer yield of about 1.6 x 10 7 TU/mL, or about 1.7 x 10 7 TU/mL, or about 1.8 x 10 7 TU/mL, or about 1.9 x 10 7 TU/mL of culture medium at day 15 of culture.
  • the method results in a viral infectious titer yield of at least 2 x 10 7 TU/mL of culture medium at day 15 of culture.
  • the method results in a viral infectious titer yield of about 2.1 x 10 7 TU/mL, or about 2.2 x 10 7 TU/mL, or about 2.3 x 10 7 TU/mL, or about 2.4 x 10 7 TU/mL, or about 2.5 x 10 7 TU/mL, or about 2.6 x 10 7 TU/mL, or about 2.7 x 10 7 TU/mL, or about 2.8 x 10 7 TU/mL, or about 2.9 x 10 7 TU/mL of culture medium at day 15 of culture.
  • the method results in a viral infectious titer yield of at least 1 x
  • the method results in a viral infectious titer yield of at least 1 x 10 6 TU/mL of culture medium at day 20 of culture.
  • the method results in a viral infectious titer yield of about 1.5 x 10 6 TU/mL, or about 2 x 10 6 TU/mL, or about 5 x 10 6 TU/mL, or about 7 x
  • the method results in a viral infectious titer yield of at least 1 x 10 7 TU/mL of culture medium at day 20 of culture.
  • the method results in a viral infectious titer yield of about 1.1 x 10 7 TU/mL, or about 1.2 x 10 7 TU/mL, or about 1.3 x 10 7 TU/mL, or about 1.4 x 10 7 TU/mL, or about 1.5 x 10 7 TU/mL of culture medium at day 20 of culture.
  • the method results in a viral infectious titer yield of at least 1.5 x 10 7 TU/mL of culture medium at day 20 of culture.
  • the method results in a viral infectious titer yield of about 1.6 x 10 7 TU/mL, or about 1.7 x 10 7 TU/mL, or about 1.8 x 10 7 TU/mL, or about 1.9 x 10 7 TU/mL of culture medium at day 20 of culture.
  • the method results in a viral infectious titer yield of at least 2 x 10 7 TU/mL of culture medium at day 20 of culture.
  • the method results in a viral infectious titer yield of about 2.1 x 10 7 TU/mL, or about 2.2 x 10 7 TU/mL, or about 2.3 x 10 7 TU/mL, or about 2.4 x 10 7 TU/mL, or about 2.5 x 10 7 TU/mL, or about 2.6 x 10 7 TU/mL, or about 2.7 x 10 7 TU/mL, or about 2.8 x 10 7 TU/mL, or about 2.9 x
  • the method results in a viral infectious titer yield of at least 1 x 10 5 transducing units (TU) /mL of culture medium at day 25 of culture.
  • the method results in a viral infectious titer yield of at least 1 x 10 6 TU/mL of culture medium at day 25 of culture.
  • the method results in a viral infectious titer yield of about 1.5 x 10 6 TU/mL, or about 2 x 10 6 TU/mL, or about 5 x 10 6 TU/mL, or about 7 x
  • the method results in a viral infectious titer yield of at least 1 x 10 7 TU/mL of culture medium at day 25 of culture.
  • the method results in a viral infectious titer yield of about 1.1 x 10 7 TU/mL, or about 1.2 x 10 7 TU/mL, or about 1.3 x 10 7 TU/mL, or about 1.4 x 10 7 TU/mL, or about 1.5 x 10 7 TU/mL of culture medium at day 25 of culture.
  • the method results in a viral infectious titer yield of at least 1.5 x 10 7 TU/mL of culture medium at day 25 of culture.
  • the method results in a viral infectious titer yield of about 1.6 x 10 7 TU/mL, or about 1.7 x 10 7 TU/mL, or about 1.8 x 10 7 TU/mL, or about 1.9 x 10 7 TU/mL of culture medium at day 25 of culture.
  • the method results in a viral infectious titer yield of at least 2 x 10 7 TU/mL of culture medium at day 25 of culture.
  • the method results in a viral infectious titer yield of about 2.1 x 10 7 TU/mL, or about 2.2 x 10 7 TU/mL, or about 2.3 x 10 7 TU/mL, or about 2.4 x 10 7 TU/mL, or about 2.5 x 10 7 TU/mL, or about 2.6 x 10 7 TU/mL, or about 2.7 x 10 7 TU/mL, or about 2.8 x 10 7 TU/mL, or about 2.9 x
  • the method results in a viral infectious titer yield of at least 1 x
  • the method results in a viral infectious titer yield of at least 1 x 10 6 TU/mL of culture medium at day 30 of culture.
  • the method results in a viral infectious titer yield of about 1.5 x 10 6 TU/mL, or about 2 x 10 6 TU/mL, or about 5 x 10 6 TU/mL, or about 7 x
  • the method results in a viral infectious titer yield of at least 1 x 10 7 TU/mL of culture medium at day 30 of culture.
  • the method results in a viral infectious titer yield of about 1.1 x 10 7 TU/mL, or about 1.2 x 10 7 TU/mL, or about 1.3 x 10 7 TU/mL, or about 1.4 x 10 7 TU/mL, or about 1.5 x 10 7 TU/mL of culture medium at day 30 of culture.
  • the method results in a viral infectious titer yield of at least 1.5 x 10 7 TU/mL of culture medium at day 30 of culture.
  • the method results in a viral infectious titer yield of about 1.6 x 10 7 TU/mL, or about 1.7 x 10 7 TU/mL, or about 1.8 x 10 7 TU/mL, or about 1.9 x 10 7 TU/mL of culture medium at day 30 of culture.
  • the method results in a viral infectious titer yield of at least 2 x 10 7 TU/mL of culture medium at day 30 of culture.
  • the method results in a viral infectious titer yield of about 2.1 x 10 7 TU/mL, or about 2.2 x 10 7 TU/mL, or about 2.3 x 10 7 TU/mL, or about 2.4 x 10 7 TU/mL, or about 2.5 x 10 7 TU/mL, or about 2.6 x 10 7 TU/mL, or about 2.7 x 10 7 TU/mL, or about 2.8 x 10 7 TU/mL, or about 2.9 x 10 7 TU/mL of culture medium at day 30 of culture.
  • the method results in a viral infectious titer yield of at least 1 x
  • the method results in a viral infectious titer yield of at least 1 x 10 6 TU/mL of culture medium at day 35 of culture.
  • the method results in a viral infectious titer yield of about 1.5 x 10 6 TU/mL, or about 2 x 10 6 TU/mL, or about 5 x 10 6 TU/mL, or about 7 x
  • the method results in a viral infectious titer yield of at least 1 x 10 7 TU/mL of culture medium at day 35 of culture.
  • the method results in a viral infectious titer yield of about 1.1 x 10 7 TU/mL, or about 1.2 x 10 7 TU/mL, or about 1.3 x 10 7 TU/mL, or about 1.4 x 10 7 TU/mL, or about 1.5 x 10 7 TU/mL of culture medium at day 35 of culture.
  • the method results in a viral infectious titer yield of at least 1.5 x 10 7 TU/mL of culture medium at day 35 of culture.
  • the method results in a viral infectious titer yield of about 1.6 x 10 7 TU/mL, or about 1.7 x 10 7 TU/mL, or about 1.8 x 10 7 TU/mL, or about 1.9 x 10 7 TU/mL of culture medium at day 35 of culture.
  • the method results in a viral infectious titer yield of at least 2 x 10 7 TU/mL of culture medium at day 35 of culture.
  • the method results in a viral infectious titer yield of about 2.1 x 10 7 TU/mL, or about 2.2 x 10 7 TU/mL, or about 2.3 x 10 7 TU/mL, or about 2.4 x 10 7 TU/mL, or about 2.5 x 10 7 TU/mL, or about 2.6 x 10 7 TU/mL, or about 2.7 x 10 7 TU/mL, or about 2.8 x 10 7 TU/mL, or about 2.9 x
  • the method results in a viable cell density of at least about 1 x
  • the method results in a viable cell density of at least about 1 x 10 6 cells/mL of culture medium at day 15 of culture. In one example, the method results in a viable cell density of at least about 1.5 x
  • the method results in a viable cell density of at least about 1 x 10 7 cells/mL of culture medium at day 15 of culture. In one example, the method results in a viable cell density of at least about 1.1 x 10 7 cells/mL, or about 1.2 x 10 7 cells/mL, or about 1.3 x
  • the method results in a viable cell density of at least about 1.5 x 10 7 cells/mL of culture medium at day 15 of culture. In one example, the method results in a viable cell density of at least about 1.5 x 10 7 cells/mL, or about 1.6 x 10 7 cells/mL, or about 1.7 x 10 7 cells/mL, or about 1.8 x 10 7 cells/mL, or about 1.9 x 10 7 cells/mL, or about 2.0 x 10 7 cells/mL of culture medium at day 15 of culture.
  • the method results in a viable cell density of at least about 2.0 x 10 7 cells/mL of culture medium at day 15 of culture. In one example, the method results in a viable cell density of at least about 2.1 x 10 7 cells/mL, or about 2.2 x 10 7 cells/mL, or about 2.3 x 10 7 cells/mL, or about 2.4 x 10 7 cells/mL, or about 2.5 x 10 7 cells/mL of culture medium at day 15 of culture. In one example, the method results in a viable cell density of at least about 2.5 x 10 7 cells/mL of culture medium at day 15 of culture.
  • the method results in a viable cell density of at least about 2.6 x 10 7 cells/mL, or about 2.7 x 10 7 cells/mL, or about 2.8 x 10 7 cells/mL, or about 2.9 x 10 7 cells/mL, or about 3.0 x 10 7 cells/mL of culture medium at day 15 of culture.
  • the method results in a viable cell density of at least about 1 x
  • the method results in a viable cell density of at least about 1 x 10 6 cells/mL of culture medium at day 20 of culture. In one example, the method results in a viable cell density of at least about 1.5 x
  • the method results in a viable cell density of at least about 1 x 10 7 cells/mL of culture medium at day 20 of culture. In one example, the method results in a viable cell density of at least about 1.1 x 10 7 cells/mL, or about 1.2 x 10 7 cells/mL, or about 1.3 x
  • the method results in a viable cell density of at least about 1.5 x 10 7 cells/mL of culture medium at day 20 of culture. In one example, the method results in a viable cell density of at least about 1.5 x 10 7 cells/mL, or about 1.6 x 10 7 cells/mL, or about 1.7 x 10 7 cells/mL, or about 1.8 x 10 7 cells/mL, or about 1.9 x 10 7 cells/mL, or about 2.0 x 10 7 cells/mL of culture medium at day 20 of culture.
  • the method results in a viable cell density of at least about 2.0 x 10 7 cells/mL of culture medium at day 20 of culture. In one example, the method results in a viable cell density of at least about 2.1 x 10 7 cells/mL, or about 2.2 x 10 7 cells/mL, or about 2.3 x 10 7 cells/mL, or about 2.4 x 10 7 cells/mL, or about 2.5 x 10 7 cells/mL of culture medium at day 20 of culture. In one example, the method results in a viable cell density of at least about 2.5 x 10 7 cells/mL of culture medium at day 20 of culture.
  • the method results in a viable cell density of at least about 2.6 x 10 7 cells/mL, or about 2.7 x 10 7 cells/mL, or about 2.8 x 10 7 cells/mL, or about 2.9 x 10 7 cells/mL, or about 3.0 x 10 7 cells/mL of culture medium at day 20 of culture.
  • the method results in a viable cell density of at least about 1 x 10 5 cells/mL of culture medium at day 25 of culture.
  • the method results in a viable cell density of at least about 1 x 10 6 cells/mL of culture medium at day 25 of culture.
  • the method results in a viable cell density of at least about 1.5 x
  • the method results in a viable cell density of at least about 1 x 10 7 cells/mL of culture medium at day 25 of culture. In one example, the method results in a viable cell density of at least about 1.1 x 10 7 cells/mL, or about 1.2 x 10 7 cells/mL, or about 1.3 x
  • the method results in a viable cell density of at least about 1.5 x 10 7 cells/mL of culture medium at day 25 of culture. In one example, the method results in a viable cell density of at least about 1.5 x 10 7 cells/mL, or about 1.6 x 10 7 cells/mL, or about 1.7 x 10 7 cells/mL, or about 1.8 x 10 7 cells/mL, or about 1.9 x 10 7 cells/mL, or about 2.0 x 10 7 cells/mL of culture medium at day 25 of culture.
  • the method results in a viable cell density of at least about 2.0 x 10 7 cells/mL of culture medium at day 25 of culture. In one example, the method results in a viable cell density of at least about 2.1 x 10 7 cells/mL, or about 2.2 x 10 7 cells/mL, or about 2.3 x 10 7 cells/mL, or about 2.4 x 10 7 cells/mL, or about 2.5 x 10 7 cells/mL of culture medium at day 25 of culture. In one example, the method results in a viable cell density of at least about 2.5 x 10 7 cells/mL of culture medium at day 25 of culture.
  • the method results in a viable cell density of at least about 2.6 x 10 7 cells/mL, or about 2.7 x 10 7 cells/mL, or about 2.8 x 10 7 cells/mL, or about 2.9 x 10 7 cells/mL, or about 3.0 x 10 7 cells/mL of culture medium at day 25 of culture.
  • the method results in a viable cell density of at least about 1 x
  • the method results in a viable cell density of at least about 1 x 10 6 cells/mL of culture medium at day 30 of culture. In one example, the method results in a viable cell density of at least about 1.5 x
  • the method results in a viable cell density of at least about 1 x 10 7 cells/mL of culture medium at day 30 of culture. In one example, the method results in a viable cell density of at least about 1.1 x 10 7 cells/mL, or about 1.2 x 10 7 cells/mL, or about 1.3 x
  • the method results in a viable cell density of at least about 1.5 x 10 7 cells/mL of culture medium at day 30 of culture. In one example, the method results in a viable cell density of at least about 1.5 x 10 7 cells/mL, or about 1.6 x 10 7 cells/mL, or about 1.7 x 10 7 cells/mL, or about 1.8 x 10 7 cells/mL, or about 1.9 x 10 7 cells/mL, or about 2.0 x 10 7 cells/mL of culture medium at day 30 of culture.
  • the method results in a viable cell density of at least about 2.0 x 10 7 cells/mL of culture medium at day 30 of culture. In one example, the method results in a viable cell density of at least about 2.1 x 10 7 cells/mL, or about 2.2 x 10 7 cells/mL, or about 2.3 x 10 7 cells/mL, or about 2.4 x 10 7 cells/mL, or about 2.5 x 10 7 cells/mL of culture medium at day 30 of culture. In one example, the method results in a viable cell density of at least about 2.5 x 10 7 cells/mL of culture medium at day 30 of culture.
  • the method results in a viable cell density of at least about 2.6 x 10 7 cells/mL, or about 2.7 x 10 7 cells/mL, or about 2.8 x 10 7 cells/mL, or about 2.9 x 10 7 cells/mL, or about 3.0 x 10 7 cells/mL of culture medium at day 30 of culture.
  • the method results in a viable cell density of at least about 1 x
  • the method results in a viable cell density of at least about 1 x 10 6 cells/mL of culture medium at day 35 of culture. In one example, the method results in a viable cell density of at least about 1.5 x
  • the method results in a viable cell density of at least about 1 x 10 7 cells/mL of culture medium at day 35 of culture. In one example, the method results in a viable cell density of at least about 1.1 x 10 7 cells/mL, or about 1.2 x 10 7 cells/mL, or about 1.3 x
  • the method results in a viable cell density of at least about 1.5 x 10 7 cells/mL of culture medium at day 35 of culture. In one example, the method results in a viable cell density of at least about 1.5 x 10 7 cells/mL, or about 1.6 x 10 7 cells/mL, or about 1.7 x 10 7 cells/mL, or about 1.8 x 10 7 cells/mL, or about 1.9 x 10 7 cells/mL, or about 2.0 x 10 7 cells/mL of culture medium at day 35 of culture.
  • the method results in a viable cell density of at least about 2.0 x 10 7 cells/mL of culture medium at day 35 of culture. In one example, the method results in a viable cell density of at least about 2.1 x 10 7 cells/mL, or about 2.2 x 10 7 cells/mL, or about 2.3 x 10 7 cells/mL, or about 2.4 x 10 7 cells/mL, or about 2.5 x 10 7 cells/mL of culture medium at day 35 of culture. In one example, the method results in a viable cell density of at least about 2.5 x 10 7 cells/mL of culture medium at day 35 of culture.
  • the method results in a viable cell density of at least about 2.6 x 10 7 cells/mL, or about 2.7 x 10 7 cells/mL, or about 2.8 x 10 7 cells/mL, or about 2.9 x 10 7 cells/mL, or about 3.0 x 10 7 cells/mL of culture medium at day 35 of culture.
  • the cell line has a viability of at least 70% at day 15 of culture.
  • the cell line has a viability of about 70%, or about 75% or about 80% at day 15 of culture.
  • the cell line has a viability of at least 75% at day 15 of culture.
  • the cell line has a viability of at least 80% at day 15 of culture.
  • the cell line has a viability of about 80% or about 85% or about 90% at day 15 of culture.
  • the cell line has a viability of about 80% at day 15 of culture.
  • the cell line has a viability of about 85% at day 15 of culture. For example, a viability of about 86%, or about 87%, or about 88%, or about 89% at day 15 of culture. In a further example, the cell line has a viability of about 90% at day 15 of culture. In one example, the cell line has a viability of at least 90% at day 15 of culture. For example, the cell line has a viability of about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95% at day 15 of culture. In one example, the cell line has a viability of at least 95% at day 15 of culture. For example, the cell line has a viability of about 96%, or about 97%, or about 98%, or about 99% at day 15 of culture.
  • the cell line has a viability of at least 70% at day 20 of culture.
  • the cell line has a viability of about 70%, or about 75% or about 80% at day 20 of culture.
  • the cell line has a viability of at least 75% at day 20 of culture.
  • the cell line has a viability of at least 80% at day 20 of culture.
  • the cell line has a viability of about 80% or about 85% or about 90% at day 20 of culture.
  • the cell line has a viability of about 80% at day 20 of culture.
  • the cell line has a viability of about 85% at day 20 of culture. For example, a viability of about 86%, or about 87%, or about 88%, or about 89% at day 20 of culture. In a further example, the cell line has a viability of about 90% at day 20 of culture. In one example, the cell line has a viability of at least 90% at day 20 of culture. For example, the cell line has a viability of about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95% at day 20 of culture. In one example, the cell line has a viability of at least 95% at day 20 of culture. For example, the cell line has a viability of about 96%, or about 97%, or about 98%, or about 99% at day 20 of culture.
  • the cell line has a viability of at least 70% at day 25 of culture.
  • the cell line has a viability of about 70%, or about 75% or about 80% at day 25 of culture.
  • the cell line has a viability of at least 75% at day 25 of culture.
  • the cell line has a viability of at least 80% at day 25 of culture.
  • the cell line has a viability of about 80% or about 85% or about 90% at day 25 of culture.
  • the cell line has a viability of about 80% at day 25 of culture.
  • the cell line has a viability of about 85% at day 25 of culture. For example, a viability of about 86%, or about 87%, or about 88%, or about 89% at day 25 of culture. In a further example, the cell line has a viability of about 90% at day 25 of culture. In one example, the cell line has a viability of at least 90% at day 25 of culture. For example, the cell line has a viability of about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95% at day 25 of culture. In one example, the cell line has a viability of at least 95% at day 25 of culture. For example, the cell line has a viability of about 96%, or about 97%, or about 98%, or about 99% at day 25 of culture.
  • the cell line has a viability of at least 70% at day 30 of culture.
  • the cell line has a viability of about 70%, or about 75% or about 80% at day 30 of culture.
  • the cell line has a viability of at least 75% at day 30 of culture.
  • the cell line has a viability of at least 80% at day 30 of culture.
  • the cell line has a viability of about 80% or about 85% or about 90% at day 30 of culture.
  • the cell line has a viability of about 80% at day 30 of culture.
  • the cell line has a viability of about 85% at day 30 of culture. For example, a viability of about 86%, or about 87%, or about 88%, or about 89% at day 30 of culture. In a further example, the cell line has a viability of about 90% at day 30 of culture. In one example, the cell line has a viability of at least 90% at day 30 of culture. For example, the cell line has a viability of about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95% at day 30 of culture. In one example, the cell line has a viability of at least 95% at day 30 of culture. For example, the cell line has a viability of about 96%, or about 97%, or about 98%, or about 99% at day 30 of culture.
  • the cell line has a viability of at least 70% at day 35 of culture.
  • the cell line has a viability of about 70%, or about 75% or about 80% at day 35 of culture.
  • the cell line has a viability of at least 75% at day 35 of culture.
  • the cell line has a viability of at least 80% at day 35 of culture.
  • the cell line has a viability of about 80% or about 85% or about 90% at day 35 of culture.
  • the cell line has a viability of about 80% at day 35 of culture.
  • the cell line has a viability of about 85% at day 35 of culture. For example, a viability of about 86%, or about 87%, or about 88%, or about 89% at day 35 of culture. In a further example, the cell line has a viability of about 90% at day 35 of culture. In one example, the cell line has a viability of at least 90% at day 35 of culture. For example, the cell line has a viability of about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95% at day 35 of culture. In one example, the cell line has a viability of at least 95% at day 35 of culture. For example, the cell line has a viability of about 96%, or about 97%, or about 98%, or about 99% at day 35 of culture.
  • the method increases the virus infectious titer yield by at least 2% or 3% or 4% or 5% or 10% or 15% or 20%. In one example, the method increases the virus infectious titer yield by at least 10%.
  • the suspension cell culture has a volume of greater than about 1 L, about 2 L, about 5 L, about 10 L, about 50 L, about 100 L, about 500 L, about 1000 L, about 5,000 L, about 10,000 L, or about 15,000 L.
  • the suspension cell culture has a volume of between about 1 L and 1000 L.
  • the suspension cell culture has a volume of about 1 L.
  • the suspension cell culture has a volume of about 5 L.
  • the suspension cell culture has a volume of about 10 L.
  • the suspension cell culture has a volume of about 50 L.
  • the suspension cell culture has a volume of about 100 L.
  • the suspension cell culture has a volume of about 500 L.
  • the suspension cell culture has a volume of about 1000 L. In another example, the suspension cell culture has a volume of about 5000 L. In another example, the suspension cell culture has a volume of about 10,000 L. In another example, the suspension cell culture has a volume of about 15,000 L.
  • the suspension cell culture is operated with a dissolved carbon dioxide (CO2) level of between 4% and 8%.
  • CO2 dissolved carbon dioxide
  • the suspension cell culture is operated with about 4% CO2.
  • the suspension cell culture is operated with about 5% CO2.
  • the suspension cell culture is operated with about 6% CO2.
  • the suspension cell culture is operated with about 7% CO2.
  • the suspension cell culture is operated with about 8% CO2.
  • the suspension cell culture is operated at a pH of between 6.0 and 8.0. In one example, the suspension cell culture is at a pH of between about 6.5 and 7.5. For example, the pH is between about 6.90 and about 7.3. In one example, the pH is about 7.1. In one example, the pH is about 6.5. In one example, the pH is about 6.6. In one example, the pH is about 6.7. In one example, the pH is about 6.8. In one example, the pH is about 6.9. In one example, the pH is about 7.0. In one example, the pH is about 7.1. In one example, the pH is about 7.2. In one example, the pH is about 7.3. In one example, the pH is about 7.4. In one example, the pH is about 7.5.
  • the suspension cell culture is operated at a temperature of between about 35 °C and 39 °C.
  • the suspension cell culture is at a temperature of about 35 °C, or about 35.5 °C, or about 36 °C, or about 36.5 °C, or about 37 °C, or about 37.5 °C, or about 38 °C, or about 38.5 °C, or about 39 °C.
  • the suspension cell culture is at a temperature of between about 36.5 °C and about 37.5 °C.
  • the suspension cell culture is at a temperature of about 37.0 °C.
  • the suspension cell culture is at a temperature of between about 38 °C and about 39 °C.
  • the suspension cell culture is at a temperature of about 38.5 °C.
  • the suspension cell culture is at a pH of between 6.0 and 8.0 and/or at a temperature of between 35-39 °C. In one example, the suspension cell culture is at a pH of between 6.0 and 8.0 and/or at a temperature of between 37-38.5 °C. For example, the suspension cell culture is at a pH of between about 6.8 and about 7.1 and/or at a temperature of between about 37 °C and about 38.5 °C. In one example, the suspension cell culture is at a pH of between about 6.8 and about 7.1 and at a temperature of between about 37 °C and about 38.5 °C. In one example, the suspension cell culture is at a pH of about 6.9 to about 7.0 and at a temperature of about 37.0 °C.
  • the method further comprises purifying the enveloped virus from the suspension cell culture.
  • purifying the enveloped virus comprises one or more steps selected from the group consisting of anion exchange chromatography, concentration and diafiltration.
  • a method of the disclosure additionally comprises performing sterile filtration.
  • the sterile filtration is performed prior to concentrating and diafiltering the eluted virus.
  • the sterile filtration is performed after concentrating and diafiltering the eluted virus.
  • the method additionally comprising formulating the purified enveloped virus into a pharmaceutical formulation or into a solution suitable for infecting a cell.
  • An exemplary enveloped virus is a retrovirus.
  • the retrovirus is a lentivirus.
  • the lentivirus is HIV or a derivative thereof.
  • the present disclosure also provides a purified enveloped virus produced by the method described herein. BRIEF DESCRIPTION OF THE DRAWINGS
  • Figure 1 is a schematic drawing of the acoustic wave mediated perfusion set-up for (left) bench-top bioreactors and for (right) Ambr® 250 bioreactors.
  • the acoustic field in the cell separation chamber allows to continuously collect cell-free cell culture supernatant.
  • the acoustic field is switched off and the flow direction of the harvest pump is reversed to return the separated cells into the bioreactor.
  • Figure 2 is a series of graphical representations showing the perfusion process for stable lentivirus production using acoustic wave cell separation in the Ambr® 250 high throughput bioreactor system.
  • A Harvest rates and bleed rates.
  • B Viable cell densities.
  • C Cell viabilities.
  • D Infectious virus titer.
  • F Cell specific productivities of infectious virus.
  • G Cell specific infectious virus yields. Values are shown as mean for A and as mean ⁇ SD for B-G.
  • VVD vessels volumes per day.
  • Figure 3 is a series of graphical representations showing scale-up of two perfusion processes for stable lentivirus production using acoustic wave cell separation at bench-top bioreactor scale (run 1).
  • the standard process at a harvest rate of 1 VVD was performed in a 5 L bioreactor.
  • the intensified process was performed at a harvest rate of 3 VVD in a 2 L bioreactor.
  • A Harvest rates and bleed rates.
  • B Viable cell densities.
  • C Cell viabilities.
  • D Infectious virus titer.
  • E Yield of infectious titer per reactor volume or volumetric yield.
  • F Cell specific productivities of infectious virus.
  • (G) Cell specific infectious virus yields.
  • H Cell specific productivities of vector RNA genomes.
  • I Cell specific yields of vector RNA genomes.
  • BR bleed rate
  • HR harvest rate
  • VVD vessels volumes per day.
  • Figure 4 is a series of graphical representations showing the bench-top bioreactor perfusion processes for stable lentivirus production using acoustic wave cell separation with optimized set-up (run 2).
  • the standard process at a harvest rate of 1 VVD was performed in a 5 L bioreactor.
  • the intensified process was performed at a harvest rate of 3 VVD in a 2 L bioreactor.
  • a PharmaPure® low spallation pump tubing size 17 was used to recirculate the cell suspension for operation in perfusion mode.
  • the medium was supplemented with 0.5 % poloxamer 188 and 0.4 % cholesterol lipid concentrate.
  • the run was performed with one vessel per condition.
  • C Cell viabilities.
  • E Yield of infectious titer per reactor volume or volumetric yield.
  • F Cell specific productivities of infectious virus.
  • G Cell specific infectious virus yields.
  • H Cell specific productivities of vector RNA genomes.
  • I Cell specific yields of vector RNA genomes.
  • Figure 5 is a graphical representation showing temperature- and time-dependent inactivation of WAS-T2A-GFP lentivirus.
  • the cell-free vector harvest produced using stable GPRTGs producer cells in perfusion bioreactors, was incubated at both 4 °C and 37 °C, and subsequently frozen at -80 °C at different time points.
  • a half-life of 6 hours at 37 °C and 153 hours at 4°C was determined by a non-linear regression using a one phase decay equation with a robust fit.
  • Figure 6 is a series of graphical representations showing the DNA concentrations and ratio of infectious virus to p24 capsid protein determined for cell culture supernatant sampled from bench-top bioreactors with optimized set-up (run 2).
  • A DNA concentrations.
  • B Ratio of infectious virus particles to capsid protein p24.
  • composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e., one or more) of those steps, compositions of matter, groups of steps or groups of compositions of matter.
  • any example of the present disclosure herein shall be taken to apply mutatis mutandis to any other example of the disclosure unless specifically stated otherwise. Stated another way, any specific example of the present disclosure may be combined with any other specific example of the disclosure (except where mutually exclusive). Any example of the present disclosure disclosing a specific feature or group of features or method or method steps will be taken to provide explicit support for disclaiming the specific feature or group of features or method or method steps.
  • enveloped virus refers to DNA and RNA viruses that have a viral envelope. Envelopes are typically derived from host cell membranes (e.g., phospholipids and proteins), but may include viral glycoproteins on the surface of the envelope. Enveloped viruses also comprise a “capsid”, which is a protein layer between the envelope and viral genome. In one example, the enveloped virus is a retrovirus. For example, the enveloped virus is a lentivirus, e.g., human immunodeficiency virus.
  • cell culture fluid or “cell culture medium” will be understood to encompass the fluid or medium in which cells are grown for the purpose of producing an enveloped virus.
  • the fluid or medium does not comprise the cells (e.g., the cells may have been removed, e.g., by centrifugation and/or removal of supernatant).
  • cell culture or “suspension cell culture” will be understood to refer to the collective of the cell culture fluid or medium and the cultured cells.
  • suspension in reference to the cell lines will be understood to refer to single cells or small aggregates of cells that are free-floating in the cell culture medium. For example, such cells function and multiply in an agitated growth medium, thus forming a suspension.
  • purify or “purifying” or “purification” shall be taken to mean the removal, whether completely or partially, of at least one impurity present in the cell culture fluid, which thereby improves the level of purity of enveloped virus in solution.
  • filtered cell culture fluid will be understood to encompass the cell culture fluid after it has been subjected to harvest filtration.
  • the virus is a retrovirus, for example, a lentivirus.
  • retroviruses are from alpha retroviruses (such avian leukosis virus (ALV)), from beta retroviruses (such as mouse mammary tumor virus (MMTV)), from gamma retroviruses (such as murine leukemia virus (MLV)), from delta retroviruses (such as human T- lymphotropic virus (HTLV)), from epsilon retroviruses (such as Walleye dermal sarcoma virus (WDSV)), from spumavirus (such as human foamy virus (HFV) or simian foamy virus (SFV)), from primate lentiviruses such as the different types of human immunodeficiency viruses (HIV), the different types of simian immunodeficiency viruses (SIV), or from non-primate mammal lentiviruses such as the equine infectious anemia virus (EIAV), from the feline immunodefici
  • the enveloped virus e.g., the retrovirus
  • the enveloped virus is pseudotyped, i.e., it comprises an envelope glycoprotein derived from a virus different from the virus from which it is derived, a modified envelope glycoprotein or a chimeric envelope glycoprotein.
  • the enveloped virus comprises a transgene introduced into its genome.
  • the transgene will depend on the specific use for which the enveloped viral vector is intended.
  • Exemplary transgenes include a transgene coding for a therapeutic RNA (e.g. encoding an antisense complementary RNA of a target RNA or DNA sequence), a transgene encoding for a protein that is deficient or absent in a subject affected with a pathology, or a transgene used for vaccination with DNA, i.e. a transgene coding for a protein, the expression of which will induce vaccination of the recipient body against said protein.
  • the transgene encodes a protein or nucleic acid useful for treating a hemoglobinopathy, e.g., sickle cell disease or a thalassemia. In some examples, the transgene encodes a protein or nucleic acid useful for treating a primary immunodeficiency. In some examples, the transgene encodes a protein or nucleic acid useful for treating Wiskott-Aldrich Syndrome. In some examples, the transgene encodes a protein or nucleic acid useful for treating X linked agammaglobulinemia.
  • an enveloped virus is produced by introducing the four following elements into a host cell: an expression cassette comprising a lentiviral gene gagpol, an expression cassette comprising a lentiviral gene rev, a transgene, all positioned between a lentiviral LTR-5’ and a lentiviral LTR-3’, and an expression cassette encoding envelope glycoprotein(s).
  • Producer cell lines comprising a lentiviral gene gagpol, an expression cassette comprising a lentiviral gene rev, a transgene, all positioned between a lentiviral LTR-5’ and a lentiviral LTR-3’, and an expression cassette encoding envelope glycoprotein(s).
  • the enveloped virus is produced from a stable line expressing one or several elements required for producing an enveloped virus (Miller (2001) Curr. Protoc. Hum. Genet. Chapter 12: Unit 12.5.; Rodrigues et al. 2011, supra).
  • the enveloped virus is produced from a mammal host cell transfected transiently with one or several plasmids coding for the elements required for producing the virus.
  • the elements are introduced into the cell by means of multiple plasmids: one plasmid bearing an expression cassette comprising a lentiviral gagpol gene, one plasmid bearing an expression cassette comprising a lentiviral rev gene, one plasmid bearing an expression cassette encoding the envelope glycoprotein(s), one plasmid bearing an expression cassette comprising a tetracycline transactivator (iTA) gene, and/or one plasmid bearing an expression cassette comprising a lentiviral tat gene.
  • iTA tetracycline transactivator
  • a transfer plasmid comprising an expression cassette with the transgene, comprised between a lentiviral LTR-5’ and LTR-3’, can be introduced as a concatemer along with a helper plasmid with an antibiotic resistance cassette to confer resistance to the producer cells.
  • the host cell may be selected from any cell allowing production of an enveloped virus.
  • the cell is selected from a human cell (HEK293, HEK293T, HEK293FT, HEK293OX, Te671, HT1080, CEM), a musteli cell (NIH-3T3), a mustelidae cell (Mpf), a canid cell (D17), and derivatives thereof.
  • the cell is selected from CHO cells, BHK cells, MDCK cells, C3H 10T1/2 cells, FLY I, Psi-2 cells, BOSC 23 cells, PA317 cells, WEHI cells, COS cells, BSC 1 cells, BSC 40 cells, BMT 10 cells, VERO cells, W138 cells, MRC5 cells, A549 cells, HT1080 cells, B-50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells, 211 cells, and 211 A cells, and derivatives thereof.
  • the cell is selected from the GPR, GPRG, GPRT, GPRGT, and GPRTG cell lines. In another example, the cell is selected from a cell line derived from any of the above cell lines.
  • the cell line is a suspension cell line selected from GPR, GPRG, GPRT, GPRGT, and GPRTG cell lines.
  • the suspension cell line is a cell line derived from any of the above cell lines.
  • the suspension cell line is a cell line derived from a GPRG cell line.
  • the suspension cell line is a cell line derived from a GPRGT cell line.
  • the suspension cell line is a cell line derived from a GPRTG cell line.
  • the enveloped virus is produced from stable producer cells.
  • Stable producer cells can be derived from packaging cell lines, including as any of the cell lines disclosed herein.
  • the packaging cell lines are GPRG or GPRTG cell lines (Throm et al. (2009) Blood 113(21):5104-5110; and Bonner et al. (2015) Molecular Therapy, Vol. 23, Suppl. 1, S35).
  • stable producer cell line cells are generated by synthesizing a vector by cloning one or more genes into a recombinant plasmid; forming a concatemeric array from an expression cassette excised from the synthesized vector, and an expression cassette obtained from an antibiotic resistance cassette plasmid; transfecting packaging cell line cells with the formed concatemeric array; and selecting and isolating the stable producer cell line cells.
  • Virus is produced by inducing the inducible promoters of the stable producer cell line cells.
  • the present disclosure also provides a stable producer cell clone capable of producing an enveloped virus.
  • the stable producer cell clone is cultured to produce a stable producer cell line. Methods of culturing the stable producer cell clone to generate a stable producer cell line are described herein and/or are apparent to the skilled person.
  • the cells are cultivated in a medium suitable for cultivation of mammal cells and for producing an enveloped virus.
  • the cells can be cultivated in an adherent environment, e.g., while attached to a surface, or in a suspension environment, e.g., suspended in the medium.
  • the medium may moreover be supplemented with additives known in the field such as antibiotics, serum (notably fetal calf serum, etc.) added in suitable concentrations.
  • the medium may be supplemented with GlutaMaxTM, PluronicTM F-68 (ThermoFisher), LONG® R3 IGF-I (Sigma-Aldrich), Cell BoostTM 5, and/or an antidumping agent.
  • the medium used may notably comprise serum or be serum-free.
  • Culture media for mammal cells include, for example, DMEM (Dulbecco’s Modified Eagle’s medium) medium, RPMI1640 or a mixture of various culture media, including for example DMEM/F12, or a serum-free medium like optiMEM®, optiPRO®, optiPRO-SFM®, CD293® (ThermoFisher), TransFxTM (Cytiva), BalanCD® (Irvine), Freestyle F17® (Life Technologies), or Ex-Cell® 293 (Sigma-Aldrich).
  • DMEM Dynabecco’s Modified Eagle’s medium
  • RPMI1640 a mixture of various culture media, including for example DMEM/F12, or a serum-free medium like optiMEM®, optiPRO®, optiPRO-SFM®, CD293® (ThermoFisher), TransFxTM (Cytiva), BalanCD® (Irvine), Freestyle F17® (Life Technologies), or Ex
  • the cells are cultivated in a culture media comprising TransFxTM (Cytiva).
  • the cells are supplemented with one or more additives selected from the group consisting of GlutaMaxTM, Cell BoostTM 5, poloxamer 188 and combinations thereof.
  • the cells are supplemented with GlutaMaxTM.
  • the cells are supplemented with Cell BoostTM 5.
  • the cells are supplemented with poloxamer 188.
  • the cells are supplemented with GlutaMaxTM and Cell BoostTM 5.
  • the cells are supplemented with GlutaMaxTM and poloxamer 188.
  • the cells are supplemented with Cell BoostTM 5 and poloxamer 188.
  • the cells are supplemented with GlutaMaxTM, Cell BoostTM 5 and poloxamer 188.
  • the cells are supplemented with GlutaMaxTM, Cell BoostTM 5 and poloxamer 188.
  • cells are supplemented with less than 15 mM GlutaMaxTM.
  • the cells are supplemented with about 15 mM GlutaMaxTM, or about 14 mM GlutaMaxTM, or about 13 mM GlutaMaxTM, or about 12 mM GlutaMaxTM, or about 11 mM GlutaMaxTM, or about 10 mM GlutaMaxTM.
  • the cells are supplemented with less than 10 mM GlutaMaxTM.
  • the cells are supplemented with about 10 mM GlutaMaxTM, or about 9 mM GlutaMaxTM, or about 8 mM GlutaMaxTM, or about 7 mM GlutaMaxTM, or about 6 mM GlutaMaxTM, or about 5 mM GlutaMaxTM.
  • cells are supplemented with less than 5 mM GlutaMaxTM.
  • the cells are supplemented with about 5 mM GlutaMaxTM, or about 4 mM GlutaMaxTM, or about 3 mM GlutaMaxTM, or about 2 mM GlutaMaxTM, or about 1 mM GlutaMaxTM.
  • the cells are supplemented with between 1 mM and 10 mM GlutaMaxTM.
  • the cells are supplement with between 4 mM and 8 mM GlutaMaxTM. In one example, the cells are supplemented with 4 mM GlutaMaxTM. In another example, the cells are supplemented with 5 mM GlutaMaxTM. In a further example, the cells are supplemented with 6 mM GlutaMaxTM. In one example, the cells are supplemented with 7 mM GlutaMaxTM. In a further example, the cells are supplemented with 8 mM GlutaMaxTM.
  • the cells are supplemented with between 0.01% and 1% poloxamer 188. In one example, the cells are supplemented with between 0.05% and 1% poloxamer 188. For example, the cells are supplemented with between 0.05 and 0.5% poloxamer 188. In one example, the cells are supplemented with between 0.1% and 0.5% poloxamer 188. For example, the cells are supplemented with 0.1% poloxamer 188. In another example, the cells are supplemented with 0.2% poloxamer 188. In a further example, the cells are supplemented with 0.3% poloxamer 188. In one example, the cells are supplemented with 0.4% poloxamer 188. In a further example, the cells are supplemented with 0.5% poloxamer 188.
  • the cells are supplemented with less than 10% Cell BoostTM 5.
  • the cells are supplemented with about 10% Cell BoostTM 5, or about 9% Cell BoostTM 5, or about 8% Cell BoostTM 5, or about 7% Cell BoostTM 5, or about 6% Cell BoostTM 5.
  • the cells are supplemented with less than 5% Cell BoostTM 5.
  • the cells are supplemented with about 5% Cell BoostTM 5, or about 4% Cell BoostTM 5, or about 3% Cell BoostTM 5, or about 2% Cell BoostTM 5, or about 1% Cell BoostTM 5.
  • the cells are supplemented with between 1% and 10% Cell BoostTM 5.
  • the cells are supplemented with between 2% and 8% Cell BoostTM 5.
  • the cells are supplemented with between 4% and 6% Cell BoostTM 5.
  • the cells are supplemented with about 4% Cell BoostTM 5.
  • the cells are supplemented with about 5% Cell BoostTM 5.
  • the cells are supplemented with about 6% Cell BoostTM 5.
  • any agent allowing transfection of plasmids may be used.
  • exemplary agents include calcium phosphate or polyethyleneimine.
  • the conditions e.g., amount of plasmid(s), ratio between the plasmids, ratio between the plasmid(s) and the transfection agent, the type of medium, etc.
  • the transfection time may be adapted by one skilled in the art according to the characteristics of the produced virus and/or of the transgene introduced into the transfer plasmid.
  • the culture medium used has a neutral pH (e.g. comprised between 7 and 7.4, notably 7, 7.1, 7.2, 7.3 or 7.4) conventionally used in the state of the art for cultivating cells and producing viruses.
  • the production process used comprises the cultivation of producing cells in a moderately acid medium.
  • moderately acid condition designates the pH of an aqueous solution comprised between 5 and 6.8, for example between 5.5 and 6.5, such as between 5.8 and 6.2.
  • the selected pH will also depend on the buffering power of the culture medium used, which one skilled in the art may easily determine taking into account his/her general knowledge.
  • One skilled in the art is able to modify the pH of a solution.
  • the production of the enveloped virus comprises: transient transfection of HEK293T cells or derivatives thereof by means of one or several plasmids coding for the elements required for production of said enveloped vector, or by the use of stable producing cells, e.g., GPRG or GPRTG, producing the vectors constitutively or after induction; culturing the cells in a suitable medium, for which the pH is of about 6 or of about 7; harvesting cell culture medium containing the enveloped virus.
  • the present disclosure provides a method of producing an enveloped virus in a suspension cell culture.
  • the method comprises culturing a suspension cell line expressing a tetracycline-suppressible gene expression system in a cell culture medium.
  • Methods of the disclosure are applicable to producing enveloped viruses from both small- and large-scale productions.
  • the methods are particularly useful for their ability to be scaled up for manufacturing pharmaceutical products at commercial scale.
  • production of an enveloped virus includes a cell expansion phase and a viral production phase.
  • the cell expansion phase includes a seed train cell culture.
  • seed train refers to the generation of an adequate number of cells (i.e., cell growth) for viral production.
  • seed train cell culture comprises several cultivation systems which become larger with each passage (e.g. T-flasks, roller bottles or shake flasks, small scale bioreactor systems and subsequently larger bioreactors) in order to scale the culture from a small volume of cells to a larger volume of cells suitable for virus production.
  • the cells are grown in a cell expansion phase prior to virus production.
  • the cell expansion phase is carried out in an expansion bioreactor (also termed an N-l bioreactor).
  • an expansion bioreactor also termed an N-l bioreactor.
  • the viral production phase is carried out in a production bioreactor (also termed an N bioreactor).
  • a production bioreactor also termed an N bioreactor.
  • the cell expansion phase and viral production phase are carried out in the same vessel.
  • expansion of the suspension cell line and production of the enveloped virus occur in the same vessel.
  • the cell expansion phase and viral production phase are carried out in the same bioreactor.
  • the cell expansion phase and viral production phase are carried out in different vessels.
  • the cell expansion phase is carried out in an expansion bioreactor and viral production phase is carried out in a production bioreactor, wherein the expansion bioreactor and the production bioreactor are different.
  • the cell culture is operated in a perfusion mode.
  • the cell expansion phase and/or viral production phase are operated in perfusion mode.
  • the cell expansion phase is carried out perfusion mode.
  • the viral production phase is carried out in perfusion mode.
  • the cell expansion and the viral production phases are carried out in perfusion mode.
  • a perfusion mode for a particular phase of cell culture does not mean that the entire culture phase is carried out in that mode. For example, it only means that a period of the cell culture phase (e.g., at least 1 day) is carried out in that mode. It will also be understood that the mode does not necessary commence on day 0 of the culture phase. For example, the culture may commence on day 0 and perfusion mode only commence on day 2 of the cell culture phase.
  • perfusion mode involves the constant feeding of fresh media and removal of spent media while retaining high numbers of viable cells (i.e., continuous media exchange).
  • the cells are cultured in fluidized bed bioreactors, hollow fiber bioreactors, roller bottles, shake flasks, or stirred tank bioreactors. In one example, cells are cultured in a stirred tank bioreactor. In examples, the cells are cultured in a Biostat® or Univessel® bioreactor (Sartorius).
  • the volume of the cell culture can be for example, about 0.01 L to about 0.1 L, or about 0.1 L to about 1 L, or about 1 L to about 5 L. In another example, the volume of the cell culture can be about 5 L to about 10 L, about 10 L to about 50 L, about 50 L to about 100 L, about 100 L to about 200 L, about 200 L to about 500 L, about 500 L to about 1000 L, about 1000 L to about 2000 L, or about 2000 L to about 5000 L. In one example, the volume of the cell culture is between about 35 and 150 L. In one example, the volume of the cell culture is about 35-150 L. In one example, the volume of the cell culture is about 50-70 L.
  • the suspension cell culture is operated at a temperature that permits cell growth and viral production.
  • the cell culture has a temperature conventionally used in the state of the art for cultivating cells and producing viruses.
  • the suspension cell culture is at a temperature of between 35-39°C. For example, at a temperature of 37 ⁇ 0.5°C or at a temperature of 38 ⁇ 0.5°C.
  • the suspension cell culture is operated at large-scale.
  • the suspension cell culture is operated at commercial-scale.
  • the suspension cell culture is operated for a period of at least 10 days.
  • the suspension cell cultures is operated for a period of between about 10 and 50 days.
  • the suspension cell culture is operated for a period of between 10 and 35 days, for example, about 10 days or about 12 days, or about 15 days, or about 18 days, or about 20 days, or about 22 days, or about 25 days, or about 28 days, or about 30 days or about 32 days or about 35 days.
  • the suspension cell culture is operated for at least 15 days.
  • the suspension cell culture is operated for about 20 days.
  • the suspension cell culture is operated for at least 20 days.
  • the suspension cell culture is operated for at least 25 days.
  • the suspension cell culture is operated for about 28 days. In one example, the suspension cell culture is operated for at least 30 days. In one example, the suspension cell culture is operated for at least 32 days. For example, the suspension cell culture is operated for a period of 35 days. In one example, the suspension cell culture is operated for at least 35 days.
  • the present disclosure provides methods for improving the recovery of enveloped viruses from cell culture fluid.
  • Methods of the disclosure are applicable to production of enveloped viruses from both small- and large-scale productions.
  • the methods are particularly useful for their ability to be scaled up for manufacturing pharmaceutical products at commercial scale.
  • the present disclosure provides a process for producing an enveloped virus in a suspension perfusion cell culture comprising filtering the cell culture fluid through a filter at a harvest rate of at least 2 vessel volumes per day (VVD) to produce a filtered cell culture fluid comprising the enveloped virus, wherein the filter retains cells from the suspension cell line.
  • VVD vessel volumes per day
  • perfusion harvest filtration refers to the removal of cell culture media containing virus particles from the producer cells for downstream processing, wherein the cells are retained by the filter.
  • harvest refers to the cell culture media containing virus particles that has been removed for the purpose of downstream processing.
  • filtered cell culture fluid will be understood to encompass the cell culture fluid after it has been subjected to harvest filtration.
  • VVD tissue volume(s) per day
  • perfusion rate i.e., in VVD
  • harvest rate refers to the medium exchange rate through the harvest line in VVD.
  • bleed rate or “cell discard rate” refers to the medium exchange rate of cell biomass that is removed from the cell culture in VVD.
  • the inventors determined that the recirculation of cells retained in the filter into the suspension cell culture increased the yields of envelope virus from the suspension cell culture.
  • the inventors found that they could remove enveloped virus from the suspension cell culture by retaining the suspension cell line cells using an acoustic standing wave and then returning the retained suspension cell line cells into the suspension cell culture.
  • the method further comprises recirculating cell culture fluid through the filter to return the retained suspension cell line cells into the suspension cell culture.
  • the retained suspension cell line cells are recirculated into the suspension cell culture at a recirculation rate greater than the harvest rate.
  • the term “recirculation rate” refers to the medium flow rate in the recirculation loop, i.e., from the filter back into the suspension cell culture.
  • the method comprises the use of an acoustic chamber or acoustic wave device.
  • the method comprises (i) flowing the suspension cell culture through an acoustic standing wave within an acoustic chamber, (ii) retaining the suspension cell line cells within the acoustic chamber, and (iii) recirculating the cell culture fluid through the acoustic chamber comprising the retained suspension cell line cells to return the retained suspension cell line cells into the suspension cell culture.
  • Exemplary acoustic wave devices employ ultrasonic particle separation technology as described in EP 0633049.
  • Exemplary acoustic wave devices include devices as described in US 10,773,194.
  • in-line in the context of a process step refers to a process step that is integrated into or combined with one or more other process steps, or that flows directly from or to another process step without requiring manual intervention or handling.
  • purification of the enveloped virus comprises a purification step, e.g., anion exchange chromatography, to reduce overall volume and to separate viral vector from host cell DNA, proteins, and media components, and an ultrafiltration/diafiltration step to concentrate the viral vector into a final formulation buffer.
  • the downstream step further includes a sterile filtration step for removal of microorganisms from the final product.
  • the enveloped virus is purified from the cell culture comprising one or more steps selected from the group consisting of anion exchange chromatography, concentration and diafiltration.
  • the enveloped virus is purified using anion exchange.
  • the anion exchange is performed in bind-elute mode.
  • the enveloped virus binds to the anion exchanger while contaminants flow through.
  • the virus is subsequently eluted from the anion exchanger. Performing anion exchange in this manner reduces the volume of liquid in which the virus is suspended and removes contaminants such as host cell DNA, host cell proteins, and medium components like fetal bovine serum.
  • anion exchangers will be apparent to the skilled artisan.
  • Exemplary anion exchangers are a column comprising a resin or a membrane or another suitable substrate.
  • the anion exchanger is a weak anion exchanger, e.g., comprising an ion exchange group selected from a diethylaminoethyl (DEAE) or aminoethyl group.
  • a weak anion exchanger e.g., comprising an ion exchange group selected from a diethylaminoethyl (DEAE) or aminoethyl group.
  • the anion exchanger is a strong anion exchanger, e.g., comprising an ion exchange group selected from a quaternary ammonium (Q), diethyl- 2-hydroxypropylaminoethyl (QAE), triethylaminoethyl (TEAE), or trimethyl aminoethyl group.
  • exemplary anion exchangers include MUSTANG® E, MUSTANG® Q, SARTOBIND® Q, CHROMASORB®, POSSIDYNE®, CAPTO® Q, QSFF, POROS® Q, FRACTOGEL® Q, NATRIX® Q.
  • the anion exchanger comprises a Q ion exchange group.
  • the anion exchanger is a membrane anion exchanger comprising a Q ion exchange group.
  • the anion exchanger is MUSTANG® Q.
  • an enveloped virus eluted from anion exchange column is further purified on the basis of its size.
  • the buffer in which virus was eluted from the anion exchange column is exchanged more or less at the same time.
  • tangential flow filtration is preferred. This method permits impurity removal and buffer exchange at almost the same time.
  • Tangential flow ultrafiltration/diafiltration is a method which may be used to remove residual protein and nucleic acids as well as for exchanging working buffer into a final formulation buffer. Ultrafiltration using tangential flow is preferred and different devices can be used (e.g. Proflux and LAB SCALE (ultrafiltration system) TFF System, both Millipore or the KR2i system from Repligen).
  • the particular ultrafiltration membrane selected will be of a filter pore size sufficient small to retain enveloped virus but large enough to allow penetration of impurities.
  • nominal molecular weight cut-offs between 100 and 1000 kDa may be appropriate (e.g. UFP-750-E-5A, GE Healthcare; BIO MAX (ultrafiltration device) NMWC 1000, Millipore). In one example, the molecular weight cut-off is 500kDa.
  • the membrane composition may be, but it is not limited to, regenerate cellulose, (modified) polyethersulfone, polysulfone.
  • Membranes can be of flat sheet or hollow fibre type.
  • the main parameters that must be optimized are flux rate and trans-membrane pressure. In combination with nominal molecular weight cut-off these two parameters will enable efficient purification and buffer exchange and high virus yield.
  • sterile filtration may be performed to eliminate bioburden. Therefore diluted eluate or final retentate from the ultrafiltration step may be filtered through a filter, for example a 0.22 pm filter.
  • the filter may be constructed from various materials, which may include but are not limited to polypropylene, hydrophilic PVDF, cellulose, hydrophilic regenerated cellulose, cellulose esters, wetting agent-free cellulose acetate, cellulose acetate, nylon, hydrophilic nylon membrane, polyethersulfone, hydrophilic polyethersulfone, hydrophilic asymmetric PES, or any other material which is consistent with low unspecific influenza virus binding.
  • the filter may have a single membrane layer or more than one layer or may incorporate a prefilter of the same or different material, for example a 0.45 pm prefilter. The sterile filtrated virus can be held frozen for subsequent manipulation.
  • a method of producing an enveloped virus in a suspension perfusion cell culture comprising:
  • a method of producing an enveloped virus in a suspension perfusion cell culture comprising:
  • a method of producing an enveloped virus in a suspension perfusion cell culture comprising:
  • the harvest rate is about 1 VVD from day 0 to about day 5, about 2 VVD from about day 5 and about 3VDD from about day 6 to the end of culture;
  • the recirculation rate is about 2 VVD from day 0 to about day 5, about 4 VVD from about day 5 and about 6VDD from about day 6 to the end of culture.
  • the method further comprises performing a suspension cell line cell bleed.
  • purifying the enveloped virus comprises one or more steps selected from the group consisting of anion exchange chromatography, concentration and diafiltration.
  • a Tet-off inducible polyclonal GPRTGs suspension producer cell line expressing a WAS-T2A-GFP LV upon induction was cultivated in TransFx-H medium (Cytiva) supplemented with 6 mM GlutaMAX (ThermoFisher), 0.1% poloxamer 188 (Merck), 5% Cell Boost 5 (Cytiva) and 100 pL L 1 Antidumping Agent (Thermo Fisher), hereafter called complete TransFx-H medium.
  • Cell cultivation was performed in shake flasks at a maximum relative working volume of 32% using a shaker incubator set at 37°C, 5% CO2 and 70% relative humidity.
  • VCD viable cell density
  • a 24-way Ambr® 250 high throughput bioreactor system (Sartorius) was used to investigate the effect of higher perfusion rates on LV production.
  • Each vessel was equipped with an APS -2402 system (SonoSep Technologies) consisting of a 1 mL acoustic separation chamber (ASC) and a controller, enabling LV production in perfusion mode by acoustic wave mediated cell separation (Figure 1).
  • ASC acoustic separation chamber
  • the bioreactors were operated at a stirring speed of 250 rotations per minute (rpm), a temperature of 37°C, a DO level of 50% and a pH of 6.95 ⁇ 0.15 for cell cultivation. CO2 was sparged to control the upper pH limit.
  • the overlay was set to a fixed air volume flow of 1 mL min 1 .
  • the vessels were preconditioned with complete doxycycline-free TransFx-H medium. All vessels were inoculated at a VCD of 1.5 x 10 6 cells mL 1 at a final working volume of 200 mL. 200 pL of EX-CELL® antifoam (Sigma-Aldrich) were added per vessel after inoculation using the liquid handler.
  • perfusion was initiated at a harvest rate of 1 VVD for all vessels and 200 pL of antifoam was regularly added in an interval of 6 hours.
  • the liquid levels were controlled manually by adjusting the flow rates of the harvest pumps.
  • the harvest rate was increased to 2 VVD at 5 days post induction (dpi) and to 3 VVD at 6 dpi ( Figure 2A).
  • the cell densities were controlled by bolus removal of cell suspension in an interval of 6 hours using the liquid handler and subsequent bolus media addition to reach the initial bioreactor volume.
  • the bioreactors were sampled at 0 dpi and daily starting from 3 dpi by removing 7 mL of cell suspension using the liquid handler. 100 pL of the cell suspension were used to perform a cell count. The remaining volume was centrifuged for 5 minutes at 336 g and the supernatant frozen at -80°C in 1 mL aliquots for quantification of infectious titers and metabolites. Lactate and glucose concentrations in the supernatant were determined using EPOC Blood Analysis System (Siemens Healthcare).
  • a Biostat B-DCU system (Sartorius) was used for LV productions at bench-top bioreactor scale ( Figure 1). All cultivations were performed at a temperature of 37°C, a DO level of 50% and a pH of 6.95 ⁇ 0.15. CO2 was sparged to control the upper pH limit.
  • the bioreactors were equipped with an APS-107 system (SonoSep Technologies) consisting of a controller (part number: SC-107) and a 30 mL ASC (part number: SS- 30), enabling LV production in perfusion mode by acoustic wave mediated cell separation.
  • the investigation of a harvest rate of 1 VVD was performed in a 5 L glass bioreactor (Sartorius) at a final working volume of 4.5 L.
  • Cultivation was performed at a stirring speed of 100-140 rpm, and the overlay was set to a fixed air volume flow of 0.2 L min 1 .
  • the investigation of a harvest rate of 3 VVD was performed in a 2 L glass bioreactor (Sartorius) at a final working volume of 1.5- 1.8 L.
  • Cultivation was performed at a stirring speed of 114-160 rpm, and the overlay was set to a fixed air volume flow of 0.08 L min 1 .
  • Bioreactor run 1 was performed using complete TransFx-H medium.
  • a PharMed® BPT tubing size 16 (Saint-Gobain) was used as a pump tubing for cell recirculation.
  • complete TransFx-H medium was additionally supplemented with a final concentration of 0.4% cholesterol lipid concentrate (Thermo Fisher), and the poloxamer 188 concentration was increased to 0.5%.
  • a PharmaPure® low spallation pump tubing size 17 (Saint-Gobain) was used for cell recirculation.
  • the bioreactors were preconditioned with doxycycline-free medium and inoculated at a VCD of 1.5 x 10 6 cells mL 1 . After cultivation in batch mode for two days, perfusion was initiated at a harvest rate of approximately 1 VVD and the harvest was continuously collected at 4°C. For the 2 L bioreactors, the harvest rate was gradually increased to 3 VVD ( Figure 3A and 4A).
  • the VCD was controlled by a continuous cell bleed based on the daily determined VCD. EX-CELL® antifoam was added manually on demand.
  • the bioreactors were sampled daily and the cell suspension was centrifuged for 5 minutes at 336 g.
  • the supernatant was separated from the cell pellet and frozen at - 80°C in 1 mL aliquots for quantification of infectious titers and metabolites. Lactate and glucose concentrations in the supernatant were determined using EPOC Blood Analysis System (Siemens Healthcare).
  • LV containing supernatant was collected from induced cell suspensions cultivated in stirred-tank bioreactors after centrifugation for 5 minutes at 336 g.
  • the supernatants were aliquoted into 1.5 mL screwed-cap sample tubes (VWR), incubated at 4°C or 37°C and frozen at -80°C at different time points for infectious titer determination. Aliquots that were incubated at 4°C were frozen after 0 h, 6 h, 21 h, 70 h and 142 h, respectively. Aliquots that were incubated at 37°C were frozen after 0 h, 2 h, 4 h, 6 h and 21 h, respectively.
  • Adherent HEK293T/17 cells were cultivated in DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin-Streptomycin (all Thermo Fisher) at 5% CO2 using a static incubator (Thermo HERAcell 250i).
  • FBS fetal bovine serum
  • Penicillin-Streptomycin all Thermo Fisher
  • TU transducing units
  • Virus containing supernatants were thawed quickly and fivefold serial dilutions were performed in duplicates using transduction medium, starting from a 50-fold dilution. 20 pL of each dilution was added to seeded cells resulting in a final volume of 100 pL per well. Tracking controls were included by diluting a GFP LV preparation with a known virus concentration 25,000-fold using transduction medium. Negative controls were included by adding 20 pL DMEM medium with 10% FBS instead of virus containing supernatants.
  • the percentage of GFP + cells was determined by analysing 10,000 events using MACSQuant® Analyzer 16 Flow Cytometer (Miltenyi Biotec) and infectious titers were calculated in TU mL 1 using dilutions that led to a proportion of 5% to 30% GFP + cells in the sample.
  • LV RNA concentration was determined by ddPCR using QX200TM Droplet Digital PCR (Bio-Rad Laboratories) according to manufacturer’s instructions.
  • PCR was performed under the following thermocycling conditions: enzyme activation at 95°C for 10 minutes, 40 cycles of denaturation at 94°C for 30 seconds and annealing/extension at 60°C for 1 minute, and final enzyme deactivation at 98°C for 10 minutes.
  • positive and negative droplets were counted using QX200TM Droplet Reader and QuantaSoft software (BioRad).
  • the calculated WAS-T2A-GFP LV copy number was corrected using the normalization factor calculated by the calculated P-globin LV copy number.
  • primer-probe sets were used to amplify the GFP gene and the P- globin gene (rGbG) as a control for normalization (GFP: fwd 5’- CTGCTGCCCGACAACCA-3’, rev 5’-TGTGATCGCGCTTCTCGTT-3’ and probe 5’- HEX-TACCTGAGCACCCAGTCCGCCCT-3’; rGbG: fwd 5’
  • Determination of lentivirus-associated HIV p24 core protein was performed using the commercially available QuickTiterTM Lentivirus Titer Kit (Cell Biolabs) according to manufacturer’s instructions.
  • Total DNA was determined using the QubitTM double- stranded (ds) DNA Assay- Kit (Thermo Fisher). Samples and controls were diluted under light protection using the HS buffer and reagent dilutions supplied with the kit. Samples were incubated for a maximum of 15 minutes and read using a SPARK® microplate reader (Tecan) at 485 nm excitation and 530 nm emission.
  • ds QubitTM double- stranded
  • HS buffer and reagent dilutions supplied with the kit. Samples were incubated for a maximum of 15 minutes and read using a SPARK® microplate reader (Tecan) at 485 nm excitation and 530 nm emission.
  • the cell specific virus yield Q v ,k is defined as the total lentivirus amount produced per cell at a given time point tk:
  • the total virus yield produced was normalized per reactor volume to allow a comparison between different bioreactor sizes.
  • the arithmetic mean of infectious titers measured in the bioreactor was used for an approximation:
  • VY v volumetric yield or yield per reactor volume [TU mLBioreactor’ 1 ], V Bioreactor — working volume bioreactor [mL]
  • VSV-G pseudotyped LVs have a temperature-dependent finite half-life, measurable by gradual loss of infectivity over time, that can be approximated using an exponential decay function.
  • LVs carrying a WAS-T2A-GFP construct produced using GPRTGs derived stable producer cells in stirred-tank bioreactors a half-life of 153 hours at 4°C and a half-life of 6 hours at 37°C in the unprocessed cell-free harvest matrix was determined (Figure 5).
  • a novel small-scale perfusion system based on acoustic wave separation that can be connected to the Ambr® 250 high throughput bioreactor system was employed (Figure 1).
  • the capacity of the system is mainly determined by 1) the cell density in the bioreactor and 2) the target harvest rate. If the cell densities or harvest rates are increasing, the volume of concentrated cell suspension in the ASC is increasing more quickly, requiring more frequent back-flush cycles.
  • the cell viability profile seems to be comparable between a process with a harvest rate of 1 VVD and 3 VVD, reaching average cell viabilities of 80.5 + 2.8% and 79.0 + 2.1% at 11 dpi, respectively ( Figure 2C).
  • Figure 2E shows that the mean cell specific virus productivity is increasing for the 3 VVD process after 5 dpi, which marks the timepoint after increasing the harvest rate compared to the control process at 1 VVD ( Figure 2A).
  • the standard production process was performed at a constant harvest rate of 1 VVD.
  • the VCD profile of the 1 VVD and 3 VVD process seem comparable until 6 dpi, and a cell bleed was applied for the 1 VVD process to regulate the VCD at approximately 2 x 10 7 cells mL" 1 .
  • the VCD reached a plateau at 2.84 x 10 7 cells mL' 1 at 7 dpi ( Figure 3B).
  • the plateau phase was observed until 9 dpi, followed by a constant VCD decline, visible aggregate formation in the bioreactor and the presence of majorly dead cells in the harvest. Therefore, the 3 VVD process was stopped at 14 dpi.
  • the final volumetric yields were 8.53 x 10 8 TU mLs ioreactor" 1 at 20 dpi for the 1 VVD process and 6.05 x 10 8 TU mLBioreactor’ 1 at 14 dpi for the 3 VVD process.
  • the cumulative cell specific virus yield was following a similar trend like the cumulative yield per bioreactor volume, with final values of 29.7 TU cell" 1 at 14 dpi for the 1 VVD process and 53.4 TU cell" 1 at 20 dpi for the 3 VVD process ( Figure 3G).
  • the final cumulative yield at 3 VVD was 3.1 -fold higher, reaching a value of 3.31 x 10 9 TU IULB ioreactor 1 compared to 1.08 x 10 9 TU mLs ioreactor" 1 at 1 VVD ( Figure 4E).
  • the higher functional virus yields obtained by the higher perfusion rate were also verified by determining the titers in the collected harvest.
  • Cell specific productivities were higher at a harvest rate of 3 VVD except for the last day of the ferment ( Figure 4F). Highest cell specific productivities were obtained at 14 dpi in both bioreactors, showing values of 7.7 TU cell" 1 day" 1 at 1 VVD and 11.1 TU cell" 1 day” 1 at 3 VVD.
  • the final cumulative cell-specific virus yield was 61.9 TU cell" 1 at 1 VVD and 89.1 TU cell” 1 at 3 VVD ( Figure 4G). Higher cell specific productivities of RNA vector copies were obtained at 3 VVD throughout the process ( Figure 4H), resulting in a 3.0-fold higher final cumulative yield of 5.02 x 10 12 RNA copies mLsioreactor’ 1 compared to 1.66 x 10 12 RNA copies mLsioreactor’ 1 at 1 VVD.
  • the ratio of infectious particles to the capsid protein p24 was determined at elected time points as an indicator for LV quality ( Figure 6B). When comparing the ratios at 10 and 14 dpi, where the target harvest rates were reached, higher values of 2.23 x 10 4 TU ng P 24 -1 and 3.41 x 10 4 TU ng P 24 -1 were obtained at 3 VVD compared to 1.13 x 10 4 TU ng P 24 -1 and 2.25 x 10 4 TU ng P 24 -1 at 1 VVD.

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Abstract

La présente divulgation concerne de manière générale la fabrication de produits de thérapie génique et, spécifiquement, des procédés de production d'un virus enveloppé dans une culture cellulaire de perfusion en suspension comprenant le filtrage du fluide de culture cellulaire dans un filtre à un taux de récolte d'au moins 2 volumes de récipient par jour (VVD) pour produire un fluide de culture cellulaire filtré comprenant le virus enveloppé, le filtre retenant les cellules de la lignée cellulaire en suspension.
PCT/IB2025/054027 2024-04-19 2025-04-17 Procédés de production d'un virus enveloppé Pending WO2025219920A1 (fr)

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US20180298323A1 (en) * 2012-03-15 2018-10-18 Flodesign Sonics, Inc. Acoustic perfusion devices

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Publication number Priority date Publication date Assignee Title
US20180298323A1 (en) * 2012-03-15 2018-10-18 Flodesign Sonics, Inc. Acoustic perfusion devices

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Title
MAXIMILIAN KLIMPEL; BEATRICE PFLÜGER‐MÜLLER; MARTA ARRIZABALAGA CASCALLANA; SARAH SCHWINGAL; NIKKI INDRESH LAL; THOMAS NOLL; VICKY: "Perfusion Process Intensification for Lentivirus Production Using a Novel Scale‐Down Model", BIOTECHNOLOGY AND BIOENGINEERING, JOHN WILEY, HOBOKEN, USA, vol. 122, no. 2, 13 November 2024 (2024-11-13), Hoboken, USA, pages 344 - 360, XP072794281, ISSN: 0006-3592, DOI: 10.1002/bit.28880 *
RICHELLE ANNE, CORBETT BRANDON, AGARWAL PIYUSH, VERNERSSON ANTON, TRYGG JOHAN, MCCREADY CHRIS: "Model-based intensification of CHO cell cultures: One-step strategy from fed-batch to perfusion", FRONTIERS IN BIOENGINEERING AND BIOTECHNOLOGY, FRONTIERS RESEARCH FOUNDATION, CH, vol. 10, CH , XP093367309, ISSN: 2296-4185, DOI: 10.3389/fbioe.2022.948905 *
WU ET AL.: "High cell density perfusion process for high yield of influenza A virus production using MDCK suspension cells", APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 30 January 2021 (2021-01-30), pages 1421 - 1434, XP038162805, DOI: 10.1007/s00253-020-11050- 8 *

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