Methods of producing an enveloped virus

Abstract

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 5 fluid comprising the enveloped virus, wherein the filter retains cells from the suspension cell line.

Description

[0001] METHODS OF PRODUCING AN ENVELOPED VIRUS

[0002] RELATED APPLICATION DATA

[0003] The present application claims priority from United States Patent Application No. 63 / 636,283 filed 19 April 2024 entitled “Methods of producing an enveloped virus”. The entire contents of this application are hereby incorporated by reference.

[0004] SEQUENCE LISTING

[0005] The present application is filed together with a Sequence Listing in electronic form. The entire contents of the Sequence Listing is hereby incorporated by reference.

[0006] FIELD

[0007] 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.

[0008] BACKGROUND

[0009] 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.

[0010] Lentiviruses are genetically engineered and usually based on human immunodeficiency virus 1 (HIV-1). To increase safety, modern vectors contain only those HIV genes which are necessary for infection and gene delivery, but the genes necessary for replication and virulence factors have been removed. Often, the envelope protein of HIV-1 is exchanged with that of another virus to allow infection of a wide range of target cells, e.g., VSV-G protein from Vesicular stomatitis Indiana virus (VSV).

[0011] 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.

[0012] Once the virus production has been induced, the release of the virus occurs by budding after successful assembly within the cells. 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.

[0013] The costs for lentiviral therapies are limiting their accessibility, with manufacturing costs being a contributing factor due to established production methods like transient transfection and only moderate process yields.

[0014] Therefore, the skilled person will appreciate from the foregoing that there is a need in the art for improved methods of virus production.

[0015] SUMMARY

[0016] In work leading up to the present invention, the inventors sought to produce a method of producing enveloped viruses, e.g., for gene therapy, at commercial scale and suitable for regulatory requirements.

[0017] 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. In developing the methods of the disclosure, 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. In addition, 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.

[0018] Thus, the findings by the inventors provide methods of producing enveloped viruses.

[0019] The present disclosure provides a method of producing an enveloped virus in a suspension perfusion cell culture, the method comprising:

[0020] (i) culturing a suspension cell line in a cell culture medium to produce a cell culture fluid comprising the enveloped virus; and (ii) filtering the cell culture fluid through a filter at a harvest rate of at least 2 vessel volume per day (VVD) to produce a filtered cell culture fluid comprising the enveloped virus, wherein the filter retains cells from the suspension cell line.

[0021] In one example, the method further comprises recirculating cell culture fluid through the filter to return the retained suspension cell line cells into the suspension cell culture.

[0022] The present disclosure further provides a method of producing an enveloped virus in a suspension perfusion cell culture, the method comprising:

[0023] (i) culturing a suspension cell line in a cell culture medium to produce a cell culture fluid comprising the enveloped virus;

[0024] (ii) filtering the cell culture fluid through a filter at a harvest rate of at least 1 vessel volume per day (VVD) to produce a filtered cell culture fluid comprising the enveloped virus, wherein the filter retains cells from the suspension cell line, and

[0025] (iii)recirculating cell culture fluid through the filter to return the retained suspension cell line cells into the suspension cell culture.

[0026] In one example, the harvest rate is greater than about 1 VVD. For example, the harvest rate is between about 1 and 10 VVD. In one example, 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. For example, the harvest rate is 2 VVD. In another example, the harvest rate is 3 VVD.

[0027] In one example, 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.

[0028] In one example, the method comprises recirculating the retained suspension cell line cells into the suspension cell culture at a recirculation rate greater than the harvest rate. For example, 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. In one example, 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 present disclosure provides a method of producing an enveloped virus in a suspension perfusion cell culture, the method comprising:

[0029] (i) culturing a suspension cell line in a cell culture medium to produce a cell culture fluid comprising the enveloped virus;

[0030] (ii) filtering the cell culture fluid using an acoustic standing wave to produce a filtered cell culture fluid comprising the enveloped virus, wherein the filter retains cells from the suspension cell line, and

[0031] (iii)recirculating cell culture fluid through the filter to return the retained suspension cell line cells into the suspension cell culture, wherein the harvest filtration is operated at a harvest rate of greater than 1 VVD; and the recirculation is operated at a recirculation rate of 2 times the harvest rate.

[0032] In one example of any method described herein, 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.

[0033] In one example, 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.

[0034] It will be apparent to the skilled person that the tetracycline-suppressible gene expression system is also known as a Tet-Off expression system.

[0035] In one example, 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.

[0036] In one example, 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.

[0037] 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 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. For example, 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. In one example, 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.

[0038] 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 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. For example, 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.

[0039] In one example, 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.

[0040] In one example, the harvest rate is about 1 VVD from day 0 to about day 5 following induction of enveloped virus production. For example, the harvest rate is about 2 VVD from about day 5 following induction of enveloped virus production. In a further example, the harvest rate is about 3 VVD from about day 6 following induction of enveloped virus production.

[0041] In one example, the recirculation rate is between about 2 VVD and 10 VVD. For example, the recirculation rate is about 2 VVD, or about 4 VVD, or about 6 VVD. In one example, the recirculation rate is 6 VVD.

[0042] In one example, 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.

[0043] In one example, following induction of enveloped virus production:

[0044] (i) 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; and

[0045] (ii)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.

[0046] In one example, the method further comprises performing a suspension cell line cell bleed. For example, the suspension cell line cell bleed is performed at a rate of 0.1 VVD to 0.5 VVD. In one example, the suspension cell line cell bleed is performed at a rate of 0.1 VVD. In another example, the suspension cell line cell bleed is performed at a rate of 0.2 VVD. In a further example, the suspension cell line cell bleed is performed at a rate of 0.3 VVD. In one example, the suspension cell line cell bleed is performed at a rate of 0.4 VVD. In another example, the suspension cell line cell bleed is performed at a rate of 0.5 VVD.

[0047] In one example, 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.

[0048] In one example, 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%.

[0049] In one example, the suspension cell line cell bleed is performed to maintain a target cell density of at least about 1 x 107cells / mL of cell culture fluid. For example, the suspension cell line cell bleed is performed to maintain a target cell density of between about 1 x 107cells / mL and 5 x 107cells / mL of cell culture fluid. In one example, the suspension cell line cell bleed is performed to maintain a target cell density of about 1 x 107cells / mL of cell culture fluid. In one example, the suspension cell line cell bleed is performed to maintain a target cell density of about 2 x 107cells / mL of cell culture fluid. In one example, the suspension cell line cell bleed is performed to maintain a target cell density of about 3 x 107cells / mL of cell culture fluid. In one example, the suspension cell line cell bleed is performed to maintain a target cell density of about 4 x 107cells / 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 107cells / mL of cell culture fluid.

[0050] In exemplary forms of the disclosure, 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.

[0051] In one example, the suspension cell line is initially seeded in the cell culture medium at a density of between about 1 x 105cells / mL and 1 x 1010cells / mL of cell culture medium. For example, the suspension cell line is initially seeded in the cell culture medium at a density of between about 1 x 105cells / mL and 1 x IO10cells / mL, or 1 x 105cells / mL and 1 x 107cells / mL, or about 0.1 x 106cells / mL and 1 x 108cells / mL, or about 0.5 x 106cells / mL and 1 x 107cells / mL, or about 0.5 x 106cells / mL and 5 x 106cells / mL, or about 0.5 x 106cells / mL and 2.5 x 106cells / mL of cell culture medium. In one example, the suspension cell line is initially seeded in the cell culture medium at a density of 1 x 105cells / mL and 1 x 107cells / mL In one example, the suspension cell line is initially seeded in the cell culture medium at a density of 0.5 x 106cells / mL to 5.0 x 106cells / mL. In one example, the suspension cell line is initially seeded in the cell culture medium at a density of between 0.8 x 106cells / mL and 1.2 x 106. In one example, the suspension cell line is initially seeded in the cell culture medium at a density of between 1 x 106cells / mL and 2.5 x 106. In one example, the suspension cell line is initially seeded in the cell culture medium at a density of between 1.5 x 106cells / mL and 2 x 106. In one example, the suspension cell line is initially seeded in the cell culture medium at a density of about 1 x 105cells / mL, or about 2 x 105cells / mL, or about 3 x 105cells / mL, or about 4 x 105cells / mL, or about 5 x 105cells / mL, or about 6 x 105cells / mL, or about 7 x 105cells / mL, or about 8 x 105cells / mL, or about 9 x 105cells / mL, or about 10 x 105cells / mL of cell culture medium. In one example, the suspension cell line is initially seeded in the cell culture medium at a density of about 1 x 106cells / mL, or about 2 x 106cells / mL, or about 3 x 106cells / mL, or about 4 x 106cells / mL, or about 5 x 106cells / mL, or about 6 x 106cells / mL, or about 7 x 106cells / mL, or about 8 x 106cells / mL, or about 9 x 106cells / mL, or about 10 x 106cells / mL of cell culture medium. In one example, the suspension cell line is initially seeded in the cell culture medium at a density of about 0.5 x 106cells / mL. In one example, the suspension cell line is initially seeded in the cell culture medium at a density of about 1 x 106cells / mL. In one example, the suspension cell line is initially seeded in the cell culture medium at a density of about 1.5 x 106cells / mL. In one example, the suspension cell line is initially seeded in the cell culture medium at a density of about 1.8 x 106cells / mL. In one example, the suspension cell line is initially seeded in the cell culture medium at a density of about 2 x 106cells / mL. In one example, the suspension cell line is initially seeded in the cell culture medium at a density of 2.5 x 106cells / mL. In one example, the suspension cell line is initially seeded in the cell culture medium at a density of 3.0 x 106cells / mL. In one example, the suspension cell line is initially seeded in the cell culture medium at a density of 3.5 x 106cells / mL. In one example, the suspension cell line is initially seeded in the cell culture medium at a density of 4.0 x 106cells / mL. In one example, the suspension cell line is initially seeded in the cell culture medium at a density of 4.5 x 106cells / mL. In one example, the suspension cell line is initially seeded in the cell culture medium at a density of 5.0 x 106cells / mL. In one example, the suspension cell line is initially seeded in the cell culture medium at a density of about 1 x 107cells / mL, or about 2 x 107cells / mL, or about 3 x 107cells / mL, or about 4 x 107cells / mL, or about 5 x 107cells / mL, or about 6 x 107cells / mL, or about 7 x 107cells / mL, or about 8 x 107cells / mL, or about 9 x 107cells / mL, or about 10 x 107cells / mL of cell culture medium. In one example, the suspension cell line is initially seeded in the cell culture medium at a density of about 1 x 108cells / mL, or about 2 x 108cells / mL, or about 3 x 108cells / mL, or about 4 x 108cells / mL, or about 5 x 108cells / mL, or about 6 x 108cells / mL, or about 7 x 108cells / mL, or about 8 x 108cells / mL, or about 9 x 108cells / mL, or about 10 x 108cells / mL of cell culture medium. In one example, the suspension cell line is initially seeded in the cell culture medium at a density of about 1 x 109cells / mL, or about 2 x 109cells / mL, or about 3 x 109cells / mL, or about 4 x 109cells / mL, or about 5 x 109cells / mL, or about 6 x 109cells / mL, or about 7 x 109cells / mL, or about 8 x 109cells / mL, or about 9 x 109cells / mL, or about 10 x 109cells / mL of cell culture medium.

[0052] In one example, the suspension cell line is grown to a viable cell density of between about 1 x 105cells / mL to about 1 x 1010cells / mL of cell culture medium. For example, the suspension cell line is grown to a viable cell density of between about 1 x

[0053] 105cells / mL and 1 x 1010cells / mL, or about 0.1 x 106cells / mL and 1 x 108cells / mL, or about 0.5 x 106cells / mL and 1 x 107cells / mL, or about 0.5 x 106cells / mL and 5 x 106cells / mL, or about 0.5 x 106cells / mL and 2.5 x 106cells / mL of cell culture medium. In one example, the suspension cell line is grown to a viable cell density of between about

[0054] 1 x 106cells / mL to about 1 x 107of cell culture medium. In another example, the suspension cell line is grown to a viable cell density of between about 6 x 106cells / mL to about 1 x 107of cell culture medium. In one example, the suspension cell line is grown to a viable cell density of between about 0.5 x 106cells / mL to 5.0 x 106cells / mL. In one example, the suspension cell line is grown to a viable cell density of between about 1 x

[0055] 106cells / mL and 2.5 x 106. In one example, the suspension cell line is grown to a viable cell density of between about 1.5 x 106cells / mL and 2 x 106. In one example, the suspension cell line is grown to a viable cell density of about 1 x 105cells / mL, or about

[0056] 2 x 105cells / mL, or about 3 x 105cells / mL, or about 4 x 105cells / mL, or about 5 x 105cells / mL, or about 6 x 105cells / mL, or about 7 x 105cells / mL, or about 8 x 105cells / mL, or about 9 x 105cells / mL, or about 10 x 105cells / mL of cell culture medium. In one example, the suspension cell line is grown to a viable cell density of about 1 x 106cells / mL, or about 2 x 106cells / mL, or about 3 x 106cells / mL, or about 4 x 106cells / mL, or about 5 x 106cells / mL, or about 6 x 106cells / mL, or about 7 x 106cells / mL, or about 8 x 106cells / mL, or about 9 x 106cells / mL, or about 10 x 106cells / mL of cell culture medium. In one example, the suspension cell line is grown to a viable cell density of about 0.5 x 106cells / mL. In one example, the suspension cell line is grown to a viable cell density of about 1 x 106cells / mL. In one example, the suspension cell line is grown to a viable cell density of about 1.5 x 106cells / mL. In one example, the suspension cell line is grown to a viable cell density of about 1.8 x 106cells / mL. In one example, the suspension cell line is grown to a viable cell density of about 2 x 106cells / mL. In one example, the suspension cell line is grown to a viable cell density of about 2.5 x 106cells / mL. In one example, the suspension cell line is grown to a viable cell density of about 3.0 x 106cells / mL. In one example, the suspension cell line is grown to a viable cell density of about 3.5 x 106cells / mL. In one example, the suspension cell line is grown to a viable cell density of about 4.0 x 106cells / mL. In one example, the suspension cell line is grown to a viable cell density of about 4.5 x 106cells / mL. In one example, the suspension cell line is grown to a viable cell density of about 5.0 x 106cells / mL. In one example, the suspension cell line is grown to a viable cell density of about 1 x 107cells / mL, or about 2 x 107cells / mL, or about 3 x 107cells / mL, or about 4 x 107cells / mL, or about 5 x 107cells / mL, or about 6 x 107cells / mL, or about 7 x 107cells / mL, or about 8 x 107cells / mL, or about 9 x 107cells / mL, or about 10 x 107cells / mL of cell culture medium. In one example, the suspension cell line is grown to a viable cell density of about 1 x 108cells / mL, or about 2 x 108cells / mL, or about 3 x 108cells / mL, or about 4 x 108cells / mL, or about 5 x 108cells / mL, or about 6 x 108cells / mL, or about 7 x 108cells / mL, or about 8 x 108cells / mL, or about 9 x 108cells / mL, or about 10 x 108cells / mL of cell culture medium. In one example, the suspension cell line is grown to a viable cell density of about 1 x 109cells / mL, or about 2 x 109cells / mL, or about 3 x 109cells / mL, or about 4 x 109cells / mL, or about 5 x 109cells / mL, or about 6 x 109cells / mL, or about 7 x 109cells / mL, or about 8 x 109cells / mL, or about 9 x 109cells / mL, or about 10 x 109cells / mL of cell culture medium.

[0057] In one example of any method described herein, the method results in a viral infectious titer yield of at least 1 x 105transducing units (TU) / mL. For example, the method results in a viral infectious titer yield of between about 1 x 105TU / mL and 1 x 1010TU / mL. In one example, the method results in a viral infectious titer yield of about 1 x 105TU / mL, or about 1.25 x 105TU / mL, or about 1.5 x 105TU / mL, or at least 1.75 x 105TU / mL, or at least 2 x 105TU / mL, or at least 2.5 x 105TU / mL, or at least 3 x 105TU / mL, or about 3.5 x 105TU / mL, or about 4 x 105TU / mL, or about 5 x 105TU / mL. In one example, the method results in a viral infectious titer yield of about 6 x 105TU / mL, or about 7 x 105TU / mL, or at least 8 x 105TU / mL, or at least 9 x 105TU / mL, or at least 10 x 105TU / mL. In another example, the method results in a viral infectious titer yield of about 1 x 106TU / mL, or about 1.5 x 106TU / mL, or about 2 x 106TU / mL, or about 5 x 106TU / mL, or about 7 x 106TU / mL, or about 10 x 106TU / mL. In a further example, the method results in a viral infectious titer yield of about 1 x 107TU / mL, or about 5 x 107TU / mL, or about 10 x 108TU / mL, or about 5 x 108TU / mL, or about 10 x 108TU / mL, or about 5 x 109TU / mL, or about 1 x 1010TU / mL.

[0058] In one example, the method results in a viral infectious titer yield of at least 1 x

[0059] 105transducing units (TU) / mL of culture medium at day 3 of culture. For example, the method results in a viral infectious titer yield of at least 1 x 106TU / mL of culture medium at day 3 of culture. For example, the method results in a viral infectious titer yield of about 1.5 x 106TU / mL, or about 2 x 106TU / mL, or about 5 x 106TU / mL, or about 7 x

[0060] 106TU / mL, or about 10 x 106TU / mL of culture medium at day 3 of culture. In another example, the method results in a viral infectious titer yield of at least 1 x 107TU / mL of culture medium at day 3 of culture. For example, the method results in a viral infectious titer yield of about 1.1 x 107TU / mL, or about 1.2 x 107TU / mL, or about 1.3 x 107TU / mL, or about 1.4 x 107TU / mL, or about 1.5 x 107TU / mL of culture medium at day 3 of culture. In one example, the method results in a viral infectious titer yield of at least 1.5 x 107TU / mL of culture medium at day 3 of culture. For example, the method results in a viral infectious titer yield of about 1.6 x 107TU / mL, or about 1.7 x 107TU / mL, or about 1.8 x 107TU / mL, or about 1.9 x 107TU / mL of culture medium at day 3 of culture. In one example, the method results in a viral infectious titer yield of at least 2 x 107TU / mL of culture medium at day 3 of culture. For example, the method results in a viral infectious titer yield of about 2.1 x 107TU / mL, or about 2.2 x 107TU / mL, or about 2.3 x 107TU / mL, or about 2.4 x 107TU / mL, or about 2.5 x 107TU / mL, or about 2.6 x 107TU / mL, or about 2.7 x 107TU / mL, or about 2.8 x 107TU / mL, or about 2.9 x 107TU / mL of culture medium at day 3 of culture.

[0061] In one example, the method results in a viral infectious titer yield of at least 1 x

[0062] 105transducing units (TU) / mL of culture medium at day 5 of culture. For example, the method results in a viral infectious titer yield of at least 1 x 106TU / mL of culture medium at day 5 of culture. For example, the method results in a viral infectious titer yield of about 1.5 x 106TU / mL, or about 2 x 106TU / mL, or about 5 x 106TU / mL, or about 7 x

[0063] 106TU / mL, or about 10 x 106TU / mL of culture medium at day 5 of culture. In another example, the method results in a viral infectious titer yield of at least 1 x 107TU / mL of culture medium at day 5 of culture. For example, the method results in a viral infectious titer yield of about 1.1 x 107TU / mL, or about 1.2 x 107TU / mL, or about 1.3 x 107TU / mL, or about 1.4 x 107TU / mL, or about 1.5 x 107TU / mL of culture medium at day 5 of culture. In one example, the method results in a viral infectious titer yield of at least 1.5 x 107TU / mL of culture medium at day 5 of culture. For example, the method results in a viral infectious titer yield of about 1.6 x 107TU / mL, or about 1.7 x 107TU / mL, or about 1.8 x 107TU / mL, or about 1.9 x 107TU / mL of culture medium at day 5 of culture. In one example, the method results in a viral infectious titer yield of at least 2 x 107TU / mL of culture medium at day 5 of culture. For example, the method results in a viral infectious titer yield of about 2.1 x 107TU / mL, or about 2.2 x 107TU / mL, or about 2.3 x 107TU / mL, or about 2.4 x 107TU / mL, or about 2.5 x 107TU / mL, or about 2.6 x 107TU / mL, or about 2.7 x 107TU / mL, or about 2.8 x 107TU / mL, or about 2.9 x 107TU / mL of culture medium at day 5 of culture.

[0064] In one example, the method results in a viral infectious titer yield of at least 1 x

[0065] 105transducing units (TU) / mL of culture medium at day 10 of culture. For example, the method results in a viral infectious titer yield of at least 1 x 106TU / mL of culture medium at day 10 of culture. For example, the method results in a viral infectious titer yield of about 1.5 x 106TU / mL, or about 2 x 106TU / mL, or about 5 x 106TU / mL, or about 7 x

[0066] 106TU / mL, or about 10 x 106TU / mL of culture medium at day 10 of culture. In another example, the method results in a viral infectious titer yield of at least 1 x 107TU / mL of culture medium at day 10 of culture. For example, the method results in a viral infectious titer yield of about 1.1 x 107TU / mL, or about 1.2 x 107TU / mL, or about 1.3 x 107TU / mL, or about 1.4 x 107TU / mL, or about 1.5 x 107TU / mL of culture medium at day 10 of culture. In one example, the method results in a viral infectious titer yield of at least 1.5 x 107TU / mL of culture medium at day 10 of culture. For example, the method results in a viral infectious titer yield of about 1.6 x 107TU / mL, or about 1.7 x 107TU / mL, or about 1.8 x 107TU / mL, or about 1.9 x 107TU / mL of culture medium at day 10 of culture. In one example, the method results in a viral infectious titer yield of at least 2 x 107TU / mL of culture medium at day 10 of culture. For example, the method results in a viral infectious titer yield of about 2.1 x 107TU / mL, or about 2.2 x 107TU / mL, or about 2.3 x 107TU / mL, or about 2.4 x 107TU / mL, or about 2.5 x 107TU / mL, or about

[0067] 2.6 x 107TU / mL, or about 2.7 x 107TU / mL, or about 2.8 x 107TU / mL, or about 2.9 x

[0068] 107TU / mL of culture medium at day 10 of culture.

[0069] In one example, the method results in a viral infectious titer yield of at least 1 x

[0070] 105transducing units (TU) / mL of culture medium at day 15 of culture. For example, the method results in a viral infectious titer yield of at least 1 x 106TU / mL of culture medium at day 15 of culture. For example, the method results in a viral infectious titer yield of about 1.5 x 106TU / mL, or about 2 x 106TU / mL, or about 5 x 106TU / mL, or about 7 x

[0071] 106TU / mL, or about 10 x 106TU / mL of culture medium at day 15 of culture. In another example, the method results in a viral infectious titer yield of at least 1 x 107TU / mL of culture medium at day 15 of culture. For example, the method results in a viral infectious titer yield of about 1.1 x 107TU / mL, or about 1.2 x 107TU / mL, or about 1.3 x 107TU / mL, or about 1.4 x 107TU / mL, or about 1.5 x 107TU / mL of culture medium at day 15 of culture. In one example, the method results in a viral infectious titer yield of at least 1.5 x 107TU / mL of culture medium at day 15 of culture. For example, the method results in a viral infectious titer yield of about 1.6 x 107TU / mL, or about 1.7 x 107TU / mL, or about 1.8 x 107TU / mL, or about 1.9 x 107TU / mL of culture medium at day 15 of culture. In one example, the method results in a viral infectious titer yield of at least 2 x 107TU / mL of culture medium at day 15 of culture. For example, the method results in a viral infectious titer yield of about 2.1 x 107TU / mL, or about 2.2 x 107TU / mL, or about 2.3 x 107TU / mL, or about 2.4 x 107TU / mL, or about 2.5 x 107TU / mL, or about 2.6 x 107TU / mL, or about 2.7 x 107TU / mL, or about 2.8 x 107TU / mL, or about 2.9 x 107TU / mL of culture medium at day 15 of culture.

[0072] In one example, the method results in a viral infectious titer yield of at least 1 x

[0073] 105transducing units (TU) / mL of culture medium at day 20 of culture. For example, the method results in a viral infectious titer yield of at least 1 x 106TU / mL of culture medium at day 20 of culture. For example, the method results in a viral infectious titer yield of about 1.5 x 106TU / mL, or about 2 x 106TU / mL, or about 5 x 106TU / mL, or about 7 x

[0074] 106TU / mL, or about 10 x 106TU / mL of culture medium at day 20 of culture. In another example, the method results in a viral infectious titer yield of at least 1 x 107TU / mL of culture medium at day 20 of culture. For example, the method results in a viral infectious titer yield of about 1.1 x 107TU / mL, or about 1.2 x 107TU / mL, or about 1.3 x 107TU / mL, or about 1.4 x 107TU / mL, or about 1.5 x 107TU / mL of culture medium at day 20 of culture. In one example, the method results in a viral infectious titer yield of at least 1.5 x 107TU / mL of culture medium at day 20 of culture. For example, the method results in a viral infectious titer yield of about 1.6 x 107TU / mL, or about 1.7 x 107TU / mL, or about 1.8 x 107TU / mL, or about 1.9 x 107TU / mL of culture medium at day 20 of culture. In one example, the method results in a viral infectious titer yield of at least 2 x 107TU / mL of culture medium at day 20 of culture. For example, the method results in a viral infectious titer yield of about 2.1 x 107TU / mL, or about 2.2 x 107TU / mL, or about 2.3 x 107TU / mL, or about 2.4 x 107TU / mL, or about 2.5 x 107TU / mL, or about 2.6 x 107TU / mL, or about 2.7 x 107TU / mL, or about 2.8 x 107TU / mL, or about 2.9 x

[0075] 107TU / mL of culture medium at day 20 of culture.

[0076] In one example, the method results in a viral infectious titer yield of at least 1 x 105transducing units (TU) / mL of culture medium at day 25 of culture. For example, the method results in a viral infectious titer yield of at least 1 x 106TU / mL of culture medium at day 25 of culture. For example, the method results in a viral infectious titer yield of about 1.5 x 106TU / mL, or about 2 x 106TU / mL, or about 5 x 106TU / mL, or about 7 x

[0077] 106TU / mL, or about 10 x 106TU / mL of culture medium at day 25 of culture. In another example, the method results in a viral infectious titer yield of at least 1 x 107TU / mL of culture medium at day 25 of culture. For example, the method results in a viral infectious titer yield of about 1.1 x 107TU / mL, or about 1.2 x 107TU / mL, or about 1.3 x 107TU / mL, or about 1.4 x 107TU / mL, or about 1.5 x 107TU / mL of culture medium at day 25 of culture. In one example, the method results in a viral infectious titer yield of at least 1.5 x 107TU / mL of culture medium at day 25 of culture. For example, the method results in a viral infectious titer yield of about 1.6 x 107TU / mL, or about 1.7 x 107TU / mL, or about 1.8 x 107TU / mL, or about 1.9 x 107TU / mL of culture medium at day 25 of culture. In one example, the method results in a viral infectious titer yield of at least 2 x 107TU / mL of culture medium at day 25 of culture. For example, the method results in a viral infectious titer yield of about 2.1 x 107TU / mL, or about 2.2 x 107TU / mL, or about 2.3 x 107TU / mL, or about 2.4 x 107TU / mL, or about 2.5 x 107TU / mL, or about 2.6 x 107TU / mL, or about 2.7 x 107TU / mL, or about 2.8 x 107TU / mL, or about 2.9 x

[0078] 107TU / mL of culture medium at day 25 of culture.

[0079] In one example, the method results in a viral infectious titer yield of at least 1 x

[0080] 105transducing units (TU) / m of culture medium at day 30 of culture. For example, the method results in a viral infectious titer yield of at least 1 x 106TU / mL of culture medium at day 30 of culture. For example, the method results in a viral infectious titer yield of about 1.5 x 106TU / mL, or about 2 x 106TU / mL, or about 5 x 106TU / mL, or about 7 x

[0081] 106TU / mL, or about 10 x 106TU / mL of culture medium at day 30 of culture. In another example, the method results in a viral infectious titer yield of at least 1 x 107TU / mL of culture medium at day 30 of culture. For example, the method results in a viral infectious titer yield of about 1.1 x 107TU / mL, or about 1.2 x 107TU / mL, or about 1.3 x 107TU / mL, or about 1.4 x 107TU / mL, or about 1.5 x 107TU / mL of culture medium at day 30 of culture. In one example, the method results in a viral infectious titer yield of at least 1.5 x 107TU / mL of culture medium at day 30 of culture. For example, the method results in a viral infectious titer yield of about 1.6 x 107TU / mL, or about 1.7 x 107TU / mL, or about 1.8 x 107TU / mL, or about 1.9 x 107TU / mL of culture medium at day 30 of culture. In one example, the method results in a viral infectious titer yield of at least 2 x 107TU / mL of culture medium at day 30 of culture. For example, the method results in a viral infectious titer yield of about 2.1 x 107TU / mL, or about 2.2 x 107TU / mL, or about 2.3 x 107TU / mL, or about 2.4 x 107TU / mL, or about 2.5 x 107TU / mL, or about 2.6 x 107TU / mL, or about 2.7 x 107TU / mL, or about 2.8 x 107TU / mL, or about 2.9 x 107TU / mL of culture medium at day 30 of culture.

[0082] In one example, the method results in a viral infectious titer yield of at least 1 x

[0083] 105transducing units (TU) / m of culture medium at day 35 of culture. For example, the method results in a viral infectious titer yield of at least 1 x 106TU / mL of culture medium at day 35 of culture. For example, the method results in a viral infectious titer yield of about 1.5 x 106TU / mL, or about 2 x 106TU / mL, or about 5 x 106TU / mL, or about 7 x

[0084] 106TU / mL, or about 10 x 106TU / mL of culture medium at day 35 of culture. In another example, the method results in a viral infectious titer yield of at least 1 x 107TU / mL of culture medium at day 35 of culture. For example, the method results in a viral infectious titer yield of about 1.1 x 107TU / mL, or about 1.2 x 107TU / mL, or about 1.3 x 107TU / mL, or about 1.4 x 107TU / mL, or about 1.5 x 107TU / mL of culture medium at day 35 of culture. In one example, the method results in a viral infectious titer yield of at least 1.5 x 107TU / mL of culture medium at day 35 of culture. For example, the method results in a viral infectious titer yield of about 1.6 x 107TU / mL, or about 1.7 x 107TU / mL, or about 1.8 x 107TU / mL, or about 1.9 x 107TU / mL of culture medium at day 35 of culture. In one example, the method results in a viral infectious titer yield of at least 2 x 107TU / mL of culture medium at day 35 of culture. For example, the method results in a viral infectious titer yield of about 2.1 x 107TU / mL, or about 2.2 x 107TU / mL, or about 2.3 x 107TU / mL, or about 2.4 x 107TU / mL, or about 2.5 x 107TU / mL, or about 2.6 x 107TU / mL, or about 2.7 x 107TU / mL, or about 2.8 x 107TU / mL, or about 2.9 x

[0085] 107TU / mL of culture medium at day 35 of culture.

[0086] In one example, the method results in a viable cell density of at least about 1 x

[0087] 105cells / mL of culture medium at day 15 of culture. For example, the method results in a viable cell density of at least about 1 x 106cells / 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

[0088] 106cells / mL, or about 2 x 106cells / mL, or about 5 x 106cells / mL, or about 7 x 106cells / mL, or about 10 x 106cells / 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 x 107cells / 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 107cells / mL, or about 1.2 x 107cells / mL, or about 1.3 x

[0089] 107cells / mL, or about 1.4 x 107cells / mL, or about 1.5 x 107cells / 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 107cells / 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 107cells / mL, or about 1.6 x 107cells / mL, or about 1.7 x 107cells / mL, or about 1.8 x 107cells / mL, or about 1.9 x 107 cells / mL, or about 2.0 x 107cells / mL of culture medium at day 15 of culture. In one example, the method results in a viable cell density of at least about 2.0 x 107cells / 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 107cells / mL, or about 2.2 x 107cells / mL, or about 2.3 x 107cells / mL, or about 2.4 x 107cells / mL, or about 2.5 x 107cells / 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 107cells / mL of culture medium at day 15 of culture. In one example, the method results in a viable cell density of at least about 2.6 x 107cells / mL, or about 2.7 x 107cells / mL, or about 2.8 x 107cells / mL, or about 2.9 x 107cells / mL, or about 3.0 x 107cells / mL of culture medium at day 15 of culture.

[0090] In one example, the method results in a viable cell density of at least about 1 x

[0091] 105cells / mL of culture medium at day 20 of culture. For example, the method results in a viable cell density of at least about 1 x 106cells / 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

[0092] 106cells / mL, or about 2 x 106cells / mL, or about 5 x 106cells / mL, or about 7 x 106cells / mL, or about 10 x 106cells / 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 x 107cells / 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 107cells / mL, or about 1.2 x 107cells / mL, or about 1.3 x

[0093] 107cells / mL, or about 1.4 x 107cells / mL, or about 1.5 x 107cells / 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 107cells / 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 107cells / mL, or about 1.6 x 107cells / mL, or about 1.7 x 107cells / mL, or about 1.8 x 107cells / mL, or about 1.9 x 107cells / mL, or about 2.0 x 107cells / mL of culture medium at day 20 of culture. In one example, the method results in a viable cell density of at least about 2.0 x 107cells / 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 107cells / mL, or about 2.2 x 107cells / mL, or about 2.3 x 107cells / mL, or about 2.4 x 107cells / mL, or about 2.5 x 107cells / 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 107cells / mL of culture medium at day 20 of culture. In one example, the method results in a viable cell density of at least about 2.6 x 107cells / mL, or about 2.7 x 107cells / mL, or about 2.8 x 107cells / mL, or about 2.9 x 107cells / mL, or about 3.0 x 107cells / mL of culture medium at day 20 of culture.

[0094] In one example, the method results in a viable cell density of at least about 1 x 105cells / mL of culture medium at day 25 of culture. For example, the method results in a viable cell density of at least about 1 x 106cells / 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

[0095] 106cells / mL, or about 2 x 106cells / mL, or about 5 x 106cells / mL, or about 7 x 106cells / mL, or about 10 x 106cells / 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 x 107cells / 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 107cells / mL, or about 1.2 x 107cells / mL, or about 1.3 x

[0096] 107cells / mL, or about 1.4 x 107cells / mL, or about 1.5 x 107cells / 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 107cells / 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 107cells / mL, or about 1.6 x 107cells / mL, or about 1.7 x 107cells / mL, or about 1.8 x 107cells / mL, or about 1.9 x 107cells / mL, or about 2.0 x 107cells / mL of culture medium at day 25 of culture. In one example, the method results in a viable cell density of at least about 2.0 x 107cells / 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 107cells / mL, or about 2.2 x 107cells / mL, or about 2.3 x 107cells / mL, or about 2.4 x 107cells / mL, or about 2.5 x 107cells / 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 107cells / mL of culture medium at day 25 of culture. In one example, the method results in a viable cell density of at least about 2.6 x 107cells / mL, or about 2.7 x 107cells / mL, or about 2.8 x 107cells / mL, or about 2.9 x 107cells / mL, or about 3.0 x 107cells / mL of culture medium at day 25 of culture.

[0097] In one example, the method results in a viable cell density of at least about 1 x

[0098] 105cells / mL of culture medium at day 30 of culture. For example, the method results in a viable cell density of at least about 1 x 106cells / 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

[0099] 106cells / mL, or about 2 x 106cells / mL, or about 5 x 106cells / mL, or about 7 x 106cells / mL, or about 10 x 106cells / 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 x 107cells / 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 107cells / mL, or about 1.2 x 107cells / mL, or about 1.3 x

[0100] 107cells / mL, or about 1.4 x 107cells / mL, or about 1.5 x 107cells / 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 107cells / 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 107cells / mL, or about 1.6 x 107cells / mL, or about 1.7 x 107cells / mL, or about 1.8 x 107cells / mL, or about 1.9 x 107 cells / mL, or about 2.0 x 107cells / mL of culture medium at day 30 of culture. In one example, the method results in a viable cell density of at least about 2.0 x 107cells / 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 107cells / mL, or about 2.2 x 107cells / mL, or about 2.3 x 107cells / mL, or about 2.4 x 107cells / mL, or about 2.5 x 107cells / 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 107cells / mL of culture medium at day 30 of culture. In one example, the method results in a viable cell density of at least about 2.6 x 107cells / mL, or about 2.7 x 107cells / mL, or about 2.8 x 107cells / mL, or about 2.9 x 107cells / mL, or about 3.0 x 107cells / mL of culture medium at day 30 of culture.

[0101] In one example, the method results in a viable cell density of at least about 1 x

[0102] 105cells / mL of culture medium at day 35 of culture. For example, the method results in a viable cell density of at least about 1 x 106cells / 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

[0103] 106cells / mL, or about 2 x 106cells / mL, or about 5 x 106cells / mL, or about 7 x 106cells / mL, or about 10 x 106cells / 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 x 107cells / 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 107cells / mL, or about 1.2 x 107cells / mL, or about 1.3 x

[0104] 107cells / mL, or about 1.4 x 107cells / mL, or about 1.5 x 107cells / 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 107cells / 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 107cells / mL, or about 1.6 x 107cells / mL, or about 1.7 x 107cells / mL, or about 1.8 x 107cells / mL, or about 1.9 x 107cells / mL, or about 2.0 x 107cells / mL of culture medium at day 35 of culture. In one example, the method results in a viable cell density of at least about 2.0 x 107cells / 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 107cells / mL, or about 2.2 x 107cells / mL, or about 2.3 x 107cells / mL, or about 2.4 x 107cells / mL, or about 2.5 x 107cells / 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 107cells / mL of culture medium at day 35 of culture. In one example, the method results in a viable cell density of at least about 2.6 x 107cells / mL, or about 2.7 x 107cells / mL, or about 2.8 x 107cells / mL, or about 2.9 x 107cells / mL, or about 3.0 x 107cells / mL of culture medium at day 35 of culture.

[0105] In one example, the cell line has a viability of at least 70% at day 15 of culture. For example, the cell line has a viability of about 70%, or about 75% or about 80% at day 15 of culture. In one example, the cell line has a viability of at least 75% at day 15 of culture. In another example, the cell line has a viability of at least 80% at day 15 of culture. For example, the cell line has a viability of about 80% or about 85% or about 90% at day 15 of culture. In one example, the cell line has a viability of about 80% at day 15 of culture. For example, a viability of about 81%, or about 82%, or about 83%, or about 84%. In another example, 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.

[0106] In one example, the cell line has a viability of at least 70% at day 20 of culture. For example, the cell line has a viability of about 70%, or about 75% or about 80% at day 20 of culture. In one example, the cell line has a viability of at least 75% at day 20 of culture. In another example, the cell line has a viability of at least 80% at day 20 of culture. For example, the cell line has a viability of about 80% or about 85% or about 90% at day 20 of culture. In one example, the cell line has a viability of about 80% at day 20 of culture. For example, a viability of about 81%, or about 82%, or about 83%, or about 84%. In another example, 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.

[0107] In one example, the cell line has a viability of at least 70% at day 25 of culture. For example, the cell line has a viability of about 70%, or about 75% or about 80% at day 25 of culture. In one example, the cell line has a viability of at least 75% at day 25 of culture. In another example, the cell line has a viability of at least 80% at day 25 of culture. For example, the cell line has a viability of about 80% or about 85% or about 90% at day 25 of culture. In one example, the cell line has a viability of about 80% at day 25 of culture. For example, a viability of about 81%, or about 82%, or about 83%, or about 84%. In another example, 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.

[0108] In one example, the cell line has a viability of at least 70% at day 30 of culture. For example, the cell line has a viability of about 70%, or about 75% or about 80% at day 30 of culture. In one example, the cell line has a viability of at least 75% at day 30 of culture. In another example, the cell line has a viability of at least 80% at day 30 of culture. For example, the cell line has a viability of about 80% or about 85% or about 90% at day 30 of culture. In one example, the cell line has a viability of about 80% at day 30 of culture. For example, a viability of about 81%, or about 82%, or about 83%, or about 84%. In another example, 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.

[0109] In one example, the cell line has a viability of at least 70% at day 35 of culture. For example, the cell line has a viability of about 70%, or about 75% or about 80% at day 35 of culture. In one example, the cell line has a viability of at least 75% at day 35 of culture. In another example, the cell line has a viability of at least 80% at day 35 of culture. For example, the cell line has a viability of about 80% or about 85% or about 90% at day 35 of culture. In one example, the cell line has a viability of about 80% at day 35 of culture. For example, a viability of about 81%, or about 82%, or about 83%, or about 84%. In another example, 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.

[0110] In one example, 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%.

[0111] In one example, 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. For example, the suspension cell culture has a volume of between about 1 L and 1000 L. In one example, the suspension cell culture has a volume of about 1 L. In another example, the suspension cell culture has a volume of about 5 L. In a further example, the suspension cell culture has a volume of about 10 L. In one example, the suspension cell culture has a volume of about 50 L. In another example, the suspension cell culture has a volume of about 100 L. In a further example, the suspension cell culture has a volume of about 500 L. In one example, 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.

[0112] In one example, the suspension cell culture is operated with a dissolved carbon dioxide (CO2) level of between 4% and 8%. For example, the suspension cell culture is operated with about 4% CO2. In another example, the suspension cell culture is operated with about 5% CO2. In a further example, the suspension cell culture is operated with about 6% CO2. In a further example, the suspension cell culture is operated with about 7% CO2. In a further example, the suspension cell culture is operated with about 8% CO2.

[0113] In one example, 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. In one example, the suspension cell culture is operated at a temperature of between about 35 °C and 39 °C. For example, 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. In one example, the suspension cell culture is at a temperature of between about 36.5 °C and about 37.5 °C. For example, the suspension cell culture is at a temperature of about 37.0 °C. In one example, the suspension cell culture is at a temperature of between about 38 °C and about 39 °C. For example, the suspension cell culture is at a temperature of about 38.5 °C.

[0114] 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 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.

[0115] In one example, the method further comprises purifying the enveloped virus from the suspension cell culture.

[0116] Methods of purifying the enveloped virus from the suspension cell culture will be apparent to the skilled person and / or are described herein. In one example, purifying the enveloped virus comprises one or more steps selected from the group consisting of anion exchange chromatography, concentration and diafiltration.

[0117] In one example, a method of the disclosure additionally comprises performing sterile filtration. For example, the sterile filtration is performed prior to concentrating and diafiltering the eluted virus. In an alternative example, the sterile filtration is performed after concentrating and diafiltering the eluted virus.

[0118] In one example, the method additionally comprising formulating the purified enveloped virus into a pharmaceutical formulation or into a solution suitable for infecting a cell.

[0119] An exemplary enveloped virus is a retrovirus. For example, the retrovirus is a lentivirus. For example, the lentivirus is HIV or a derivative thereof.

[0120] The present disclosure also provides a purified enveloped virus produced by the method described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0121] 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. In the harvest mode, the acoustic field in the cell separation chamber allows to continuously collect cell-free cell culture supernatant. In the backflush mode, the acoustic field is switched off and the flow direction of the harvest pump is reversed to return the separated cells into the bioreactor.

[0122] 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. (E) Yield of infectious titer per reactor volume or volumetric yield, p = 0.0396 by an unpaired t-test. (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. BR = bleed rate; HR = harvest rate; VVD = vessels volumes per day.

[0123] 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.

[0124] 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. (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.

[0125] 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.

[0126] 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.

[0127] DETAILED DESCRIPTION

[0128] General

[0129] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, 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.

[0130] Those skilled in the art will appreciate that the present disclosure is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications. The disclosure also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.

[0131] The present disclosure is not to be limited in scope by the specific examples described herein, which are intended for the purpose of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the present disclosure.

[0132] 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.

[0133] Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (for example, in immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0134] Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques utilised in the present disclosure are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J.E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present).

[0135] The term “and / or”, e.g., “X and / or Y” shall be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for either meaning.

[0136] Throughout this specification the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0137] All publications cited herein are hereby incorporated by reference in their entirety. Where reference is made to a URL or other such identifier or address, it is understood that such identifiers can change and particular information on the internet can come and go, but equivalent information can be found by searching the internet. Reference thereto evidences the availability and public dissemination of such information.

[0138] Any discussion of documents, acts, materials, devices, articles or the like that has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

[0139] Selected Definitions

[0140] As used herein, the term “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.

[0141] As used herein, the term “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).

[0142] As used herein, the term “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.

[0143] The term “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.

[0144] The term “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.

[0145] As used herein, the term “filtered cell culture fluid” will be understood to encompass the cell culture fluid after it has been subjected to harvest filtration.

[0146] Production of Enveloped Viruses

[0147] Methods for the production of enveloped viruses will be apparent to the skilled artisan and / or described, for example, in Ansorge et al., (2010) Biochem. Eng. J. 48: 362- 377; Schweizer and Merten (2010) Curr. Gene Ther. 10: 474-486; and Rodrigues et al., (2011) Viral Gene Therapy. Xu, InTech. Chapter 2: 15-40.

[0148] Enveloped viruses

[0149] In one example, the virus is a retrovirus, for example, a lentivirus. Exemplary 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 immunodeficiency virus (FIV), the caprine arthritis-encephalitis virus (CAEV), or the ovine visna-maedi virus (VMV).

[0150] In some examples, the enveloped virus, e.g., the retrovirus, 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.

[0151] Trans gene expression

[0152] In some examples, 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. In some examples, 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.

[0153] In some examples, 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

[0154] In some examples, 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). In one example, 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. According to an alternative example, 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. 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.

[0155] The host cell may be selected from any cell allowing production of an enveloped virus. According to one example, 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. According to one example, 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.

[0156] According to one example, 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.

[0157] In one example, the cell line is a suspension cell line selected from GPR, GPRG, GPRT, GPRGT, and GPRTG cell lines. For example, the suspension cell line is a cell line derived from any of the above cell lines. In one example, the suspension cell line is a cell line derived from a GPRG cell line. In another example, the suspension cell line is a cell line derived from a GPRGT cell line. In a further example, the suspension cell line is a cell line derived from a GPRTG cell line.

[0158] Methods of adapting an adherent cell line to grow in suspension will be apparent to the skilled person and / or described herein. In one example, 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. In some embodiments 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). In one example, 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.

[0159] The present disclosure also provides a stable producer cell clone capable of producing an enveloped virus.

[0160] In one example, 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.

[0161] Cell culture medium

[0162] 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 GlutaMax™, Pluronic™ F-68 (ThermoFisher), LONG® R3 IGF-I (Sigma-Aldrich), Cell Boost™ 5, and / or an antidumping agent. The medium used may notably comprise serum or be serum-free. Culture media for mammal cells are known and 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), TransFx™ (Cytiva), BalanCD® (Irvine), Freestyle F17® (Life Technologies), or Ex-Cell® 293 (Sigma-Aldrich).

[0163] In one example, the cells are cultivated in a culture media comprising TransFx™ (Cytiva).

[0164] In one example, the cells are supplemented with one or more additives selected from the group consisting of GlutaMax™, Cell Boost™ 5, poloxamer 188 and combinations thereof. For example, the cells are supplemented with GlutaMax™. In a further example, the cells are supplemented with Cell Boost™ 5. In one example, the cells are supplemented with poloxamer 188. In another example, the cells are supplemented with GlutaMax™ and Cell Boost™ 5. In a further example, the cells are supplemented with GlutaMax™ and poloxamer 188. In another example, the cells are supplemented with Cell Boost™ 5 and poloxamer 188. In one example, the cells are supplemented with GlutaMax™, Cell Boost™ 5 and poloxamer 188.

[0165] In one example, cells are supplemented with less than 15 mM GlutaMax™. For example, the cells are supplemented with about 15 mM GlutaMax™, or about 14 mM GlutaMax™, or about 13 mM GlutaMax™, or about 12 mM GlutaMax™, or about 11 mM GlutaMax™, or about 10 mM GlutaMax™. In one example, the cells are supplemented with less than 10 mM GlutaMax™. For example, the cells are supplemented with about 10 mM GlutaMax™, or about 9 mM GlutaMax™, or about 8 mM GlutaMax™, or about 7 mM GlutaMax™, or about 6 mM GlutaMax™, or about 5 mM GlutaMax™. In one example, cells are supplemented with less than 5 mM GlutaMax™. For example, the cells are supplemented with about 5 mM GlutaMax™, or about 4 mM GlutaMax™, or about 3 mM GlutaMax™, or about 2 mM GlutaMax™, or about 1 mM GlutaMax™. In one example, the cells are supplemented with between 1 mM and 10 mM GlutaMax™. For example, the cells are supplement with between 4 mM and 8 mM GlutaMax™. In one example, the cells are supplemented with 4 mM GlutaMax™. In another example, the cells are supplemented with 5 mM GlutaMax™. In a further example, the cells are supplemented with 6 mM GlutaMax™. In one example, the cells are supplemented with 7 mM GlutaMax™. In a further example, the cells are supplemented with 8 mM GlutaMax™.

[0166] In one example, 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.

[0167] In one example, the cells are supplemented with less than 10% Cell Boost™ 5. For example, the cells are supplemented with about 10% Cell Boost™ 5, or about 9% Cell Boost™ 5, or about 8% Cell Boost™ 5, or about 7% Cell Boost™ 5, or about 6% Cell Boost™ 5. In one example, the cells are supplemented with less than 5% Cell Boost™ 5. For example, the cells are supplemented with about 5% Cell Boost™ 5, or about 4% Cell Boost™ 5, or about 3% Cell Boost™ 5, or about 2% Cell Boost™ 5, or about 1% Cell Boost™ 5. In one example, the cells are supplemented with between 1% and 10% Cell Boost™ 5. For example, the cells are supplemented with between 2% and 8% Cell Boost™ 5. In one example, the cells are supplemented with between 4% and 6% Cell Boost™ 5. For example, the cells are supplemented with about 4% Cell Boost™ 5. In another example, the cells are supplemented with about 5% Cell Boost™ 5. In a further example, the cells are supplemented with about 6% Cell Boost™ 5.

[0168] In a process using transiently transfected cells, 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.) and 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.

[0169] According to some examples, 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. In other examples, the production process used comprises the cultivation of producing cells in a moderately acid medium. The expression “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.

[0170] In one example, 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. Suspension cell culture

[0171] The present disclosure provides a method of producing an enveloped virus in a suspension cell culture. For example, the method comprises culturing a suspension cell line expressing a tetracycline-suppressible gene expression system in a cell culture medium.

[0172] 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.

[0173] It will be apparent to the skilled person that production of an enveloped virus includes a cell expansion phase and a viral production phase.

[0174] The skilled person will understand that the cell expansion phase includes a seed train cell culture. As used herein, the term “seed train” refers to the generation of an adequate number of cells (i.e., cell growth) for viral production. The skilled person will understand that 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.

[0175] It will be apparent to the skilled person that in any method described herein, that during the cell expansion phase the cells are cultured in the presence of tetracycline or a derivative thereof to suppress virus production but permit cell growth.

[0176] In one example, the cells are grown in a cell expansion phase prior to virus production.

[0177] In one example, the cell expansion phase is carried out in an expansion bioreactor (also termed an N-l bioreactor).

[0178] In one example, the viral production phase is carried out in a production bioreactor (also termed an N bioreactor).

[0179] In one example, the cell expansion phase and viral production phase are carried out in the same vessel. For example, expansion of the suspension cell line and production of the enveloped virus occur in the same vessel. For example, the cell expansion phase and viral production phase are carried out in the same bioreactor.

[0180] In one example, the cell expansion phase and viral production phase are carried out in different vessels. For example, 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.

[0181] As described herein, the cell culture is operated in a perfusion mode. For example, the cell expansion phase and / or viral production phase are operated in perfusion mode. In one example, the cell expansion phase is carried out perfusion mode. In another example, the viral production phase is carried out in perfusion mode. In a further example, the cell expansion and the viral production phases are carried out in perfusion mode.

[0182] It will be apparent to the skilled person that reference to a perfusion mode for a particular phase of cell culture (i.e., cell expansion and / or viral production) 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.

[0183] It will be apparent to the skilled person that “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).

[0184] In one example, 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).

[0185] In one example, 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.

[0186] In one example, the suspension cell culture is operated at a temperature that permits cell growth and viral production. For example, the cell culture has a temperature conventionally used in the state of the art for cultivating cells and producing viruses. In one example, 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.

[0187] In one example, the suspension cell culture is operated at large-scale. For example, the suspension cell culture is operated at commercial-scale.

[0188] In one example, the suspension cell culture is operated for a period of at least 10 days. For example, the suspension cell cultures is operated for a period of between about 10 and 50 days. In one example, 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. In one example, the suspension cell culture is operated for at least 15 days. For example, the suspension cell culture is operated for about 20 days. In another example, the suspension cell culture is operated for at least 20 days. In a further example, the suspension cell culture is operated for at least 25 days. For example, 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.

[0189] Perfusion Harvest of Enveloped Viruses

[0190] The present disclosure provides methods for improving the recovery of enveloped viruses from cell culture fluid.

[0191] 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.

[0192] 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. It will be apparent to the skilled person from the disclosure herein that the process of filtering the cell culture fluid through a filter to produce a filtered cell culture fluid is a perfusion harvest filtration step.

[0193] As used herein, “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. The term “harvest” refers to the cell culture media containing virus particles that has been removed for the purpose of downstream processing.

[0194] As used herein, the term “filtered cell culture fluid” will be understood to encompass the cell culture fluid after it has been subjected to harvest filtration.

[0195] As used herein, the term “vessel volume(s) per day” or “VVD” refers to the medium exchange rate in the cell culture. It will be apparent to the skilled person that the “perfusion rate” (i.e., in VVD) is the total amount of medium exchanged daily in the cell culture and includes the spent medium or “harvest rate”, in addition to any sampling or “bleed rate”. The term “harvest rate” refers to the medium exchange rate through the harvest line in VVD. The “bleed rate” or “cell discard rate” refers to the medium exchange rate of cell biomass that is removed from the cell culture in VVD.

[0196] As discussed herein, 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.

[0197] Accordingly, the method further comprises recirculating cell culture fluid through the filter to return the retained suspension cell line cells into the suspension cell culture. For example, the retained suspension cell line cells are recirculated into the suspension cell culture at a recirculation rate greater than the harvest rate. As used herein, 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.

[0198] In one example, the method comprises the use of an acoustic chamber or acoustic wave device. For example, 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.

[0199] Acoustic chambers or acoustic wave devices suitable for use in the present disclosure will be apparent to the skilled person and / or described herein. 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.

[0200] It will be apparent to the skilled person that the use of an acoustic chamber as described herein facilitates in-line processing. Thus, in some examples, the method is performed in-line.

[0201] As used herein, the term “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. Purifying Enveloped Viruses

[0202] In some examples of any method described herein, 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. In some examples, the downstream step further includes a sterile filtration step for removal of microorganisms from the final product.

[0203] In one example, 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.

[0204] Following harvest, the enveloped virus is purified using anion exchange.

[0205] In one example, the anion exchange is performed in bind-elute mode. In this regard, 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.

[0206] Suitable 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.

[0207] In one example, the anion exchanger is a weak anion exchanger, e.g., comprising an ion exchange group selected from a diethylaminoethyl (DEAE) or aminoethyl group.

[0208] In another example, 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.

[0209] In one example, the anion exchanger comprises a Q ion exchange group.

[0210] In one example, the anion exchanger is a membrane anion exchanger comprising a Q ion exchange group. For example, the anion exchanger is MUSTANG® Q.

[0211] In one example, an enveloped virus eluted from anion exchange column is further purified on the basis of its size. In one example, the buffer in which virus was eluted from the anion exchange column, is exchanged more or less at the same time. In the process of the disclosure, 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. Depending on the manufacturer and membrane type, 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.

[0212] As an additional step 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.

[0213] Embodiments of the Disclosure

[0214] The invention is further disclosed in the following numbered paragraphs:

[0215] 1. A method of producing an enveloped virus in a suspension perfusion cell culture, the method comprising:

[0216] (i) culturing a suspension cell line in a cell culture medium to produce a cell culture fluid comprising the enveloped virus; and (ii) filtering the cell culture fluid through a filter at a harvest rate of at least 2 vessel volume per day (VVD) to produce a filtered cell culture fluid comprising the enveloped virus, wherein the filter retains cells from the suspension cell line.

[0217] 2. The method of paragraph 1, wherein the method further comprises recirculating cell culture fluid through the filter to return the retained suspension cell line cells into the suspension cell culture.

[0218] 3. A method of producing an enveloped virus in a suspension perfusion cell culture, the method comprising:

[0219] (i) culturing a suspension cell line in a cell culture medium to produce a cell culture fluid comprising the enveloped virus;

[0220] (ii) filtering the cell culture fluid through a filter at a harvest rate of at least 1 vessel volume per day (VVD) to produce a filtered cell culture fluid comprising the enveloped virus, wherein the filter retains cells from the suspension cell line, and

[0221] (iii)recirculating cell culture fluid through the filter to return the retained suspension cell line cells into the suspension cell culture.

[0222] 4. The method of paragraph 3, wherein the harvest rate is greater than about 1 VVD.

[0223] 5. The method of paragraphs 3 or 4, wherein the harvest rate is between about 1 and 10 VVD.

[0224] 6. The method of paragraphs 1 or 2, wherein the harvest rate is about 2 VVD or about 3 VVD.

[0225] 7. The method of any one of paragraphs 1 to 6, wherein the harvest rate is 2 VVD.

[0226] 8. The method of any one of paragraphs 1 to 6, wherein the harvest rate is 3 VVD.

[0227] 9. The method of any one of paragraphs 1 to 8, wherein the filter is an acoustic standing wave.

[0228] 10. The method of any one of paragraphs 1 to 9, wherein the method comprises recirculating the retained suspension cell line cells into the suspension cell culture at a recirculation rate greater than the harvest rate. 11. The method of paragraph 10, wherein the recirculation rate is about 1.5 times, or about 2 times, or about 2.5 times, or about 3 times the harvest rate.

[0229] 12. The method of paragraph 10, wherein the recirculation rate is 2 times the harvest rate.

[0230] 13. A method of producing an enveloped virus in a suspension perfusion cell culture, the method comprising:

[0231] (i) culturing a suspension cell line in a cell culture medium to produce a cell culture fluid comprising the enveloped virus;

[0232] (ii) filtering the cell culture fluid using an acoustic standing wave to produce a filtered cell culture fluid comprising the enveloped virus, wherein the filter retains cells from the suspension cell line, and

[0233] (iii)recirculating cell culture fluid through the filter to return the retained suspension cell line cells into the suspension cell culture, wherein the harvest filtration is operated at a harvest rate of greater than 1 VVD; and the recirculation is operated at a recirculation rate of 2 times the harvest rate.

[0234] 14. The method of any one of paragraphs 1 to 13, wherein 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.

[0235] 15. The method of paragraph 14, wherein the method comprises inducing production of the enveloped virus.

[0236] 16. The method of paragraph 15, wherein the harvest rate is about 1 VVD from day 0 to about day 5 following induction of enveloped virus production.

[0237] 17. The method of paragraphs 15 or 16, wherein the harvest rate is about 2 VVD from about day 5 following induction of enveloped virus production.

[0238] 18. The method of any one of paragraphs 15 to 17, wherein the harvest rate is about 3 VVD from about day 6 following induction of enveloped virus production. 19. The method of any one of paragraphs 10 to 18, wherein the recirculation rate is between about 2 VVD and 10 VVD.

[0239] 20. The method of any one of paragraphs 10 to 19, wherein the recirculation rate is about 2 VVD, or about 4 VVD, or about 6 VVD.

[0240] 21. The method of any one of paragraphs 10 to 20, wherein the recirculation rate is 6 VVD.

[0241] 22. The method of any one of paragraphs 10 to 21, wherein the harvest rate is 3 VVD and the recirculation rate is 6 VVD.

[0242] 23. The method of any one of paragraphs 15 to 22, wherein following induction of enveloped virus production:

[0243] (i) 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; and

[0244] (ii)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.

[0245] 24. The method of any one of paragraphs 1 to 23, the method further comprises performing a suspension cell line cell bleed.

[0246] 25. The method of paragraph 19, wherein the suspension cell line cell bleed is performed at a rate of 0.1 VVD to 0.5 VVD.

[0247] 26. The method of paragraphs 24 or 25, wherein the suspension cell line cell bleed is performed at a rate of 0.2 VVD.

[0248] 27. The method of paragraphs 24 or 25, wherein the suspension cell line cell bleed is performed at a rate of 0.3 VVD.

[0249] 28. The method of any one of paragraphs 24 to 27, wherein the suspension cell line cell bleed is performed to maintain a target cell viability of at least 80%. 29. The method of any one of paragraphs 24 to 28, wherein the suspension cell line cell bleed is performed to maintain a target cell density of at least about 1 x 107cells / mL of cell culture fluid.

[0250] 30. The method of any one of paragraphs 24 to 29, wherein the suspension cell line cell bleed is performed to maintain a target cell density of between about 1 x 107cells / mL and 5 x 107cells / mL of cell culture fluid.

[0251] 31. The method of any one of paragraphs 24 to 30, wherein the suspension cell line cell bleed is performed to maintain a target cell density of about 2 x 107cells / mL of cell culture fluid.

[0252] 32. The method of any one of paragraphs 24 to 31, wherein the suspension cell line cell bleed is performed to maintain a target cell density of about 4 x 107cells / mL of cell culture fluid.

[0253] 33. The method of any one of paragraphs 1 to 32, wherein the suspension cell line is a stable producer cell line.

[0254] 34. The method of any one of paragraphs 1 to 33, wherein the suspension cell line is initially seeded in the cell culture medium at a density of between about 1 x 105cells / mL and 1 x 1010cells / mL of cell culture medium.

[0255] 35. The method of paragraph 34, wherein the suspension cell line is initially seeded in the cell culture medium at a density of between about 1 x 105cells / mL and 1 x 107cells / mL of cell culture medium.

[0256] 36. The method of paragraphs 34 or 35, wherein the suspension cell line is initially seeded in the cell culture medium at a density of between about 0.8 x 106cells / mL and 1.2 x 106cells / mL of cell culture medium.

[0257] 37. The method of any one of paragraphs 34 to 36, wherein the suspension cell line is initially seeded in the cell culture medium at a density of about 1 x 106cells / mL of cell culture medium. 38. The method of any one of paragraphs 15 to 37, wherein the suspension cell line is grown to a viable cell density of between about 1 x 105cells / mL to about 1 x 1010cells / mL of cell culture fluid prior to induction of enveloped virus production.

[0258] 39. The method of any one of paragraphs 1 to 38, wherein the suspension cell culture is operated for a period of at least 15 days.

[0259] 40. The method of any one of paragraphs 1 to 39, wherein the method results in a viral infectious titer yield of at least 1 x 107transducing units (TU) / m of cell culture fluid.

[0260] 41. The method of any one of paragraphs 1 to 40, wherein the method results in a cell viability of at least 75% for at least 15 days.

[0261] 42. The method of any one of paragraphs 1 to 41, wherein the method results in a viability of at least 80% for at least 12 days.

[0262] 43. The method of any one of paragraphs 1 to 42, wherein the method results in a viable cell density of at least 1.0 x 107cells / mL of cell culture fluid for at least 10 days, or for at least 15 days, or for at least 20 days.

[0263] 44. The method of any one of paragraphs 1 to 43, wherein the cell culture medium comprises a non-ionic surfactant.

[0264] 45. The method of paragraph 44, wherein the non-ionic surfactant is in the cell culture medium at a concentration of between about 0.01% (v / v) and 2% (v / v).

[0265] 46. The method of paragraph 45, wherein the non-ionic surfactant is in the cell culture medium at a concentration of between about 0.1% (v / v) and 1% (v / v).

[0266] 47. The method of paragraphs 45 or 46, wherein the non-ionic surfactant is in the cell culture medium at a concentration of about 0.5% (v / v).

[0267] 48. The method of any one of paragraphs 44 to 47, wherein the non-ionic surfactant is poloxamer 188. 49. The method of any one of paragraphs 1 to 48, wherein 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, or about 1,000 L.

[0268] 50. The method of any one of paragraphs 1 to 49, wherein 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.

[0269] 51. The method of any one of paragraphs 1 to 50, wherein the method further comprises purifying the enveloped virus from the filtered cell culture fluid.

[0270] 52. The method of paragraph 51, wherein purifying the enveloped virus comprises one or more steps selected from the group consisting of anion exchange chromatography, concentration and diafiltration.

[0271] 53. The method of paragraphs 51 or 52, additionally comprising formulating the purified enveloped virus into a pharmaceutical formulation or into a solution suitable for infecting a cell.

[0272] 54. The method of any one of paragraphs 1 to 53, wherein the enveloped virus is a retrovirus.

[0273] 55. The method of paragraph 54, wherein the retrovirus is a lentivirus.

[0274] 56. A purified enveloped virus produced by a method according to any one of paragraphs 1 to 55.

[0275] 57. The method of any of paragraphs 1 to 55, wherein the virus is pseudotyped with VSV-G. SEQUENCES OF THE DISCLOSURE

[0276] The present disclosure is described further in the following non-limiting examples.

[0277] EXAMPLES

[0278] Example 1: Materials and Methods

[0279] Cell culture

[0280] A Tet-off inducible polyclonal GPRTGs suspension producer cell line expressing a WAS-T2A-GFP LV upon induction (Klimpel et al. (2023) Biotechnol. Bioeng. 120(9), 2622-2638) 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 L1Antidumping 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.

[0281] For seed expansion cells were seeded at a viable cell density (VCD) of 4 x 105cells mL1for a three-day split and at 3 x 105cells mL1for a four-day split in complete TransFx-H medium supplemented with a final concentration of 2.5 ng mL1doxycycline (MP Biomedicals). Four days before inoculation for virus production, cells were seeded at a VCD of 1 x 106cells mL1at a doxycycline concentration of 1 ng mL1. 24 hours before inoculation for virus production, a medium exchange was performed after centrifuging the cell suspension at 100 g for 10 minutes and replacing 66% of the spent medium with complete TransFx-H medium supplemented with 1 ng mL1doxycycline. Induction of LV production in all bioreactors was performed by direct inoculation into doxycycline-free medium according to a previously developed dilution method (Klimpel et al. (2023) Biotechnol. Bioeng. 120(9), 2622-2638). Comparison of different medium exchange rates in Ambr® 250 bioreactors

[0282] 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).

[0283] 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 min1. On the day before inoculation, the vessels were preconditioned with complete doxycycline-free TransFx-H medium. All vessels were inoculated at a VCD of 1.5 x 106cells mL1at 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. After a three-day batch cultivation, 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. For the investigation of a higher perfusion rate, 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).

[0284] Scale-up in bench-top bioreactors

[0285] 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 min1. 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 min1.

[0286] Two bioreactor runs were performed. 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. For bioreactor run 2, 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.

[0287] The bioreactors were preconditioned with doxycycline-free medium and inoculated at a VCD of 1.5 x 106cells mL1. 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).

[0288] LV stability study

[0289] 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.

[0290] Infectious titer determination

[0291] Adherent HEK293T / 17 cells (ATCC) 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). For the quantification of transducing units (TU), cells were seeded in 96-well plates at a VCD of 2.5 x 105cells mL1at a volume of 80 pL per well using DMEM medium supplemented with 10% FBS and 100 pg / mL polybrene (Merck), hereafter called transduction medium. 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. Cells were trypsinized four days post transduction, washed with PBS (Thermo Fisher) and resuspended in cold PBS supplemented with 0.05% glutaraldehyde solution (Sigma- Aldrich). Samples were incubated for 20 minutes at 4°C, centrifuged for 2 minutes at 800 g, and resuspended in 100 pL autoMACS® Running Buffer (Miltenyi Biotec). 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 mL1using dilutions that led to a proportion of 5% to 30% GFP+cells in the sample.

[0292] Digital droplet PCRfor determination of vector RNA concentration

[0293] For the determination of the WAS-T2A-GFP LV RNA concentration, all samples were spiked with a P-globin LV with a known vector concentration prior to the extraction process. Samples were then treated with DNAse (Qiagen) and incubated for 10 minutes at room temperature to digest host cell DNA. Vector RNA extraction was performed using QIAamp Viral RNA Mini Kit and Quicube connect (Qiagen) according to manufacturer’s instructions. The RT-PCR step was done using the High-Capacity cDNA Reverse Transcription Kit (Thermo Scientific). LV RNA concentration was determined by ddPCR using QX200™ Droplet Digital PCR (Bio-Rad Laboratories) according to manufacturer’s instructions. In brief, 20 pL of the reaction mixture containing 50 ng template DNA, lx ddPCR Supermix for Probes (No dUTPs) (Bio-Rad), 50 units pL1of Haelll restriction enzyme (New England Biolabs), 900 nM of each primer, and 250 nM of each probe (Bio-Rad) was loaded into the sample wells in the QX100 Droplet Generator (Bio-Rad). A total of 40 pl of oil-water emulsion, containing approximately 20,000 droplets, was generated with the droplet generator, and transferred into a separate well of a 96-well PCR plate. 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. After PCR amplification, positive and negative droplets were counted using QX200™ 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.

[0294] The following 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’

[0295] CCCCATACCATCAGTACAAATTGCT-3’, rev 5’-

[0296] TGTTAGAGGACACATGCTCACATACAT-3’ and probe 5’-FAM- CCTCCTTTGCAAGTGTATTTACGACGGT-3 ’ ).

[0297] Lentivirus-Associated. HIV p24 ELISA

[0298] Determination of lentivirus-associated HIV p24 core protein was performed using the commercially available QuickTiter™ Lentivirus Titer Kit (Cell Biolabs) according to manufacturer’s instructions.

[0299] Quantification of total DNA concentration

[0300] Total DNA was determined using the Qubit™ 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.

[0301] Equations for process comparison

[0302] The perfusion rate is defined as the sum of the harvest and bleed rate: (1) P = H + B(1)

[0303] P = perfusion rate [d1]; H = harvest rate [d1]; B = bleed rate [d1]

[0304] The equation for the cell specific virus productivity qv was adapted from Coronel et al. (2019) Biotechnol. Prog. 36(10). The calculation at a time point ti was estimated by using determined infectious titers in the bioreactor: qv= cell specific virus productivity [TU cell1d1], cv= infectious titer [TU mL1], t = time post induction [h], X = cells density [cells mL1]

[0305] The cell specific virus yield Qv,k is defined as the total lentivirus amount produced per cell at a given time point tk:

[0306] (3) Qv,k = SLi qv,i

[0307] Qv= cell specific virus yield [TU cell1]

[0308] 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:

[0309] VYv= volumetric yield or yield per reactor volume [TU mLBioreactor’1], V Bioreactor — working volume bioreactor [mL]

[0310] The following equation was used to describe the continuous dilution of a substance in an ideally mixed continuous stirred tank reactor:

[0311] (5) c£= c0* e-p*fid = relative concentration at ti [%], co = relative concentration at to [%], h = time after to [d]

[0312] Statistical analysis

[0313] Statistical analysis was performed using GraphPad Prism 10. The LV half-life was determined by a nonlinear regression using a one phase decay equation with a robust fit. Multiple unpaired t-tests were performed to compare virus production using the Ambr® 250 between a harvest rate of 1 VVD and 3 VVD with respect to cell specific yields and volumetric yields. Differences were considered significant when *p <0.05.

[0314] Example 2: Results

[0315] 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. For 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). To investigate whether higher perfusion rates have an impact on LV yields, 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). All bioreactors were inoculated at a VCD of 1.50 + 0.09 x 106cells mL1and perfusion was initiated after a 3-day batch phase (Figure 2A). The first set of bioreactors was used to mimic the previously established standard LV production process (Klimpel et al., 2023), which is performed at a harvest rate of 1 VVD with a variable cell bleed to maintain a VCD of approximately 2 x 107cells mL'1(Figure 2B). To simulate the continuous cell bleed at bench-top bioreactor scale, which is performed using a peristaltic pump at a fixed flow rate that is adjusted based on the daily determined VCD, a bolus bleed was performed every 6 hours in the Ambr® 250 system using the liquid handler and a subsequent bolus addition of fresh medium. For the second set of bioreactors, the perfusion rate was gradually increased to 2 VVD at 5 dpi and to 3 VVD at 6 dpi (Figure 2A). The initiation of the cell bleed was delayed, resulting in an average VCD of 3.99 + 0.34 x 107cells mL1at 7 dpi (Figure 2B). As the volume of concentrated cell culture in the ASC is continuously increasing during every harvest cycle, the set-up requires a repetitive back flush into the bioreactor to avoid cell loss into the harvest stream. 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). A similar profile of infectious titers was found for both processes, reaching maximum values of 7.27 + 1.74 x 107TU mL'1at a harvest rate of 1 VVD and 8.47 + 5.26 x 107TU mL'1at a harvest rate of 3 VVD both at 10 dpi (Figure 2D). As the flow rates in the harvest line are rather low due to the small bioreactor volume of the Ambr® vessels, the increased residence time of the virus in the harvest line at room temperature may lead to increased virus degradation. The resulting cumulative virus yield per bioreactor volume was found to be significantly higher at a harvest rate of 3 VVD at 11 dpi (2.27 + 0.28 x 108TU mLsioreactor’1vs. 6.91 + 2.54 x 108TU mLsioreactor’1; p = 0.0396) (Figure 2E). As a higher target VCD has been applied for a LV production at 3 VVD which is affecting volumetric virus yields, to investigate the effect of higher perfusion rates on the virus recovery, the virus yields should be normalized per cell. Figure 2F 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 cumulative cell specific yield at the end of the process was 17.1 ± 1.2 TU cell1at a harvest rate of 1 VVD and 24.2 ± 5.0 TU cell1at a harvest rate of 3 VVD (p = 0.0745; Figure 2G). Although the final cumulative cell specific yield between the processes was not found to be significant, significant differences were determined when comparing the cell specific virus yields at 6-9 dpi (all p values <0.002), supporting the hypothesis that increased perfusion rates are increasing LV yields.

[0316] To verify the findings from the small-scale model, the effect of higher perfusion rates was tested at bench-top scale using a 5 L bioreactor for the standard process at a harvest rate of 1 VVD, and a 2 L bioreactor to investigate a harvest rate of 3 VVD. Since the 3 VVD process is aiming for higher target VCDs, the acoustic separation capacity exhausts faster within a harvest cycle at the same bioreactor and ASC scale (Figure 1). For LV production at higher perfusion rates, a similar process strategy like in the Ambr® 250 was applied by gradually increasing the perfusion rate after an initial batch phase. The peak harvest rate of 3 VVD was applied starting from 6 dpi (Figure 3A). 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 107cells mL"1. However, for the process at 3 VVD the VCD reached a plateau at 2.84 x 107cells mL'1at 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. Cell viability started to decline earlier for the 3 VVD process compared to the 1 VVD process (Figure 3C). Comparable infectious titers were determined until 7 dpi for both processes (Figure 3D). In the following course, infectious titers of 4-6 x 107TU mL'1were maintained for the 1 VVD process, while infectious titers for the 3 VVD process were gradually declining. The cumulative virus yield per bioreactor volume at 3 VVD initially showed an increasing trend compared to the process at 1 VVD, but the trend reversed in the later course (Figure 3E). The final volumetric yields were 8.53 x 108TU mLs ioreactor"1at 20 dpi for the 1 VVD process and 6.05 x 108TU mLBioreactor’1at 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"1at 14 dpi for the 1 VVD process and 53.4 TU cell"1at 20 dpi for the 3 VVD process (Figure 3G). At the beginning of the process, similar to the small-scale findings, the cell specific productivities tended to be higher for the process at 3 VVD, peaking at 5.1 TU cell"1day"1at 7 dpi (Figure 3F). However, in the following course cell specific productivities at 3 VVD were dropping below those obtained at 1 VVD. Interestingly, the cell specific productivity of vector genomes, representing the productivity of physical vector particles, was higher for the process at 3 VVD throughout the investigated production period (Figure 3H). In contrast to the cumulative yield of functional LV, the trend for the cumulative yield of vector genomes per bioreactor volume didn’t reverse in the same way (Figure 31).

[0317] For the second bioreactor run of LV production at higher harvest rates, a similar approach was applied by gradually increasing the harvest rate up to 3 VVD. In contrast to the first bioreactor run, cells were growing to a VCD of approximately 4.29 x 107cells mL"1at 7 dpi, and the cell density was controlled and maintained by applying a continuous cell bleed (Figure 4A and 4B). A similar viability profile was obtained between a harvest rate of 1 VVD and 3 VVD with a stronger viability decline at the end of the ferment, reaching 71.4% for 1 VVD at 17 dpi and 57.7 % for 3 VVD at 16 dpi (Figure 4C). At the same time, a turbidity increase of the cell free harvest can be observed (data not shown). As the cell growth is slowing down shortly before, the cell bleed was turned off at 16 dpi for the 1 VVD process and at 15 dpi for the 3 VVD process (Figure 4A). A similar infectious titer profile was observed between a harvest rate of 1 VVD and 3 VVD, with slightly higher titers for the process at 3 VVD (Figure 4D). The highest infectious titer determined was 1.24 x 108TU mL1at 12 dpi at 1 VVD, and 1.74 x 108TU mL1at 14 dpi at 3 VVD. The final cumulative yield at 3 VVD was 3.1 -fold higher, reaching a value of 3.31 x 109TU IULB ioreactor1compared to 1.08 x 109TU mLs ioreactor"1at 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"1day"1at 1 VVD and 11.1 TU cell"1day"1at 3 VVD. The final cumulative cell-specific virus yield was 61.9 TU cell"1at 1 VVD and 89.1 TU cell"1at 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 1012RNA copies mLsioreactor’1compared to 1.66 x 1012RNA copies mLsioreactor’1at 1 VVD. DNA concentrations in the supernatant were increasing over process time, reaching a peak concentration of 9452 ng mL"1at 12 dpi for the 1 VVD process and 9522 ng mL"1at 16 dpi for the 3 VVD process (Figure 6A), indicating a similar impurity profile between both processes. The results indicate that the change of the pump tubing for recirculation of the cell suspension and the increase of the poloxamer 188 concentration reduced the induced shear stress on the producer cells. Higher volumetric and cell-specific LV productivities and yields obtained at higher harvest rates support the hypothesis of increasing LV recovery by decreasing the LV residence time in the bioreactor due to a low vector halflife of 6 hours at 37°C (Figure 5).

[0318] To control for a specific target VCD, the application of a cell bleed may be beneficial. The decrease in virus degradation could affect not only the yields of the LV process but also the quality of the produced LV. 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 104TU ngP24-1and 3.41 x 104TU ngP24-1were obtained at 3 VVD compared to 1.13 x 104TU ngP24-1and 2.25 x 104TU ngP24-1at 1 VVD.

Claims

CLAIMS1. A method of producing an enveloped virus in a suspension perfusion cell culture, the method comprising:(i) culturing a suspension cell line in a cell culture medium to produce a cell culture fluid comprising the enveloped virus; and(ii) filtering the cell culture fluid through a filter at a harvest rate of at least 2 vessel volume per day (VVD) to produce a filtered cell culture fluid comprising the enveloped virus, wherein the filter retains cells from the suspension cell line.

2. The method of claim 1, wherein the method further comprises recirculating cell culture fluid through the filter to return the retained suspension cell line cells into the suspension cell culture.

3. A method of producing an enveloped virus in a suspension perfusion cell culture, the method comprising:(i) culturing a suspension cell line in a cell culture medium to produce a cell culture fluid comprising the enveloped virus;(ii) filtering the cell culture fluid through a filter at a harvest rate of at least 1 vessel volume per day (VVD) to produce a filtered cell culture fluid comprising the enveloped virus, wherein the filter retains cells from the suspension cell line, and(iii)recirculating cell culture fluid through the filter to return the retained suspension cell line cells into the suspension cell culture.

4. The method of claim 3, wherein the harvest rate is greater than about 1 VVD.

5. The method of claim 3, wherein the harvest rate is between about 1 and 10 VVD.

6. The method of claim 1, wherein the harvest rate is about 2 VVD or about 3 VVD.

7. The method of claim 1, wherein the harvest rate is 2 VVD.

8. The method of claim 1, wherein the harvest rate is 3 VVD.

9. The method of claim 1, wherein the filter is an acoustic standing wave.

10. The method of claim 1, wherein the method comprises recirculating the retained suspension cell line cells into the suspension cell culture at a recirculation rate greater than the harvest rate.

11. The method of claim 10, wherein the recirculation rate is about 1.5 times, or about 2 times, or about 2.5 times, or about 3 times the harvest rate.

12. The method of claim 10, wherein the recirculation rate is 2 times the harvest rate.

13. A method of producing an enveloped virus in a suspension perfusion cell culture, the method comprising:(i) culturing a suspension cell line in a cell culture medium to produce a cell culture fluid comprising the enveloped virus;(ii) filtering the cell culture fluid using an acoustic standing wave to produce a filtered cell culture fluid comprising the enveloped virus, wherein the filter retains cells from the suspension cell line, and(iii)recirculating cell culture fluid through the filter to return the retained suspension cell line cells into the suspension cell culture, wherein the harvest filtration is operated at a harvest rate of greater than 1 VVD; and the recirculation is operated at a recirculation rate of 2 times the harvest rate.

14. The method of claim 1, wherein 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.

15. The method of claim 14, wherein the method comprises inducing production of the enveloped virus.

16. The method of claim 15, wherein the harvest rate is about 1 VVD from day 0 to about day 5 following induction of enveloped virus production.

17. The method of claim 15, wherein the harvest rate is about 2 VVD from about day5 following induction of enveloped virus production.

18. The method of claim 15, wherein the harvest rate is about 3 VVD from about day6 following induction of enveloped virus production.

19. The method of claim 10, wherein the recirculation rate is between about 2 VVD and 10 VVD.

20. The method of claim 10, wherein the recirculation rate is about 2 VVD, or about 4 VVD, or about 6 VVD.

21. The method of claim 10, wherein the recirculation rate is 6 VVD.

22. The method of claim 10, wherein the harvest rate is 3 VVD and the recirculation rate is 6 VVD.

23. The method of claim 15, wherein following induction of enveloped virus production:(i) 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; and(ii)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.

24. The method of claim 1, the method further comprises performing a suspension cell line cell bleed.

25. The method of claim 19, wherein the suspension cell line cell bleed is performed at a rate of 0.1 VVD to 0.5 VVD.

26. The method of claim 24, wherein the suspension cell line cell bleed is performed at a rate of 0.2 VVD.

27. The method of claim 24, wherein the suspension cell line cell bleed is performed at a rate of 0.3 VVD.

28. The method of claim 24, wherein the suspension cell line cell bleed is performed to maintain a target cell viability of at least 80%.

29. The method of claim 24, wherein the suspension cell line cell bleed is performed to maintain a target cell density of at least about 1 x 107cells / mL of cell culture fluid.

30. The method of claim 24, wherein the suspension cell line cell bleed is performed to maintain a target cell density of between about 1 x 107cells / mL and 5 x 107cells / mL of cell culture fluid.

31. The method of claim 24, wherein the suspension cell line cell bleed is performed to maintain a target cell density of about 2 x 107cells / mL of cell culture fluid.

32. The method of claim 24, wherein the suspension cell line cell bleed is performed to maintain a target cell density of about 4 x 107cells / mL of cell culture fluid.

33. The method of claim 1, wherein the suspension cell line is a stable producer cell line.

34. The method of claim 1, wherein the suspension cell line is initially seeded in the cell culture medium at a density of between about 1 x 105cells / mL and 1 x IO10cells / mL of cell culture medium.

35. The method of claim 34, wherein the suspension cell line is initially seeded in the cell culture medium at a density of between about 1 x 105cells / mL and 1 x 107cells / mL of cell culture medium.

36. The method of claim 34, wherein the suspension cell line is initially seeded in the cell culture medium at a density of between about 0.8 x 106cells / mL and 1.2 x 106cells / mL of cell culture medium.

37. The method of claim 34, wherein the suspension cell line is initially seeded in the cell culture medium at a density of about 1 x 106cells / mL of cell culture medium.

38. The method of claim 15, wherein the suspension cell line is grown to a viable cell density of between about 1 x 105cells / mL to about 1 x 1010cells / mL of cell culture fluid prior to induction of enveloped virus production.

39. The method of claim 1 , wherein the suspension cell culture is operated for a period of at least 15 days.

40. The method of claim 1, wherein the method results in a viral infectious titer yield of at least 1 x 107transducing units (TU) / mL of cell culture fluid.

41. The method of claim 1, wherein the method results in a cell viability of at least 75% for at least 15 days.

42. The method of claim 1, wherein the method results in a viability of at least 80% for at least 12 days.

43. The method of claim 1, wherein the method results in a viable cell density of at least 1.0 x 107cells / mL of cell culture fluid for at least 10 days, or for at least 15 days, or for at least 20 days.

44. The method of claim 1, wherein the cell culture medium comprises a non-ionic surfactant.

45. The method of claim 44, wherein the non-ionic surfactant is in the cell culture medium at a concentration of between about 0.01% (v / v) and 2% (v / v).

46. The method of claim 45, wherein the non-ionic surfactant is in the cell culture medium at a concentration of between about 0.1% (v / v) and 1% (v / v).

47. The method of claim 45, wherein the non-ionic surfactant is in the cell culture medium at a concentration of about 0.5% (v / v).

48. The method of claim 44, wherein the non-ionic surfactant is poloxamer 188.

49. The method of claim 1, wherein 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, or about 1,000 L.

50. The method of claim 1, wherein 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.

51. The method of claim 1, wherein the method further comprises purifying the enveloped virus from the filtered cell culture fluid.

52. The method of claim 51, wherein purifying the enveloped virus comprises one or more steps selected from the group consisting of anion exchange chromatography, concentration and diafiltration.

53. The method of claim 51, additionally comprising formulating the purified enveloped virus into a pharmaceutical formulation or into a solution suitable for infecting a cell.

54. The method of claim 1, wherein the enveloped virus is a retrovirus.

55. The method of claim 54, wherein the retrovirus is a lentivirus.

56. A purified enveloped virus produced by a method according to claim 1.

57. The method of claim 1, wherein the virus is pseudotyped with VSV-G.

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