Method for producing an enveloped virus

Abstract

The present disclosure relates to a method for producing an enveloped virus in a suspension cell culture, which includes culturing a suspension cell line expressing a tetracycline-repressible gene expression system in a cell culture medium. The present disclosure also relates to a stable production cell clone capable of producing an enveloped virus in a suspension cell culture.

Description

Technical Field

[0001] Related Application Data This application claims priority to U.S. Patent Application No. 63 / 366,408, titled "Methods of producing an enveloped virus," filed on June 15, 2022, and U.S. Patent Application No. 63 / 498,041, titled "Methods of producing an enveloped virus," filed on April 25, 2023. The entire contents of both are incorporated herein by reference.

[0002] The present disclosure generally relates to the production of gene therapy products, and specifically to methods for producing enveloped viruses from suspension cell cultures that express a tetracycline-inducible gene expression system.

Background Art

[0003] Retroviruses, such as lentiviruses, are one of the most studied viral vectors for gene therapy. Generally, retroviruses are RNA viruses that permanently integrate their genetic information into the chromosomes of target cells. The advantages of retroviruses include long-term transgene expression in target cells, low immunogenic potential, and the ability to transduce both dividing and non-dividing cells.

[0004] Lentiviruses are genetically modified and typically based on human immunodeficiency virus 1 (HIV-1). To increase safety, current vectors contain only the HIV genes necessary for infection and gene delivery, while genes required for replication and toxic factors are removed. Often, the envelope protein of HIV-1 is replaced with the VSV-G protein of another virus, such as vesicular stomatitis virus Indiana (VSV), to enable infection of a wide range of target cells.

[0005] To produce lentivirus, cells such as human embryonic kidney cells HEK293T are transfected with three to four plasmids. These include a transfer plasmid carrying the gene of interest, as well as several packaging plasmids encoding vesicular stomatitis G protein (VSV-G) and essential viral proteins involved in gene integration or self-organization. These plasmids are transiently transfected into cells, or producer cell lines are created by stably integrating a plasmid with an inducible promoter that can induce lentivirus production.

[0006] Once virus production is induced, after successful assembly within the cell, virus release by budding occurs. Lentivirus is harvested from producer cells and then purified and concentrated in downstream processes.

[0007] Clinical-grade lentiviral vectors are often produced by transient transfection of adherent cell lines. Such production methods are costly, require large amounts of GMP-grade plasmids, and inhibit process expansion and reproducibility.

[0008] Therefore, in the art, there is a need for an efficient process for producing lentivirus in cell culture systems, for example, for gene therapy. Summary of the Invention Problems to be Solved by the Invention

[0009] In the research leading to the present invention, the inventors have attempted to develop a method for producing enveloped viruses for gene therapy, for example, on a commercial scale and in a form suitable for regulatory requirements. Means for Solving the Problems

[0010] An upstream process for producing an enveloped virus in a suspension cell culture expressing a tetracycline-repressible (i.e., Tet-Off) gene expression system developed by the inventors includes reducing the concentration of tetracycline or its derivative (e.g., doxycycline) from the cell culture to induce virus production.

[0011] In adherent cell lines, since cells grow on the surface, medium exchange is easy and the medium can be removed without disturbing the cells. In adherent cell lines, tetracycline or its derivative can be removed by performing a medium exchange in which the cell culture medium containing tetracycline or the derivative is replaced with a cell culture medium not containing tetracycline or the derivative. However, in suspension cell lines, to induce virus production, usually, tetracycline or its derivative is removed from the cell culture medium by centrifugation to pellet the cells (i.e., remove the cells from the suspension), then a cell culture medium not containing tetracycline or the derivative is added, and then the cells are resuspended in the cell culture medium not containing tetracycline or the derivative by stirring the cells.

[0012] In developing these methods, the inventors determined that the usual methods of reducing the concentration of tetracycline or its derivative from the cell culture (e.g., centrifugation and resuspension) degrade the quality of the cells due to the high shear forces applied to the cells and increase the risk of contamination due to the open and manual processes.

[0013] To solve this problem, the inventors confirmed that the concentration of tetracycline or its derivatives in cell cultures can be reduced by using dilution or acoustic standing wave methods. In one example, the inventors found that the concentration of tetracycline or its derivatives in cell cultures can be reduced by diluting the suspension cell culture with a cell culture medium that does not contain tetracycline or its derivatives. In another example, the inventors used acoustic standing waves to hold suspension cell line cells, removed a portion of the cell culture medium containing tetracycline or derivatives from the suspension cell culture, and contacted the held suspension cell line cells with a cell culture medium that does not contain tetracycline or derivatives, and found that the concentration of tetracycline or its derivatives in the cell culture can be reduced.

[0014] Thus, the discovery by the inventors provides a method for producing enveloped viruses.

[0015] In one example, the present disclosure provides a method for producing an enveloped virus in a suspension cell culture, the method comprising culturing a suspension cell line expressing a tetracycline-inducible gene expression system in a cell culture medium.

[0016] It will be apparent to those skilled in the art that the tetracycline-inducible gene expression system is also known as the Tet-Off expression system.

[0017] In an exemplary form of the present disclosure, the suspension cell line is a stable production cell line, i.e., a cell in which the genetic material required to produce lentivirus is stably integrated therein. Such cells are distinguished from cells having transiently integrated genetic elements.

[0018] Exemplary enveloped viruses are retroviruses. For example, the retrovirus is a lentivirus. For example, the lentivirus is HIV or a derivative thereof.

[0019] In one example, the suspension cell line is cultured in a cell culture medium containing an amount of tetracycline or a derivative thereof sufficient to suppress the production of the enveloped virus and to allow for the expansion of the suspension cell line.

[0020] In one example, the first cell culture is an expanding cell culture. For example, the first cell culture is performed in an expanding (or N-1) bioreactor.

[0021] In one example, a sufficient amount of tetracycline or its derivative in the cell culture medium is at least 0.1 ng / mL of the cell culture medium. In one example, a sufficient amount of tetracycline or its derivative in the cell culture medium is from about 0.1 ng / mL to about 10,000 ng / mL of the cell culture medium. In one example, a sufficient amount of tetracycline or its derivative in the cell culture medium is from about 0.1 ng / mL to about 1,000 ng / mL of the cell culture medium. In one example, a sufficient amount of tetracycline or its derivative in the cell culture medium is from about 0.1 ng / mL to about 100 ng / mL of the cell culture medium. In one example, a sufficient amount of tetracycline or its derivative in the cell culture medium is from about 0.1 ng / mL to about 10 ng / mL of the cell culture medium. For example, a sufficient amount of tetracycline or its derivative 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 the cell culture medium. In one example, a sufficient amount of tetracycline or its derivative in the cell culture medium is at least 0.2 ng / mL. In one example, a sufficient amount of tetracycline or its derivative in the cell culture medium is at least 0.5 ng / mL. In one example, a sufficient amount of tetracycline or its derivative is from about 0.5 ng / mL to about 5 ng / mL, or from about 1 ng / mL to about 5 ng / mL, or from about 1.5 ng / mL to about 5 ng / mL, or from about 2 ng / mL to about 5 ng / mL of the cell culture medium. In one example, a sufficient amount of tetracycline or its derivative in the cell culture medium is from 0.5 ng / mL to 5 ng / mL of the cell culture medium. For example, a sufficient amount of tetracycline or its derivative in the cell culture medium is 0.5 ng / mL, or 1 ng / mL, or 1.5 ng / mL, or 2 ng / mL, or 2.5 ng / mL, or 3 ng / mL, or 3.5 ng / mL, or 4 ng / mL, or 4.5 ng / mL, or 5 ng / mL of the cell culture medium. For example, a sufficient amount of tetracycline or its derivative in the cell culture medium is 0.1 ng / mL of the cell culture medium.In one example, a sufficient amount of tetracycline or its derivative in the cell culture medium is 1 ng / mL of the cell culture medium. In one example, a sufficient amount of tetracycline or its derivative in the cell culture medium is 1.5 ng / mL of the cell culture medium.

[0022] In one example, the method includes reducing the concentration of tetracycline or its derivative in the cell culture medium such that production of the enveloped virus is induced.

[0023] In one example, the concentration of tetracycline or its derivative is reduced by at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 85%, or at least 90%, or at least 95%. In one example, the concentration of tetracycline or its derivative is reduced by at least 50%. In one example, the concentration of tetracycline or its derivative is reduced by at least 60%. In one example, the concentration of tetracycline or its derivative is reduced by at least 70%. In one example, the concentration of tetracycline or its derivative is reduced by at least 80%. In one example, the concentration of tetracycline or its derivative is reduced by at least 85%. In one example, the concentration of tetracycline or its derivative is reduced by at least 90%. In one example, the concentration of tetracycline or its derivative is reduced by at least 95%.

[0024] In one example, the concentration of tetracycline or its derivative in the cell culture medium is reduced to a concentration of 0.5 ng / mL or less of the cell culture medium. In one example, the concentration of tetracycline or its derivative in the cell culture medium is reduced to a concentration of 0.5 ng / mL to 0.001 ng / mL of the cell culture medium. For example, the concentration of tetracycline or its derivative 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 the cell culture medium. In one example, the concentration of tetracycline or its derivative in the cell culture medium is reduced to a concentration of 0.2 ng / mL or less of the cell culture medium. In one example, the concentration of tetracycline or its derivative in the cell culture medium is reduced to a concentration of 0.1 ng / mL or less in the cell culture medium. In one example, the concentration of tetracycline or its derivative in the cell culture medium is reduced to a concentration of 0.1 ng / mL to 0.001 ng / mL in the cell culture medium. For example, the concentration of tetracycline or its derivative in the cell culture medium is reduced to a concentration of about 0.1 ng / mL, or about 0.05 ng / mL, or about 0.01 ng / mL, or about 0.005 ng / mL, or about 0.001 ng / mL of the cell culture medium. In one example, the concentration of tetracycline or its derivative in the cell culture medium is reduced to a concentration of about 0.5 ng / mL of the cell culture medium. In one example, the concentration of tetracycline or its derivative in the cell culture medium is reduced to a concentration of about 0.25 ng / mL of the cell culture medium. In one example, the concentration of tetracycline or its derivative in the cell culture medium is reduced to a concentration of about 0.2 ng / mL of the cell culture medium. In one example, the concentration of tetracycline or its derivative in the cell culture medium is reduced to a concentration of about 0.1 ng / mL of the cell culture medium. In one example, the concentration of tetracycline or its derivative in the cell culture medium is reduced to a concentration of about 0.05 ng / mL of the cell culture medium. In one example, the concentration of tetracycline or its derivative in the cell culture medium is reduced to a concentration of about 0.01 ng / mL of the cell culture medium.In one example, the concentration of tetracycline or its derivative in the cell culture medium is reduced to a concentration of about 0.005 ng / mL of the cell culture medium. In one example, the concentration of tetracycline or its derivative in the cell culture medium is reduced to a concentration of about 0.001 ng / mL of the cell culture medium.

[0025] In one example, prior to reducing the concentration of tetracycline or its derivative in the cell culture medium such that production of the enveloped virus is induced, the amount of tetracycline or its derivative in the cell culture medium sufficient to suppress production of the enveloped virus and allow for an increase in the suspension cell line is adjusted. For example, the concentration of tetracycline or its derivative in the growth bioreactor is set to a first concentration for a first period and then adjusted to a second concentration lower than the first concentration for a second period.

[0026] For example, the first concentration of tetracycline or its derivative in the cell culture medium is at least 1 ng / mL of the cell culture medium. In one example, the first concentration of tetracycline or its derivative in the cell culture medium is from about 1 ng / mL to about 10 ng / mL of the cell culture medium. For example, the first concentration of tetracycline or its derivative in the cell culture medium is about 1 ng / mL, or about 2 ng / mL, or about 3 ng / mL, or about 4 ng / mL, or about 5 ng / mL, or about 6 ng / mL, or about 7 ng / mL, or about 8 ng / mL, or about 9 ng / mL, or about 10 ng / mL of the cell culture medium. In one example, the first concentration of tetracycline or its derivative in the cell culture medium is from about 1 ng / mL to about 5 ng / mL, or from about 1.5 ng / mL to about 5 ng / mL, or from about 2 ng / mL to about 5 ng / mL, or from about 2.5 ng / mL to about 5 ng / mL of the cell culture medium. For example, the first concentration of tetracycline or its derivative in the cell culture medium is 1 ng / mL, or 1.5 ng / mL, or 2 ng / mL, or 2.5 ng / mL, or 3 ng / mL, or 3.5 ng / mL, or 4 ng / mL, or 4.5 ng / mL, or 5 ng / mL of the cell culture medium. For example, the first concentration of tetracycline or its derivative in the cell culture medium is 2.5 ng / mL of the cell culture medium.

[0027] For example, the concentration of tetracycline or its derivative in the increasing bioreactor is set to the first concentration for a period of about 1 to about 8 days. In one example, the first period is about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, or about 8 days.

[0028] For example, the second concentration of tetracycline or its derivative in the cell culture medium is at least 0.1 ng / mL of the cell culture medium. In one example, the second concentration of tetracycline or its derivative in the cell culture medium is from about 0.1 ng / mL to about 2.5 ng / mL of the cell culture medium. For example, the second concentration of tetracycline or its derivative 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 the cell culture medium. In one example, the second concentration of tetracycline or its derivative in the cell culture medium is from about 0.1 ng / mL to about 2.5 ng / mL of the cell culture medium, or from about 0.5 ng / mL to about 2.5 ng / mL of the cell culture medium, or from about 1 ng / mL to about 2.5 ng / mL of the cell culture medium, or from about 1.5 ng / mL to about 2.5 ng / mL of the cell culture medium. For example, the second concentration of tetracycline or its derivative in the cell culture medium is 1 ng / mL, or 1.5 ng / mL, or 2 ng / mL, or 2.5 ng / mL of the cell culture medium. For example, the second concentration of tetracycline or its derivative in the cell culture medium is 1 ng / mL of the cell culture medium.

[0029] For example, the concentration of tetracycline or its derivative in the increasing bioreactor is set to the second concentration for a period of about 1 day to about 4 days. In one example, the first period is about 1 day, about 2 days, about 3 days, or about 4 days.

[0030] In one example, the concentration of tetracycline or its derivative in the increasing bioreactor is set to a concentration of 2.5 ng / mL for about 2 days and then adjusted to a concentration of 1.0 ng / mL for about 2 days.

[0031] In one example, in the cell culture medium, the concentration of tetracycline or its derivative is: (i) diluting the suspension cell culture with a cell culture medium that does not contain tetracycline or a derivative; or (ii) Using acoustic standing waves to hold the suspension cell line cells, removing a portion of the cell culture medium containing tetracycline or a derivative from the suspension cell culture, and contacting the held suspension cell line cells with a cell culture medium that does not contain tetracycline or a derivative is reduced by.

[0032] In one example, the concentration of tetracycline or a derivative in the cell culture medium is reduced by diluting the suspension cell culture with a cell culture medium that does not contain tetracycline or a derivative.

[0033] In one example, diluting the suspension cell culture includes directly adding a cell culture medium that does not contain tetracycline or a derivative to a cell culture medium that contains tetracycline or a derivative.

[0034] In one example, diluting the suspension cell culture includes adding a cell culture medium that does not contain tetracycline or a derivative to the suspension cell culture.

[0035] In one example, the suspension cell culture is diluted at a ratio of about 1:1 to 1:20 in a cell culture medium that does not contain tetracycline or a derivative. For example, the suspension cell culture is diluted at a ratio of 1:2 to 1:10, or a ratio of 1:4 to 1:7, in a cell culture medium that does not contain tetracycline or a derivative. In one example, the suspension cell culture is diluted at a ratio of 1:2 to 1:10 in a cell culture medium that does not contain tetracycline or a derivative. For example, the suspension cell culture is diluted at a ratio of about 1:2, or about 1:3, or about 1:4, or about 1:5, or about 1:6, or about 1:7, or about 1:8, or about 1:9, or about 1:10 in a cell culture medium that does not contain tetracycline or a derivative. In one example, the suspension cell culture is diluted at a ratio of 1:4 to 1:7 in a cell culture medium that does not contain tetracycline or a derivative. In one example, the suspension cell culture is diluted at a ratio of about 1:4 in a cell culture medium that does not contain tetracycline or a derivative. In another example, the suspension cell culture is diluted at a ratio of about 1:5 in a cell culture medium that does not contain tetracycline or a derivative. In yet another example, the suspension cell culture is diluted at a ratio of about 1:6 in a cell culture medium that does not contain tetracycline or a derivative. In one example, the suspension cell culture is diluted at a ratio of about 1:7 in a cell culture medium that does not contain tetracycline or a derivative.

[0036] In one example, in the cell culture medium, the concentration of tetracycline or a derivative is reduced by using acoustic standing waves to hold suspension cell line cells, removing a portion of the cell culture medium containing tetracycline or its derivative from the suspension cell culture, and contacting the held suspension cell line cells with a cell culture medium that does not contain tetracycline or a derivative.

[0037] In one example, the suspension cell line is 5 seeded initially in the cell culture medium at a density of about 1×10 10 cells / mL to 1×10 5 cells / mL. For example, the suspension cell line is about 1×10 10cells / mL, or 1×10 5 cells / mL to 1×10 7 cells / mL, or approximately 0.1×10 6 cells / mL to 1×10 8 cells / mL, or approximately 0.5×10 6 cells / mL to 1×10 7 cells / mL, or approximately 0.5×10 6 cells / mL to 5×10 6 cells / mL, or approximately 0.5×10 6 cells / mL to 2.5×10 6 cells / mL at the density of the cell culture medium and is first seeded into the cell culture medium. In one example, the suspension cell line is 1×10 5 cells / mL and 1×10 7 cells / mL and is first seeded into the cell culture medium. In one example, the suspension cell line is 0.5×10 6 cells / mL to 5.0×10 6 cells / mL and is first seeded into the cell culture medium. In one example, the suspension cell line is 0.8×10 6 cells / mL to 1.2×10 6 cells / mL and is first seeded into the cell culture medium. In one example, the suspension cell line is 1×10 6 cells / mL to 2.5×10 6 cells / mL and is first seeded into the cell culture medium. In one example, the suspension cell line is 1.5×10 6 cells / mL to 2×10 6 cells / mL and is first seeded into the cell culture medium. In one example, the suspension cell line is approximately 1×10 5 cells / mL, or approximately 2×10 5 cells / mL, or approximately 3×10 5 cells / mL, or approximately 4×10 5 cells / mL, or approximately 5×10 5 cells / mL, or approximately 6×10 5 cells / mL, or approximately 7×10 5 cells / mL, or approximately 8×10 5 cells / mL, or approximately 9×10 5 cells / mL, or approximately 10×10 5It is initially seeded into the cell culture medium at a density of cells / mL of the cell culture medium. In one example, the suspension cell line is about 1×10 6 cells / mL, or about 2×10 6 cells / mL, or about 3×10 6 cells / mL, or about 4×10 6 cells / mL, or about 5×10 6 cells / mL, or about 6×10 6 cells / mL, or about 7×10 6 cells / mL, or about 8×10 6 cells / mL, or about 9×10 6 cells / mL, or about 10×10 6 It is initially seeded into the cell culture medium at a density of cells / mL of the cell culture medium. In one example, the suspension cell line is about 0.5×10 6 cells / mL, and is initially seeded into the cell culture medium. In one example, the suspension cell line is about 1×10 6 cells / mL, and is initially seeded into the cell culture medium. In one example, the suspension cell line is about 1.5×10 6 cells / mL, and is initially seeded into the cell culture medium. In one example, the suspension cell line is about 1.8×10 6 cells / mL, and is initially seeded into the cell culture medium. In one example, the suspension cell line is about 2×10 6 cells / mL, and is initially seeded into the cell culture medium. In one example, the suspension cell line is 2.5×10 6 cells / mL, and is initially seeded into the cell culture medium. In one example, the suspension cell line is 3.0×10 6 cells / mL, and is initially seeded into the cell culture medium. In one example, the suspension cell line is 3.5×10 6 cells / mL, and is initially seeded into the cell culture medium. In one example, the suspension cell line is 4.0×10 6 cells / mL, and is initially seeded into the cell culture medium. In one example, the suspension cell line is 4.5×10 6 cells / mL, and is initially seeded into the cell culture medium. In one example, the suspension cell line is 5.0×10 6It is initially seeded into the cell culture medium at a density of cells / mL. In one example, the suspension cell line is about 1×10 7 cells / mL, or about 2×10 7 cells / mL, or about 3×10 7 cells / mL, or about 4×10 7 cells / mL, or about 5×10 7 cells / mL, or about 6×10 7 cells / mL, or about 7×10 7 cells / mL, or about 8×10 7 cells / mL, or about 9×10 7 cells / mL, or about 10×10 7 It is initially seeded into the cell culture medium at a density of cells / mL of the cell culture medium. In one example, the suspension cell line is about 1×10 8 cells / mL, or about 2×10 8 cells / mL, or about 3×10 8 cells / mL, or about 4×10 8 cells / mL, or about 5×10 8 cells / mL, or about 6×10 8 cells / mL, or about 7×10 8 cells / mL, or about 8×10 8 cells / mL, or about 9×10 8 cells / mL, or about 10×10 8 It is initially seeded into the cell culture medium at a density of cells / mL of the cell culture medium. In one example, the suspension cell line is about 1×10 9 cells / mL, or about 2×10 9 cells / mL, or about 3×10 9 cells / mL, or about 4×10 9 cells / mL, or about 5×10 9 cells / mL, or about 6×10 9 cells / mL, or about 7×10 9 cells / mL, or about 8×10 9 cells / mL, or about 9×10 9 cells / mL, or about 10×10 9 It is initially seeded into the cell culture medium at a density of cells / mL of the cell culture medium.

[0038] In one example, the suspension cell line is grown to a viable cell density of about 1×10 5 cells / mL to about 1×10 10 cells / mL before diluting the suspension cell culture in a cell culture medium that does not contain tetracycline or a derivative. For example, the suspension cell line is about 1×10 5 cells / mL to 1×10 10 cells / mL, or about 0.1×10 6 cells / mL to 1×10 8 cells / mL, or about 0.5×10 6 cells / mL to 1×10 7 cells / mL, or about 0.5×10 6 cells / mL to 5×10 6 cells / mL, or about 0.5×10 6 cells / mL to 2.5×10 6 cells / mL viable cell density of the cell culture medium. In one example, the suspension cell line is grown to a viable cell density of about 1×10 6 cells / mL to about 1×10 7 cells / mL before diluting the suspension cell culture in a cell culture medium that does not contain tetracycline or a derivative. In another example, the suspension cell line is grown to a viable cell density of about 6×10 6 cells / mL to about 1×10 7 cells / mL before diluting the suspension cell culture in a cell culture medium that does not contain tetracycline or a derivative. In one example, the suspension cell line is grown to a viable cell density of about 0.5×10 6 cells / mL to 5.0×10 6 cells / mL. In one example, the suspension cell line is grown to a viable cell density of about 1×10 6 cells / mL to 2.5×10 6 cells / mL. In one example, the suspension cell line is grown to a viable cell density of about 1.5×10 6 cells / mL to 2×10 6 cells / mL. In one example, the suspension cell line is about 1×10 5 cells / mL, or about 2×10 5 cells / mL, or about 3×10 5 cells / mL, or about 4×10 5 cells / mL, or about 5×105 cells / mL, or about 6×10 5 cells / mL, or about 7×10 5 cells / mL, or about 8×10 5 cells / mL, or about 9×10 5 cells / mL, or about 10×10 5 cells / mL is grown to the viable cell density of the cell culture medium. In one example, the suspension cell line is about 1×10 6 cells / mL, or about 2×10 6 cells / mL, or about 3×10 6 cells / mL, or about 4×10 6 cells / mL, or about 5×10 6 cells / mL, or about 6×10 6 cells / mL, or about 7×10 6 cells / mL, or about 8×10 6 cells / mL, or about 9×10 6 cells / mL, or about 10×10 6 cells / mL is grown to the viable cell density of the cell culture medium. In one example, the suspension cell line is about 0.5×10 6 cells / mL is grown to the viable cell density. In one example, the suspension cell line is about 1×10 6 cells / mL is grown to the viable cell density. In one example, the suspension cell line is about 1.5×10 6 cells / mL is grown to the viable cell density. In one example, the suspension cell line is about 1.8×10 6 cells / mL is grown to the viable cell density. In one example, the suspension cell line is about 2×10 6 cells / mL is grown to the viable cell density. In one example, the suspension cell line is about 2.5×10 6 cells / mL is grown to the viable cell density. In one example, the suspension cell line is about 3.0×10 6 cells / mL is grown to the viable cell density. In one example, the suspension cell line is about 3.5×10 6 cells / mL is grown to the viable cell density. In one example, the suspension cell line is about 4.0×10 6 cells / mL is grown to the viable cell density. In one example, the suspension cell line is about 4.5×10 6It grows to a viable cell density of cells / mL. In one example, the suspension cell line grows to a viable cell density of about 5.0×10 6 It grows to a viable cell density of cells / mL. In one example, the suspension cell line is about 1×10 7 cells / mL, or about 2×10 7 cells / mL, or about 3×10 7 cells / mL, or about 4×10 7 cells / mL, or about 5×10 7 cells / mL, or about 6×10 7 cells / mL, or about 7×10 7 cells / mL, or about 8×10 7 cells / mL, or about 9×10 7 cells / mL, or about 10×10 7 It grows to a viable cell density of cells / mL in the cell culture medium. In one example, the suspension cell line is about 1×10 8 cells / mL, or about 2×10 8 cells / mL, or about 3×10 8 cells / mL, or about 4×10 8 cells / mL, or about 5×10 8 cells / mL, or about 6×10 8 cells / mL, or about 7×10 8 cells / mL, or about 8×10 8 cells / mL, or about 9×10 8 cells / mL, or about 10×10 8 It grows to a viable cell density of cells / mL in the cell culture medium. In one example, the suspension cell line is about 1×10 9 cells / mL, or about 2×10 9 cells / mL, or about 3×10 9 cells / mL, or about 4×10 9 cells / mL, or about 5×10 9 cells / mL, or about 6×10 9 cells / mL, or about 7×10 9 cells / mL, or about 8×10 9 cells / mL, or about 9×10 9 cells / mL, or about 10×10 9 It grows to a viable cell density of cells / mL in the cell culture medium.

[0039] In one example, the suspension cell line has a viability of at least 60% before diluting the suspension cell culture in a cell culture medium that does not contain tetracycline or a derivative thereof. For example, the suspension cell line has a viability of at least 60%, or 70%, or 80%, or 85%, or 90%, or 95%, or 99% before diluting the suspension cell culture in a cell culture medium that does not contain tetracycline or a derivative thereof. In one example, the suspension cell line has a viability of at least 70% before diluting the suspension cell culture in a cell culture medium that does not contain tetracycline or a derivative thereof. In one example, the suspension cell line has a viability of at least 80% before diluting the suspension cell culture in a cell culture medium that does not contain tetracycline or a derivative thereof. In one example, the suspension cell line has a viability of at least 85% before diluting the suspension cell culture in a cell culture medium that does not contain tetracycline or a derivative thereof. In one example, the suspension cell line has a viability of at least 90% before diluting the suspension cell culture in a cell culture medium that does not contain tetracycline or a derivative thereof. In one example, the suspension cell line has a viability of at least 95% before diluting the suspension cell culture in a cell culture medium that does not contain tetracycline or a derivative thereof. In one example, the suspension cell line has a viability of at least 99% before diluting the suspension cell culture in a cell culture medium that does not contain tetracycline or a derivative thereof.

[0040] In one example, the suspension cell culture is operated in batch, fed-batch, continuous, semi-continuous, or perfusion mode. In one example, the cell culture is operated in batch mode. In another example, the cell culture is operated in fed-batch mode. In a further example, the cell culture is operated in semi-continuous mode. In another example, the cell culture is operated in perfusion mode. In one example, the cell culture is operated in batch and perfusion modes. For example, the cell culture is initially operated in batch mode and then in perfusion mode.

[0041] In one example of any of the methods described herein, the method results in a viral infectivity titer yield of at least 1×10 5 transducing units (TU) / mL. For example, the method results in a viral infectivity titer yield of about 1×10 5 TU / mL to 1×10 10 TU / mL. In one example, the method results in a viral infectivity titer yield of about 1×10 5 TU / mL, or about 1.25×10 5 TU / mL, or about 1.5×10 5 TU / mL, or at least 1.75×10 5 TU / mL, or at least 2×10 5 TU / mL, or at least 2.5×10 5 TU / mL, or at least 3×10 5 TU / mL, or about 3.5×10 5 TU / mL, or about 4×10 5 TU / mL, or about 5×10 5 TU / mL. In one example, the method results in a viral infectivity titer yield of about 6×10 5 TU / mL, or about 7×10 5 TU / mL, or at least 8×10 5 TU / mL, or at least 9×10 5 TU / mL, or at least 10×10 5 TU / mL. In another example, the method results in a viral infectivity titer yield of about 1×10 6 TU / mL, or about 1.5×10 6 TU / mL, or about 2×10 6 TU / mL, or about 5×10 6 TU / mL, or about 7×10 6 TU / mL, or about 10×10 6 TU / mL. In a further example, the method results in a viral infectivity titer yield of about 1×10 7 TU / mL, or about 5×10 7 TU / mL, or about 10×10 8 TU / mL, or about 5×10 8 TU / mL, or about 10×10 8TU / mL, or about 5×10 9 TU / mL, or about 1×10 10 TU / mL.

[0042] In one example, by this method, the viral infectivity titer yield is at least 1×10 5 transducing units (TU) / mL of culture medium. For example, by this method, the viral infectivity titer yield is at least 1×10 6 TU / mL of culture medium. For example, by this method, the viral infectivity titer yield is about 1.5×10 6 TU / mL, or about 2×10 6 TU / mL, or about 5×10 6 TU / mL, or about 7×10 6 TU / mL, or about 10×10 6 TU / mL of culture medium. In another example, by this method, the viral infectivity titer yield is at least 1×10 7 TU / mL of culture medium. For example, by this method, the viral infectivity titer yield is about 1.1×10 7 TU / mL, or about 1.2×10 7 TU / mL, or about 1.3×10 7 TU / mL, or about 1.4×10 7 TU / mL, or about 1.5×10 7 TU / mL of culture medium. In one example, by this method, the viral infectivity titer yield is at least 1.5×10 7 TU / mL of culture medium. For example, by this method, the viral infectivity titer yield is about 1.6×10 7 TU / mL, or about 1.7×10 7 TU / mL, or about 1.8×10 7 TU / mL, or about 1.9×10 7 TU / mL of culture medium. In one example, by this method, the viral infectivity titer yield is at least 2×10 7 TU / mL of culture medium. For example, by this method, the viral infectivity titer yield is about 2.1×107 TU / mL, or about 2.2×10 7 TU / mL, or about 2.3×10 7 TU / mL, or about 2.4×10 7 TU / mL, or about 2.5×10 7 TU / mL, or about 2.6×10 7 TU / mL, or about 2.7×10 7 TU / mL, or about 2.8×10 7 TU / mL, or about 2.9×10 7 becomes a culture medium of TU / mL.

[0043] In one example, by this method, the viral infectivity titer yield is at least 1×10 5 transducing units (TU) / mL of the culture medium. For example, by this method, the viral infectivity titer yield is at least 1×10 6 TU / mL of the culture medium. For example, by this method, the viral infectivity titer yield is about 1.5×10 6 TU / mL, or about 2×10 6 TU / mL, or about 5×10 6 TU / mL, or about 7×10 6 TU / mL, or about 10×10 6 TU / mL of the culture medium. In another example, by this method, the viral infectivity titer yield is at least 1×10 7 TU / mL of the culture medium. For example, by this method, the viral infectivity titer yield is about 1.1×10 7 TU / mL, or about 1.2×10 7 TU / mL, or about 1.3×10 7 TU / mL, or about 1.4×10 7 TU / mL, or about 1.5×10 7 TU / mL of the culture medium. In one example, by this method, the viral infectivity titer yield is at least 1.5×10 7 TU / mL of the culture medium. For example, by this method, the viral infectivity titer yield is about 1.6×10 7 TU / mL, or about 1.7×107 TU / mL, or about 1.8×10 7 TU / mL, or about 1.9×10 7 TU / mL of the culture medium. In one example, by this method, the viral infectivity titer yield is at least 2×10 7 TU / mL of the culture medium. For example, by this method, the viral infectivity titer yield is about 2.1×10 7 TU / mL, or about 2.2×10 7 TU / mL, or about 2.3×10 7 TU / mL, or about 2.4×10 7 TU / mL, or about 2.5×10 7 TU / mL, or about 2.6×10 7 TU / mL, or about 2.7×10 7 TU / mL, or about 2.8×10 7 TU / mL, or about 2.9×10 7 TU / mL of the culture medium.

[0044] In one example, by this method, the viral infectivity titer yield is at least 1×10 5 transducing units (TU) / mL of the culture medium. For example, by this method, the viral infectivity titer yield is at least 1×10 6 TU / mL of the culture medium. For example, by this method, the viral infectivity titer yield is about 1.5×10 6 TU / mL, or about 2×10 6 TU / mL, or about 5×10 6 TU / mL, or about 7×10 6 TU / mL, or about 10×10 6 TU / mL of the culture medium. In another example, by this method, the viral infectivity titer yield is at least 1×10 7 TU / mL of the culture medium. For example, by this method, the viral infectivity titer yield is about 1.1×10 7 TU / mL, or about 1.2×10 7 TU / mL, or about 1.3×10 7 TU / mL, or about 1.4×107 TU / mL, or about 1.5×10 7 TU / mL culture medium. In one example, by this method, the viral infectivity titer yield is at least 1.5×10 7 TU / mL culture medium on the 25th day of culture. For example, by this method, the viral infectivity titer yield is about 1.6×10 7 TU / mL, or about 1.7×10 7 TU / mL, or about 1.8×10 7 TU / mL, or about 1.9×10 7 TU / mL culture medium on the 25th day of culture. In one example, by this method, the viral infectivity titer yield is at least 2×10 7 TU / mL culture medium on the 25th day of culture. For example, by this method, the viral infectivity titer yield is about 2.1×10 7 TU / mL, or about 2.2×10 7 TU / mL, or about 2.3×10 7 TU / mL, or about 2.4×10 7 TU / mL, or about 2.5×10 7 TU / mL, or about 2.6×10 7 TU / mL, or about 2.7×10 7 TU / mL, or about 2.8×10 7 TU / mL, or about 2.9×10 7 TU / mL culture medium.

[0045] In one example, by this method, the viral infectivity titer yield is at least 1×10 5 transducing units (TU) / mL culture medium on the 30th day of culture. For example, by this method, the viral infectivity titer yield is at least 1×10 6 TU / mL culture medium on the 30th day of culture. For example, by this method, the viral infectivity titer yield is about 1.5×10 6 TU / mL, or about 2×10 6 TU / mL, or about 5×10 6 TU / mL, or about 7×10 6 TU / mL, or about 10×10 6It becomes a culture medium of TU / mL. In another example, by this method, the viral infectivity titer yield is at least 1×10 7 TU / mL in the culture medium. For example, by this method, the viral infectivity titer yield is about 1.1×10 7 TU / mL, or about 1.2×10 7 TU / mL, or about 1.3×10 7 TU / mL, or about 1.4×10 7 TU / mL, or about 1.5×10 7 TU / mL in the culture medium. In one example, by this method, the viral infectivity titer yield is at least 1.5×10 7 TU / mL in the culture medium. For example, by this method, the viral infectivity titer yield is about 1.6×10 7 TU / mL, or about 1.7×10 7 TU / mL, or about 1.8×10 7 TU / mL, or about 1.9×10 7 TU / mL in the culture medium. In one example, by this method, the viral infectivity titer yield is at least 2×10 7 TU / mL in the culture medium. For example, by this method, the viral infectivity titer yield is about 2.1×10 7 TU / mL, or about 2.2×10 7 TU / mL, or about 2.3×10 7 TU / mL, or about 2.4×10 7 TU / mL, or about 2.5×10 7 TU / mL, or about 2.6×10 7 TU / mL, or about 2.7×10 7 TU / mL, or about 2.8×10 7 TU / mL, or about 2.9×10 7 TU / mL in the culture medium.

[0046] In one example, by this method, the viral infectivity titer yield is at least 1×10 5It becomes transduction unit (TU) / mL of the culture medium. For example, by this method, the viral infectivity titer yield becomes at least 1×10 6 TU / mL on the 35th day of culture. For example, by this method, the viral infectivity titer yield becomes about 1.5×10 6 TU / mL, or about 2×10 6 TU / mL, or about 5×10 6 TU / mL, or about 7×10 6 TU / mL, or about 10×10 6 TU / mL of the culture medium. In another example, by this method, the viral infectivity titer yield becomes at least 1×10 7 TU / mL of the culture medium. For example, by this method, the viral infectivity titer yield becomes about 1.1×10 7 TU / mL, or about 1.2×10 7 TU / mL, or about 1.3×10 7 TU / mL, or about 1.4×10 7 TU / mL, or about 1.5×10 7 TU / mL of the culture medium. In one example, by this method, the viral infectivity titer yield becomes at least 1.5×10 7 TU / mL of the culture medium. For example, by this method, the viral infectivity titer yield becomes about 1.6×10 7 TU / mL, or about 1.7×10 7 TU / mL, or about 1.8×10 7 TU / mL, or about 1.9×10 7 TU / mL of the culture medium. In one example, by this method, the viral infectivity titer yield becomes at least 2×10 7 TU / mL of the culture medium. For example, by this method, the viral infectivity titer yield becomes about 2.1×10 7 TU / mL, or about 2.2×10 7 TU / mL, or about 2.3×10 7 TU / mL, or about 2.4×10 7 TU / mL, or about 2.5×10 7 TU / mL, or about 2.6×10 7TU / mL, or about 2.7×10 7 TU / mL, or about 2.8×10 7 TU / mL, or about 2.9×10 7 will be the culture medium of TU / mL.

[0047] In one example, by this method, the viable cell density will be at least about 1×10 5 cells / mL of the culture medium. For example, by this method, the viable cell density will be at least about 1×10 6 cells / mL of the culture medium. In one example, by this method, the viable cell density will be at least about 1.5×10 6 cells / mL, or about 2×10 6 cells / mL, or about 5×10 6 cells / mL, or about 7×10 6 cells / mL, or about 10×10 6 cells / mL of the culture medium. In one example, by this method, the viable cell density will be at least about 1×10 7 cells / mL of the culture medium. In one example, by this method, the viable cell density will be at least about 1.1×10 7 cells / mL, or about 1.2×10 7 cells / mL, or about 1.3×10 7 cells / mL, or about 1.4×10 7 cells / mL, or about 1.5×10 7 cells / mL of the culture medium. In one example, by this method, the viable cell density will be at least about 1.5×10 7 cells / mL of the culture medium. In one example, by this method, the viable cell density will be at least about 1.5×10 7 cells / mL, or about 1.6×10 7 cells / mL, or about 1.7×10 7 cells / mL, or about 1.8×10 7 cells / mL, or about 1.9×10 7 cells / mL, or about 2.0×10 7It becomes cells / mL of the culture medium. In one example, by this method, the viable cell density becomes at least about 2.0×10 7 cells / mL of the culture medium. In one example, by this method, the viable cell density becomes at least about 2.1×10 7 cells / mL, or about 2.2×10 7 cells / mL, or about 2.3×10 7 cells / mL, or about 2.4×10 7 cells / mL, or about 2.5×10 7 cells / mL of the culture medium. In one example, by this method, the viable cell density becomes at least about 2.5×10 7 cells / mL of the culture medium. In one example, by this method, the viable cell density becomes at least about 2.6×10 7 cells / mL, or about 2.7×10 7 cells / mL, or about 2.8×10 7 cells / mL, or about 2.9×10 7 cells / mL, or about 3.0×10 7 cells / mL of the culture medium.

[0048] In one example, by this method, the viable cell density becomes at least about 1×10 5 cells / mL of the culture medium. For example, by this method, the viable cell density becomes at least about 1×10 6 cells / mL of the culture medium. In one example, by this method, the viable cell density becomes at least about 1.5×10 6 cells / mL, or about 2×10 6 cells / mL, or about 5×10 6 cells / mL, or about 7×10 6 cells / mL, or about 10×10 6 cells / mL of the culture medium. In one example, by this method, the viable cell density becomes at least about 1×10 7 cells / mL of the culture medium. In one example, by this method, the viable cell density becomes at least about 1.1×10 7 cells / mL, or about 1.2×10 7cells / mL, or about 1.3×10 7 cells / mL, or about 1.4×10 7 cells / mL, or about 1.5×10 7 cells / mL of the culture medium. In one example, by this method, the viable cell density becomes at least about 1.5×10 7 cells / mL of the culture medium. In one example, by this method, the viable cell density becomes at least about 1.5×10 7 cells / mL, or about 1.6×10 7 cells / mL, or about 1.7×10 7 cells / mL, or about 1.8×10 7 cells / mL, or about 1.9×10 7 cells / mL, or about 2.0×10 7 cells / mL of the culture medium. In one example, by this method, the viable cell density becomes at least about 2.0×10 7 cells / mL of the culture medium. In one example, by this method, the viable cell density becomes at least about 2.1×10 7 cells / mL, or about 2.2×10 7 cells / mL, or about 2.3×10 7 cells / mL, or about 2.4×10 7 cells / mL, or about 2.5×10 7 cells / mL of the culture medium. In one example, by this method, the viable cell density becomes at least about 2.5×10 7 cells / mL of the culture medium. In one example, by this method, the viable cell density becomes at least about 2.6×10 7 cells / mL, or about 2.7×10 7 cells / mL, or about 2.8×10 7 cells / mL, or about 2.9×10 7 cells / mL, or about 3.0×10 7 cells / mL of the culture medium.

[0049] In one example, by this method, the viable cell density becomes at least about 1×10 5cells / mL of the culture medium. For example, by this method, the viable cell density becomes at least about 1×10 6 cells / mL of the culture medium. In one example, by this method, the viable cell density becomes at least about 1.5×10 6 cells / mL, or about 2×10 6 cells / mL, or about 5×10 6 cells / mL, or about 7×10 6 cells / mL, or about 10×10 6 cells / mL of the culture medium. In one example, by this method, the viable cell density becomes at least about 1×10 7 cells / mL of the culture medium. In one example, by this method, the viable cell density becomes at least about 1.1×10 7 cells / mL, or about 1.2×10 7 cells / mL, or about 1.3×10 7 cells / mL, or about 1.4×10 7 cells / mL, or about 1.5×10 7 cells / mL of the culture medium. In one example, by this method, the viable cell density becomes at least about 1.5×10 7 cells / mL of the culture medium. In one example, by this method, the viable cell density becomes at least about 1.5×10 7 cells / mL, or about 1.6×10 7 cells / mL, or about 1.7×10 7 cells / mL, or about 1.8×10 7 cells / mL, or about 1.9×10 7 cells / mL, or about 2.0×10 7 cells / mL of the culture medium. In one example, by this method, the viable cell density becomes at least about 2.0×10 7 cells / mL of the culture medium. In one example, by this method, the viable cell density becomes at least about 2.1×10 7 cells / mL, or about 2.2×10 7 cells / mL, or about 2.3×10 7 cells / mL, or about 2.4×10 7cells / mL, or about 2.5×10 7 cells / mL of the culture medium. In one example, by this method, the viable cell density becomes at least about 2.5×10 7 cells / mL of the culture medium. In one example, by this method, the viable cell density becomes at least about 2.6×10 7 cells / mL, or about 2.7×10 7 cells / mL, or about 2.8×10 7 cells / mL, or about 2.9×10 7 cells / mL, or about 3.0×10 7 cells / mL of the culture medium.

[0050] In one example, by this method, the viable cell density becomes at least about 1×10 5 cells / mL of the culture medium. For example, by this method, the viable cell density becomes at least about 1×10 6 cells / mL of the culture medium. In one example, by this method, the viable cell density becomes at least about 1.5×10 6 cells / mL, or about 2×10 6 cells / mL, or about 5×10 6 cells / mL, or about 7×10 6 cells / mL, or about 10×10 6 cells / mL of the culture medium. In one example, by this method, the viable cell density becomes at least about 1×10 7 cells / mL of the culture medium. In one example, by this method, the viable cell density becomes at least about 1.1×10 7 cells / mL, or about 1.2×10 7 cells / mL, or about 1.3×10 7 cells / mL, or about 1.4×10 7 cells / mL, or about 1.5×10 7 cells / mL of the culture medium. In one example, by this method, the viable cell density becomes at least about 1.5×10 7 cells / mL of the culture medium. In one example, by this method, the viable cell density becomes at least about 1.5×10 7cells / mL, or about 1.6×10 7 cells / mL, or about 1.7×10 7 cells / mL, or about 1.8×10 7 cells / mL, or about 1.9×10 7 cells / mL, or about 2.0×10 7 cells / mL of the culture medium. In one example, by this method, the viable cell density becomes at least about 2.0×10 7 cells / mL of the culture medium. In one example, by this method, the viable cell density becomes at least about 2.1×10 7 cells / mL, or about 2.2×10 7 cells / mL, or about 2.3×10 7 cells / mL, or about 2.4×10 7 cells / mL, or about 2.5×10 7 cells / mL of the culture medium. In one example, by this method, the viable cell density becomes at least about 2.5×10 7 cells / mL of the culture medium. In one example, by this method, the viable cell density becomes at least about 2.6×10 7 cells / mL, or about 2.7×10 7 cells / mL, or about 2.8×10 7 cells / mL, or about 2.9×10 7 cells / mL, or about 3.0×10 7 cells / mL of the culture medium.

[0051] In one example, by this method, the viable cell density becomes at least about 1×10 5 cells / mL of the culture medium. For example, by this method, the viable cell density becomes at least about 1×10 6 cells / mL of the culture medium. In one example, by this method, the viable cell density becomes at least about 1.5×10 6 cells / mL, or about 2×10 6 cells / mL, or about 5×10 6 cells / mL, or about 7×10 6 cells / mL, or about 10×10 6cells / mL of the culture medium. In one example, by this method, the viable cell density is at least about 1×10 7 cells / mL of the culture medium. In one example, by this method, the viable cell density is at least about 1.1×10 7 cells / mL, or about 1.2×10 7 cells / mL, or about 1.3×10 7 cells / mL, or about 1.4×10 7 cells / mL, or about 1.5×10 7 cells / mL of the culture medium. In one example, by this method, the viable cell density is at least about 1.5×10 7 cells / mL of the culture medium. In one example, by this method, the viable cell density is at least about 1.5×10 7 cells / mL, or about 1.6×10 7 cells / mL, or about 1.7×10 7 cells / mL, or about 1.8×10 7 cells / mL, or about 1.9×10 7 cells / mL, or about 2.0×10 7 cells / mL of the culture medium. In one example, by this method, the viable cell density is at least about 2.0×10 7 cells / mL of the culture medium. In one example, by this method, the viable cell density is at least about 2.1×10 7 cells / mL, or about 2.2×10 7 cells / mL, or about 2.3×10 7 cells / mL, or about 2.4×10 7 cells / mL, or about 2.5×10 7 cells / mL of the culture medium. In one example, by this method, the viable cell density is at least about 2.5×10 7 cells / mL of the culture medium. In one example, by this method, the viable cell density is at least about 2.6×10 7 cells / mL, or about 2.7×10 7 cells / mL, or about 2.8×10 7 cells / mL, or about 2.9×10 7cells / mL, or about 3.0×10 7 cells / mL of culture medium.

[0052] In one example, the cell line has a viability of at least 70% on day 15 of culture. For example, the cell line has a viability of about 70%, or about 75%, or about 80% on day 15 of culture. In one example, the cell line has a viability of at least 75% on day 15 of culture. In another example, the cell line has a viability of at least 80% on day 15 of culture. For example, the cell line has a viability of about 80%, or about 85%, or about 90% on day 15 of culture. In one example, the cell line has a viability of about 80% on day 15 of culture. For example, the viability is about 81%, or about 82%, or about 83%, or about 84%. In another example, the cell line has a viability of about 85% on day 15 of culture. For example, the viability is about 86%, or about 87%, or about 88%, or about 89% on day 15 of culture. In a further example, the cell line has a viability of about 90% on day 15 of culture. In one example, the cell line has a viability of at least 90% on 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% on day 15 of culture. In one example, the cell line has a viability of at least 95% on day 15 of culture. For example, the cell line has a viability of about 96%, or about 97%, or about 98%, or about 99% on day 15 of culture.

[0053] In one example, the cell line has a viability of at least 70% on the 20th day of culture. For example, the cell line has a viability of about 70%, or about 75%, or about 80% on the 20th day of culture. In one example, the cell line has a viability of at least 75% on the 20th day of culture. In another example, the cell line has a viability of at least 80% on the 20th day of culture. For example, the cell line has a viability of about 80% or about 85% or about 90% on the 20th day of culture. In one example, the cell line has a viability of about 80% on the 20th day of culture. For example, the viability is about 81%, or about 82%, or about 83%, or about 84%. In another example, the cell line has a viability of about 85% on the 20th day of culture. For example, the viability is about 86%, or about 87%, or about 88%, or about 89% on the 20th day of culture. In a further example, the cell line has a viability of about 90% on the 20th day of culture. In one example, the cell line has a viability of at least 90% on the 20th day 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% on the 20th day of culture. In one example, the cell line has a viability of at least 95% on the 20th day of culture. For example, the cell line has a viability of about 96%, or about 97%, or about 98%, or about 99% on the 20th day of culture.

[0054] In one example, the cell line has a viability of at least 70% on the 25th day of culture. For example, the cell line has a viability of about 70%, or about 75%, or about 80% on the 25th day of culture. In one example, the cell line has a viability of at least 75% on the 25th day of culture. In another example, the cell line has a viability of at least 80% on the 25th day of culture. For example, the cell line has a viability of about 80%, or about 85%, or about 90% on the 25th day of culture. In one example, the cell line has a viability of about 80% on the 25th day of culture. For example, the viability is about 81%, or about 82%, or about 83%, or about 84%. In another example, the cell line has a viability of about 85% on the 25th day of culture. For example, the viability is about 86%, or about 87%, or about 88%, or about 89% on the 25th day of culture. In a further example, the cell line has a viability of about 90% on the 25th day of culture. In one example, the cell line has a viability of at least 90% on the 25th day 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% on the 25th day of culture. In one example, the cell line has a viability of at least 95% on the 25th day of culture. For example, the cell line has a viability of about 96%, or about 97%, or about 98%, or about 99% on the 25th day of culture.

[0055] In one example, the cell line has a viability of at least 70% on the 30th day of culture. For example, the cell line has a viability of about 70%, or about 75%, or about 80% on the 30th day of culture. In one example, the cell line has a viability of at least 75% on the 30th day of culture. In another example, the cell line has a viability of at least 80% on the 30th day of culture. For example, the cell line has a viability of about 80% or about 85% or about 90% on the 30th day of culture. In one example, the cell line has a viability of about 80% on the 30th day of culture. For example, the viability is about 81%, or about 82%, or about 83%, or about 84%. In another example, the cell line has a viability of about 85% on the 30th day of culture. For example, on the 30th day of culture, the viability is about 86%, or about 87%, or about 88%, or about 89%. In a further example, the cell line has a viability of about 90% on the 30th day of culture. In one example, the cell line has a viability of at least 90% on the 30th day 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% on the 30th day of culture. In one example, the cell line has a viability of at least 95% on the 30th day of culture. For example, the cell line has a viability of about 96%, or about 97%, or about 98%, or about 99% on the 30th day of culture.

[0056] In one example, the cell line has a viability of at least 70% on day 35 of culture. For example, the cell line has a viability of about 70%, or about 75%, or about 80% on day 35 of culture. In one example, the cell line has a viability of at least 75% on day 35 of culture. In another example, the cell line has a viability of at least 80% on day 35 of culture. For example, the cell line has a viability of about 80%, or about 85%, or about 90% on day 35 of culture. In one example, the cell line has a viability of about 80% on day 35 of culture. For example, the viability is about 81%, or about 82%, or about 83%, or about 84%. In another example, the cell line has a viability of about 85% on day 35 of culture. For example, on day 35 of culture, the viability is about 86%, or about 87%, or about 88%, or about 89%. In a further example, the cell line has a viability of about 90% on day 35 of culture. In one example, the cell line has a viability of at least 90% on 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% on day 35 of culture. In one example, the cell line has a viability of at least 95% on day 35 of culture. For example, the cell line has a viability of about 96%, or about 97%, or about 98%, or about 99% on day 35 of culture.

[0057] In one example, the method increases the viral infectivity 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 viral infectivity titer yield by at least 10%.

[0058] In one example, the tetracycline derivative is selected from the group consisting of minocycline, doxycycline, demeclocycline, oxytetracycline, and tigecycline. In one example, the tetracycline derivative is doxycycline.

[0059] In one example, the suspension cell culture has a volume of 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 more than about 15,000 L. For example, the suspension cell culture has a volume of about 1 L to about 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 yet another 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 yet another 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.

[0060] In one example, the suspension cell culture is operated at dissolved carbon dioxide (CO2) levels of 4% to 8%. For example, the suspension cell culture is operated at about 4% CO2. In another example, the suspension cell culture is operated at about 5% CO2. In a further example, the suspension cell culture is operated at about 6% CO2. In a further example, the suspension cell culture is operated at about 7% CO2. In a further example, the suspension cell culture is operated at about 8% CO2.

[0061] In one example, the suspension cell culture is operated at a pH of 6.0 to 8.0. In one example, the suspension cell culture has a pH of about 6.5 to 7.5. For example, the pH is from about 6.90 to 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.

[0062] In one example, the suspension cell culture is operated at a temperature of about 35°C to about 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 has a temperature of about 36.5°C to 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 has a temperature of about 38°C to about 39°C. For example, the suspension cell culture is at a temperature of about 38.5°C.

[0063] In one example, the suspension cell culture has a pH of 6.0 to 8.0 and / or a temperature of 35 to 39°C. In one example, the suspension cell culture has a pH of about 6.0 to about 8.0 and / or a temperature of about 37°C to about 38.5°C. For example, the suspension cell culture has a pH of about 6.8 to about 7.1 and / or a temperature of about 37°C to about 38.5°C. In one example, the suspension cell culture has a pH of about 6.8 to about 7.1 and a temperature of about 37°C to about 38.5°C. In one example, the suspension cell culture has a pH of about 6.9 to about 7.0 and a temperature of about 37.0°C.

[0064] In one example, the method further includes selecting a cell clone that can produce an enveloped virus with a high infectivity titer and survival rate for at least 15 days. For example, the method selects a cell clone that can produce an enveloped virus with an infectivity titer of at least 5×10 6 TU / mL of the culture medium and / or a survival rate of at least 75% and / or a viable cell density of at least 1×10 7 cells / mL of the culture medium.

[0065] In one example, the method further includes selecting a cell clone that can produce an enveloped virus with a high infectivity titer and survival rate for at least 20 days. For example, the method selects a cell clone that can produce an enveloped virus with an infectivity titer of at least 5×10 6 TU / mL of the culture medium and / or a survival rate of at least 75% and / or a viable cell density of at least 1×10 7 cells / mL of the culture medium.

[0066] In one example, the method further includes selecting a cell clone that can produce an enveloped virus with a high infectivity titer and survival rate for at least 25 days. For example, the method selects a cell clone that can produce an enveloped virus with an infectivity titer of at least 5×10 6 TU / mL of the culture medium and / or a survival rate of at least 75% and / or a viable cell density of at least 1×10 7 cells / mL of the culture medium.

[0067] In one example, the method further includes selecting a cell clone that can produce an enveloped virus with a high infectivity titer and survival rate for at least 30 days. For example, the method selects a cell clone that can produce an enveloped virus with an infectivity titer of at least 5×10 6Infectivity titer of the TU / mL culture medium and / or at least 75% viability and / or at least 1×10 7 Further comprising selecting a cell clone capable of producing an enveloped virus at a viable cell density of cells / mL culture medium.

[0068] In one example, the method further comprises selecting a cell clone capable of producing an enveloped virus with a high infectivity titer and viability for at least 35 days. For example, the method is at least 35 days, at least 5×10 6 Infectivity titer of the TU / mL culture medium and / or at least 75% viability and / or at least 1×10 7 Further comprising selecting a cell clone capable of producing an enveloped virus at a viable cell density of cells / mL culture medium.

[0069] Additional methods for selecting suitable cell clones will be apparent to those skilled in the art and / or are described herein and include, for example, the stability and / or mutation status of the cell clone and the ability of the purified virus to transduce hematopoietic stem cells.

[0070] In one example, the method further comprises purifying the enveloped virus from a suspension cell culture.

[0071] Methods for purifying an enveloped virus from a suspension cell culture will be apparent to those skilled in the art and / or are described herein. In one example, purifying the enveloped virus includes one or more steps selected from the group consisting of clarification filtration, anion exchange chromatography, concentration, and diafiltration.

[0072] In one example, the method of the present disclosure further comprises performing sterile filtration. For example, sterile filtration is performed before concentrating and diafiltering the eluted virus. In an alternative example, sterile filtration is performed after concentrating and diafiltering the eluted virus.

[0073] In one example, the method further includes formulating the purified enveloped virus into a solution suitable for infecting a pharmaceutical formulation or cells.

[0074] The present disclosure also provides a purified enveloped virus produced by the methods described herein.

Brief Description of the Drawings

[0075]

Figure 1

Figure 2

Figure 3-1

Figure 3-2

Figure 3-3

Figure 4

Modes for Carrying Out the Invention

[0076] General Throughout this specification, unless otherwise specifically stated or required by the context, references to a single step, composition, group of steps, or group of compositions include one and more (i.e., one or more) of that step, composition, group of steps, or group of compositions. Thus, as used herein, the singular forms “a,” “an,” and “the” include plural aspects unless the context clearly dictates otherwise. For example, reference to “a” includes one and two or more; reference to “an” includes one and two or more; reference to “the” includes one and two or more, etc.

[0077] One of ordinary skill in the art will recognize that the present disclosure is susceptible to variations and modifications other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications. The present disclosure includes all steps, configurations, compositions, and compounds individually or collectively mentioned or shown herein, and any combination or any two or more combinations of said steps or configurations.

[0078] The present disclosure should not be limited in scope by the specific examples described herein, which are for illustrative purposes only. Functionally equivalent products, compositions, and methods are clearly within the scope of the present disclosure.

[0079] Unless otherwise specifically stated, any example of the present disclosure herein shall be applicable mutatis mutandis to any other example of the present disclosure. In other words, any particular example of the present disclosure can be combined (except in the case of exclusivity) with any other particular example of the present disclosure.

[0080] Any example of the present disclosure that discloses a particular configuration or group of configurations or method or method step shall not provide express support for negating a particular configuration or group of configurations or method or method step.

[0081] Unless otherwise defined, all technical and scientific terms used in this specification shall have the same meaning as commonly understood by one of ordinary skill in the art (e.g., molecular biology, bacteriology, virology).

[0082] Unless indicated otherwise, conventional techniques of molecular biology, bacteriology, virology, recombinant DNA technology, peptide synthesis in solution, solid-phase peptide synthesis, and immunology utilized in this disclosure are standard procedures and are well known to those of ordinary skill 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 (ed.), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D. M. Glover and B. D. Hames (eds.), DNA Cloning: A Practical Approach, Volumes 1 - 4, IRL Press (1995 and 1996), as well as F. M. Ausubel et al. (eds.), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley - Interscience (1988, including all revisions to date), Ed Harlow and David Lane (eds.) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J. E. Coligan et al. (eds.) Current Protocols in Immunology, John Wiley & Sons (including all revisions to date).

[0083] The term "and / or", e.g., "X and / or Y", is understood to mean either "X and Y" or "X or Y", and is intended to provide clear support for both meanings or either meaning.

[0084] The term "about", unless otherwise specified, refers to + / - 20% of the specified value, more particularly, for example, + / - 10%. To avoid misunderstanding, the specified value following the term "about" should be construed to include the exact specified value itself (e.g., "about 10" includes exactly 10).

[0085] As used herein, the term "from" is intended to indicate that the specified integer is obtained from a particular source, but not necessarily directly from that source (i.e., including those obtained by recombination).

[0086] Throughout this specification, the word "comprise", or variations such as "comprises" or "comprising", is intended to mean the inclusion of the 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.

[0087] Selected Definitions As used herein, the term "enveloped virus" refers to DNA and RNA viruses that have a viral envelope. The envelope is typically derived from the host cell membrane (e.g., phospholipids and proteins), but may also contain viral glycoproteins on the envelope surface. Enveloped viruses also include a "capsid", which is a protein layer between the envelope and the viral genome. In one example, an enveloped virus is a retrovirus. For example, an enveloped virus is a lentivirus, such as the human immunodeficiency virus.

[0088] As used herein, the term "cell culture fluid" or "cell culture medium" is to be understood to encompass the liquid or medium in which cells are grown to produce enveloped viruses. The liquid or medium does not contain cells (e.g., the cells may have been removed by centrifugation and / or removal of the supernatant).

[0089] As used herein, the term "cell culture" or "suspension cell culture" is to be understood to refer to the aggregate of cell culture fluid or medium and cultured cells.

[0090] The term "suspension" with respect to a cell line is to be understood to refer to single cells or small aggregates of cells freely floating in the cell culture medium. For example, such cells function and propagate in the agitated growth medium to form a suspension.

[0091] The term "purify" or "purifying" or "purification" means removing at least one impurity present in the cell culture medium, whether completely or partially, thereby improving the purity level of the enveloped virus in the solution.

[0092] The term "impurity" or "impurities" is to be understood to include one or more components in the cell culture medium other than the enveloped virus. For example, impurities include process-related impurities such as host cell DNA, host cell proteins, and medium components (e.g., fetal bovine serum).

[0093] Production of Enveloped Viruses Methods for producing enveloped viruses will be apparent to those skilled in the art and / or are 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.

[0094] Enveloped virus In one example, the virus is a retrovirus, such as a lentivirus. Exemplary retroviruses include alpharetroviruses (such as avian leukosis virus (ALV)), betaretroviruses (such as mouse mammary tumor virus (MMTV)), gammaretroviruses (such as mouse leukemia virus (MLV)), deltaretroviruses (such as human T-lymphotropic virus (HTLV)), epsilonretroviruses (such as walleye dermal sarcoma virus (WDSV)), spumaviruses (such as human foamy virus (HFV) or simian foamy virus (SFV)), primate lentiviruses such as different types of human immunodeficiency virus (HIV), different types of simian immunodeficiency virus (SIV), or non-primate mammalian lentiviruses such as equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), caprine arthritis encephalitis virus (CAEV), or visna maedi virus (VMV).

[0095] In some examples, the enveloped virus, such as a retrovirus, is pseudotyped, i.e., the pseudotype contains an envelope glycoprotein, a modified envelope glycoprotein, or a chimeric envelope glycoprotein derived from a virus different from the virus from which it is derived.

[0096] Gene expression In some examples, an enveloped virus contains a transgene introduced into its genome. The transgene will be determined by the specific use for which the enveloped viral vector is intended. Exemplary transgenes include transgenes encoding therapeutic RNAs (e.g., antisense complementary RNAs to target RNA or DNA sequences), transgenes encoding proteins that are deficient or deleted in a subject with a lesion, or transgenes used for DNA vaccination, i.e., transgenes encoding 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 an abnormal hemoglobinopathy, such as sickle cell disease or thalassemia. In some examples, the transgene encodes a protein or nucleic acid useful for treating 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.

[0097] In some examples, an enveloped virus is produced by introducing into a host cell four elements: an expression cassette containing the lentiviral gene gagpol, an expression cassette containing the lentiviral gene rev, all transgenes located between the lentiviral LTR-5’ and the lentiviral LTR-3’, and an expression cassette encoding an envelope glycoprotein.

[0098] Producer cell line In some examples, an enveloped virus is produced from a stable system that expresses one or several elements necessary to produce the enveloped virus (Miller (2001) Curr. Protoc. Hum. Genet. Chapter 12: Unit 12.5.; Rodrigues et al. 2011, supra). In one example, an enveloped virus is produced from mammalian host cells transiently transfected with one or several plasmids encoding the elements necessary to produce the virus. According to an alternative example, the elements are introduced into cells using multiple plasmids: one plasmid having an expression cassette containing the lentiviral gagpol gene, one plasmid having an expression cassette containing the lentiviral rev gene, one plasmid having an expression cassette encoding an envelope glycoprotein, one plasmid having an expression cassette containing the tetracycline transactivator (tTA) gene, and / or one plasmid having an expression cassette containing the lentiviral tat gene. A transfer plasmid containing an expression cassette with a transgene contained between the LTR-5’ and LTR-3’ of a lentivirus can be introduced into producer cells as a concatemer together with a helper plasmid having an antibiotic resistance cassette conferring resistance.

[0099] The host cell is selected from any cell capable of producing an enveloped virus. According to one example, the cell is selected from human cells (HEK293, HEK293T, HEK293FT, HEK293OX, Te671, HT1080, CEM), mouse cells (NIH-3T3), ferret family cells (Mpf), canine family cells (D17), and derivatives thereof. According to one example, the cell is selected from CHO cells, BHK cells, MDCK cells, C3H10T1 / 2 cells, FLYI, Psi-2 cells, BOSC23 cells, PA317 cells, WEHI cells, COS cells, BSC1 cells, BSC40 cells, BMT10 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 211A cells, and derivatives thereof.

[0100] 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 cell lines derived from any of the above cell lines.

[0101] In one example, the cell line is a suspension cell line selected from the GPR, GPRG, GPRT, GPRGT, 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 the GPRG cell line. In another example, the suspension cell line is a cell line derived from the GPRGT cell line. In a further example, the suspension cell line is a cell line derived from the GPRTG cell line.

[0102] The method of adapting adherent cell lines to grow in suspension will be apparent to those skilled in the art and is described herein.

[0103] In one example, an enveloped virus is produced from stable producer cells. The stable producer cells can be derived from a packaging cell line comprising any of the cell lines disclosed herein. In some embodiments, the packaging cell line is a GPRG or GPRTG cell line (Throm et al., (2009) Blood 113(21):5104-5110; and Bonner et al., (2015) Molecular Therapy, Volume 23, Supplement 1, S35). In one example, the stable producer cell line cells synthesize a vector by cloning one or more genes into a recombinant plasmid; form a concatemer array from an expression cassette excised from the synthesized vector and an expression cassette obtained from an antibiotic resistance cassette plasmid; transfect the packaging cell line cells with the formed concatemer array; and generate the stable producer cell line cells by selecting and isolating them. The virus is produced by inducing an inducible promoter of the stable producer cell line cells.

[0104] The present disclosure also provides stable producer cell clones capable of producing an enveloped virus.

[0105] In one example, stable producer cell clones are cultured to produce a stable producer cell line. Methods of culturing stable producer cell clones to generate a stable producer cell line are described herein and / or will be apparent to those skilled in the art.

[0106] The present disclosure provides stable producer cell clones capable of producing an enveloped virus at an infectious titer of at least 5×10 6 TU / mL culture medium and / or a viability of at least 75% and / or a viable cell density of at least 1.0×10 7 cells / mL culture medium. For example, the stable producer cell clones have an infectious titer of at least 5×10 6 TU / mL culture medium, and / or a viability of at least 75%, and / or a viable cell density of at least 1.0×10 7It is possible to produce an enveloped virus at a viable cell density of cells / mL of the culture medium.

[0107] The present disclosure provides, for at least 20 days, at least 5×10 6 the infectivity titer of TU / mL of the culture medium and / or at least 75% viability and / or at least 1.0×10 7 It also provides a stable producing cell clone that can produce an enveloped virus at a viable cell density of cells / mL of the culture medium. For example, the stable producing cell clone has, on the 20th day of culture, at least 5×10 6 the infectivity titer of TU / mL of the culture medium, and / or at least 75% viability, and / or at least 1.0×10 7 It is possible to produce an enveloped virus at a viable cell density of the culture medium of cells / mL of the culture medium.

[0108] The present disclosure provides, for at least 25 days, at least 5×10 6 the infectivity titer of TU / mL of the culture medium and / or at least 75% viability and / or at least 1.0×10 7 It also provides a stable producing cell clone that can produce an enveloped virus at a viable cell density of cells / mL of the culture medium. For example, the stable producing cell clone has, on the 25th day of culture, at least 5×10 6 the infectivity titer of TU / mL of the culture medium, and / or at least 75% viability, and / or at least 1.0×10 7 It is possible to produce an enveloped virus at a viable cell density of cells / mL of the culture medium.

[0109] The present disclosure provides, for at least 30 days, at least 5×10 6 the infectivity titer of TU / mL of the culture medium and / or at least 75% viability and / or at least 1.0×10 7 It also provides a stable producing cell clone that can produce an enveloped virus at a viable cell density of cells / mL of the culture medium. For example, the stable producing cell clone has, on the 30th day of culture, at least 5×10 6An infectious titer of TU / mL of the culture medium, and / or at least 75% viability, and / or at least 1.0×10 7 viable cell density of cells / mL of the culture medium, an enveloped virus can be produced.

[0110] The present disclosure provides a stable producing cell clone that can produce an enveloped virus at an infectious titer of TU / mL of the culture medium of at least 5×10 6 and / or at least 75% viability and / or at least 1.0×10 7 viable cell density of cells / mL of the culture medium for at least 35 days. For example, the stable producing cell clone can produce an enveloped virus at an infectious titer of at least 5×10 6 TU / mL of the culture medium, and / or at least 75% viability, and / or at least 1.0×10 7 viable cell density of cells / mL of the culture medium on day 35 of culture.

[0111] The present disclosure provides a stable producing cell line that can produce an enveloped virus at an infectious titer of TU / mL of the culture medium of at least 5×10 6 and / or at least 75% viability and / or at least 1.0×10 7 viable cell density of cells / mL of the culture medium for at least 15 days. For example, the stable producing cell line can produce an enveloped virus at an infectious titer of at least 5×10 6 TU / mL of the culture medium, and / or at least 75% viability, and / or at least 1.0×10 7 viable cell density of cells / mL of the culture medium on day 15 of culture.

[0112] The present disclosure provides a stable producing cell line that can produce an enveloped virus at an infectious titer of TU / mL of the culture medium of at least 5×10 6 and / or at least 75% viability and / or at least 1.0×10 7A stable producer cell line that can produce an enveloped virus at a viable cell density of cells / mL of the culture medium is also provided. For example, the stable producer cell line has an infectious titer of at least 5×10 6 TU / mL, and / or a viability of at least 75%, and / or a viable cell density of at least 1.0×10 7 cells / mL of the culture medium on day 20 of culture, and can produce an enveloped virus.

[0113] The present disclosure provides a stable producer cell line that can produce an enveloped virus at an infectious titer of at least 5×10 6 TU / mL of the culture medium and / or a viability of at least 75% and / or a viable cell density of at least 1.0×10 7 cells / mL of the culture medium for at least 25 days. For example, the stable producer cell line has an infectious titer of at least 5×10 6 TU / mL of the culture medium, and / or a viability of at least 75%, and / or a viable cell density of at least 1.0×10 7 cells / mL of the culture medium on day 25 of culture, and can produce an enveloped virus.

[0114] The present disclosure provides a stable producer cell line that can produce an enveloped virus at an infectious titer of at least 5×10 6 TU / mL of the culture medium and / or a viability of at least 75% and / or a viable cell density of at least 1.0×10 7 cells / mL of the culture medium for at least 30 days. For example, the stable producer cell line has an infectious titer of at least 5×10 6 TU / mL of the culture medium, and / or a viability of at least 75%, and / or a viable cell density of at least 1.0×10 7 cells / mL of the culture medium on day 30 of culture, and can produce an enveloped virus.

[0115] The present disclosure provides a stable producer cell line that can produce an enveloped virus at an infectious titer of at least 5×10 6An infectious titer of TU / mL in the culture medium and / or at least 75% viability and / or at least 1.0×10 7 A stable producer cell line capable of producing an enveloped virus is also provided at a viable cell density of cells / mL in the culture medium. For example, the stable producer cell line has, on day 35 of culture, at least 5×10 6 An infectious titer of TU / mL in the culture medium, and / or at least 75% viability, and / or at least 1.0×10 7 A viable cell density of cells / mL in the culture medium and is capable of producing an enveloped virus.

[0116] Methods for determining cell viability will be apparent to those skilled in the art and / or are described herein. For example, cell viability is determined using a hemocytometer after trypan blue staining. For example, cell viability is determined by performing trypan blue dye exclusion to determine the ratio of viable cells to total cells in the sample.

[0117] Methods for measuring infectious titer will be apparent to those skilled in the art and / or are described herein. In one example, the infectious titer is determined by performing flow cytometry following transduction with GFP LV. For example, the method involves incubating a virus-containing supernatant with HEK293T cells seeded in a plate, followed by trypsinization and washing, determining the percentage of GFP-positive cells using flow cytometry, and calculating the infectious titer in terms of transduction units (TU) / mL of the medium.

[0118] Methods for determining viable cell density will be apparent to those skilled in the art and / or are described herein. In one example, viable cell density is determined using a hemocytometer after trypan blue staining. For example, viable cell density is determined by performing trypan blue dye exclusion to determine the total number of viable cells per 1 mL of the sample.

[0119] In one example, a stable production cell clone and / or a stable production cell line comprises the characteristics of the cell lines described herein.

[0120] In one example, a stable production cell clone and / or a stable production cell line expresses a tetracycline-inducible gene expression system in a cell culture medium.

[0121] In one example, a stable production cell clone and / or a stable production cell line is adapted to grow in suspension cell culture.

[0122] In one example, a stable production cell clone and / or a stable production cell line is selected from or derived from a GPR, GPRG, GPRT, GPRGT or GPRTG cell line.

[0123] The present disclosure provides a method for producing a stable production cell clone capable of producing an enveloped virus in a suspension cell culture, the method comprising culturing a stable production cell clone in a suspension cell culture for at least 15 days, detecting a cell viability and / or an infectious titer yield and / or a viable cell density, and selecting a stable production cell clone, wherein the stable production cell clone, after at least 15 days of culture, meets one or more or all of the following criteria: (i) an infectious titer of at least 5×10 6 TU / mL of culture medium; (ii) a viability of at least 75%; and (iii) a viable cell density of at least 1.0×10 7 cells / mL of culture medium and is produced when one or more or all of the above are met.

[0124] The present disclosure provides a method for producing a stable production cell clone capable of producing an enveloped virus in a suspension cell culture, comprising culturing the stable production cell clone in a suspension cell culture for at least 20 days, detecting cell viability and / or infectious titer yield and / or viable cell density, and selecting a stable production cell clone, wherein the stable production cell clone, after at least 20 days of culture, meets one or more or all of the following criteria: (i) an infectious titer of at least 5×10 6 TU / mL of culture medium; (ii) a viability of at least 75%; and (iii) a viable cell density of at least 1.0×10 7 cells / mL of culture medium and is produced when one or more or all of the above are satisfied.

[0125] The present disclosure provides a method for producing a stable production cell clone capable of producing an enveloped virus in a suspension cell culture, comprising culturing the stable production cell clone in a suspension cell culture for at least 25 days, detecting cell viability and / or infectious titer yield and / or viable cell density, and selecting a stable production cell clone, wherein the stable production cell clone, after at least 25 days of culture, meets one or more or all of the following criteria: (i) an infectious titer of at least 5×10 6 TU / mL of culture medium; (ii) a viability of at least 75%; and (iii) a viable cell density of at least 1.0×10 7 cells / mL of culture medium and is produced when one or more or all of the above are satisfied.

[0126] The present disclosure provides a method for generating stable producer cell clones capable of producing enveloped viruses in suspension cell cultures, the method comprising culturing the stable producer cell clones in suspension cell culture for at least 30 days, detecting cell viability and / or infectious titer yield and / or viable cell density, and selecting stable producer cell clones, wherein the stable producer cell clones, after culturing for at least 30 days, meet one or more or all of the following criteria: (i) an infectious titer of at least 5×10 6 TU / mL of culture medium; (ii) a viability of at least 75%; and (iii) a viable cell density of at least 1.0×10 7 cells / mL of culture medium and is produced when one or more or all of the above are met.

[0127] The present disclosure provides a method for generating stable producer cell clones capable of producing enveloped viruses in suspension cell cultures, the method comprising culturing the stable producer cell clones in suspension cell culture for at least 35 days, detecting cell viability and / or infectious titer yield and / or viable cell density, and selecting stable producer cell clones, wherein the stable producer cell clones, after culturing for at least 35 days, meet one or more or all of the following criteria: (i) an infectious titer of at least 5×10 6 TU / mL of culture medium; (ii) a viability of at least 75%; and (iii) a viable cell density of at least 1.0×10 7 cells / mL of culture medium and is produced when one or more or all of the above are met.

[0128] Cell culture medium The cells are cultured in a medium suitable for culturing mammalian cells and producing enveloped viruses. The cells can be cultured while attached to an attachment environment, for example, a surface, or in a suspension environment, for example, suspended in a medium. The medium can be further supplemented with additives known in the art such as antibiotics, serum (especially fetal bovine serum, etc.) added at appropriate concentrations. The medium is supplemented with GlutaMax™, Pluronic™ F-68 (ThermoFisher), LONG® R3 IGF-I (Sigma-Aldrich), CellBoost™ 5, and / or an anti-aggregation agent. The medium used may or may not contain serum in particular. Culture media for mammalian cells are known, for example, DMEM (Dulbecco's Modified Eagle Medium) medium, RPMI 1640, or a mixture of various culture media including, for example, DMEM / F12, or serum-free media such as optiMEM®, optiPRO®, optiPRO-SFM®, CD293® (ThermoFisher), TransFx™ (Cytiva), BalanCD® (Irvine), Freestyle F17® (Life Technologies) or Ex-Cell® 293 (Sigma-Aldrich).

[0129] In one example, the cells are cultured in a medium containing TransFx™ (Cytiva).

[0130] 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 yet another 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.

[0131] In one example, the 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, the cells are supplemented with 5 mM or less 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 1 mM to 10 mM GlutaMax™. For example, the cells are supplemented with 4 mM to 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™.

[0132] In one example, the cells are supplemented with 0.05% to 1% poloxamer 188. For example, the cells are supplemented with 0.05% to 0.5% poloxamer 188. In one example, the cells are supplemented with 0.08% to 0.2% poloxamer 188. For example, the cells are supplemented with 0.08% poloxamer 188. In another example, the cells are supplemented with 0.09% poloxamer 188. In a further example, the cells are supplemented with 0.1% poloxamer 188. In one example, the cells are supplemented with 0.15% poloxamer 188. In a further example, the cells are supplemented with 0.2% poloxamer 188.

[0133] In one example, the cells are supplemented with less than 10% Cell Boost™ 5. For example, the cells are supplemented with approximately 10% Cell Boost™ 5, or approximately 9% Cell Boost™ 5, or approximately 8% Cell Boost™ 5, or approximately 7% Cell Boost™ 5, or approximately 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 approximately 5% Cell Boost™ 5, or approximately 4% Cell Boost™ 5, or approximately 3% Cell Boost™ 5, or approximately 2% Cell Boost™ 5, or approximately 1% Cell Boost™ 5. In one example, the cells are supplemented with 1% to 10% Cell Boost™ 5. For example, the cells are supplemented with 2% to 8% Cell Boost™ 5. In one example, the cells are supplemented with 4% to 6% Cell Boost™ 5. For example, the cells are supplemented with approximately 4% Cell Boost™ 5. In another example, the cells are supplemented with approximately 5% Cell Boost™ 5. In yet another example, the cells are supplemented with approximately 6% Cell Boost™ 5.

[0134] In a process using transiently transfected cells, any agent that enables transfection of the plasmid can be used. Exemplary agents include calcium phosphate or polyethyleneimine. The conditions (e.g., amount of plasmid, ratio of plasmids to each other, ratio of plasmid to transfection agent, type of medium, etc.) and transfection time are adapted by those skilled in the art according to the characteristics of the virus produced and / or the transgene introduced into the transfer plasmid.

[0135] According to some examples, the culture medium used has a neutral pH (e.g., included in 7 - 7.4, particularly 7, 7.1, 7.2, 7.3 or 7.4) conventionally used for culturing cells and producing viruses. In one example, the suspension cell culture is at pH 6.0 - 8.0. For example, the pH of the culture medium is 7.1 ± 0.15. In other examples, the production process used includes culturing the production cells in a moderately acidic medium. The expression "moderately acidic conditions" refers to the pH of an aqueous solution included in 5 - 6.8, e.g., 5.8 - 6.2, 5.5 - 6.5, etc. The pH selected will depend on the buffering power of the culture medium used and can be readily determined by those skilled in the art taking into account general knowledge. Those skilled in the art can change the pH of the solution.

[0136] In one example, the production of an enveloped virus comprises: transient transfection of HEK293T cells or derivatives thereof using one or several plasmids encoding the elements required for the production of said enveloped vector, or stable production cells stably transfected with the gene of interest and producing the vector constitutively or after induction, e.g., using GPRG or GPRTG; culturing the cells in a suitable medium with a pH of about 6 or about 7; collecting the cell culture medium containing the enveloped virus.

[0137] Suspension cell culture The present disclosure provides a method for producing an enveloped virus in a suspension cell culture. For example, the method includes culturing a suspension cell line expressing a tetracycline-repressible gene expression system in a cell culture medium.

[0138] The method of the present disclosure is applicable to producing an enveloped virus both on a small scale and on a large scale. The method is particularly useful in that it can be scaled up for manufacturing pharmaceuticals on a commercial scale.

[0139] It will be apparent to those skilled in the art that there are a cell growth phase and a virus production phase in the production of an enveloped virus.

[0140] Those skilled in the art will understand that the cell growth phase includes seed train cell culture. As used herein, the term "seed train" refers to the generation (i.e., cell proliferation) of a sufficient number of cells for virus production. Those skilled in the art will understand that the seed train cell culture includes a plurality of culture systems (e.g., T-flasks, roller bottles or shake flasks, small-scale bioreactor systems, and subsequent larger-scale bioreactors) that increase in size with each passage in order to increase the culture from a small number of cells to a larger number of cells suitable for virus production.

[0141] It will be apparent to those skilled in the art that in any of the methods described herein, during the cell growth phase, the cells are cultured in the presence of tetracycline or its derivatives, which suppresses virus production but permits cell growth.

[0142] In one example, the cells are grown during the cell growth phase before virus production.

[0143] In one example, the cell growth phase is carried out in an increasing bioreactor (also referred to as an N-1 bioreactor).

[0144] In one example, the virus production phase is carried out in a production bioreactor (also referred to as an N bioreactor).

[0145] In one example, the cell growth phase and the virus production phase are carried out in the same container. For example, the growth of a suspension cell line and the production of an enveloped virus occur in the same container. For example, the cell growth phase and the virus production phase are carried out in the same bioreactor.

[0146] In one example, the cell growth phase and the virus production phase are carried out in different containers. For example, the cell growth phase is carried out in a growth bioreactor, the virus production phase is carried out in a production bioreactor, and the growth bioreactor and the production bioreactor are different.

[0147] In one example, the cell culture is operated in batch, fed-batch, continuous, semi-continuous, or perfusion mode.

[0148] In one example, the cell growth phase and / or the virus production phase are operated in batch, fed-batch, continuous, semi-continuous, or perfusion mode.

[0149] In one example, the cell growth phase is carried out in batch, fed-batch, continuous, semi-continuous, or perfusion mode. In one example, the cell growth phase is carried out in batch mode. In another example, the cell growth phase is carried out in fed-batch mode. In a further example, the cell growth phase is carried out in continuous mode. In one example, the cell growth phase is carried out in perfusion mode. In another example, the cell growth phase is carried out in batch and perfusion modes. For example, the cell growth phase is carried out in initial batch mode and then in perfusion mode.

[0150] In one example, the virus production phase is carried out in batch, fed-batch, continuous, semi-continuous, or perfusion mode. In one example, the virus production phase is carried out in batch mode. In another example, the virus production phase is carried out in fed-batch mode. In a further example, the virus production phase is carried out in continuous mode. In one example, the virus production phase is carried out in perfusion mode. In another example, the virus production phase is carried out in batch and perfusion modes. For example, the virus production phase is first carried out in batch mode and then in perfusion mode.

[0151] In one example, the cell expansion phase and the virus production phase are carried out in batch mode. In another example, the cell expansion phase and the virus production phase are carried out in perfusion mode. In a further example, the cell expansion phase is carried out in batch mode and the virus production phase is carried out in perfusion mode.

[0152] It will be apparent to those skilled in the art that references to batch, fed-batch, continuous, and / or perfusion modes at a particular stage of cell culture (i.e., cell expansion and / or virus production) do not mean that the entire culture stage is carried out in that mode. For example, it only means that a period of the cell culture stage (e.g., at least 1 day) is carried out in that mode. It will also be understood that the mode does not necessarily have to start on day 0 of the culture period. For example, the culture can start on day 0 and the perfusion mode can only start on day 2 of the cell culture period.

[0153] In one example, the suspension cell culture is operated in batch mode. It will be apparent to those skilled in the art that "batch mode" refers to a process in which cells are first cultured in a medium and this medium is neither removed, nor exchanged, nor supplemented during or before the end of the culture, i.e., no fresh medium is "fed" to the cells.

[0154] In one example, suspension cell culture is operated in fed-batch mode. It will be apparent to those skilled in the art that "fed-batch mode" refers to a process in which one or more nutrients are supplied to the bioreactor during the culture period. In one example, the cell growth phase and / or the virus production phase are operated in fed-batch mode. In one example, the cell growth phase is operated in fed-batch mode.

[0155] In one example, suspension cell culture is operated in perfusion mode. It will be apparent to those skilled in the art that "perfusion mode" involves a continuous supply of fresh medium and removal of spent medium (i.e., continuous medium exchange) while retaining a high number of viable cells. In one example, the cell growth phase and / or the virus production phase are operated in perfusion mode. In one example, the virus production phase is operated in perfusion mode. In one example, perfusion mode involves perfusing with a medium containing tetracycline or its derivative throughout the cell growth phase. In one example, perfusion mode involves first perfusing with a medium containing tetracycline or its derivative and subsequently perfusing with a medium not containing tetracycline or the derivative before induction.

[0156] In one example, the cells are cultured in a fluidized bed bioreactor, a hollow fiber bioreactor, a roller bottle, a shake flask, or a stirred tank bioreactor. In one example, the cells are cultured in a stirred tank bioreactor. In one example, the cells are cultured in a Biostat® or Univessel® bioreactor (Sartorius).

[0157] In one example, the volume of the cell culture can be, for example, from about 0.01 L to about 0.1 L, or from about 0.1 L to about 1 L, or from about 1 L to about 5 L. In another example, the volume of the cell culture can be from about 5 L to about 10 L, from about 10 L to about 50 L, from about 50 L to about 100 L, from about 100 L to about 200 L, from about 200 L to about 500 L, from about 500 L to about 1000 L, from about 1000 L to about 2000 L, or from 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 from about 35 to 150 L. In one example, the volume of the cell culture is from about 50 to 70 L.

[0158] In one example, the suspension cell culture is operated at a temperature at which cell growth and virus production are possible. For example, the cell culture has a temperature that is customarily used in the art for culturing cells and producing viruses. In one example, the suspension cell culture is at a temperature of 35 to 39 °C. For example, a temperature of 37 ± 0.5 °C, or a temperature of 38 ± 0.5 °C.

[0159] In one example, the suspension cell culture is operated on a large scale. For example, the suspension cell culture is operated on a commercial scale.

[0160] In one example, the suspension cell culture is manipulated for at least 10 days. For example, the suspension cell culture is manipulated for about 10 days to 50 days. In one example, the suspension cell culture is manipulated for 10 days to 35 days, such as 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 manipulated for at least 15 days. For example, the suspension cell culture is manipulated for about 20 days. In another example, the suspension cell culture is manipulated for at least 20 days. In a further example, the suspension cell culture is manipulated for at least 25 days. For example, the suspension cell culture is manipulated for about 28 days. In one example, the suspension cell culture is manipulated for at least 30 days. In one example, the suspension cell culture is manipulated for at least 32 days. For example, the suspension cell culture is manipulated for 35 days. In one example, the suspension cell culture is manipulated for at least 35 days.

[0161] Reduction of tetracycline and its derivatives in suspension cell cultures The methods of the present disclosure are applicable for producing enveloped viruses in both small-scale and large-scale production. The methods are particularly useful in that they can be scaled up for manufacturing pharmaceuticals on a commercial scale.

[0162] The present disclosure provides methods for improving the production of enveloped viruses from suspension cell cultures. In particular, the present disclosure provides methods for removing or reducing the concentration of tetracycline or its derivatives from suspension cell cultures for the production of enveloped viruses, where the cell line expresses a tetracycline-repressible gene expression system. It will be apparent to those skilled in the art from the disclosure herein that the methods of the present disclosure result in an increase in cell quality and virus production.

[0163] It will be apparent to those skilled in the art that from the disclosure of the specification, it is not necessary for the concentration of tetracycline or its derivatives to be completely removed or reduced. For example, the concentration of tetracycline or its derivatives is reduced by at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 85%, or at least 90%, or at least 95%.

[0164] As used herein, reference to a cell line that expresses a "tetracycline-repressible gene expression system" or "Tet-OFF" system refers to a cell line that stably expresses a tetracycline-controlled transactivator (tTA) such that transcription from a tetracycline-responsive element promoter is silenced by the presence of tetracycline or its derivatives (e.g., doxycycline).

[0165] Those skilled in the art will understand that the cells are first cultured in the presence of tetracycline or its derivatives in order to suppress virus production but allow the cell line to grow or expand.

[0166] In one example, the amount of tetracycline or its derivatives in the cell culture medium required to suppress virus production is at least 0.1 ng / mL.

[0167] In one example, tetracycline or its derivatives in the cell culture medium suppress virus production for 1 to 20 days. For example, the amount of tetracycline or its derivatives in the cell culture medium suppresses virus production for about 1 day, or about 2 days, or about 3 days, or about 4 days, or about 5 days, or about 6 days, or about 7 days, or about 8 days, or about 9 days, or about 10 days. In another example, the amount of tetracycline or its derivatives in the cell culture medium suppresses virus production for about 11 days, or about 12 days, or about 13 days, or about 14 days, or about 15 days, or about 16 days, or about 17 days, or about 18 days, or about 19 days, or about 20 days.

[0168] It will be apparent to those skilled in the art from the disclosure of this specification that tetracycline or its derivatives can be added to the cell culture medium as a single bolus supply, as multiple supplies, or continuously over the culture period. In one example, tetracycline or its derivatives are added to the cell culture medium as a single bolus supply. For example, tetracycline or its derivatives are added to the cell culture medium at the start of cell culture (i.e., day 0). In another example, tetracycline or its derivatives are added to the cell culture medium daily or every two days during the cell culture period. In one example, tetracycline or its derivatives are added to the cell culture medium by perfusion cell culture at a defined concentration. For example, tetracycline or its derivatives are added to the cell culture medium by perfusion cell culture at a concentration of 1.5 ng / mL. In a further example, tetracycline or its derivatives are added to the cell culture medium only as much as necessary to maintain the minimum concentration of tetracycline or its derivatives in the cell culture medium. For example, tetracycline or its derivatives are added to the cell culture medium to maintain a concentration of at least 0.1 ng / mL in the cell culture medium.

[0169] It will be apparent to those skilled in the art that discontinuation (or reduction in concentration) of the use of tetracycline or its derivatives results in the expression of enveloped viruses. For example, the concentration of tetracycline or its derivatives in the cell culture medium is reduced to 0.5 ng / mL or less.

[0170] The inventors have found that during the growth stage of cell cultures, as the cell density increases, the concentration of tetracycline or an equivalent required to suppress virus production also increases. The inventors have found that virus production is induced over time if the cell culture medium is not supplemented with tetracycline or an equivalent.

[0171] As discussed herein, a conventional method for removing tetracycline or its derivatives from a suspension cell culture involves centrifuging the cells, removing the supernatant, and then resuspending the cells to wash the cells with a cell culture medium that does not contain tetracycline or the derivative. However, when this method is applied to a suspension cell line, high shear forces are applied to the cells, resulting in a decrease in cell quality and an increased risk of manual contamination. In addition, these conventional methods include manual steps, are time-consuming, and can add several hours to the process time, so centrifugation cannot be easily scaled up on a commercial scale.

[0172] The inventors' solutions to these problems are to dilute the suspension cell culture with a cell culture medium that does not contain tetracycline or the derivative; or to use acoustic standing waves to hold the suspension cell line cells and remove a portion of the cell culture medium containing tetracycline or its derivative from the suspension cell culture, and to contact the held suspension cell line cells with a cell culture medium that does not contain tetracycline or its derivative. These methods both result in less stress on the cells and fewer manual steps compared to traditional centrifugation methods, thus improving cell quality. In addition, the inventors' solution reduces the risk of contamination of the culture by utilizing a closed system. The methods of the present disclosure also increase the viral infectivity titer yield.

[0173] Accordingly, the present disclosure provides a method for removing tetracycline or its derivative from a suspension cell culture for the production of an enveloped virus, the method comprising: (i) diluting the suspension cell culture with a cell culture medium that does not contain tetracycline or the derivative; or (ii) using acoustic standing waves to hold the suspension cell line cells, removing a portion of the cell culture medium containing tetracycline or its derivative from the suspension cell culture, and contacting the held suspension cell line cells with a cell culture medium that does not contain tetracycline or the derivative.

[0174] Dilution of Suspension Cell Cultures In one example, the present disclosure provides a method for removing tetracycline or a derivative thereof from a suspension cell culture for producing an enveloped virus, the method comprising diluting the suspension cell culture with a cell culture medium that does not contain tetracycline or a derivative thereof.

[0175] As used herein, the terms “diluting” or “dilute” with respect to a cell culture are understood to mean reducing the concentration of a solute (i.e., tetracycline or a derivative thereof) in the cell culture.

[0176] As discussed herein, the inventors have determined that it is not necessary to completely remove tetracycline or a derivative thereof from a cell culture to induce virus production. That is, the concentration of tetracycline or a derivative thereof need only be reduced, not completely removed. This was an unexpected result as a common protocol for inducing Tet-OFF cells requires removing all or substantially all of the tetracycline or a derivative thereof, e.g., via one or more washing steps. It was unexpected that simply diluting, rather than removing, the spent medium containing tetracycline or a derivative thereof would be effective in inducing virus production.

[0177] For example, it was only necessary to reduce the concentration of tetracycline or its derivative to a concentration of less than about 0.1 ng / mL. Therefore, the inventors determined that a tetracycline or its derivative concentration of less than about 0.1 ng / mL permits virus production. In another example, it was only necessary to reduce the concentration of tetracycline or its derivative to a concentration of less than about 0.2 ng / mL. Therefore, the inventors determined that a tetracycline or its derivative concentration of less than about 0.2 ng / mL permits virus production. In another example, it was only necessary to reduce the concentration of tetracycline or its derivative to a concentration of less than about 0.5 ng / mL. Therefore, the inventors determined that a tetracycline or its derivative concentration of less than about 0.5 ng / mL permits virus production.

[0178] In one example, the concentration of tetracycline or its derivative in the cell culture is diluted by the addition of cell culture medium that does not contain tetracycline or the derivative. For example, the cell culture medium that does not contain tetracycline or the derivative is added directly to the cell culture medium that contains tetracycline or the derivative.

[0179] In one example, the cell culture medium that does not contain tetracycline or the derivative is added to the suspension cell culture. In this embodiment of the present disclosure, it will be apparent to those skilled in the art that the cell growth phase and the virus production phase occur within the same container (i.e., bioreactor).

[0180] In one example, a suspension cell culture containing a cell culture medium containing tetracycline or a derivative is added to a cell culture medium that does not contain tetracycline or a derivative. In this embodiment of the present disclosure, it will be apparent to those skilled in the art that the cell growth phase and the virus production phase occur in separate containers (i.e., bioreactors). For example, a suspension cell culture containing a cell culture medium containing tetracycline or a derivative is cultured in a first bioreactor and added to a cell culture medium that does not contain tetracycline or a derivative in a second bioreactor. For example, the suspension cell culture is transferred from a first bioreactor containing a cell culture medium containing tetracycline or a derivative to a second bioreactor, and the second bioreactor contains a cell culture medium that does not contain tetracycline or a derivative.

[0181] It will be apparent to those skilled in the art that references in the present disclosure to the first and subsequent containers (i.e., bioreactors) are for purposes of comparison only and not references to defined or specific bioreactors. The first, second (and any subsequent) containers can be separated by any number of other containers.

[0182] In one example, a suspension cell culture containing a cell culture medium containing tetracycline or a derivative is cultured in a growth bioreactor, and a suspension cell culture containing a cell culture medium containing tetracycline or a derivative is added to a cell culture medium that does not contain tetracycline or a derivative in a production bioreactor. For example, the suspension cell culture is transferred from a growth bioreactor containing a cell culture medium containing tetracycline or a derivative to a production bioreactor, and the production bioreactor contains a cell culture medium that does not contain tetracycline or a derivative. In one example, the production bioreactor is filled with a pre-treated cell culture medium (with temperature, pH, and dissolved oxygen levels set) that does not contain tetracycline or a derivative, and the suspension cell culture is transferred from the growth bioreactor to the pre-treated medium.

[0183] In one example, the suspension cell culture is diluted at a ratio of about 1:1 to 1:20 in a cell culture medium that does not contain tetracycline or a derivative. For example, the suspension cell culture is diluted at a ratio of 1:2 to 1:10, or 1:4 to 1:7 in a cell culture medium that does not contain tetracycline or a derivative. In one example, the suspension cell culture is diluted at a ratio of 1:2 to 1:10 in a cell culture medium that does not contain tetracycline or a derivative. For example, the suspension cell culture is diluted at a ratio of about 1:2, or about 1:3, or about 1:4, or about 1:5, or about 1:6, or about 1:7, or about 1:8, or about 1:9, or about 1:10 in a cell culture medium that does not contain tetracycline or a derivative. In one example, the suspension cell culture is diluted at a ratio of 1:4 to 1:7 in a cell culture medium that does not contain tetracycline or a derivative. In one example, the suspension cell culture is diluted at a ratio of about 1:4 in a cell culture medium that does not contain tetracycline or a derivative. In another example, the suspension cell culture is diluted at a ratio of about 1:5 in a cell culture medium that does not contain tetracycline or a derivative. In yet another example, the suspension cell culture is diluted at a ratio of about 1:6 in a cell culture medium that does not contain tetracycline or a derivative. In one example, the suspension cell culture is diluted at a ratio of about 1:7 in a cell culture medium that does not contain tetracycline or a derivative.

[0184] It will be apparent to those skilled in the art from the disclosure herein that the ratio at which the suspension cell culture is diluted in a cell culture medium that does not contain tetracycline or a derivative depends on the concentration of tetracycline or its derivative in the suspension cell culture. For example, those skilled in the art will recognize that when the concentration of tetracycline or its derivative in the suspension cell culture is about 1 ng / mL, the suspension cell culture is diluted such that the concentration of tetracycline or its derivative in the cell culture medium is less than 0.1 ng / mL. In a further example, those skilled in the art will recognize that if the concentration of tetracycline or its derivative in the suspension cell culture is about 0.1 ng / mL, the suspension cell culture need not be diluted prior to virus production.

[0185] It will be apparent to those skilled in the art from the disclosure herein that the amount of tetracycline or its derivative in which virus production is induced also depends on the cell density. Higher density cells require a higher concentration of tetracycline or its derivative to suppress induction.

[0186] Acoustic standing wave separation In one example, the present disclosure provides a method for removing tetracycline or its derivative from a suspension cell culture for the production of an enveloped virus, comprising retaining suspension cell line cells using an acoustic standing wave, removing a portion of the cell culture medium containing tetracycline or the derivative from the suspension cell culture, and contacting the retained suspension cell line cells with a cell culture medium that does not contain tetracycline or the derivative.

[0187] Removing a portion of the cell culture medium containing tetracycline or the derivative from the suspension cell culture includes removing at least about 10% of the cell culture medium containing tetracycline or the derivative. In one example, removing a portion of the cell culture medium containing tetracycline or the derivative from the suspension cell culture includes removing all or substantially all of the cell culture medium containing tetracycline or the derivative. In one example, removing a portion of the cell culture medium containing tetracycline or the derivative from the suspension cell culture includes removing about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% of the cell culture medium containing tetracycline or the derivative.

[0188] In one example, after removing a portion of the cell culture medium containing tetracycline or the derivative from the suspension cell culture, the cell line cells remain in suspension. In another example, after removing a portion of the cell culture medium containing tetracycline or the derivative from the suspension cell culture, the cell line cells are not yet in suspension and need to be resuspended.

[0189] In one example, contacting the retained suspension cell line cells with a cell culture medium that does not contain tetracycline or a derivative includes resuspending the retained suspension cell line cells in a cell culture medium that does not contain tetracycline or a derivative. In one example, contacting the retained suspension cell line cells with a cell culture medium that does not contain tetracycline or a derivative includes flowing the suspension cell line cells into a container containing the cell culture medium that does not contain tetracycline or a derivative.

[0190] As described above, the inventors have shown that when using acoustic standing waves to separate suspension cell line cells from a cell culture medium containing tetracycline or a derivative, compared to centrifugation, cell stress is reduced, and as a result, cell quality and virus titer production are improved.

[0191] In one example, the method includes the use of an acoustic chamber or an acoustic device. For example, the method includes (i) flowing a suspension cell culture containing a cell culture medium containing tetracycline or a derivative through an acoustic standing wave in an acoustic chamber, (ii) retaining the suspension cell line cells in the acoustic chamber, and (iii) flowing a cell culture medium that does not contain tetracycline or a derivative through the acoustic chamber containing the retained suspension cell line cells to contact the cell line cells with the cell culture medium that does not contain tetracycline or a derivative.

[0192] Acoustic chambers or acoustic wave devices suitable for use in the present disclosure will be apparent to those skilled in the art and / or are described herein. Exemplary acoustic devices employ ultrasonic particle separation techniques such as those described in EP0633049. Exemplary acoustic wave devices include those described in US10,773,194.

[0193] In one example, the method includes (i) flowing a suspension cell culture comprising a cell culture medium containing tetracycline or a derivative through an acoustic standing wave from a first container into an acoustic chamber, (ii) retaining suspension cell line cells within the acoustic chamber, (iii) flowing a cell culture medium not containing tetracycline or a derivative through the acoustic chamber containing the retained suspension cell line cells to contact the cell line cells with the cell culture medium not containing tetracycline or a derivative, and (iv) flowing the suspension cell line cells and the cell culture medium not containing tetracycline or a derivative into a second container.

[0194] In one example, the method includes (i) flowing a suspension cell culture comprising a cell culture medium containing tetracycline or a derivative through an acoustic standing wave from an increase bioreactor into an acoustic chamber, (ii) retaining suspension cell line cells within the acoustic chamber, and (iii) flowing a cell culture medium not containing tetracycline or a derivative through the acoustic chamber containing the retained suspension cell line cells to contact the cell line cells with the cell culture medium not containing tetracycline or a derivative, and (iv) flowing the suspension cell line cells and the cell culture medium not containing tetracycline or a derivative into a production bioreactor.

[0195] It will be apparent to those skilled in the art that using an acoustic chamber as described herein facilitates in-line processing. Thus, in some embodiments, the method is performed in-line.

[0196] As used herein, the term "in-line" in the context of a process step refers to a process step that is integrated with or combined with one or more other process steps, or that flows directly from or to other process steps without the need for manual intervention or handling.

[0197] In one example, the acoustic standing wave is present in-line between the first and second containers.

[0198] In one example, the method includes removing a portion of a suspension cell culture containing a cell culture medium containing tetracycline or a derivative from a first container, retaining suspension cell line cells from the portion of the suspension cell culture using an acoustic standing wave, and contacting the retained suspension cell line cells with a cell culture medium not containing tetracycline or a derivative in a second container.

[0199] In one example, the method includes removing a portion of a suspension cell culture containing a cell culture medium containing tetracycline or a derivative from an increase bioreactor, retaining suspension cell line cells from the portion of the suspension cell culture using an acoustic standing wave, and contacting the retained suspension cell line cells with a cell culture medium not containing tetracycline or a derivative in a production bioreactor.

[0200] Purification of enveloped virus In one example, an enveloped virus is purified from a cell culture by a method comprising one or more steps selected from the group consisting of clarification filtration, anion exchange chromatography, concentration, and diafiltration.

[0201] Downstream processes for purifying and concentrating a viral vector from a cell culture include a harvest filtration step (also known as "clarification filtration" or "harvest clarification filtration" or "bioburden reduction") for removing cell debris and components from the harvest, a purification step for reducing the overall volume and separating the viral vector from host cell DNA, proteins, and media components, such as anion exchange chromatography, and an ultrafiltration / diafiltration step for concentrating the viral vector into the final formulation buffer. In some examples, the downstream process further includes a filtration sterilization step for removing microorganisms from the final product.

[0202] As used herein, "harvesting" refers to removing the cell culture medium containing virus particles from the production cells for downstream processing, and "harvest" refers to the cell culture medium containing virus particles removed for downstream processing. The harvesting process may include collecting one or more harvests. "Harvest filtration" refers to either the filtered harvest or the cell culture medium containing virus particles that has been filtered for downstream processing and from which the production cells have been removed.

[0203] In one example, the harvested cell culture fluid is filtered after production of an enveloped virus.

[0204] As used herein, the term "filtered cell culture fluid" will be understood to encompass the cell culture fluid after being subjected to harvest filtration.

[0205] After harvest filtration, the enveloped virus is purified using anion exchange.

[0206] In one example, anion exchange is performed in a bind - elute mode. In this case, the enveloped virus binds to the anion exchanger and the contaminants pass through. The virus is then eluted from the anion exchanger. By performing anion exchange in this manner, the volume of the liquid in which the virus is suspended is reduced and contaminants such as host cell DNA, host cell proteins, and medium components such as fetal bovine serum are removed.

[0207] Suitable anion exchangers will be apparent to those skilled in the art. Exemplary anion exchangers are columns containing a resin or a membrane or another suitable substrate.

[0208] In one example, the anion exchanger is a weak anion exchanger and includes, for example, an ion exchange group selected from diethylaminoethyl (DEAE) or aminoethyl groups.

[0209] In another example, the anion exchanger is a strong anion exchanger and includes, for example, an ion exchange group selected from a quaternary ammonium (Q), diethyl-2-hydroxypropylaminoethyl (QAE), triethylaminoethyl (TEAE), or trimethylaminoethyl group. Exemplary anion exchangers include MUSTANG® E, MUSTANG® Q, SARTOBIND® Q, CHROMASORB® , POSSIDYNE® , CAPTO® Q, QSFF, POROS® Q, FRACTOGEL® Q, NATRIX® Q.

[0210] In one example, the anion exchanger includes a Q ion exchange group.

[0211] In one example, the anion exchanger is a membrane anion exchanger that includes a Q ion exchange group. For example, the anion exchanger is MUSTANG® Q.

[0212] In one example, the enveloped virus eluted from the anion exchange column is further purified based on its size. In one example, the buffer in which the virus is eluted from the anion exchange column is exchanged approximately simultaneously. Tangential flow filtration is preferred in the process of the present disclosure. This method enables impurity removal and buffer exchange almost simultaneously.

[0213] Tangential flow ultrafiltration / diafiltration is a method used to remove residual proteins and nucleic acids and to exchange the working buffer with the final formulation buffer. Ultrafiltration using tangential flow is preferred, and different devices can also be used (e.g., Proflux and LABSCALE (ultrafiltration system) TFF systems, both from Millipore, or the Repligen KR2i system). The specific ultrafiltration membrane selected will have a filter pore size that is small enough to retain enveloped viruses but large enough for impurities to pass through. Depending on the manufacturer and type of membrane, a nominal molecular weight cut-off of 100 to 1000 kDa is appropriate (e.g., UFP-750-E-5A, GE Healthcare; BIOMAX (ultrafiltration device) NMWC1000, Millipore). In one example, the molecular weight cut-off is 500 kDa. The membrane composition may be, but is not limited to, regenerated cellulose, (modified) polyethersulfone, polysulfone. The membrane can be of flat sheet or hollow fiber type. The main parameters that must be optimized are the flow rate and the transmembrane pressure. In combination with the nominal molecular weight cut-off, these two parameters enable efficient purification and buffer exchange and high virus yields.

[0214] As an additional step, filtration sterilization can be performed to eliminate biological contamination. Thus, the diluted eluate or final residue from the ultrafiltration step is filtered by a filter, such as a 0.22 μm filter. Filters can be constructed from a variety of materials, including, but not limited to, polypropylene, hydrophilic PVDF, cellulose, hydrophilic regenerated cellulose, cellulose ester, cellulose acetate without wetting agents, cellulose acetate, nylon, hydrophilic nylon membrane, polyethersulfone, hydrophilic polyethersulfone, hydrophilic asymmetric PES, or any other material consistent with low non-specific influenza virus binding. The filter can have a single membrane layer or one or more layers, or can incorporate a pre-filter of the same or different material, such as a 0.45 μm pre-filter. The virus filtered aseptically can then be frozen and held for subsequent processing.

[0215] The present disclosure is further described in the following non-limiting examples.

Example

[0216] Adaptation of a Stable Adhesive Packaging Cell Line for Expandable Lentivirus Production Stable producer pools were generated by stable concatemer array transfection using the WASp-T2A-GFP construct and subsequent antibiotic selection. The parental cell line for all packaging and producer cell lines is the adherent HEK293T / 17 cell line. The original adherent GPRG and GPRTG packaging cell lines were established by stably introducing all genetic elements required for lentivirus production, except for the gene of interest (transfer gene).

[0217] Subsequently, the packaging cell lines GPRG and GPRTG were adapted for growth in suspension using different serum-free media. Briefly, each cell line was thawed from the master cell bank. Both cell lines were cultured adherently using 2.5 ng / mL of doxycycline and 2 μg / mL of puromycin. The cells were passaged twice in a static phase using T-flasks in D10 medium (DMEM + 10% FBS). During the static-phase culture, the medium was completely changed every 48 - 96 hours, and the cell density was maintained above 1E6 cells / mL until the cells reached an appropriate doubling time (less than 50 hours) and viability (more than 90%). A seeding density of 0.3 - 0.4E6 cells / mL, which is low, was applied for seeding increase. After cell recovery, a direct switch to serum-free medium was performed. Subsequent cultures were carried out in a shaking environment at 120 rpm (19 mm orbit) using non-baffled shake flasks, at 32% of the maximum working volume, under 5 - 8% CO2.

Example

[0218] Establishment of Suspension Cell Cultures The packaging and production cell lines used were based on the inducible TET-OFF expression system, where the presence of doxycycline inhibits the expression of lentiviral components, and the removal of doxycycline induces the production of lentiviruses.

[0219] The experiments were carried out using CAL-H production clones adapted to suspension. The cells for inoculation into the production bioreactor were expanded in an N-1 seed train bioreactor using doxycycline-supplemented medium.

[0220] Briefly, seeding increase was achieved under the following conditions:

Table 1

Example

[0221] Removal of Doxycycline by Acoustic Wave Method To remove doxycycline from the cell culture medium, the cells were washed with a medium not containing doxycycline using a device based on acoustic separation before inoculation into the production bioreactor.

[0222] An acoustic wave device was connected in-line between the N-1 bioreactor and the production bioreactor. Cells in the medium containing doxycycline flowed into the device and were retained in the acoustic wave field while the medium flowed out into the waste liquid bag. Fresh medium not containing doxycycline was fed in, and the cells were released into the production bioreactor. This process was repeated until all the cells were transferred to the production bioreactor in a cell culture medium not containing doxycycline.

[0223] The viable cell density and infectivity titer of the samples were measured. Briefly, to measure the infectivity titer, cells were collected at various time points during the culture period, stained with an antibody against human gamma globulin, and the infectivity titer of the sample measured in transducing units (TU) / mL was determined.

[0224] As shown in Figure 1, removal of doxycycline using the acoustic wave method yielded a viable cell density comparable to that of the normal centrifugation method. By applying daily cell discharge from the 4th day using an acoustic wave washing device, the viable cell density was maintained at the same level as that of slow-growing centrifugal culture. The titer yield was also equal to or higher than that of the centrifugation method.

Example

[0225] Removal of doxycycline using the dilution method Cells were grown to a density of approximately 5-fold in the N-1 bioreactor, achieving a viable cell density of 6 - 10×10 6 cells / mL and a viability of 80% or more.

[0226] Subsequently, the cells were directly transferred to the production bioreactor containing a medium not containing doxycycline, achieving a target cell concentration of approximately 1.5 - 1.8×10 6 cells / mL and a volume of 4.5 kg in the production bioreactor.

[0227] To examine the effect of doxycycline concentration on virus production, cells were cultured in an N-1 bioreactor for 5 days (i.e., working days -5 to 0) in the presence of 0.1 ng / mL, 0.5 ng / mL, 1 ng / ml, 2 ng / mL, 2.5 ng / mL, or 5 ng / mL of doxycycline. Note that doxycycline was not replenished during N-1 culture. However, medium exchanges were performed on working days -2 and -1 using media containing each concentration of doxycycline.

[0228] On day 0, cells were directly transferred to the production bioreactor containing medium without doxycycline at a ratio of approximately 1:5. After transfer to the production bioreactor, to prevent stress to the cells, the bioreactor was operated in batch mode for 2 days and then in perfusion mode with fresh medium.

[0229] As shown in Figure 2, in the production bioreactor, a low concentration of doxycycline at 0.1 ng / mL suppressed virus production for 2 days, while concentrations above 0.5 ng / mL suppressed virus production until at least day 1. Cultures with doxycycline above 1.0 ng / mL showed a large increase in virus production in the production bioreactor around day 2.

[0230] At the time of seeding into the production bioreactor, theoretically, it would be 0.2 ng / ml of doxycycline at a dilution of approximately 1:5. Surprisingly, virus induction was initiated in the presence of low concentrations of doxycycline before perfusion with fresh doxycycline-free medium.

Example

[0231] Comparison of virus induction strategies Initially, cells were grown in a seed train bioreactor in the presence of 1 ng / mL of doxycycline. After 65 hours of culture, perfusion was performed with 1 ng / mL of doxycycline. As shown in Figure 3A, high viable cell density and cell viability were achieved.

[0232] Three different virus induction strategies (i.e., dilution method, acoustic wave method, centrifugation method) were compared side by side.

[0233] Cells grew to approximately 10 - 15×10 6 cells / mL, and then continuous cell discharge was performed at a constant flow rate to maintain the cell density at ~15×10 6 cells / mL. As shown in Figure 3B, high cell viability was maintained in the production bioreactor until day 10.

[0234] When the infectivity titer from the bioreactor was evaluated daily, as shown in Figures 3C and 3D, the three methods were equivalent in terms of total virus yield, and the yield of centrifugation was slightly lower. Another drawback of centrifugation is that cells are exposed to more shear force.

[0235] As shown in Figure 3E, cell-specific productivity increased rapidly over time. The pH was also measured over time, and the pH remained within the range of the non-moving zone where base addition was not required (data not shown). As shown in Figures 3F and 3G, the metabolite (i.e., lactate (Figure 3G) and glucose (Figure 3F)) concentrations were stable over time.

[0236] The operation of one production bioreactor was extended beyond day 10 until day 12. As shown in Figure 4, the infectivity titer and cell-specific productivity continued to increase after day 12, indicating that production can be extended. To prevent overgrowth and maintain the cell density at approximately 1.5E7 cells / mL, cell discharge was applied. The cell discharge strategy prevents nutrient depletion and the increase in turbidity of the harvest that would interfere with the downstream process for virus purification.

Example

[0237] Evaluation of the possibility of large-scale production To evaluate the scale-up and robustness of the suspension harvest, suspension cell lines GPRG and GPRTG were grown in a production bioreactor until day 35. When the viable cell density, viability, and infectious titer of the stable production cell clones were measured, high viable cell density, cell viability, and infectious titer were achieved until day 35.

[0238] These studies have shown that stable suspension production cell lines can produce enveloped viruses at commercial scale (i.e., for at least 15 days).

Claims

1. A method for producing enveloped viruses in a suspension cell culture, comprising culturing a suspension cell line expressing a tetracycline-repressive gene expression system in a cell culture medium.

2. The method according to claim 1, wherein the suspended cell line is a stable producing cell line.

3. The method according to claim 1 or 2, wherein the suspended cell line is first cultured in a cell culture medium containing a sufficient amount of tetracycline or a derivative thereof to suppress the production of enveloped viruses and allow for an increase in the suspended cell line.

4. The method according to claim 3, wherein the amount of tetracycline or its derivative in the cell culture medium is at least 0.5 ng / mL.

5. The method according to claim 4, comprising reducing the concentration of tetracycline or a derivative thereof in a cell culture medium so as to induce the production of enveloped viruses.

6. The method according to claim 5, wherein the concentration of tetracycline or its derivative in the cell culture medium is reduced to a concentration of 0.5 ng / mL or less.

7. The method according to claim 5, wherein the concentration of tetracycline or its derivatives in the cell culture medium is reduced by diluting the suspended cell culture with a cell culture medium that does not contain tetracycline or its derivatives, and diluting the suspended cell culture includes (i) directly adding a cell culture medium that does not contain tetracycline or its derivatives to a cell culture medium that contains tetracycline or its derivatives, or adding a cell culture medium that does not contain tetracycline or its derivatives to the suspended cell culture.

8. Before diluting the suspended cell culture with a cell culture medium that does not contain tetracycline or its derivatives, the suspended cell line should be approximately 1 × 10⁻⁶ 6 cells / mL ~ approx. 1 x 10 7 The method according to claim 7, wherein cells are grown to a viable cell density of cells / mL.

9. The method according to claim 7, wherein the suspended cell culture is diluted in a cell culture medium that does not contain tetracycline or its derivatives in a ratio of about 1:1 to about 1:

20.

10. The method according to claim 1, wherein the suspended cell culture is operated in perfusion mode.

11. The method according to claim 1, wherein the suspended cell culture is manipulated for at least 15 days.

12. The method according to claim 3, wherein the tetracycline derivative is doxycycline.

13. The method according to claim 1, wherein the suspended cell culture has a volume of approximately 1 L, approximately 2 L, approximately 5 L, approximately 10 L, approximately 50 L, approximately 100 L, approximately 500 L, approximately 1,000 L, approximately 5,000 L, approximately 10,000 L, or more than approximately 15,000 L.

14. The method according to claim 1, further comprising purifying enveloped viruses from a suspended cell culture.

15. The method according to claim 14, further comprising formulating a purified enveloped virus into a pharmaceutical formulation or a solution suitable for infecting cells.

16. The method according to claim 1, wherein the enveloped virus is a lentivirus.

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