Methods of producing enveloped viruses

Abstract

The present disclosure relates to a method of producing an enveloped virus in a suspension cell culture, the method comprising culturing a suspension cell line expressing a tetracycline suppressor gene expression system in a cell culture medium. The disclosure also relates to stable producing cell clones capable of producing enveloped viruses in suspension cell cultures.

Description

[0001]

Related application data

[0002] This application claims priority to U.S. Patent Application No. 63 / 366,408, entitled “Methods for Producing Envelope Viruses,” filed on June 15, 2022, and U.S. Patent Application No. 63 / 498,041, entitled “Methods for Producing Envelope Viruses,” filed on April 25, 2023. The entire contents of each are hereby incorporated by reference. [Technical field]

[0003] The present disclosure relates generally to the manufacture of gene therapy products, and in particular to methods for producing enveloped viruses from suspension cell cultures expressing a tetracycline-repressible gene expression system. [Background of the invention]

[0005] Retroviruses, such as lentiviruses, are one of the most studied viral vectors for gene therapy. Retroviruses are typically RNA-based viruses that permanently integrate their genetic information into target cell chromosomes. 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.

[0006] Lentiviruses are genetically engineered and are usually based on the human immunodeficiency virus 1 (HIV-1). To improve safety, modern vectors contain only the HIV genes necessary for infection and gene delivery, but genes essential for replication and virulence factors have been removed. Often, the envelope protein of HIV-1 is swapped with that of another virus to allow infection of a variety of target cells, for example, the VSV-G protein from Indiana vesicular sputum virus (VSV).

[0007] To produce lentivirus, cells such as human embryonic kidney HEK 293T are transfected with 3-4 plasmids. These include a transfer plasmid with the gene of interest and several packaging plasmids encoding, vesicular stomatitis G protein (VSV-G), and essential viral proteins responsible for gene integration or self-assembly. These plasmids can be transiently transfected into cells, or a producer cell line can be generated by stably integrating the plasmids with an inducible promoter to induce lentivirus production.

[0008] Once virus production is induced, virus release occurs via budding after successful intracellular assembly. Lentivirus is harvested from producer cells and then purified and concentrated in downstream processes.

[0009] Clinical-grade lentiviral vectors are most commonly produced by transient transfection of adherent cell lines. These production methods are costly, require large amounts of GMP-grade plasmid, and hinder process scalability and reproducibility.

[0010] Therefore, there is a need in the art for an efficient method of producing lentivirus in a cell culture system, for example for gene therapy. [Summary of the Invention]

[0011] In the work leading to the present invention, the inventors sought to produce a method for producing enveloped viruses, e.g. for use in gene therapy, on a commercial scale and suitable for regulatory requirements.

[0012] The upstream process for producing enveloped viruses in suspension cell cultures expressing a tetracycline-repressible (ie, Tet-Off) gene expression system generated by the inventors includes a step of reducing the concentration of tetracycline or its derivatives (eg, doxycycline) in the cell culture to induce virus production.

[0013] In adherent cell lines, medium exchange is simple because cells grow on the surface and the medium can be removed without disturbing the cells. In adherent cell lines, tetracycline or its derivatives can be removed by performing a medium exchange to replace a cell culture medium containing tetracycline or its derivatives with a cell culture medium without tetracycline or its derivatives. However, in suspension cell lines, inducing virus production generally requires removing tetracycline or its derivatives from the cell culture medium by centrifugation to pellet the cells (i.e., removing the cells from the suspension), then adding a cell culture medium without tetracycline or its derivatives, and then resuspending the cells in a cell culture medium without tetracycline or its derivatives by agitating the cells.

[0014] In developing these methods, the inventors determined that typical methods for reducing the concentration of tetracycline or its derivatives in cell cultures (e.g., centrifugation and resuspension) reduce cell quality due to high shear forces on the cells and also increase the risk of contamination due to open manual steps.

[0015] To address this problem, the inventors determined that they could reduce the concentration of tetracycline or its derivatives in cell culture by diluting or using an acoustic standing wave method. In one example, the inventors found that they could reduce the concentration of tetracycline or its derivatives in cell culture by diluting a suspension cell culture with a cell culture medium without tetracycline or its derivatives. In another example, the inventors found that they could reduce the concentration of tetracycline or its derivatives in cell culture by retaining cells of a suspension cell line using an acoustic standing wave, removing a portion of the cell culture medium containing tetracycline or its derivatives from the suspension cell culture, and contacting the retained suspension cell line cells with a cell culture medium without tetracycline or its derivatives.

[0016] Thus, the inventors' discoveries provide methods for producing enveloped viruses.

[0017] In one example, the present disclosure provides a method of producing an enveloped virus in a suspension cell culture, the method comprising culturing a suspension cell line expressing a tetracycline-repressible gene expression system in a cell culture medium.

[0018] It will be apparent to the skilled person that the tetracycline-repressible gene expression system is also referred to as the Tet-Off expression system.

[0019] In an exemplary form of the present disclosure, the suspension cell line is a stable production cell line, i.e., a cell having genetic material required for producing a lentivirus stably incorporated therein. Such a cell is different from a cell having genetic elements transiently incorporated therein.

[0020] A typical enveloped virus is a retrovirus. For example, the retrovirus is a lentivirus. For example, the lentivirus is HIV or a derivative thereof.

[0021] In one example, the suspension cell line is initially cultured in a cell culture medium containing a sufficient amount of tetracycline or a derivative thereof to inhibit the production of enveloped viruses and to allow expansion of the suspension cell line.

[0022] In one example, the initial cell culture is an expansion cell culture. For example, the initial cell culture is conducted in an expansion (or N-1) bioreactor.

[0023] In one example, a sufficient amount of tetracycline or a derivative thereof in a cell culture medium is at least 0.1 ng / mL of cell culture medium. In one example, a sufficient amount of tetracycline or a derivative thereof in a cell culture medium is between about 0.1 ng / mL and about 10,000 ng / mL of cell culture medium. In one example, a sufficient amount of tetracycline or a derivative thereof in a cell culture medium is between about 0.1 ng / mL and about 1,000 ng / mL of cell culture medium. In one example, a sufficient amount of tetracycline or a derivative thereof in a cell culture medium is between about 0.1 ng / mL and about 100 ng / mL of cell culture medium. In one example, a sufficient amount of tetracycline or a derivative thereof in a cell culture medium is between about 0.1 ng / mL and about 10 ng / mL of 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 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 in the cell culture medium is between about 0.5 ng / mL and about 5 ng / mL, or about 1 ng / mL and about 5 ng / mL, or about 1.5 ng / mL and about 5 ng / mL, or about 2 ng / mL and about 5 ng / mL of cell culture medium. In one example, a sufficient amount of tetracycline or its derivative in the cell culture medium is between 0.5 ng / mL and 5 ng / mL of 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. For example, a sufficient amount of tetracycline or its derivative in the cell culture medium is 0.1 ng / mL cell culture medium. In one example, a sufficient amount of tetracycline or its derivative in the cell culture medium is 1 ng / mL cell culture medium. In one example, a sufficient amount of tetracycline or its derivative in the cell culture medium is 1.5 ng / mL cell culture medium.

[0024] In one example, the method includes reducing the concentration of tetracycline or a derivative thereof in a cell culture medium such that production of enveloped virus is induced.

[0025] In one 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%. In one example, the concentration of tetracycline or its derivatives is reduced by at least 50%. In one example, the concentration of tetracycline or its derivatives is reduced by at least 60%. In one example, the concentration of tetracycline or its derivatives is reduced by at least 70%. In one example, the concentration of tetracycline or its derivatives is reduced by at least 80%. In one example, the concentration of tetracycline or its derivatives is reduced by at least 85%. In one example, the concentration of tetracycline or its derivatives is reduced by at least 90%. In one example, the concentration of tetracycline or its derivatives is reduced by at least 95%.

[0026] In one example, the concentration of tetracycline or its derivatives in the cell culture medium is reduced to a concentration of 0.5 ng / mL cell culture medium or less. In one example, the concentration of tetracycline or its derivatives in the cell culture medium is reduced to a concentration of 0.5 ng / mL to 0.001 ng / mL cell culture medium. For example, the concentration of tetracycline or its derivatives in the cell culture medium is reduced to a concentration of about 0.5 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 0.2 ng / mL, or about 0.1 ng / mL cell culture medium. In one example, the concentration of tetracycline or its derivatives in the cell culture medium is reduced to a concentration of 0.2 ng / mL cell culture medium or less. In one example, the concentration of tetracycline or its derivatives in the cell culture medium is reduced to a concentration of 0.1 ng / mL cell culture medium or less. In one example, the concentration of tetracycline or its derivatives in the cell culture medium is reduced to a concentration of 0.1 ng / mL to 0.001 ng / mL of the cell culture medium. For example, the concentration of tetracycline or its derivatives 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 derivatives 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 derivatives 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 derivatives 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 derivatives 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 derivatives 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 derivatives 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 derivatives 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 derivatives in the cell culture medium is reduced to a concentration of about 0.001 ng / mL of the cell culture medium.

[0027] In one example, before reducing the concentration of tetracycline or its derivative in the cell culture medium, a sufficient amount of tetracycline or its derivative is adjusted in the cell culture medium to inhibit the production of enveloped viruses and allow expansion of the suspension cell line, thereby inducing the production of enveloped viruses. For example, in an expansion bioreactor, the concentration of tetracycline or its derivative is set to a first concentration for a first period of time, and then adjusted to a second concentration lower than the first concentration for a second period of time.

[0028] For example, the first concentration of tetracycline or its derivatives in the cell culture medium is at least 1 ng / mL of cell culture medium. In one example, the first concentration of tetracycline or its derivatives in the cell culture medium is between about 1 ng / mL and about 10 ng / mL of cell culture medium. For example, the first concentration of tetracycline or its derivatives 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 cell culture medium. In one example, the first concentration of tetracycline or its derivatives in the cell culture medium is between about 1 ng / mL and about 5 ng / mL, or about 1.5 ng / mL and about 5 ng / mL, or about 2 ng / mL and about 5 ng / mL of cell culture medium, or about 2.5 ng / mL and about 5 ng / mL of cell culture medium. For example, the first concentration of tetracycline or its derivative in the cell culture medium is 1 ng / mL, 1.5 ng / mL, 2 ng / mL, 2.5 ng / mL, 3 ng / mL, or 3.5 ng / mL, 4 ng / mL, 4.5 ng / mL or 5 ng / mL cell culture medium. For example, the first concentration of tetracycline or its derivative in the cell culture medium is 2.5 ng / mL cell culture medium.

[0029] For example, the concentration of tetracycline or its derivative is set to a first concentration in the expansion bioreactor for a period of between about 1 and about 8 days. In one example, the first period of time 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.

[0030] For example, the second concentration of tetracycline or its derivatives in the cell culture medium is at least 0.1 ng / mL cell culture medium. In one example, the second concentration of tetracycline or its derivatives in the cell culture medium is between about 0.1 ng / mL and about 2.5 ng / mL cell culture medium. For example, the second concentration of tetracycline or its derivatives 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 cell culture medium. In one example, the second concentration of tetracycline or its derivatives in the cell culture medium is between about 0.1 ng / mL and about 2.5 ng / mL, or about 0.5 ng / mL and about 2.5 ng / mL, or about 1 ng / mL and about 2.5 ng / mL cell culture medium, or about 1.5 ng / mL and about 2.5 ng / mL cell culture medium. For example, the second concentration of tetracycline or its derivative in the cell culture medium is 1 ng / mL, 1.5 ng / mL, 2 ng / mL or 2.5 ng / mL of cell culture medium. For example, the second concentration of tetracycline or its derivative in the cell culture medium is 1 ng / mL of cell culture medium.

[0031] For example, the concentration of tetracycline or its derivative is set to a second concentration in the expansion bioreactor for a period of time between about 1 and about 4 days. In one example, the first period of time is about 1 day, about 2 days, about 3 days, or about 4 days.

[0032] In one example, the concentration of tetracycline or its derivative in the expansion 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.

[0033] In one example, the concentration of tetracycline or its derivative in the cell culture medium is reduced by:

[0034] (i) diluting the suspension cell culture with cell culture medium without tetracycline or its derivatives; or

[0035] (ii) Retaining the suspension cell line cells using acoustic standing waves, removing a portion of the cell culture medium containing tetracycline or a derivative from the suspension cell culture, and contacting the retained suspension cell line cells with a cell culture medium without tetracycline or a derivative.

[0036] In one example, the concentration of tetracycline or its derivative in the cell culture medium is reduced by diluting the suspension cell culture with cell culture medium without tetracycline or its derivative.

[0037] In one example, diluting the suspension cell culture includes adding cell culture medium without tetracycline or a derivative directly to cell culture medium containing tetracycline or a derivative.

[0038] In one example, diluting the suspension cell culture includes adding a cell culture medium without tetracycline or a derivative to the suspension cell culture.

[0039] In one example, the suspension cell culture is diluted with a cell culture medium without tetracycline or derivatives at a ratio between about 1:1 and 1:20. For example, the suspension cell culture is diluted with a cell culture medium without tetracycline or derivatives at a ratio between 1:2 and 1:10 or between 1:4 and 1:7. In one example, the suspension cell culture is diluted with a cell culture medium without tetracycline or derivatives at a ratio between 1:2 and 1:10. For example, the suspension cell culture is diluted with a cell culture medium without tetracycline or derivatives at a ratio of about 1:2, about 1:3, about 1:4, or 1:5, about 1:6, or about 1:7, or 1:8, or 1:9, or about 1:10. In one example, the suspension cell culture is diluted with a cell culture medium without tetracycline or derivatives at a ratio between 1:4 and 1:7. In one example, the suspension cell culture is diluted with a cell culture medium without tetracycline or derivatives at a ratio of about 1:4. In another example, the suspension cell culture is diluted with a cell culture medium without tetracycline or a derivative at a ratio of about 1:5. In another example, the suspension cell culture is diluted with a cell culture medium without tetracycline or a derivative at a ratio of about 1:6. In one example, the suspension cell culture is diluted with a cell culture medium without tetracycline or a derivative at a ratio of about 1:7.

[0040] In one example, the concentration of tetracycline or its derivative in the cell culture medium is reduced by retaining the suspension cell line cells using acoustic standing waves, removing a portion of the cell culture medium containing tetracycline or its derivative from the suspension cell culture, and contacting the retained suspension cell line cells with a cell culture medium without tetracycline or its derivative.

[0041] In one example, a suspension cell line was initially grown at approximately 1 × 10 5 cells / mL and 1×10 10 Cells are plated in cell culture medium at a density between 1 × 10 cells / mL. For example, suspension cell lines are initially plated at a density of approximately 1 × 10 5 cells / mL~1×10 10 cells / mL or 1×10 5 cells / mL~1×10 7 cells / mL, or about 0.1×10 6 cells / mL~1×10 8 cells / mL, or about 0.5×106 cells / mL and 1×10 7 cells / mL, or about 0.5×10 6 cells / mL and 5×10 6 cells / mL, or about 0.5×10 6 cells / mL and 2.5×10 6 In one example, suspension cell lines were initially seeded at a density of 1 × 10 5 cells / mL~1×10 7 Cells are plated in cell culture medium at a density of 10 cells / mL. In one example, suspension cell lines are initially plated at 0.5 × 10 6 cells / mL~5.0×10 6 Cells are plated in cell culture medium at a density of 0.8 × 10 cells / mL of cell culture medium. In one example, suspension cell lines are initially plated at 0.8 × 10 6 cells / mL and 1.2×10 6 Cells are plated in cell culture medium at a density between 1 × 10 cells / mL of cell culture medium. In one example, suspension cell lines are initially plated at 1 × 10 6 cells / mL and 2.5×10 6 Cells are plated in cell culture medium at a density between 10 cells / mL. In one example, suspension cell lines are initially plated at 1.5 × 10 6 cells / mL and 2×10 6 In one example, suspension cell lines are initially seeded at a density of approximately 1 × 10 cells / mL of cell culture medium. 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 5×10 5 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 In one example, suspension cell lines are initially seeded at a density of approximately 1 × 10 cells / mL of cell culture medium. 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 5×106 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 In one example, suspension cell lines are initially seeded at a density of approximately 0.5 × 10 cells / mL of cell culture medium. 6 In one example, suspension cell lines are initially plated at a density of approximately 1 × 10 cells / mL in cell culture medium. 6 In one example, suspension cell lines were initially plated at a density of approximately 1.5 × 10 6 In one example, suspension cell lines were initially plated at a density of approximately 1.8 × 10 6 In one example, suspension cell lines were initially plated at a density of approximately 2 × 10 cells / mL in cell culture medium. 6 In one example, suspension cell lines were initially plated at a density of 2.5 × 10 cells / mL in cell culture medium. 6 In one example, a suspension cell line was initially seeded at a density of 3.0 × 10 cells / mL in cell culture medium. 6 Cell culture is seeded at a density of 10 cells / mL. In one example, a suspension cell line is initially seeded at 3.5 × 10 6 In one example, a suspension cell line was initially seeded at a density of 4.0 × 10 6 In one example, a suspension cell line was initially seeded at a density of 4.5 × 10 cells / mL in cell culture medium. 6 In one example, suspension cell lines were initially plated at a density of 5.0 × 10 cells / mL in cell culture medium. 6 In one example, suspension cell lines are initially plated at a density of approximately 1 × 10 cells / mL in cell culture medium. 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 7cells / mL, or about 10×10 7 In one example, suspension cell lines are initially seeded at a density of approximately 1 × 10 cells / mL of cell culture medium. 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 In one example, suspension cell lines are initially seeded at a density of approximately 1 × 10 cells / mL of cell culture medium. 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 The cells were seeded in cell culture medium at a density of 10 cells / mL cell culture medium.

[0042] In one example, the suspension cell line was grown to approximately 1 × 10 5 cells / mL~about 1×10 10 For example, suspension cell lines should grow to a viable cell density of approximately 1 × 10 cells / mL. 5 cells / mL~1×10 10 cells / mL, or about 0.1×10 6 cells / mL~1×10 8 cells / mL, or about 0.5×10 6 cells / mL~1×10 7 cells / mL, or about 0.5×10 6 cells / mL~5×10 6 cells / mL, or about 0.5×10 6cells / mL and 2.5×10 6 In one example, the suspension cell line was grown to a viable cell density of approximately 1 × 10 cells / mL of cell culture medium before diluting the suspension cell culture with cell culture medium without tetracycline or its derivatives. 6 cells / mL~about 1×10 7 In another example, the suspension cell line was grown to a viable cell density of approximately 6 × 10 cells / mL before diluting the suspension cell culture with cell culture medium without tetracycline or its derivatives. 6 cells / mL~about 1×10 7 In one example, suspension cell lines were grown to a viable cell density of approximately 0.5 × 10 6 cells / mL~5.0×10 6 In one example, suspension cell lines were grown to a viable cell density of approximately 1 × 10 6 cells / mL~2.5×10 6 In one example, suspension cell lines were grown to a viable cell density of approximately 1.5 × 10 6 cells / mL~2×10 6 In one example, suspension cell lines were grown to a viable cell density of approximately 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 5×10 5 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 In one example, a suspension cell line was grown to a viable cell density of approximately 1 × 10 cells / mL of cell culture medium. 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 6cells / mL, or about 10×10 6 In one example, a suspension cell line was grown to a viable cell density of approximately 0.5 × 10 cells / mL of cell culture medium. 6 In one example, a suspension cell line was grown to a viable cell density of approximately 1 × 10 cells / mL. 6 In one example, a suspension cell line was grown to a viable cell density of approximately 1.5 × 10 cells / mL. 6 In one example, a suspension cell line was grown to a viable cell density of approximately 1.8 × 10 6 In one example, a suspension cell line was grown to a viable cell density of approximately 2 × 10 cells / mL. 6 In one example, a suspension cell line was grown to a viable cell density of approximately 2.5 × 10 cells / mL. 6 In one example, a suspension cell line was grown to a viable cell density of approximately 3.0 × 10 cells / mL. 6 In one example, a suspension cell line was grown to a viable cell density of approximately 3.5 × 10 cells / mL. 6 In one example, a suspension cell line was grown to a viable cell density of approximately 4.0 × 10 6 In one example, a suspension cell line was grown to a viable cell density of approximately 4.5 × 10 6 In one example, a suspension cell line was grown to a viable cell density of approximately 5.0 × 10 cells / mL. 6 In one example, a suspension cell line was grown to a viable cell density of approximately 1 × 10 cells / mL. 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 In one example, a suspension cell line was grown to a viable cell density of approximately 1 × 10 cells / mL of cell culture medium. 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 8cells / 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 In one example, a suspension cell line was grown to a viable cell density of approximately 1 × 10 cells / mL of cell culture medium. 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 The viable cell density was 10 cells / mL of cell culture medium.

[0043] In one example, the suspension cell line has at least 60% viability before diluting the suspension cell culture with a cell culture medium without tetracycline or derivatives. For example, before diluting the suspension cell culture with a cell culture medium without tetracycline or derivatives, the viability of the suspension cell line is at least 60%, 70% or 80%, 85%, 90%, 95% or 99%. In one example, before diluting the suspension cell culture with a cell culture medium without tetracycline or derivatives, the viability of the suspension cell line is at least 70%. In one example, before diluting the suspension cell culture with a cell culture medium without tetracycline or derivatives, the viability of the suspension cell line is at least 80%. In one example, before diluting the suspension cell culture with a cell culture medium without tetracycline or derivatives, the viability of the suspension cell line is at least 85%. In one example, before diluting the suspension cell culture with a cell culture medium without tetracycline or derivatives, the viability of the suspension cell line is at least 90%. In one example, before diluting the suspension cell culture with a cell culture medium without tetracycline or derivatives, the viability of the suspension cell line is at least 95%. In one example, the viability of the suspension cell line is at least 99% prior to diluting the suspension cell culture with cell culture medium without tetracycline or a derivative thereof.

[0044] In one example, the suspension cell culture is operated in batch, batch fed, 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 batch fed mode. In another 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 carried out in batch and perfusion mode. For example, the cell culture is initially operated in batch mode and then in perfusion mode.

[0045] In one embodiment of any of the methods described herein, the method results in a viral infectious titer yield of at least 1×10 5 Transducing units (TU) / mL. For example, the method produces a virus infection titer yield of about 1×10 5 TU / mL and 1×10 10 TU / mL. In one example, the method produces a virus infection titer yield of approximately 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 resulted in a viral infection titer yield of approximately 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 produces a virus infection 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 another example, the method produces a virus infection titer yield of about 1×10 7 TU / mL, or about 5×107 TU / mL, or about 10×10 8 TU / mL, or about 5×10 8 TU / mL, or about 10×10 8 TU / mL, or about 5×10 9 TU / mL, or about 1×10 10 TU / mL.

[0046] In one example, the method results in the production of at least 1×10 5 The method can produce at least 1×10 transducing units (TU) / mL of culture medium. 6 TU / mL of culture medium. For example, the method produces 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 In another example, the method produces at least 1×10 TU / mL culture medium on day 15 of culture. 7 TU / mL of culture medium. For example, the virus infection titer yield produced by the method on the 15th day of culture 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 culture medium. In one example, the method produces at least 1.5×10 7 TU / mL of culture medium. For example, the virus infection titer yield produced by the method on the 15th day of culture 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. In one example, the method produces at least 2×10 7 TU / mL of the virus infection titer yield of the culture medium. For example, the virus infection titer yield produced by the method on the 15th day of culture is about 2.1×10 7 TU / mL, or about 2.2×10 7 TU / mL, or about 2.3×107 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.

[0047] In one example, the method results in the production of at least 1×10 5 The method can produce at least 1×10 transducing units (TU) / mL of culture medium. 6 TU / mL of culture medium. For example, the method produces 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, the method produces at least 1×10 7 TU / mL of culture medium. For example, the method produces about 1.1×10 7 TU / mL, about 1.2×10 7 TU / mL, 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, the method produces at least 1.5×10 7 TU / mL of culture medium. For example, the method produces 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, the method produces at least 2×10 7 TU / mL of culture medium. For example, the method produces about 2.1×10 7 TU / mL, or about 2.2×107 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 Virus infection titer yield in TU / mL culture medium.

[0048] In one example, the method results in the production of at least 1×10 5 The method can produce at least 1×10 transducing units (TU) / mL of culture medium. 6 TU / mL of culture medium. For example, the virus infection titer yield produced by the method on the 25th day of culture 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 In another example, the method produces at least 1×10 TU / mL culture medium on day 25 of culture. 7 TU / mL of the virus infection titer yield of the culture medium. For example, the virus infection titer yield produced by the method on the 25th day of culture 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 culture medium. In one example, the method produces at least 1.5×10 7 TU / mL of culture medium. For example, the virus infection titer yield produced by the method on the 25th day of culture 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. In one example, the method produces at least 2×10 7TU / mL of the virus infection titer yield of the culture medium. For example, the virus infection titer yield produced by the method on the 25th day of culture 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.

[0049] In one example, the method results in the production of at least 1×10 5 The virus infection titer yield of the method is at least 1×10 transduction units (TU) / mL culture medium. For example, the virus infection titer yield produced by the method on the 30th day of culture is at least 1×10 6 TU / mL culture medium. For example, the virus infection titer yield produced by the method on the 30th day of culture 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 In another example, the method produces at least 1×10 TU / mL culture medium on day 30 of culture. 7 TU / mL of the virus infection titer yield of the culture medium. For example, the virus infection titer yield produced by the method on the 30th day of culture 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 culture medium. In one example, the method produces at least 1.5×10 7 TU / mL of culture medium. For example, the method produces 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 7TU / mL culture medium. In one example, the method produces at least 2×10 7 TU / mL of the virus infection titer yield of the culture medium. For example, the virus infection titer yield produced by the method on the 30th day of culture 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.

[0050] In one example, the method results in the production of at least 1×10 5 The method can produce at least 1×10 transducing units (TU) / mL of culture medium. 6 TU / mL of culture medium. For example, the method produces about 1.5×10 6 TU / mL, 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, the method produces at least 1×10 7 TU / mL of culture medium. For example, the virus infection titer yield produced by the method on the 35th day of culture 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 culture medium. In one example, the method produces at least 1.5×10 7 TU / mL of culture medium. For example, the method produces about 1.6×10 7 TU / mL, or about 1.7 × 10 7 TU / mL, or about 1.8×107 TU / mL, or about 1.9×10 7 TU / mL of culture medium. In one example, the method produces at least 2×10 7 TU / mL of the virus infection titer yield of the culture medium. For example, the virus infection titer yield produced by the method on the 35th day of culture 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.

[0051] In one example, the method results in at least about 1×10 5 For example, the method produces a viable cell density of at least about 1×10 cells / mL of culture medium on day 15 of culture. 6 In one example, the method produces at least about 1.5×10 cells / mL of culture medium on day 15 of culture. 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 In one example, the method produces at least about 1×10 cells / mL of viable cell density on day 15 of culture. 7 In one example, the method produces at least about 1.1×10 cells / mL of viable cell density on day 15 of culture. 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 In one example, the method produces at least about 1.5×10 cells / mL of viable cell density on day 15 of culture. 7In one example, the method produces a viable cell density of at least about 1.5×10 cells / mL of culture medium on day 15 of culture. 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 In one example, the method produces at least about 2.0×10 cells / mL of culture medium on day 15 of culture. 7 In one example, the method produces at least about 2.1×10 cells / mL of viable cell density on day 15 of culture. 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 In one example, the method produces at least about 2.5×10 cells / mL of viable cell density on day 15 of culture. 7 In one example, the method produces at least about 2.6×10 cells / mL of viable cell density on day 15 of culture. 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 The viable cell density was 1 cell / mL culture medium.

[0052] In one example, the method produces at least about 1×10 5 For example, the method produces a viable cell density of at least about 1×10 cells / mL of culture medium on day 20 of culture. 6 In one example, the method produces at least about 1.5×10 cells / mL of culture medium on day 20 of culture. 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 In one example, the method produces at least about 1×10 cells / mL of viable cell density on day 20 of culture.7 In one example, the method produces at least about 1.1×10 cells / mL of viable cell density on day 20 of culture. 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 In one example, the method produces at least about 1.5×10 cells / mL of viable cell density on day 20 of culture. 7 In one example, the method produces a viable cell density of at least about 1.5×10 cells / mL of culture medium on day 20 of culture. 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 In one example, the method produces at least about 2.0×10 cells / mL of culture medium on day 20 of culture. 7 In one example, the method produces at least about 2.1×10 cells / mL of viable cell density on day 20 of culture. 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 In one example, the method produces at least about 2.5×10 cells / mL of viable cell density on day 20 of culture. 7 In one example, the method produces at least about 2.6×10 cells / mL of viable cell density on day 20 of culture. 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 The viable cell density was 1 cell / mL culture medium.

[0053] In one example, the method produces at least about 1×10 5For example, the method produces at least about 1×10 cells / mL of viable cell density on day 25 of culture. 6 In one example, the method produces at least about 1.5×10 cells / mL of viable cell density on day 25 of culture. 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 In one example, the method produces at least about 1×10 cells / mL of viable cell density on day 25 of culture. 7 In one example, the method produces at least about 1.1×10 cells / mL of viable cell density on day 25 of culture. 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 In one example, the method produces at least about 1.5×10 cells / mL of viable cell density on day 25 of culture. 7 In one example, the method produces a viable cell density of at least about 1.5×10 cells / mL of culture medium on day 25 of culture. 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 In one example, the method produces at least about 2.0×10 cells / mL of culture medium on day 25 of culture. 7 In one example, the method produces at least about 2.1×10 cells / mL of viable cell density on day 25 of culture. 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 In one example, the method produces at least about 2.5×10 cells / mL of viable cell density on day 25 of culture. 7In one example, the method produces at least about 2.6×10 cells / mL of viable cell density on day 25 of culture. 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 The viable cell density was 1 cell / mL culture medium.

[0054] In one example, the method produces at least about 1×10 5 For example, the method produces a viable cell density of at least about 1×10 cells / mL of culture medium on day 30 of culture. 6 In one example, the method produces at least about 1.5×10 cells / mL of culture medium on day 30 of culture. 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 In one example, the method produces at least about 1×10 cells / mL of viable cell density on day 30 of culture. 7 In one example, the method produces at least about 1.1×10 cells / mL of viable cell density on day 30 of culture. 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 In one example, the method produces at least about 1.5×10 cells / mL of viable cell density on day 30 of culture. 7 In one example, the method produces a viable cell density of at least about 1.5×10 cells / mL of culture medium on day 30 of culture. 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 In one example, the method produces at least about 2.0×10 cells / mL of culture medium on day 30 of culture.7 In one example, the method produces at least about 2.1×10 cells / mL of viable cell density on day 30 of culture. 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 In one example, the method produces at least about 2.5×10 cells / mL of viable cell density on day 30 of culture. 7 In one example, the method produces a viable cell density of at least about 2.6×10 cells / mL of culture medium on day 30 of culture. 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 culture medium.

[0055] In one example, the method produces at least about 1×10 5 For example, the method produces a viable cell density of at least about 1×10 cells / mL of culture medium on day 35 of culture. 6 In one example, the method produces at least about 1.5×10 cells / mL of culture medium on day 35 of culture. 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 In one example, the method produces at least about 1×10 cells / mL of viable cell density on day 35 of culture. 7 In one example, the method produces at least about 1.1×10 cells / mL of viable cell density on day 35 of culture. 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 In one example, the method produces at least about 1.5×10 cells / mL of viable cell density on day 35 of culture. 7In one example, the method produces a viable cell density of at least about 1.5×10 cells / mL of culture medium on day 35 of culture. 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 In one example, the method produces at least about 2.0×10 cells / mL of culture medium on day 35 of culture. 7 In one example, the method produces at least about 2.1×10 cells / mL of viable cell density on day 35 of culture. 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 In one example, the method produces at least about 2.5×10 cells / mL of culture medium on day 35 of culture. 7 In one example, the method produces at least about 2.6×10 cells / mL of viable cell density on day 35 of culture. 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 The viable cell density was 1 cell / mL culture medium.

[0056] In one example, the cell line has at least 70% viability on day 15 of culture. For example, the cell line has a viability of about 70% on day 15 of culture, or about 75% or about 80%. 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 at least 80% viability 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, on day 15 of culture, the viability is about 86%, or about 87%, or about 88%, or about 89%. In another example, the cell line has a viability of about 90% on day 15 of culture. In one example, the cell line has at least 90% viability on day 15 of culture. For example, the cell line has a viability of about 90%, about 91%, about 92%, about 93%, about 94%, or about 95% on culture day 15. In one example, the cell line has a viability of at least 95% on culture day 15. For example, the cell line has a viability of about 96%, about 97%, or about 98%, or about 99% on culture day 15.

[0057] In one example, the cell line has at least 70% viability on day 20 of culture. For example, the cell line has a viability of about 70% on day 20 of culture, or about 75% or about 80%. In one example, the cell line has a viability of at least 75% on day 20 of culture. In another example, the cell line has at least 80% viability on day 20 of culture. For example, the cell line has a viability of about 80% or about 85% or about 90% on day 20 of culture. In one example, the cell line has a viability of about 80% on day 20 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 20 of culture. For example, on day 20 of culture, the viability is about 86%, or about 87%, or about 88%, or about 89%. In another example, the cell line has a viability of about 90% on day 20 of culture. In one example, the cell line has at least 90% viability on day 20 of culture. For example, the cell line has a viability of about 90%, about 91%, about 92%, or about 93%, about 94%, or about 95% on culture day 20. In one example, the cell line has a viability of at least 95% on culture day 20. For example, the cell line has a viability of about 96%, about 97%, or about 98%, or about 99% on culture day 20.

[0058] In one example, the cell line has at least 70% viability on day 25 of culture. For example, the cell line has a viability of about 70%, about 75%, or about 80% on day 25 of culture. In one example, the cell line has at least 75% viability on day 25 of culture. In another example, the cell line has at least 80% viability on day 25 of culture. For example, the cell line has a viability of about 80%, about 85%, or about 90% on day 25 of culture. In one example, the cell line has a viability of about 80% on day 25 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 25 of culture. For example, on day 25 of culture, the viability is about 86%, or about 87%, or about 88%, or about 89%. In another example, the cell line has a viability of about 90% on day 25 of culture. In one example, the cell line has a viability of at least 90% on day 25 of culture. For example, the cell line has a viability of about 90%, about 91%, about 92%, or about 93%, about 94%, or about 95% on culture day 25. In one example, the cell line has a viability of at least 95% on culture day 25. For example, the cell line has a viability of about 96%, about 97%, or about 98%, or about 99% on culture day 25.

[0059] In one example, the cell line has at least 70% viability on day 30 of culture. For example, the cell line has about 70%, about 75%, or about 80% viability on day 30 of culture. In one example, the cell line has at least 75% viability on day 30 of culture. In another example, the cell line has at least 80% viability on day 30 of culture. For example, the cell line has about 80% or about 85% or about 90% viability on day 30 of culture. In one example, the cell line has about 80% viability on day 30 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 about 85% viability on day 30 of culture. For example, on day 30 of culture, the viability is about 86%, or about 87%, or about 88%, or about 89%. In another example, the cell line has about 90% viability on day 30 of culture. In one example, the cell line has at least 90% viability on day 30 of culture. For example, the cell line has a viability of about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95% on culture day 30. In one example, the cell line has a viability of at least 95% on culture day 30. For example, the cell line has a viability of about 96%, about 97%, or about 98%, or about 99% on culture day 30.

[0060] In one example, the cell line has at least 70% viability on day 35 of culture. For example, the cell line has a viability of about 70%, about 75%, or about 80% on day 35 of culture. In one example, the cell line has at least 75% viability on day 35 of culture. In another example, the cell line has at least 80% viability on day 35 of culture. For example, the cell line has a viability of about 80%, 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 another example, the cell line has a viability of about 90% on day 35 of culture. In one example, the cell line has at least 90% viability on day 35 of culture. For example, the cell line has a viability of about 90%, about 91%, about 92%, or about 93%, about 94%, or about 95% on culture day 35. In one example, the cell line has a viability of at least 95% on culture day 35. For example, the cell line has a viability of about 96%, about 97%, about 98%, or about 99% on culture day 35.

[0061] In one example, the method increases the viral infection 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 infection titer yield by at least 10%.

[0062] 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.

[0063] In one example, the suspension cell culture has a volume greater than about 1L, about 2L, about 5L, about 10L, about 50L, about 100L, about 500L, about 1000L, about 5,000L, about 10,000L, or about 15,000L. For example, the volume of the suspension cell culture is about 1L to 1000L. In one example, the volume of the suspension cell culture is about 1L. In another example, the volume of the suspension cell culture is about 5L. In another example, the volume of the suspension cell culture is about 10L. In one example, the volume of the suspension cell culture is about 50L. In another example, the volume of the suspension cell culture is about 100L. In another example, the volume of the suspension cell culture is about 500L. In one example, the volume of the suspension cell culture is about 1000L. In another example, the volume of the suspension cell culture is about 5000L. In another example, the volume of the suspension cell culture is about 10,000L. In another example, the volume of the suspension cell culture is about 15,000L.

[0064] In one example, the suspension cell culture is operated at a dissolved carbon dioxide (CO2) level 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 using about 5% CO2. In another example, the suspension cell culture is operated at about 6% CO2. In another example, the suspension cell culture is operated at about 7% CO2. In another example, the suspension cell culture is operated at about 8% CO2.

[0065] In one example, the suspension cell culture is operated at a pH between 6.0 and 8.0. In one example, the pH of the suspension cell culture is about 6.5 to 7.5. For example, the pH is between about 6.90 and about 7.3. In one example, the pH is about 7.1. In one example, the pH is about 6.5. In one example, the pH is about 6.6. In one example, the pH is about 6.7. In one example, the pH is about 6.8. In one example, the pH is about 6.9. In one example, the pH is about 7.0. In one example, the pH is about 7.1. In one example, the pH is about 7.2. In one example, the pH is about 7.3. In one example, the pH is about 7.4. In one example, the pH is about 7.5.

[0066] In one example, the suspension cell culture is operated at a temperature between about 35°C and 39°C. For example, the temperature of the suspension cell culture is about 35°C, about 35.5°C, about 36°C, about 36.5°C, or 37°C, about 37.5°C, or about 38°C, about 38.5°C, or about 39°C. In one example, the temperature of the suspension cell culture is between about 36.5°C and about 37.5°C. For example, the temperature of the suspension cell culture is about 37.0°C. In one example, the temperature of the suspension cell culture is between 38°C and 39°C. For example, the temperature of the suspension cell culture is about 38.5°C.

[0067] In one example, the pH of the suspension cell culture is between 6.0 and 8.0 and / or the temperature is between 35 and 39° C. In one example, the pH of the suspension cell culture is between 6.0 and 8.0 and / or between 37 and 38.5° C. For example, the pH of the suspension cell culture is between about 6.8 and about 7.1 and / or the temperature is between about 37° C. and about 38.5° C. In one example, the pH of the suspension cell culture is between about 6.8 and about 7.1 and the temperature is between about 37° C. and about 38.5° C. In one example, the pH of the suspension cell culture is between about 6.9 and about 7.0 and the temperature is about 37.0° C.

[0068] In one example, the method further comprises selecting a cell clone capable of producing enveloped virus at high infection titers and viability for at least 15 days. For example, the method further comprises selecting a cell clone capable of producing enveloped virus at a high infection titer and viability for at least 5×10 6 TU / mL of culture medium and / or at least 75% of the viability of cell clones producing enveloped virus for at least 15 days and / or at least 1 × 10 7 The viable cell density was 1 cell / mL culture medium.

[0069] In one example, the method further comprises selecting a cell clone capable of producing enveloped virus at high infection titers and viability for at least 20 days. For example, the method further comprises selecting a cell clone capable of producing enveloped virus at a high infection titer and viability for at least 5×10 6 TU / mL of culture medium with an infectious titer of at least 75% viability producing enveloped virus for at least 20 days and / or at least 1 × 10 7 Cell cloning medium with a viable cell density of 10 cells / mL.

[0070] In one example, the method further comprises selecting a cell clone capable of producing enveloped virus at high infection titers and viability for at least 25 days. For example, the method further comprises selecting a cell clone capable of producing enveloped virus at a high infection titer and viability for at least 5×10 6 TU / mL of culture medium and / or at least 75% viability and / or at least 1×10 7 Cell clones that produce enveloped virus for at least 25 days at a viable cell density of 10 cells / mL of culture medium.

[0071] In one example, the method further comprises selecting a cell clone capable of producing enveloped virus at high infection titers and viability for at least 30 days. For example, the method further comprises selecting a cell clone capable of producing enveloped virus at a high infection titer and viability for at least 5×10 6 TU / mL of culture medium and / or at least 75% viability and / or at least 1×10 7 Cell clones that produce enveloped virus for at least 30 days at a viable cell density of 10 cells / mL of culture medium.

[0072] In one example, the method further comprises selecting a cell clone capable of producing enveloped virus at high infection titers and viability for at least 35 days. For example, the method further comprises selecting a cell clone capable of producing enveloped virus at a high infection titer and viability for at least 5×10 6 TU / mL of culture medium and / or at least 75% viability and / or at least 1×10 7 Cell clones that produce enveloped virus for at least 35 days at a viable cell density of 10 cells / mL of culture medium.

[0073] Additional methods for selecting an appropriate cell clone will be apparent to the skilled artisan and / or described herein, and include, for example, the stability and / or mutational status of the cell clone and the ability of the purified virus to transduce hematopoietic stem cells.

[0074] In one example, the method further comprises purifying the enveloped virus from the suspension cell culture.

[0075] Methods for purifying enveloped viruses from suspension cell cultures are obvious to the skilled artisan and / or described herein. For example, purifying enveloped viruses comprises one or more steps selected from the group consisting of clarifying filtration, anion exchange chromatography, concentration, and diafiltration.

[0076] In one example, the method of the present disclosure also includes performing sterile filtration. For example, sterile filtration is performed before concentrating and diafiltration eluted viruses. In another example, sterile filtration is performed after concentrating and diafiltration eluted viruses.

[0077] In one example, the method further comprises formulating the purified enveloped virus into a pharmaceutical preparation or a solution suitable for infecting cells.

[0078] The present disclosure also provides purified enveloped viruses produced by the methods described herein.

Brief Description of the Figures

[0080] Figure 1 Shown are (A) viable cell number and viability and (B) infectious titer yield of cells 6 days after doxycycline removal in a production bioreactor using sonication (black) compared to infectious titers generated by centrifugation (grey).

[0081] Figure 2 Graphs showing the changes in virus yield over time by measuring (A) viable cell numbers and (B) infectious titers using the doxycycline-depleting dilution method (doxycycline concentrations were varied in the N-1 bioreactor).

[0082] Figure 3 is a series of graphs showing (A) viable cell density and (B) cell viability in a seed culture bioreactor supplemented with 1 ng / mL doxycycline and in a production bioreactor after doxycycline removal using dilution, sonication, and centrifugation. (C) infectious titer over time in a production bioreactor after doxycycline removal using dilution, sonication, and centrifugation, (D) total virus yield at harvest, (E) cell-specific productivity, and (F) glucose and (G) lactate concentrations.

[0083] Figure 4 are graphs showing (A) infectious titer development and cell-specific productivity, and (B) viable cell density and percent viability after extended production bioreactor fermentation. [Detailed description of the invention]

[0085]

General description

[0086] In this specification, reference to a single step, composition of matter, group of steps, or group of combinations of matter shall be deemed to include one and multiple (i.e., one or more) of such steps, compositions of matter, groups of steps, or groups of combinations of matter, unless expressly stated otherwise or the context requires otherwise. Thus, as used herein, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise. For example, reference to "a" includes one as well as two or more; reference to "an" includes single as well as two or more; reference to "the" includes single as well as two or more, and so on.

[0087] Those skilled 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 disclosure includes all such variations and modifications. The present disclosure also includes all steps, features, compositions and compounds mentioned or indicated in this specification, either individually or collectively, and any and all combinations or any two or more of the steps or features.

[0088] The scope of the present disclosure should not be limited by the specific examples described herein, which are provided for illustrative purposes only. Functionally equivalent products, compositions and methods are clearly within the scope of the present disclosure.

[0089] Unless otherwise specifically stated, any example of the present disclosure herein should be applicable to any other example of the present disclosure. In other words, any specific example of the present disclosure can be combined with any other specific example of the disclosure (unless mutually exclusive).

[0090] Any example of a particular feature or group of features or a method or method step disclosed in the present disclosure is not intended to provide explicit support for disclaiming the particular feature or group of features or method or method step.

[0091] Unless explicitly defined otherwise, all technical and scientific terms used herein should be construed to have the same meanings as commonly understood by one of ordinary skill in the art (eg, molecular biology, microbiology, virology).

[0092] Unless otherwise indicated, conventional techniques of molecular biology, microbiology, virology, recombinant DNA technology, peptide synthesis in solution, solid phase peptide synthesis, and immunology used in this disclosure are standard procedures well known to those skilled in the art. These techniques are described and explained in the literature, for example, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T. A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D. M. Glover and B. D. Humes (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F. M. Ausubel et al., (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Press (1991). Laboratory, (1988), and JE Coligan et al., (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates to date).

[0093] The term "and / or", such as "X and / or Y", should be understood as "X and Y" or "X or Y", and should be considered to provide clear support for both meanings or either meaning.

[0094] The term "about", unless otherwise specified, refers to + / -20% of the specified value, more preferably + / -10%. For the avoidance of doubt, the term "about" followed by a specified value should be interpreted as also including the exact specified value itself (e.g., "about 10" also includes exactly 10).

[0095] As used herein, the term "from" should be taken to mean that the specified integer can be obtained from a particular source, although not necessarily directly from that source (ie, including combinatorially obtained).

[0096] In this specification, the word "comprise" or variations such as "comprises" or "comprising" will be understood to mean the inclusion of stated elements, integers or steps, or groups of elements, integers or steps, but not the exclusion of any other elements, integers or steps, or groups of elements, integers or steps.

[0097] Definition of Choice

[0098] As used herein, the term "enveloped virus" refers to DNA and RNA viruses with a viral envelope. The envelope is usually derived from the host cell membrane (e.g., phospholipids and proteins), but may also include viral glycoproteins on the surface of the envelope. The enveloped virus also includes a "capsid," which is a protein layer between the envelope and the viral genome. In one example, the enveloped virus is a retrovirus. For example, an enveloped virus is a lentivirus, such as human immunodeficiency virus.

[0099] As used herein, the term "cell culture fluid" or "cell culture medium" will be understood to include a liquid or medium in which cells are grown for the purpose of producing enveloped viruses. The liquid or medium does not contain cells (e.g., the cells may have been removed, e.g., by centrifugation and / or removal of the supernatant).

[0100] As used herein, the term "cell culture" or "suspension cell culture" will be understood to refer to the combination of a cell culture fluid or medium and cultured cells.

[0101] The term "suspension" when referring to a cell line can be understood to mean a single cell or small aggregate of cells that are free-floating in a cell culture medium. For example, such cells function and propagate in an agitated growth medium, thereby forming a suspension.

[0102] The term "purification" or "purification" or "purification" should be understood as the complete or partial removal of at least one impurity present in the cell culture fluid, thereby increasing the purity level of the enveloped virus in the solution.

[0103] The term "impurities" or "impurities" shall include one or more components of the cell culture medium other than enveloped viruses. For example, impurities may include process-related impurities such as host cell DNA, host cell proteins, and culture medium components (e.g., fetal bovine serum).

[0104] [Generation of enveloped viruses]

[0105] Methods for producing enveloped viruses will be apparent to those skilled in the art and / or described, for example, in Ansorge et al., (2010) Biochem. Eng. J. 48:362-377; Schweizer and Merten (2010) Curr. Gene Ther. 10:474-486; and Rodrigues et al., (2011) Viral Gene Therapy. Xu, InTech. Chapter 2:15-40.

[0106]

Enveloped viruses

[0107] In one example, the virus is a retrovirus, such as a lentivirus. Example retroviruses are from: alpha retroviruses (such as avian leukosis virus (ALV)), beta retroviruses (such as mouse mammary tumor virus (MMTV)), gamma retroviruses (such as murine leukemia virus (MLV)), delta retroviruses (such as human T-lymphotropic virus (HTLV)), epsilon retroviruses (such as large eyed dermatosarcoma virus (WDSV)), sepsis viruses (such as human foamy virus (HFV) or simian foamy virus (SFV)), from primate lentiviruses, such as different types of human immunodeficiency virus (HIV), different types of simian immunodeficiency virus (SIV), or from non-primate mammalian lentiviruses, such as equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), caprine arthritis-encephalitis virus (CAEV), or ovine visna-maedi virus (VMV).

[0108] In some cases, an enveloped virus, such as a retrovirus, is pseudotyped, that is, it is composed of an envelope glycoprotein from a virus different from the virus from which it was derived, a modified envelope glycoprotein, or a chimeric envelope glycoprotein.

[0109] Transgenic expression

[0110] In some examples, the enveloped virus contains a transgene introduced into its genome. The transgene will depend on the specific use of the enveloped viral vector. Exemplary transgenes include transgenes encoding therapeutic RNA (e.g., antisense complementary RNA encoding a target RNA or DNA sequence), transgenes encoding proteins that are lacking or absent in subjects affected by the disease, or transgenes for DNA vaccination, i.e., transgenes encoding proteins, the expression of which will induce vaccination of the recipient body against the protein. In some examples, the transgene encodes a protein or nucleic acid that can be used to treat hemoglobinopathies (e.g., sickle cell disease or thalassemia). In some examples, the transgene encodes a protein or nucleic acid that can be used to treat primary immunodeficiency. In some examples, the transgene encodes a protein or nucleic acid that can be used to treat Wiskott-Aldrich syndrome. In some examples, the transgene encodes a protein or nucleic acid that can be used to treat X-linked agammaglobulinemia.

[0111] In some examples, enveloped viruses are produced by introducing the following four elements into a host cell: an expression cassette comprising the lentiviral gene gagpol, an expression cassette comprising the lentiviral gene rev, a transgene both located between the lentiviral LTR-5' and the lentiviral LTR-3', and an expression cassette encoding an envelope glycoprotein.

[0112]

Production cell lines

[0113] In some examples, the enveloped virus is produced by a stable line that expresses one or more elements required for the production of enveloped viruses (Miller (2001) Curr. Protoc. Hum. Genet. Chapter 12: Unit 12.5.; Rodrigues et al., 2011, supra). In one example, the enveloped virus is produced by a mammalian host cell that is transiently transfected with one or more plasmids encoding elements required for the production of the virus. According to another example, the elements are introduced into the cell via multiple plasmids: a plasmid with an expression cassette containing a lentiviral gagpol gene, a plasmid with an expression cassette containing a lentiviral rev gene, a plasmid with an expression cassette encoding an envelope glycoprotein, a plasmid with a gene containing a tetracycline transactivator (tTA), and / or a plasmid with an expression cassette containing a lentiviral tat gene. A transfer plasmid containing the transgene expression cassette, consisting of lentiviral LTR-5' and LTR-3', can be introduced as a conjugate together with a helper plasmid with an antibiotic resistance cassette to confer resistance to producer cells.

[0114] The host cell can be selected from any cell that allows the production of enveloped viruses. According to one example, the cell is selected from: human cells (HEK293, HEK293T, HEK293FT, HEK293OX, Te671, HT1080, CEM), mustelid cells (NIH-3T3), mustelid cells (Mpf), canine cells (D17) and derivatives thereof. According to one example, the cells are selected from: CHO cells, BHK cells, MDCK cells, C3H 10T1 / 2 cells, FLY I, Psi-2 cells, BOSC 23 cells, PA317 cells, WEHI cells, COS cells, BSC 1 cells, BSC 40 cells, BMT 10 cells, VERO cells, W138 cells, MRC5 cells, A549 cells, HT1080 cells, B-50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells, 211 cells and 211A cells and their derivatives.

[0115] According to one example, the cell is selected from GPR, GPRG, GPRT, GPRGT and GPRTG cell lines. In another example, the cell is selected from a cell line derived from any of the above cell lines.

[0116] In one example, the cell line is a suspension cell line selected from GPR, GPRG, GPRT, GPRGT and GPRTG cell lines. For example, the suspension cell line is a cell line derived from any of the above cell lines. In one example, the suspension cell line is a cell line derived from the GPRG cell line. In another example, the suspension cell line is a cell line derived from the GPRGT cell line. In another example, the suspension cell line is a cell line derived from the GPRTG cell line.

[0117] Methods for adapting adherent cell lines to growth in suspension are obvious to the skilled person and / or described herein. In one example, enveloped viruses are produced by stable production cells. Stable production cells can be derived from packaging cell lines, including any cell line 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, Vol. 23, Suppl. 1, S35). In one example, a stable production cell line cell is produced by synthesizing a vector by cloning one or more genes into a recombinant plasmid; the expression cassette excised from the synthetic vector forms a concatemeric array, and the expression cassette is obtained from an antibiotic resistance cassette plasmid; the packaging cell line cell is transfected with the formed Concatemeric Array; and the stable production cell line cell is selected and isolated. The virus is produced by inducing an inducible promoter of a stable production cell line cell.

[0118] The present disclosure also provides a stable producer cell clone capable of producing enveloped virus.

[0119] In one example, stable producer cell clones are cultured to generate stable producer cell lines. Methods of culturing stable producer cell clones to generate stable producer cell lines are described herein and / or will be apparent to the skilled artisan.

[0120] The present disclosure also provides a stable production cell clone capable of producing an infection titer of at least 5×10 6 TU / mL medium and / or viability of 75% and / or viable cell density of at least 1.0 × 10 7 For example, a stable producer cell clone can produce enveloped virus with an infectious titer of at least 5 × 10 cells / mL of culture medium on day 15 of culture. 6 TU / mL medium and / or viability of at least 75% and / or viable cell density of at least 1.0 × 10 7 cells / mL.

[0121] The present disclosure also provides a stable production cell clone capable of producing an infectious titer of at least 5×10 6 TU / mL medium and / or viability of 75% and / or viable cell density of at least 1.0 × 10 7 For example, a stable producer cell clone can produce an infectious titer of at least 5 × 10 cells / mL of culture medium on day 20. 6TU / mL medium and / or viability of at least 75% and / or viable cell density of at least 1.0 × 10 7 cells / mL of culture medium.

[0122] The present disclosure also provides a stable production cell clone capable of producing enveloped virus for at least 25 days with an infectious titer of at least 5×10 6 TU / mL medium and / or at least 75% viability and / or at least 1.0×10 7 For example, on day 25 of culture, a stable producer clone should be able to produce an infectious titer of at least 5 × 10 6 TU / mL medium and / or viability of at least 75% and / or viable cell density of at least 1.0 × 10 7 cells / mL of culture medium.

[0123] The present disclosure also provides a stable production cell clone capable of producing an infectious titer of at least 5×10 6 TU / mL medium and / or at least 75% viability and / or at least 1.0×10 7 For example, a stable producer cell clone can produce an infectious titer of at least 5 × 10 cells / mL of culture medium on day 30. 6 TU / mL medium and / or viability of at least 75% and / or viable cell density of at least 1.0 × 10 7 cells / mL of culture medium.

[0124] The present disclosure also provides a stable production cell clone capable of producing an infectious titer of at least 5×10 6 TU / mL medium and / or viability of 75% and / or viable cell density of at least 1.0 × 10 7 For example, a stable producer cell clone can produce an infectious titer of at least 5 × 10 cells / mL of culture medium on day 35. 6 TU / mL medium and / or viability of at least 75% and / or viable cell density of at least 1.0 × 10 7 cells / mL of culture medium.

[0125] The present disclosure also provides a stable production cell line capable of producing an infectious titer of at least 5×10 6 TU / mL medium and / or at least 75% viability and / or at least 1.0×10 7 For example, on day 15 of culture, a stable producer cell line should be able to produce an infectious titer of at least 5 × 106 TU / mL medium and / or viability of at least 75% and / or viable cell density of at least 1.0 × 10 7 cells / mL of culture medium.

[0126] The present disclosure also provides a stable production cell line capable of producing an infectious titer of at least 5×10 6 TU / mL medium and / or at least 75% viability and / or at least 1.0×10 7 For example, a stable producer cell line can produce an infectious titer of at least 5 × 10 cells / mL of culture on day 20. 6 TU / mL medium and / or viability of at least 75% and / or viable cell density of at least 1.0 × 10 7 cells / mL of culture medium.

[0127] The present disclosure also provides a stable production cell line capable of producing an infectious titer of at least 5×10 6 TU / mL medium and / or at least 75% viability and / or at least 1.0×10 7 For example, a stable producer cell line can produce an infectious titer of at least 5 × 10 cells / mL of culture medium on day 25. 6 TU / mL medium and / or viability of at least 75% and / or viable cell density of at least 1.0 × 10 7 cells / mL of culture medium.

[0128] The present disclosure also provides a stable production cell line capable of producing enveloped virus for at least 30 days with an infectious titer of at least 5×10 6 TU / mL medium and / or at least 75% viability and / or at least 1.0×10 7 For example, a stable production cell line should be able to produce an infectious titer of at least 5 × 10 cells / mL of culture medium on day 30. 6 TU / mL medium and / or viability of at least 75% and / or viable cell density of at least 1.0 × 10 7 cells / mL of culture medium.

[0129] The present disclosure also provides a stable production cell line capable of producing enveloped virus for at least 35 days with an infectious titer of at least 5×10 6 TU / mL medium and / or at least 75% viability and / or at least 1.0×10 7 For example, a stable producer cell line should be able to produce an infectious titer of at least 5 × 10 cells / mL of culture medium on day 35.6 TU / mL medium and / or viability of at least 75% and / or viable cell density of at least 1.0 × 10 7 cells / mL of culture medium.

[0130] Methods for determining cell viability are apparent to the skilled artisan 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 trypan blue exclusion, and the percentage of live cells to total cells in the sample is determined.

[0131] Methods for measuring infectious titers are apparent to the skilled artisan and / or described herein. In one example, the infectious titer is determined by transduction with GFP LV followed by flow cytometry. For example, the method comprises incubating the supernatant containing the virus with HEK293T cells seeded on a plate, followed by trypsinization and washing, and determining the percentage of GFP positive cells using flow cytometry and calculating the infectious titer in transduction units (TU) / mL of culture medium.

[0132] Methods for determining viable cell density are apparent to the skilled person 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 trypan blue exclusion, and the total number of viable cells per mL sample is determined.

[0133] In one example, the stable producer cell clone and / or stable producer cell line comprises the characteristics of any of the cell lines described herein.

[0134] In one example, the stable producer cell clone and / or the stable producer cell line expresses a tetracycline repressible gene expression system in the cell culture medium.

[0135] In one example, the stable producer cell clone and / or the stable producer cell line is adapted to grow in suspension cell culture.

[0136] In one example, the stable producer cell clone and / or the stable producer cell line is selected from or derived from a GPR, GPRG, GPRT, GPRGT or GPRTG cell line.

[0137] The present disclosure also provides a method for generating a stable producer cell clone capable of producing an enveloped virus in a suspension cell culture, the method comprising culturing the stable producer cell clone in the suspension cell culture for at least 15 days, detecting cell viability and / or infectious titer yield and / or viable cell density, and selecting a stable producer cell clone, wherein a stable producer cell clone is generated if one or more or all of the following criteria are met after at least 15 days of culturing:

[0138] (i) The infectious titer is at least 5×10 6 TU / mL culture medium;

[0139] (ii) a viability of at least 75%; and

[0140] (iii) At least 1.0×10 7 The viable cell density was 1 cell / mL culture medium.

[0141] The present disclosure also provides a method for producing a stable producer cell clone capable of producing an enveloped virus in a suspension cell culture, the method comprising culturing the stable producer cell clone in the 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 producer cell clone, wherein a stable producer cell clone is produced if one or more or all of the following criteria are met after at least 20 days of culturing:

[0142] (i) The infectious titer is at least 5×10 6 TU / mL culture medium;

[0143] (ii) a viability of at least 75%; and

[0144] (iii) At least 1.0×10 7 The viable cell density was 1 cell / mL culture medium.

[0145] The present disclosure also provides a method for producing a stable producer cell clone capable of producing an enveloped virus in a suspension cell culture, the method comprising culturing the stable producer cell clone in the 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 producer cell clone, wherein a stable producer cell clone is produced if one or more or all of the following criteria are met after at least 25 days of culturing:

[0146] (i) The infectious titer is at least 5×10 6 TU / mL culture medium;

[0147] (ii) a viability of at least 75%; and

[0148] (iii) The viable cell density is at least 1.0×10 7 cells / mL of culture medium.

[0149] The present disclosure also provides a method for producing a stable producer cell clone capable of producing an enveloped virus in a suspension cell culture, the method comprising culturing the stable producer cell clone in a suspension cell culture for at least 30 days, detecting cell viability and / or infectious titer yield and / or viable cell density, and selecting a stable producer cell clone, wherein a stable producer cell clone is generated if one or more or all of the following criteria are met after culturing for at least 30 days:

[0150] (i) The infectious titer is at least 5×10 6 TU / mL culture medium;

[0151] (ii) a viability of at least 75%; and

[0152] (iii) At least 1.0×10 7 The viable cell density was 1 cell / mL culture medium.

[0153] The present disclosure also provides a method for producing a stable producer cell clone capable of producing an enveloped virus in a suspension cell culture, the method comprising culturing the stable producer cell clone in the suspension cell culture for at least 35 days, detecting cell viability and / or infectious titer yield and / or viable cell density, and selecting a stable producer cell clone, wherein a stable producer cell clone is produced if one or more or all of the following criteria are met after at least 35 days of culturing:

[0154] (i) The infectious titer is at least 5×10 6 TU / mL culture medium;

[0155] (ii) a viability of at least 75%; and

[0156] (iii) At least 1.0×10 7 The viable cell density was 1 cell / mL culture medium.

[0157]

Cell culture medium

[0158] The cells are cultured in a medium suitable for culturing mammalian cells and producing enveloped viruses. The cells can be cultured in an adherent environment, e.g., when the cells are attached to a surface, or in a suspended environment, e.g., suspended in a culture medium. In addition, the culture medium can be supplemented with additives known in the art, such as appropriate concentrations of antibiotics, serum (particularly fetal bovine serum, etc.). The culture medium can be supplemented with GlutaMax TM 、Pluronic TM F-68 (ThermoFisher), R3IGF-I(Sigma-Aldrich), Cell Boost TM5 and / or anti-clotting agents. The culture medium used may in particular contain serum or be serum-free. Culture media for mammalian cells are known and include, for example, DMEM (Dulbecco's Modified Eagle Medium) medium, RPMI1640 or a mixture of various culture media, including DMEM / F12, or serum-free medium, such as optiPRO, (ThermoFisher), TransFx TM (Cytiva), (Irvine), Freestyle (Life Technologies) or 293 (Sigma-Aldrich).

[0159] In one example, cells were grown in the presence of TransFx TM (Cytiva) medium.

[0160] In one example, the cells are supplemented with one or more additives selected from the group consisting of GlutaMax TM 、Cell Boost TM 5. A group consisting of poloxamer 188 and combinations thereof. For example, cells supplemented with GlutaMax TM In another example, cells were supplemented with Cell Boost TM 5. In one example, cells were supplemented with Poloxamer 188. In another example, cells were supplemented with GlutaMax TM and Cell Boost TM 5. In another example, cells were supplemented with GlutaMax TM and poloxamer 188. In another example, cells were supplemented with Cell Boost TM 5 and poloxamer 188. In one example, cells were supplemented with GlutaMax TM 、Cell Boost TM 5 and poloxamer 188.

[0161] In one example, cells were supplemented with less than 15 mM GlutaMax TM For example, cells are supplemented with about 15 mM GlutaMax TM , or about 14mM GlutaMax TM , or about 13mM GlutaMax TM , or about 12mM GlutaMax TM , or about 11mMGlutaMaxTM , or about 10mM GlutaMax TM In one example, cells were supplemented with less than 10 mM GlutaMax TM For example, cells are supplemented with approximately 10 mM GlutaMax TM , or about 9mM GlutaMax TM , or about 8mM GlutaMax TM , or about 7mMGlutaMax TM , or about 6mM GlutaMax TM , or about 5mM GlutaMax TM In one example, cells were supplemented with less than 5 mM GlutaMax TM For example, cells are supplemented with approximately 5 mM GlutaMax TM , or about 4mM GlutaMax TM , or about 3mMGlutaMax TM , or about 2mM GlutaMax TM , or about 1mM GlutaMax TM In one example, cells were supplemented with 1 mM to 10 mM GlutaMax TM For example, cells were supplemented with 4mM to 8mM GlutaMax TM In one example, cells were supplemented with 4 mM GlutaMax TM In another example, cells were supplemented with 5 mM GlutaMax TM In another example, cells were supplemented with 6 mM GlutaMax TM In one example, cells were supplemented with 7 mM GlutaMax TM In another example, cells were supplemented with 8 mM GlutaMax TM .

[0162] 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 another example, the cells are supplemented with 0.1% poloxamer 188. In one example, the cells are supplemented with 0.15% poloxamer 188. In another example, the cells are supplemented with 0.2% poloxamer 188.

[0163] In one example, cells were replenished with less than 10% of Cell Boost TM 5. For example, Cell Boost is supplemented with about 10% TM 5, or about 9% Cell Boost TM 5, or about 8% Cell Boost TM 5, or about 7% CellBoost TM 5, or about 6% Cell Boost TM 5. In one example, the cells were replenished with less than 5% of Cell Boost TM 5. For example, Cell Boost is supplemented with about 5% TM 5, or about 4% Cell Boost TM 5, or about 3% CellBoost TM 5, or about 2% Cell Boost TM 5, or about 1% Cell Boost TM 5. In one example, cells were supplemented with 1% to 10% Cell Boost TM 5. For example, cells are supplemented with 2% to 8% Cell Boost TM 5. In one example, cells were supplemented with 4% to 6% Cell Boost TM 5. For example, Cell Boost supplemented with about 4% TM 5. In another example, cells were supplemented with approximately 5% Cell Boost TM 5. In another example, cells were supplemented with about 6% Cell Boost TM 5.

[0164] In the process of using transient transfection cells, any reagent that allows transfection of plasmids can be used. Example reagents include calcium phosphate or polyethyleneimine. Conditions (e.g., amount of plasmids, ratio between plasmids, ratio between plasmid and transfection reagent, type of culture medium, etc.) and transfection time can be adjusted by those skilled in the art according to the characteristics of the virus produced and / or the transgene introduced into the transfer plasmid.

[0165] According to some examples, the culture medium used has a neutral pH value (e.g., between 7 and 7.4, in particular 7, 7.1, 7.2, 7.3 or 7.4), which is typically used for culturing cells and producing viruses. In one example, the pH value of the suspended cell culture is between 6.0 and 8.0. For example, the pH value 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. "Moderately acidic conditions" refers to a pH value of an aqueous solution between 5 and 6.8, for example, between 5.5 and 6.5, between 5.8 and 6.2. The selected pH value will also depend on the buffering capacity of the culture medium used, which can be easily determined by a person skilled in the art after considering his / her common sense. A person skilled in the art is able to change the pH value of the solution.

[0166] In one example, the production of enveloped viruses includes: producing the vector constitutively or after induction by one or more plasmids encoding the elements required for producing the enveloped vector, or using stable production cells, for example, GPRG or GPRTG stably transfected with the target gene, transiently transfecting HEK293T cells or their derivatives; culturing the cells in a suitable culture medium with a pH of about 6 or about 7; and harvesting the cell culture medium containing the enveloped virus.

[0167]

Suspension cell culture

[0168] The present disclosure provides a method for producing enveloped viruses in 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.

[0169] The disclosed methods are suitable for producing enveloped viruses from small-scale and large-scale production. These methods are particularly useful because they can be scaled up to produce commercial-scale pharmaceuticals.

[0170] For the skilled person, the production of enveloped viruses includes a cell amplification stage and a virus production stage.

[0171] Those skilled in the art will appreciate that the cell expansion stage includes seed culture cell culture. The term "seed culture" as used herein refers to the production of a sufficient number of cells (i.e., cell growth) for virus production. Those skilled in the art will appreciate that seed culture cell culture includes multiple culture systems that become larger (e.g., T-flasks, roller bottles or shake flasks, small bioreactor systems, and subsequent large bioreactors) each time they are passed down the generations, so as to amplify the culture from a small volume of cells to a larger volume of cells suitable for virus production.

[0172] It will be apparent to the skilled person that in any of the methods described herein, during the cell expansion phase, the cells are cultured in the presence of tetracycline or a derivative thereof to inhibit virus production but allow cell growth.

[0173] In one example, cells are grown in a cell expansion phase prior to virus production.

[0174] In one example, the cell expansion phase is performed in an expansion bioreactor (also referred to as an N-1 bioreactor).

[0175] In one example, the virus production phase is carried out in a production bioreactor (also called an N bioreactor).

[0176] In one example, the cell expansion phase and the virus production phase are performed in the same container. For example, the expansion of a suspension cell line and the production of enveloped viruses occur in the same container. For example, the cell expansion phase and the virus production phase are performed in the same bioreactor.

[0177] In one example, the cell expansion phase and the virus production phase are performed in different containers. For example, the cell expansion phase is performed in an expansion bioreactor and the virus production phase is performed in a production bioreactor, wherein the expansion bioreactor and the production bioreactor are different.

[0178] In one example, the cell culture is conducted in batch, fed-batch, continuous, semi-continuous, or perfusion mode.

[0179] In one example, the cell expansion phase and / or the virus production phase is performed in batch, fed-batch, continuous, semi-continuous, or perfusion mode.

[0180] In one example, the cell expansion phase is performed in batch, batch-fed, continuous, semi-continuous, or perfusion mode. In one example, the cell expansion phase is performed in batch mode. In another example, the cell expansion phase is performed in fed-batch mode. In another example, the cell expansion phase is performed in continuous mode. In one example, the cell expansion phase is performed in perfusion mode. In another example, the cell expansion phase is performed in batch and perfusion mode. For example, the cell expansion phase is initially performed in batch mode and then performed in perfusion mode.

[0181] In one example, the virus production phase is performed in batch, batch fed, continuous, semi-continuous, or perfusion mode. In one example, the virus production phase is performed in batch mode. In another example, the virus production phase is performed in fed batch mode. In another example, the virus production phase is performed in continuous mode. In one example, the virus production phase is performed in perfusion mode. In another example, the virus production phase is performed in batch and perfusion mode. For example, the virus production phase is initially performed in batch mode and then performed in perfusion mode.

[0182] In one example, the cell expansion and virus production phases are performed in batch mode. In another example, the cell expansion and virus production phases are performed in perfusion mode. In another example, the cell expansion phase is performed in batch mode and the virus production phase is performed in perfusion mode.

[0183] It will be apparent to the skilled person that reference to batch, fed-batch, continuous and / or perfusion mode for a particular phase of cell culture (i.e., cell expansion and / or virus production) does not mean that the entire culture phase is performed in that mode. For example, it only means that a portion of the cell culture phase (e.g., at least 1 day) is performed in that mode. It will also be understood that the mode does not necessarily start on day 0 of the culture phase. For example, culture may start on day 0, and perfusion mode only starts on day 2 of the cell culture phase.

[0184] In one example, the suspension cell culture is operated in batch mode. For the skilled artisan, "batch mode" refers to a process in which cells are initially cultured in a culture medium, and the culture medium is neither removed, replaced, nor replenished, i.e., the cells are not "fed" with new culture medium during or before the culture.

[0185] In one example, the suspension cell culture is operated in fed-batch mode. For a skilled artisan, "fed-batch mode" refers to a process in which one or more nutrients are fed into a bioreactor during the culture period. In one example, the cell expansion phase and / or the virus production phase are run in fed-batch mode. In one example, the cell expansion phase is run in fed-batch mode.

[0186] In one example, the suspension cell culture is operated in perfusion mode. For technicians, "perfusion mode" obviously involves constantly feeding fresh culture medium and removing spent culture medium while retaining a large number of viable cells (i.e., continuous culture medium replacement). In one example, the cell expansion stage and / or the virus production stage are operated in perfusion mode. In one example, the virus production stage is carried out in perfusion mode. In one example, the perfusion mode involves perfusion of culture medium containing tetracycline or derivatives throughout the cell expansion stage. In one example, the perfusion mode includes initially perfusing with culture medium containing tetracycline or derivatives, and then perfusing with culture medium without tetracycline or derivatives before induction.

[0187] 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 an 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. or The cells were cultured in a bioreactor (Sartorius).

[0188] For example, the volume of the cell culture may be about 0.01 L to about 0.1 L, or about 0.1 L to about 1 L, or about 1 L to about 5 L. In another example, the volume of the cell culture may be about 5 L to about 10 L, about 10 L to about 50 L, about 50 L to about 100 L, about 100 L to about 200 L, about 200 L to about 500 L, about 500 L to about 1000 L, about 1000 L to about 2000 L, or about 2000 L to about 5000 L. In one example, the volume of the cell culture is about 35 to 150 L. In one example, the volume of the cell culture is about 35 to 150 L. In one example, the volume of the cell culture is about 50 to 70 L.

[0189] In one example, the suspension cell culture is operated at a temperature that allows cell growth and virus production. For example, the cell culture has a state-of-the-art temperature that is commonly used to grow cells and produce viruses. In one example, the temperature of the suspension cell culture is between 35 and 39°C. For example, at a temperature of 37±0.5°C or at a temperature of 38±0.5°C.

[0190] In one example, the suspension cell culture is operated on a large scale. For example, the suspension cell culture is performed on a commercial scale.

[0191] In one example, the suspension cell culture is operated for at least 10 days. For example, the operation time of the suspension cell culture is about 10 to 50 days. In one example, the operation time of the suspension cell culture is between 10 and 35 days, for example, about 10 days or about 12 days, or about 15 days, or about 18 days, or about 20 days, or about 22 days, or about 25 days, or about 28 days, or about 30 days or about 32 days or about 35 days. In one example, the suspension cell culture is operated for at least 15 days. For example, the suspension cell culture is operated for about 20 days. In another example, the suspension cell culture is operated for at least 20 days. In another example, the suspension cell culture is operated for at least 25 days. For example, the suspension cell culture is operated for about 28 days. In one example, the suspension cell culture is operated for at least 30 days. In one example, the suspension cell culture is operated for at least 32 days. For example, the operation time of the suspension cell culture is 35 days. In one example, the suspension cell culture is operated for at least 35 days.

[0192]

Degradation of tetracycline and its derivatives in suspension cell culture

[0193] The disclosed methods are suitable for producing enveloped viruses from small-scale to large-scale production. These methods are particularly useful because they can be scaled up to produce commercial-scale pharmaceuticals.

[0194] 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 to produce enveloped viruses, wherein the cell line expresses a tetracycline-repressible gene expression system. From the disclosure herein, it will be apparent to the skilled person that the disclosed methods result in an increase in cell quality and virus production.

[0195] From the disclosure herein, it will be apparent to the skilled person that it is not necessary to completely remove or reduce the concentration of tetracycline or its derivatives. 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%.

[0196] As used herein, reference to a cell line expressing a "tetracycline repressible gene expression system" or a "Tet-OFF" system refers to a cell line that stably expresses a tetracycline-controlled transactivator (tTA) such that the presence of tetracycline or a derivative thereof (e.g., doxycycline) silences transcription from a tetracycline response element promoter.

[0197] The skilled artisan will appreciate that initially the cells are cultured in the presence of tetracycline or a derivative thereof to inhibit viral production but to allow cell growth or expansion of cell lines.

[0198] In one example, the amount of tetracycline or its derivative required in the cell culture medium to inhibit virus production is at least 0.1 ng / mL.

[0199] In one example, tetracycline or its derivatives in the cell culture medium inhibits virus production for 1 to 20 days. For example, the amount of tetracycline or its derivatives in the cell culture medium inhibits 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 inhibits 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.

[0200] From the disclosure herein, it is obvious to the skilled person that tetracycline or its derivatives are added to the cell culture medium as a single bolus feed, multiple feeds or continuously during the culture process. In one example, tetracycline or its derivatives are added to the cell culture medium as a single bolus feed. For example, tetracycline or its derivatives are added to the cell culture medium at the beginning of the cell culture (i.e., day 0). In another example, tetracycline or its derivatives are added to the cell culture medium every day or every other day during the cell culture. In one example, tetracycline or its derivatives are added to the cell culture medium by perfusing the cell culture at a specified concentration. For example, tetracycline or its derivatives are added to the cell culture medium at a concentration of 1.5ng / mL by perfusing the cell culture. In another example, tetracycline or its derivatives are added to the cell culture medium as needed 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 0.1ng / mL in the cell culture medium.

[0201] It is obvious to the skilled person that discontinuation (or reduction of concentration) of tetracycline or its derivatives will result in 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.

[0202] The inventors have found that during the growth phase of the cell culture, as the cell density increases, the concentration of tetracycline or an equivalent required to inhibit virus production also increases. The inventors have found that if tetracycline or an equivalent is not supplemented in the cell culture, virus production is induced over time.

[0203] As described herein, typical methods for removing tetracycline or its derivatives from suspension cell cultures involve washing the cells with cell culture medium without tetracycline or its derivatives by centrifuging the cells, removing the supernatant, and subsequently resuspending the cells. However, applying this method to suspension cell lines results in high shear forces on the cells, which can lead to reduced cell quality and increased risk of contamination due to manual steps. In addition, centrifugation methods cannot be easily scaled up to commercial scale because these typical methods involve manual handling steps and are very time-consuming, which can add hours to the processing time.

[0204] The inventors' solution to these problems is to dilute the suspension cell culture with a cell culture medium without tetracycline or derivatives; or to retain the suspension cell line cells using acoustic standing waves, remove a portion of the cell culture medium containing tetracycline or derivatives from the suspension cell culture, and contact the retained suspension cell line cells with a cell culture medium without tetracycline or derivatives. Both methods improve cell quality, are less stressful on cells, and require fewer manual steps compared to traditional centrifugation methods. The inventors' solution also reduces the risk of culture contamination by using a closed system. The disclosed method also results in an increase in viral infection titer yield.

[0205] Therefore, the present disclosure provides a method for removing tetracycline or its derivatives from a suspension cell culture to produce an enveloped virus, wherein the method comprises: (i) diluting the suspension cell culture with a cell culture medium without tetracycline or its derivatives; or (ii) retaining suspension cell line cells using acoustic standing waves, removing a portion of the cell culture medium containing tetracycline or its derivatives from the suspension cell culture, and contacting the retained suspension cell line cells with a cell culture medium without tetracycline or its derivatives.

[0206]

Dilution of suspension cell cultures

[0207] In one example, the present disclosure provides a method for removing tetracycline or a derivative thereof from a suspension cell culture to produce an enveloped virus, comprising diluting the suspension cell culture with a cell culture medium without tetracycline or a derivative thereof.

[0208] As used herein, the term "diluting" or "dilute" when referring to a cell culture is understood to mean reducing the concentration of a solute (ie, tetracycline or a derivative thereof) in the cell culture.

[0209] As described herein, the inventors determined that it is not necessary to completely remove tetracycline or its derivatives from the cell culture to induce virus production. That is, the concentration of tetracycline or its derivatives only needs to be reduced, rather than completely eliminated. This is an unexpected result because common protocols for inducing Tet-OFF cells require that all or most of the tetracycline or its derivatives be removed, such as by one or more washing steps. Unexpectedly, only diluting the spent culture medium containing tetracycline or its derivatives, rather than removing it, is effective in inducing virus production.

[0210] For example, the concentration of tetracycline or its derivatives only needs to be reduced to a concentration below about 0.1 ng / mL. Therefore, the inventors determine that a tetracycline or its derivatives with a concentration below about 0.1 ng / mL allows the production of viruses. In another example, the concentration of tetracycline or its derivatives only needs to be reduced to a concentration below about 0.2 ng / mL. Therefore, the inventors determine that a tetracycline or its derivatives with a concentration below about 0.2 ng / mL allows the production of viruses. In another example, the concentration of tetracycline or its derivatives only needs to be reduced to a concentration below about 0.5 ng / mL. Therefore, the inventors determine that a tetracycline or its derivatives with a concentration less than about 0.5 ng / mL allows the production of viruses.

[0211] In one example, the concentration of tetracycline or its derivative in the cell culture is diluted by adding a cell culture medium without tetracycline or its derivative. For example, the cell culture medium without tetracycline or its derivative is directly added to the cell culture medium containing tetracycline or its derivative.

[0212] In one example, cell culture medium without tetracycline or derivatives is added to the suspension cell culture. It will be apparent to the skilled person that in embodiments of the present disclosure, the cell expansion phase and the virus production phase occur in the same container (ie, bioreactor).

[0213] In one example, a suspension cell culture containing a cell culture medium containing tetracycline or a derivative is added to a cell culture medium without tetracycline or a derivative. It is obvious to the skilled person that in the embodiments of the present disclosure, the cell expansion phase and the virus production phase occur in different 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 without tetracycline or a derivative in a second bioreactor. For example, a suspension cell culture is transferred from a first bioreactor containing a cell culture medium containing tetracycline or a derivative to a second bioreactor, wherein the second bioreactor contains a cell culture medium without tetracycline or a derivative.

[0214] It is apparent from the disclosure that reference to a first and subsequent container (i.e., bioreactor) does not refer to a defined or specific bioreactor, but is only used for comparison purposes. The first, second (and any subsequent) container may only be separated by any number of other containers.

[0215] In one example, a suspension cell culture containing a cell culture medium containing tetracycline or a derivative is cultured in an expansion bioreactor, and the suspension cell culture containing a cell culture medium containing tetracycline or a derivative is added to a cell culture medium without tetracycline or a derivative in a production bioreactor. For example, the suspension cell culture is transferred from an expansion bioreactor containing a cell culture medium containing tetracycline or a derivative to a production bioreactor, wherein the production bioreactor contains a cell culture medium without tetracycline or a derivative. In one example, the production bioreactor is filled with a pretreated cell culture medium without tetracycline or a derivative (with a set temperature, pH value, and dissolved oxygen level), and the suspension cell culture is transferred from the expansion bioreactor to the pretreatment culture medium.

[0216] In one example, the suspension cell culture is diluted with a cell culture medium without tetracycline or derivatives at a ratio between about 1:1 and 1:20. For example, the suspension cell culture is diluted with a cell culture medium without tetracycline or derivatives at a ratio between 1:2 and 1:10 or between 1:4 and 1:7. In one example, the suspension cell culture is diluted with a cell culture medium without tetracycline or derivatives at a ratio between 1:2 and 1:10. For example, the suspension cell culture is diluted with a cell culture medium without tetracycline or derivatives at a ratio of about 1:2, about 1:3, about 1:4, or 1:5, about 1:6, or about 1:7, or 1:8, or 1:9, or about 1:10. In one example, the suspension cell culture is diluted with a cell culture medium without tetracycline or derivatives at a ratio between 1:4 and 1:7. In one example, the suspension cell culture is diluted with a cell culture medium without tetracycline or derivatives at a ratio of about 1:4. In another example, the suspension cell culture is diluted with a cell culture medium without tetracycline or a derivative at a ratio of about 1:5. In another example, the suspension cell culture is diluted with a cell culture medium without tetracycline or a derivative at a ratio of about 1:6. In one example, the suspension cell culture is diluted with a cell culture medium without tetracycline or a derivative at a ratio of about 1:7.

[0217] From the disclosure herein, it will be apparent to the skilled person that the ratio of dilution of the suspension cell culture with the cell culture medium without tetracycline or derivatives depends on the concentration of tetracycline or derivatives thereof in the suspension cell culture. For example, the skilled person will recognize that if the concentration of tetracycline or derivatives thereof in the suspension cell culture is about 1 ng / mL, the suspension cell culture is diluted so that the concentration of tetracycline or derivatives thereof in the cell culture medium is less than 0.1 ng / mL. In another example, the skilled person will recognize that if the concentration of tetracycline or derivatives thereof in the suspension cell culture is about 0.1 ng / mL, the suspension cell culture does not need to be diluted prior to virus production.

[0218] From the disclosure herein, it is obvious to the skilled person that the amount of tetracycline or its derivatives that induce virus production also depends on the cell density. Higher density cells require higher concentrations of tetracycline or its derivatives to inhibit induction.

[0219]

Acoustic standing wave separation

[0220] In one example, the present disclosure provides a method for removing tetracycline or its derivatives from a suspension cell culture to produce an enveloped virus, the method comprising retaining suspension cell line cells using an acoustic standing wave, removing a portion of a cell culture medium containing tetracycline or its derivative from the suspension cell culture, and contacting the retained suspension cell line cells with a cell culture medium without tetracycline or its derivative.

[0221] Removing a portion of the cell culture medium containing tetracycline or a derivative from the suspension cell culture comprises removing at least 10% of the cell culture medium containing tetracycline or a derivative. In one example, removing a portion of the cell culture medium containing tetracycline or a derivative from the suspension cell culture comprises removing all or most of the cell culture medium containing tetracycline or a derivative. In one example, removing a portion of the cell culture medium containing tetracycline or a derivative from the suspension cell culture comprises 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 a derivative.

[0222] In one example, after removing a portion of the cell culture medium containing tetracycline or a 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 a derivative from the suspension cell culture, the cell line cells do not remain in suspension and must be resuspended.

[0223] In one example, contacting the retained suspension cell line cells with a cell culture medium without tetracycline or a derivative comprises resuspending the retained suspension cell line cells in a cell culture medium without tetracycline or a derivative. In one example, contacting the retained suspension cell line cells with a cell culture medium without tetracycline or a derivative comprises flowing the suspension cell line cells into a container containing a cell culture medium without tetracycline or a derivative.

[0224] As described above, the inventors have shown that the use of acoustic standing waves to separate cells of suspension cell lines from cell culture media containing tetracycline or derivatives reduces cell stress and thereby improves cell quality and virus titer production compared to centrifugation methods.

[0225] In one example, the method includes using an acoustic chamber or acoustic wave 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 the retained suspension cell line cells through the acoustic chamber to contact the cell culture medium without tetracycline or a derivative.

[0226] Acoustic chambers or acoustic wave devices suitable for use in the present disclosure will be apparent to the skilled artisan and / or described herein.Exemplary acoustic wave devices employ the ultrasonic particle separation technique described in EP 0633049. Exemplary acoustic wave devices include those described in US 10,773,194.

[0227] In one example, the method includes: (i) flowing a suspension cell culture containing a cell culture medium containing tetracycline or a derivative from a first container through an acoustic standing wave in an acoustic chamber, (ii) retaining the suspension cell line cells in the acoustic chamber, (iii) flowing through the acoustic chamber containing the retained suspension cell line cells to contact the cell culture medium without tetracycline or a derivative, and (iv) flowing the suspension cell line cells and the cell culture medium without tetracycline or a derivative into a second container.

[0228] In one example, the method includes: (i) flowing a suspension cell culture containing a cell culture medium containing tetracycline or a derivative from an expansion bioreactor through an acoustic standing wave in an acoustic chamber, (ii) retaining suspension cell line cells in the acoustic chamber, and (iii) flowing a cell culture medium without 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 without tetracycline or a derivative, and (iv) flowing the suspension cell line cells and the cell culture medium without tetracycline or a derivative into a production bioreactor.

[0229] It will be apparent to the skilled person that the use of the acoustic chambers described herein facilitates inline processing. Thus, in some examples, the method is performed inline.

[0230] As used herein, in the context of a process step, the term "in-line" refers to a process step that is integrated into or combined with one or more other process steps, or that flows directly from or to another process step without human intervention or handling.

[0231] In one example, the standing acoustic wave is located between the first container and the second container.

[0232] In one example, the method includes removing a portion of a suspension cell culture comprising a cell culture medium containing tetracycline or a derivative from a first container, retaining suspension cell line cells from the suspension cell culture portion using an acoustic standing wave, and contacting the retained suspension cell line cells with a cell culture medium without tetracycline or a derivative in a second container.

[0233] In one example, the method includes removing a portion of a suspension cell culture comprising a cell culture medium containing tetracycline or a derivative from an expansion 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 without tetracycline or a derivative in a production bioreactor.

[0234] Purification of enveloped viruses

[0235] In one example, enveloped viruses are purified from cell culture, including one or more steps selected from the group consisting of clarification filtration, anion exchange chromatography, concentration, and diafiltration.

[0236] Downstream processes for purifying and concentrating viral vectors from cell culture include a harvest filtration step (also called "clarification filtration" or "harvest clarification filtration" or "bioburden reduction") to remove cell debris and components from the harvest, purification steps such as anion exchange chromatography to reduce the total volume and separate the viral vectors from host cell DNA, proteins and media components, and ultrafiltration / diafiltration steps to concentrate the viral vectors into the final formulation buffer. In some examples, the downstream steps also include a sterile filtration step to remove microorganisms from the final product.

[0237] As used herein, "harvesting" refers to the removal of cell culture medium containing viral particles from producer cells for downstream processing, while "harvest" refers to cell culture medium containing viral particles that has been removed for downstream processing. The harvesting process may include harvesting one or more harvests. "Harvest filtration" refers to the filtered harvest or cell culture medium containing viral particles that has been filtered to remove producer cells for downstream processing.

[0238] In one example, the harvested cell culture fluid is filtered after enveloped virus production.

[0239] As used herein, the term "filtered cell culture fluid" will be understood to include the cell culture fluid after harvesting and filtration.

[0240] After filtration of the harvest, enveloped viruses were purified using anion exchange.

[0241] In one example, anion exchange is performed in bind-elute mode. In this regard, enveloped viruses bind to the anion exchanger while contaminants flow through. The viruses are subsequently eluted from the anion exchanger. Performing anion exchange in this manner reduces the volume of liquid in which the viruses are suspended and removes contaminants such as host cell DNA, host cell proteins, and media components such as fetal bovine serum.

[0242] Suitable anion exchangers will be apparent to those skilled in the art. Typical anion exchangers are columns comprising a resin or a membrane or other suitable substrate.

[0243] In one example, the anion exchanger is a weak anion exchanger, for example, comprising an ion exchange group selected from diethylaminoethyl (DEAE) or aminoethyl.

[0244] In another example, the anion exchanger is a strong anion exchanger, for example, comprising an ion exchange group selected from quaternary ammonium (Q), diethyl-2-hydroxypropylaminoethyl (QAE), triethylaminoethyl (TEAE) or trimethylaminoethyl. Typical anion exchangers include E. Q. Q. Q, QSF F, Q. Q. Q.

[0245] In one example, the anion exchanger includes Q ion exchange groups.

[0246] In one example, the anion exchanger is a membrane anion exchanger comprising Q ion exchange groups. For example, the anion exchanger is Q.

[0247] In one example, enveloped viruses eluted from an anion exchange column are further purified based on their size. In one example, the buffers for eluting viruses from an anion exchange column are exchanged more or less simultaneously. In the methods disclosed herein, tangential flow filtration is preferred. This method allows for the removal of impurities and the exchange of buffers almost simultaneously.

[0248] Tangential flow ultrafiltration / diafiltration is a method that can be used to remove residual proteins and nucleic acids and exchange working buffer for final formulation buffer.Ultrafiltration using tangential flow is preferred, and different equipment (such as Proflux and LABSCALE (ultrafiltration system) TFF systems, including Millipore or Repligen's KR2i system) can be used. The filter pore size of the specific ultrafiltration membrane selected is small enough to retain enveloped viruses, but large enough to allow impurities to penetrate. Depending on the manufacturer and membrane type, a nominal molecular weight cutoff between 100 and 1000 kDa may be suitable (such as UFP-750-E-5A, GE Healthcare; BIOMAX (ultrafiltration device) NMWC 1000, Millipore). In one example, the molecular weight cutoff is 500 kDa. The membrane composition can be, but is not limited to, regenerated cellulose, (modified) polyethersulfone, polysulfone. The membrane can be a flat plate or hollow fiber type. The main parameters that must be optimized are flux rate and transmembrane pressure. Combined with the nominal molecular weight cutoff, these two parameters will enable efficient purification and buffer exchange as well as high virus yields.

[0249] As an additional step, sterile filtration can be performed to eliminate bioburden. Therefore, the diluted eluate or final retentate in the ultrafiltration step can be filtered through a filter, such as a 0.22 μm filter. The filter can be made of various materials, including but not limited to polypropylene, hydrophilic PVDF, cellulose, hydrophilic regenerated cellulose, cellulose esters, cellulose acetate without wetting agents, cellulose acetate, nylon, hydrophilic nylon membranes, 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 multiple membrane layers, or can include a pre-filter of the same or different materials, such as a 0.45 μm pre-filter. The sterile filtered virus can be stored frozen for subsequent operations.

[0250] The following non-limiting examples further describe the present disclosure. [Example]

[0251] [Example 1: Adaptability of adherent stable packaging cell lines for scalable lentiviral production]

[0252] Stable producer pools were generated by stable tandem array transfection with the WASp-T2A-GFP construct and subsequent antibiotic selection. The parental cell line for all packaging and production cell lines is the adherent HEK293T / 17 cell line. The original adherent GPRG and GPRTG packaging cell lines were established by stable introduction of all genetic elements required for lentiviral production, except the gene of interest (transfer gene).

[0253] The packaging cell lines GPRG and GPRTG were subsequently adapted to growth in suspension using different serum-free media. Briefly, each cell line was thawed from a master cell bank. Both cell lines were cultured in adherent culture using 2.5 ng / mL doxycycline and 2 μg / mL puromycin. The cells were cultured in a static phase for two passages using T flasks in D10 medium (DMEM + 10% FBS). The static phase culture consisted of replacing the complete medium every 48 to 96 hours and maintaining a cell density of >1×10 6 cells / mL until the cells reach an appropriate doubling time (<50 hours) and viability (>90%). 6 The cells were seeded at a low inoculation density of 10 cells / mL. After the cells were recovered, serum-free medium was used directly. The cells were then cultured in a shaking environment at 120 rpm (19 mm orbit) using a non-baffled shake flask with a maximum working volume of 32% and 5-8% CO2.

[0254] [Example 2: Establishment of suspension cell culture]

[0255] The packaging and production cell lines used are based on an inducible TET-OFF expression system, in which the presence of doxycycline represses the expression of lentiviral components, whereas removal of doxycycline induces lentiviral production.

[0256] Experiments were performed using suspension adapted CAL-H production clones. Cells used for production bioreactor inoculation were expanded in N-1 seed culture bioreactors using media supplemented with doxycycline.

[0257] Briefly, seed amplification was achieved using the following conditions:

[0258]

[0259] [Example 3: Removal of doxycycline using sonication]

[0260] Prior to inoculating the production bioreactor, cells were washed with doxycycline-free medium using a sonication-based separation device to remove doxycycline from the cell culture medium.

[0261] An acoustic wave device is connected in series between the N-1 bioreactor and the production bioreactor. Cells in medium containing doxycycline flow into the device and are retained in the acoustic wave field, while medium flows out to a waste bag. Fresh medium without doxycycline is pumped in, releasing the cells into the production bioreactor. This process is repeated until all cells have been transferred to the production bioreactor in cell culture medium without doxycycline.

[0262] Measuring viable cell density and infectious titer of samples. Briefly, to determine infectious titer, cells were sampled at various time points throughout the culture and stained with human γ-globin antibody to determine the infectious titer of the sample measured in transducing units (TU) / mL.

[0263] like Figure 1 As shown, the removal of doxycycline using sonication yielded viable cell densities comparable to those of typical centrifugation. Using a sonication wash unit, daily cell loss was performed starting on day 4 to maintain viable cell densities comparable to slower growing centrifuged cultures. Titer yields were also comparable to or higher than those of centrifugation.

[0264] [Example 4: Removal of doxycycline using the dilution method]

[0265] The cells were grown in the N-1 bioreactor to a density approximately 5 times higher, to reach 6-10×10 6 The viable cell density was 10 cells / mL and the viability was ≥ 80%.

[0266] The cells were then transferred directly to a production bioreactor containing medium without doxycycline to reach approximately 1.5 to 1.8 × 10 6 The target cell concentration was 10 cells / mL and the volume was 4.5 kg.

[0267] To investigate the effect of doxycycline concentration on virus production, cells were cultured in the 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 doxycycline. Note that doxycycline was not supplemented throughout the N-1 culture. However, medium changes were performed on working days -2 and -1 using medium containing each corresponding doxycycline concentration.

[0268] On day 0, cells were transferred directly 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.

[0269] like Figure 2 As shown, doxycycline concentrations as low as 0.1 ng / mL in the production bioreactor inhibited viral production for 2 days, while concentrations above 0.5 ng / mL inhibited viral production at least until day 1. Cultures using >1.0 ng / mL doxycycline showed a large increase in viral production around day 2 in the production bioreactor.

[0270] A dilution of about 1:5 would theoretically yield 0.2 ng / ml dox when inoculating the production bioreactor. Surprisingly, viral induction was initiated before starting perfusion with fresh doxycycline-free medium and in the presence of low concentrations of doxycycline.

[0271] [Example 5: Comparison of virus-induced strategies]

[0272] Initially, cells were grown in a seed culture bioreactor in the presence of 1 ng / mL doxycycline. After 65 hours of culture, 1 ng / mL doxycycline was perfused. Figure 3 As shown in A, high viable cell density and cell viability were achieved.

[0273] Three different virus induction strategies (i.e., dilution, sonication, and centrifugation) were compared side by side.

[0274] The cells grew to about 10-15×10 6 cells / mL, and then continuous cell loss was performed at a fixed flow rate to maintain ~15×10 6 Cell density of cells / mL. Figure 3 As shown in B, high cell viability was maintained until day 10 in the production bioreactor.

[0275] Infectious titers were assessed daily from the bioreactor, e.g. Figure 3 As shown in C and D, the three methods were comparable in terms of total virus yield, with slightly lower centrifugation yields. Another disadvantage of centrifugation is that the cells are subjected to greater shear forces.

[0276] like Figure 3 As shown in E, the cell-specific productivity increased rapidly over time. The pH was also measured throughout the duration and remained within the dead band, so no base addition was required (data not shown). Figure 3 F and 3G, metabolites (i.e., lactate ( Figure 3 G) and glucose ( Figure 3 F)) concentrations remain stable over time.

[0277] The operation time of a production bioreactor was extended from day 10 to day 12. Figure 4 As shown, the infection titer and cell-specific productivity continued to increase after 12 days, indicating that production can be prolonged. Cell loss was applied to prevent overgrowth and maintain approximately 1.5 × 10 7 The cell loss strategy prevents nutrient depletion and increases in harvest turbidity, which can hamper downstream virus purification processes.

[0278] [Example 6: Large-scale manufacturability evaluation]

[0279] To assess the scale-up and robustness of suspension harvests, suspension cell lines GPRG and GPRTG were grown in production bioreactors until day 35. Viable cell density, viability and infectious titers of stable producer cell clones were measured until day 35 when high viable cell density, cell viability and infectious titers were achieved.

[0280] These studies demonstrated that the suspension stable producer cell line is capable of producing enveloped virus on a commercial scale (i.e., for at least 15 days).

Claims

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

2. The method according to claim 1, wherein: The suspension cell line is a stable production cell line.

3. The method according to claim 1 or 2, wherein: The suspension cell line is initially cultured in a cell culture medium containing a sufficient amount of tetracycline or its derivatives to inhibit the production of enveloped viruses and to allow expansion of the suspension cell line.

4. The method according to claim 3, wherein: The sufficient amount of tetracycline or its derivative in the cell culture medium is at least 0.1 ng / mL.

5. The method according to claim 3 or 4, wherein: The sufficient amount of tetracycline or its derivative in the cell culture medium is at least 0.2 ng / mL.

6. The method according to any one of claims 3 to 5, wherein: The sufficient amount of tetracycline or its derivative in the cell culture medium is at least 0.5 ng / mL.

7. The method according to any one of claims 3 to 6, wherein: The method comprises reducing the concentration of tetracycline or a derivative thereof in the cell culture medium so as to induce the production of enveloped virus.

8. The method according to claim 7, wherein: Reduce the concentration of tetracycline or its derivatives in the cell culture medium to 0.5 ng / mL or less.

9. The method according to claim 7 or 8, wherein: Reduce the concentration of tetracycline or its derivatives in the cell culture medium to 0.2 ng / mL or less.

10. The method according to any one of claims 7 to 9, wherein: Reduce the concentration of tetracycline or its derivatives in the cell culture medium to 0.1 ng / mL or less.

11. The method according to any one of claims 7 to 10, wherein: The concentration of tetracycline or its derivatives in the cell culture medium can be reduced by: (i) diluting the suspension cell culture with cell culture medium without tetracycline or its derivatives; or (ii) Retaining the suspension cell line cells using acoustic standing waves, removing a portion of the cell culture medium containing tetracycline or a derivative from the suspension cell culture, and contacting the retained suspension cell line cells with a cell culture medium without tetracycline or a derivative.

12. The method according to claim 11, wherein: Diluting the suspension cell culture comprises adding the cell culture medium without tetracycline or derivative directly to the cell culture medium containing tetracycline or derivative.

13. The method according to claim 11, wherein: Diluting the suspension cell culture comprises adding the cell culture medium without tetracycline or derivative to the suspension cell culture.

14. The method according to any one of claims 11 to 13, wherein: Expansion of suspension cell lines and production of enveloped viruses occur in the same vessel.

15. The method according to any one of claims 11 to 14, wherein: Grow suspension cell lines to approximately 1 × 10 before diluting the suspension cell cultures with cell culture medium without tetracycline or its derivatives. 5 cells / mL~about 1×10 10 The viable cell density was between 10 cells / mL.

16. The method according to claim 15, wherein: Grow suspension cell lines to approximately 1 × 10 before diluting the suspension cell cultures with cell culture medium without tetracycline or its derivatives. 6 cells / mL~about 1×10 7 The viable cell density was between 10 cells / mL.

17. The method according to claim 15 or 16, wherein: Grow suspension cell lines to approximately 6 × 10 before diluting the suspension cell cultures with cell culture medium without tetracycline or its derivatives. 6 cells / mL~about 1×10 7 The viable cell density was between 10 cells / mL.

18. The method according to any one of claims 11 to 17, wherein: The suspension cell culture is diluted with cell culture medium without tetracycline or its derivatives at a ratio of about 1:1 to 1:

20.

19. The method according to claim 18, wherein: The suspension cell culture is diluted with cell culture medium without tetracycline or its derivatives at a ratio of about 1:4 to 1:

7.

20. The method according to any one of claims 1 to 19, wherein: Suspension cell lines were initially grown at approximately 1 × 10 5 cells / mL~1×10 10 The cells were seeded at a density between 10 cells / mL in cell culture medium.

21. The method according to claim 20, wherein: Suspension cell lines were initially grown at approximately 1 × 10 5 cells / mL~1×10 7 The cells were seeded at a density between 10 cells / mL in cell culture medium.

22. The method according to claim 20 or 21, wherein: The suspension cell line was initially grown at approximately 0.8 × 10 6 cells / mL~1.2×10 6 The cells were seeded at a density between 10 cells / mL in cell culture medium.

23. The method according to any one of claims 20 to 22, wherein: Suspension cell lines were initially grown at approximately 1 × 10 6 The cells were seeded at a density of 10 cells / mL in cell culture medium.

24. The method according to any one of claims 1 to 23, wherein: The suspension cell cultures were operated in perfusion mode.

25. The method according to any one of claims 1 to 24, wherein: The suspension cell cultures were operated for at least 15 days.

26. The method according to any one of claims 1 to 25, wherein: The method results in a virus infection titer yield of at least 1 × 10 5 transduction units (TU) / mL.

27. The method according to any one of claims 3 to 26, wherein: The tetracycline derivative is doxycycline.

28. The method according to any one of claims 1 to 27, wherein: The volume of the suspension cell culture is greater than about 1 L, about 2 L, about 5 L, about 10 L, about 50 L, about 100 L, about 500 L, about 1,000 L, about 5,000 L, about 10,000 L, or about 15,000 L.

29. The method according to any one of claims 1 to 28, wherein: The pH value of the suspension cell culture is between 6.0 and 8.0 and / or the temperature is between 35 and 39°C.

30. The method according to any one of claims 1 to 29, wherein: The method further includes purifying the enveloped virus from the suspension cell culture.

31. The method according to claim 30, wherein: Purification of enveloped viruses comprises one or more steps selected from the group consisting of clarifying filtration, anion exchange chromatography, concentration and diafiltration.

32. The method of claim 30 or 31, further comprising formulating the purified enveloped virus into a pharmaceutical preparation or a solution suitable for infecting cells.

33. The method according to any one of claims 1 to 32, wherein: The enveloped virus is a retrovirus.

34. The method of claim 33, wherein: The retrovirus is a lentivirus.

35. A purified enveloped virus produced by the method of any one of claims 1 to 34.

36. A stable producer cell clone capable of producing enveloped virus in suspension cell culture, wherein the enveloped virus is stable for at least 15 days. The infectious titer is at least 5 × 10 6 TU / mL culture medium, and / or Vitality is at least 75%, and / or The viable cell density is at least 1.0 × 10 7 cells / mL of culture medium.

37. The stable producer cell clone according to claim 35, wherein The cell clone is capable of producing enveloped virus for at least 20 days, or at least 25 days, or at least 30 days, or at least 35 days The infectious titer is at least 5 × 10 6 TU / mL culture medium, and / or Vitality is 75%, and / or The viable cell density is at least 1.0 × 10 7 cells / mL of culture medium.

38. A method for producing a stable producer cell clone capable of producing an enveloped virus in suspension cell culture, the method comprising: Stable producer cell clones were grown in suspension cell culture for at least 15 days. Detection of cell viability and / or infectious titer yield and / or viable cell density, and Select stable producing cell clones, A stable producer cell clone is generated if one or more or all of the following criteria are met after at least 15 days of culture: (i) The infectious titer is at least 5×10 6 TU / mL culture medium; (ii) a viability of at least 75%; and (iii) The viable cell density is at least 1.0×10 7 cells / mL of culture medium.

Images