Production of viral stocks

Abstract

Methods of producing Minute Mouse Virus (MMV) stock are described herein. Methods of producing xenotropic murine leukemia virus (xMuLV) stock are described herein.

Description

[0001] 10953-W001-SEC

[0002] PRODUCTION OF VIRAL STOCKS

[0003] Cross-Reference to Related Applications

[0004] The present application claims the benefit of U.S. Provisional Application 63 / 691 ,847, entitled “Production of Viral Stocks,” filed September 6, 2024.

[0005] Background

[0006] Biopharmaceutical manufacturing typically includes steps and processes specifically designed to clear (remove or inactivate) viruses, which can help to ensure product safety and quality. Viral Clearance (VC) studies assess the capacity to clear adventitious viruses from bioproduction processes, and are useful for process design and development, and to ensure process quality. In some jurisdictions, government agencies may also require VC studies for biopharmaceutical manufacturing operations. VC studies may also be performed in small-scale unit operations to assess the VC capability of each unit operation, including viral inactivation, viral filtration, and / or chromatography units.

[0007] Typically, VC studies include challenging a manufacturing process or unit, for example a filtration or chromatography process or unit, with a model virus. Downstream viral clearance can then be assessed. Minute Mouse Virus (MMV) and Xenotropic Murine Leukemia Virus (xMuLV) are two viruses that are often used as model viruses. To facilitate the performance of VC studies, high concentrations of model viruses are generated as a raw material. These concentrated viruses are referred to as “virus stocks” or “viral stocks.”

[0008] Summary

[0009] 1 . In this paragraph 1 , a method of producing Minute Mouse Virus (MMV) stock is described. The method comprises infecting mammalian cells with MMV, incubating the infected mammalian cells in a chamber for 5-15 days, 8-12 days, preferably 9-11 days, or, more preferably 10 days, isolating clarification supernatant from the mammalian cells, wherein the clarification supernatant comprises MMV, incubating the clarification 10953-W001-SEC supernatant with an endonuclease, thereby digesting nucleic acid in the clarification supernatant; and purifying MMV after the incubating with the endonuclease, thereby producingthe MMV stock.

[0010] 2. In a method of producing MMV described herein, such as the method of paragraph 1 , purifying the MMV comprises performing ultracentrifugation to produce a pellet comprising MMV and an ultracentrifugation supernatant, optionally wherein the ultracentrifugation is at 75,000 xg to 150,000 xg for 4- 10 hours; and replacing the ultracentrifugation supernatant with buffer, swelling the pelleted virus, and resuspending the swelled pelleted virus. Optionally, the resuspending is by sonication. Optionally, the buffer comprises 10 mM Tris + 100 mM NaCl + 1 mM EDTA tetrasodium dihydrate (TNE).

[0011] 3. In a method of producing MMV described herein, such as the method of paragraph 1 or 2, the purifying further comprises mixed modal chromatography, such as Capto Core 400 chromatography, after resuspending the swelled pelleted MMV.

[0012] 4. In a method of producing MMV described herein, such as the method of any one of paragraphs 1-3, the method further comprises, after incubating with the endonuclease, and prior to the purifying, concentrating the MMV by tangential flow filtration (TFF).

[0013] 5. In a method of producing MMV described herein, such as the method of any one of 1-4, the MMV stock comprises an MMV titer of at least 7 log10TCID5o / mL, such as at least 7.5 logic TCID5o / mL, at least 9 logic TCID5o / mL, or 7-12 logwTCID5o / mL, or 9-12 logic TCIDso / mL, or 7-15 logic TCID5o / mL or 9-12 logic TCID5o / mL.

[0014] 6. In a method of producing MMV described herein, such as the method of any one of paragraphs 1 -5, the MMV stock comprises less than 350 pg / mL protein, and less than 1 pg / mL DNA.

[0015] 7. In a method of producing MMV described herein, such as the method of any one of paragraphs 1-6, the MMV comprises a DNA genome, the DNA genome comprising a genetic barcode sequence specific to the MMV’s strain. 10953-W001-SEC

[0016] 8. Optionally, in the method of paragraph 7, the barcode sequence is positioned adjacent to a stop codon. The stop codon is at an end of a coding sequence for the nonstructural proteins of said MMV, optionally, the 3’ end.

[0017] 9. In a method of producing MMV described herein, such as the method of any one of paragraphs 1-8, the mammalian cells are an embryonic kidney cell line, preferably 324K cells.

[0018] 10. In a method of producing MMV described herein, such as the method of any one of paragraphs 1-9, the mammalian cells in the chamber are at a density of 1-5 x 104cells / cm2, such as 2 x 104cells / cm2, prior to the infecting.

[0019] 11. In a method of producing MMV described herein, such as the method of any one of paragraphs 1-10, the mammalian cells are infected with MMV at a multiplicity of transfection (MOI) of 0.00001 -0.1 , such as 0.0001 - 0.01.

[0020] 12. In a method of producing MMV described herein, such as the method of any one of paragraphs 1-11, the chamber is a multi-layer cell stack chamber, such as a 10-layer cell stack chamber.

[0021] 13. In a method of producing MMV described herein, such as the method of any one of paragraphs 1-12, isolating the clarification supernatant comprises centrifugation to separate the mammalian cells and debris of the mammalian cells from clarification supernatant comprisingthe MMV. Byway of example, the centrifugation may be at 5,000 xg to 20,000 xg, or 5,000 xg to 10,000 x g.

[0022] 14. In a method of producing MMV described herein, such as the method of any one of paragraphs 1-13, the endonuclease comprises benzonase, or a combination of DNase and RNase.

[0023] 15. In a method of producing MMV described herein, such as the method of any one of paragraphs 1-14, the method further comprises performing a viral clearance study in a biologies manufacturing process. The viral clearance study may comprise applying the viral stock to an upstream portion of a biologies manufacturing process and detecting a 10953-W001 -SEC presence or amount of MMV in a downstream portion of said biologies manufacturing process.

[0024] 16. Also described is an MMV stock produced by a method of producing MMV described herein, such as the method of any one of paragraphs 1-15.

[0025] 17. A method of producing xenotropic murine leukemia virus (xMuLV) stock is described herein. The method comprises expanding xMuLV-expressing mammalian cells to produce a seed culture. The method further comprises introducing the seed culture to a single-chamber closed bioreactor. The single chamber comprising fibrous discs, and the said single chamber has a volume of 3-5 L and a surface area of at least 150,000 cm2, such as 150,000 - 200,000 cm2, or 180,000 cm2. The method further comprises incubating the seed culture in the single chamber of the bioreactorfor at least 10 days to produce xMuLV in the single chamber. The method further comprises performing at least four harvests of xMuLV from the bioreactor within 10 days of said introducing. Each harvest collects virus at a titer of at least 7 Logi0TCID5o / mL, such as 7-9 Logi0TCID5o / mL. Each harvest is on a different day. The method further comprises purifying xMuLV from the harvests, thus producing xMuLV stock.

[0026] 18. In a method of producing xMuLV described herein, such as the method of paragraph 17, wherein the fibrous disks comprise a first layer of nonwoven polyester and polypropylene, and a second layer of nonwoven polyester and polypropylene, wherein the first and second layer are immobilized upon each, such as immobilization by sonication.

[0027] 19. In a method of producing xMuLV described herein, such as the method of paragraph 17 or 18, the xMuLV-expressing mammalian cells comprise mink lung (Mv1 Lu) cells.

[0028] 20. In a method of producing xMuLV described herein, such as the method of any one of paragraphs 17-19, the seed culture is introduced to the single-chamber closed bioreactor at a seeding density of 1 - 5 x 109cells / cm2 10953-W001 -SEC

[0029] 21 . In a method of producing xMuLV described herein, such as the method of any one of paragraphs 17-20, the at least four harvests comprise harvests on days 4, 6, 8, and 10 after the introducing.

[0030] 22. In a method of producing xMuLV described herein, such as the method of any one of paragraphs 17-21 , the purifying comprises isolating, by centrifugation, clarification supernatant from the mammalian cells and debris thereof, wherein the clarification supernatant comprises xMuLV, optionally wherein the centrifugation is at 5,000 xg to 15000xg. The purifying further comprises performing ultracentrifugation on the clarification supernatant, to produce a pellet comprising xMuLV and an ultracentrifugation supernatant, optionally wherein the ultracentrifugation is at 25,000 xg to 75,000 xg for 4- 10 hours. The purifying further comprises replacing the ultracentrifugation supernatant with buffer, swelling the pelleted virus, and resuspending the swelled pelleted xMuLV, and optionally wherein the buffer comprises 10 mM Tris + 100 mN NaCl + 1 mM EDTA tetrasodium dihydrate (TNE).

[0031] 23. In a method of producing xMuLV described herein, such as the method of paragraph 22, the ultracentrifugation is at 50,000 xg to 75,000 xg for 6 - 8 hours.

[0032] 24. In a method of producing xMuLV described herein, such as the method of any one of paragraphs 22 or 23, the purifying further comprises filtering the resuspended virus, optionally with a 0.1 - 0.5 pm, 0.2 - 0.5 pm, or 0.2 pm filter.

[0033] 25. In a method of producing xMuLV described herein, such as the method of any one of paragraphs 17-24, the xMuLV stock has a xMuLV titer of > 7.0 logi0TCID5o / mL, such as such as 7-9 Log TCIDso / mL.

[0034] 26. In a method of producing xMuLV described herein, such as the method of any one of paragraphs 15-25, the method further comprising performing a viral clearance study in a biologies manufacturing process. The viral clearance study comprises applying the viral stockto an upstream portion of a biologies manufacturing process and detecting a presence or amount of xMuLV is a downstream portion of said biologies manufacturing process. 10953-W001 -SEC

[0035] 27. Also described herein is an xMuLV stock produced by the method of any one of paragraphs 17-26.

[0036] Brief lotion of

[0037] FIG. 1 is a flow diagram illustrating embodiments of a method of producing MMV stock 100.

[0038] FIG. 2 is a flow diagram illustrating embodiments of a method of producing xMuLV stock 200.

[0039] FIG. 3 is a graph showing the results of a viral clearance study of MMV stock.

[0040] Detailed description

[0041] Viral stocks such as MMV and xMuLV are useful for VC studies, which can help ensure that biopharmaceutical manufacturing processes and units are clearing virus from downstream process intermediates and products, for example through viral filters and / or chromatography columns. VC can include removal and inactivation of virus. Described herein are methods for producing MMV and xMuLV stocks. These methods, which may be referred to as “next generation” methods include producing MMV stocks with higher yield and fewer impurities compared to conventional methods, and resource-efficient production of xMuLV stocks that produces a greater yield of viral stock from a single production run compared to conventional methods.

[0042] Conventional methods for making MMV stocks can result in low titers or impurities that can negatively impact the performance of small-scale viral filters during Viral Clearance (VC) studies. Moreover, it has been observed herein that a general increase in the yield of MMV stocks can be accompanied by an increase in the concentration of impurities in the stocks, including nucleic acid and protein impurities. Nucleic acid impurities in particular can lead to filter clogging in VC studies (See Example 2), an artifact which can lead to lower 10953-W001-SEC performance of the process or unit (for example, clogging of a viral filtration unit) in the VC study than would be observed in the corresponding online biopharmaceutical manufacturing process or unit. Accordingly, it has been appreciated herein that there is a need for methods of producing high-concentration MMV stocks that themselves have low impurity content, and in particular low nucleic acid impurity content. Described herein are methods of producing MMV stocks, in which the MMV stocks are high-concentration, for example at least 7.5 logic TCID50 / mL, but nucleic acid impurities concentrations are low, for example below < 1 pg / mL. These MMV stocks, in turn, support more accurate VC studies, and permit the re-use of viral filters in biopharmaceutical manufacturing processes, which can enhance the efficiency of the biopharmaceutical manufacturing processes. The method of producing MMV stocks can comprise infecting mammalian cells, such as newborn human embryonic kidney cells with MMV (e.g., 324K cells). Conventional methods for making MMV stocks typically utilize rodent cell lines such as NRK cells, and include 4 days of culture. However, it has been observed herein that human embryonic kidney cells, such as 324K cells produce higher MMV yield than rat embryonic kidney cells such as NRK cells. Without being limited by theory it has been appreciated herein that longer culture durations can produce greater yields, but also greater amounts nucleic acid impurities that interfere with VC studies. The method of producing MMV stocks described herein can comprise culturing the cells for 5 days or more, for example 5-15 days. In some aspect of the method, the culturing is for 8-12 days, or about 10 days. The method of producing MMV stocks can further comprise isolating clarification supernatant from the mammalian cells and debris of mammalian cells, in which the clarification supernatant comprises MMV. The method of producing MMV stocks can further comprise incubating the clarification supernatant with an endonuclease, thus digesting nucleic acids in the endonuclease. Without being limited by theory, it is further contemplated that endonuclease incubation as described herein can remove nucleic acid impurities, while still enjoying the advantage of higher yield viral stocks obtained through longer incubation periods. The method of producing MMV stocks can comprise purification of MMV following incubation of the clarification supernatant with the endonuclease. By way of example, the purification can 10953-W001-SEC comprise ultracentrifugation. Optionally, tangential flow filtration (TFF) is performed prior to the ultracentrifugation. TFF concentrates the MMV, thus minimizingthe amount of liquid that needs to be handled and ultracentrifuged. The purification may further comprise mixed mode chromatography after the ultracentrifugation, for example Capto Core 400 chromatography, further purifying the MMV.

[0043] Furthermore, conventional methods of producingxMuLV stocks are time-consuming, extensively utilize manual and open vessel procedures, occupy a significant amount of space, and require large media volumes. Described herein are methods of producingxMuLV stocks that utilize smaller footprint, less media, and faster production cycle than conventional methods of making xMuLV stocks. The method of producing xMuLV can comprise culturing xMuLV-expressing mammalian cells in a 3-5L closed bioreactor comprising fibrous discs. Without being limited by theory, it is contemplated that such a bioreactor configuration advantageously provides a relatively high ratio of surface area-to- volume, providing more surface for the virus-producing cells to adhere upon. The method can comprise culturing the xMuLV-expressing mammalian cells for about 10 days, and performing four harvests, for example at days 4, 6, 8, and 10. Conventional methods that utilize cell stacks for producingxMuLV stocks typically perform fewer harvests over a shorter duration (e.g., three harvests during 8 days of culture). The method can comprise purifying xMuLV to produce a stock. The purifying can comprise ultracentrifugation.

[0044] Methods of producing MMV stock

[0045] Described herein are methods of producing Minute Mouse Virus (MMV) stock. FIG. 1 illustrates embodiments of methods of making MMC stock 100. As shown in block 110, the method of producing MMV stock can comprise infecting mammalian cells with MMV. By way of example, the mammalian cells may comprise an embryonic kidney cell line, such as a human embryonic kidney cell line. Preferably the mammalian cells comprise or consist of 324K cells. In some methods, the mammalian cells in the chamber are at a density of 1-5 x 104cells / cm2, such as 2 x 104cells / cm2, prior to the infecting. In some methods, the 10953-W001-SEC mammalian cells are infected with MMV at a multiplicity of transfection (MOI) of 0.00001 - 0.1 , preferably at an MOI of 0.0001 - 0.01. Turning to block 120, the method further comprises incubating the infected mammalian cells in a chamber for 5-15 days. For example, the incubation may be for 8-12 days, preferably 9-11 days, or, more preferably 10 days. In some methods, the chamber is a multi-layer cell stack chamber, such as a 10-layer cell stack chamber. Turning to block 130, the method further comprises isolating clarification supernatant from the mammalian cells, in which the clarification supernatant comprises MMV. In some methods, isolating the clarification supernatant comprises centrifugation to separate the mammalian cells and debris thereof from clarification supernatant comprising the MMV, such as centrifugation at 5,000 xg to 20,000 xg, or preferably at 5,000 xgto 10,000 x g. The filtration may obtain greater clarification. Turningto block 140, the method further comprises incubating the clarification supernatant with an endonuclease, thus digesting nucleic acids in the clarification supernatant. By way of example, the endonuclease may comprise benzonase or a combination of DNase and RNase. Preferably, the endonuclease comprises benzonase. Without being limited by theory, it is contemplated that endonuclease digestion removes nucleic acid (DNA and RNA) impurities that have been implicated in filter clogging in VC studies (See Example 2). In some methods, the method optionally further comprises filtration of the clarification supernatant after incubation with the endonuclease, for example by 0.22 micron filtration. Turning to block 150, the method optionally comprises concentrating the MMV by tangential flow filtration (TFF). The TFF may be performed comprising after incubating with the endonuclease 140, and prior to purifying 160. Advantageously, the TFF concentrates the MMV prior to purification 160, such as ultracentrifugation 162, which alleviates time and resource requirements of the subsequent purification (e.g., ultracentrifugation). Turning to block 160, the method further comprises purifying MMV, . The purifying 160 may be performed after the incubating with the endonuclease 140, and after the optional concentrating the MMV by TFF 150. As shown in block 162, in some implementations of the method, the purifying 160 comprises performing ultracentrifugation to produce a pellet comprising MMV and an ultracentrifugation supernatant. Optionally, the ultracentrifugation 10953-W001 -SEC is at 75,000 xg to 150,000 xg for 4-10 hours, such as 110,000 xg for 6 hours. As shown in block 164, in some implementations of the method, the purifying 160 further comprises, after the ultracentrifugation 162, replacing the ultracentrifugation supernatant with buffer, swelling the pelleted virus, and resuspending the swelled pelleted virus. Optionally, the buffer comprises or consists of 10 mM Tris + 100 mM NaCl + 1 mM EDTA tetrasodium dihydrate (TNE). Optionally, the resuspending may be by sonication. Optionally, in some methods, following the resuspending, filtration is performed, such as 0.22 micron filtration. Without being limited by theory, it is contemplated that swelling the virus pellets allows the virus pellet to soften, facilitating easier virus re-suspension. Resuspension by sonication may further break up larger virus aggregates into a homogenous virus solution. As shown in block 166, in some implementations of the method, the purifying 160 further comprises, after replacingthe ultracentrifugation supernatant with buffer 164, performing mixed modal chromatography, such as Capto Core 400 chromatography. The mixed modal chromatography is performed after resuspending the swelled pelleted MMV. Advantageously, Capto Core 400 chromatography removes impurities based on both size and charge, and in particular can further remove nucleic acid impurities. Turning next to block 170, MMV stock is thus produced. As shown in Example 1 , MMV stocks according to the methods described herein produce MMV titers of at least 7 log10TCID5o / mL, including, in some examples, MMV titers of 10.2 log™ TCID50 / mL. In some methods, the MMV stock comprises an MMV titer of at least 7 log TCID5o / mL, such as at least 7.5 log10TCID5o / mL, at least 9 logi0TCID50 / mL, 7-12 logic TCID5o / mL, 9-12 log TCID50 / mL, 9-12 logi0TCID50 / mL, or 7- 15 logw TCIDso / mL. In some methods of making MMV stock described herein, the MMV stock comprises less than 350pg / mL protein, and less than 1 pg / mL DNA. In some methods of making MMV stock described herein, the MMV stock comprises less than 50pg / mL protein, and less than 0.1 pg / mL. In some methods of making MMV stock described herein, the MMV stock comprises less than 40 pg / mL protein, and less than 0.05 pg / mL. Optionally, the MMV stock produced may be subjected to quality control tests. Optionally, the MMV stock may be placed in containers such as vials and stored. 10953-W001 -SEC

[0046] Some methods of making MMV stock described herein further comprise performing a viral clearance study in a biologies manufacturing process. The viral clearance study may be performed after the MMV stock is produced 170. The viral clearance study comprises applying the produced viral stock to an upstream portion of a biologies manufacturing process and detecting a presence or amount of MMV in a downstream portion of said biologies manufacturing process. By way of example, the biologies manufacturing process may befor manufacturing a therapeutic protein, such as a monoclonal antibody or derivative of a monoclonal antibody. By way of example, the biologies manufacturing process may comprise a viral filter, and the viral clearance study comprises challenging the viral filter with the MMV stock.

[0047] For any of the methods of making an MMV stock described herein (and any of the MMV stocks described herein), the MMV may comprise a genetic barcode. Advantageously, genetic barcodes may permit the identification of produced MMV stocks, in order to differentiate MMV stocks generated for biosafety and contamination control purposes (such as VC studies) from exogenous MMV infections. As such, in any of the methods of making an MMV stock described herein (and any of the MMV stocks described herein), the MMV may comprise a DNA genome, in which the DNA genome comprises a genetic barcode sequence specific to the MMV’s strain. The barcode sequence may be positioned adjacent to a stop codon, in which the stop codon is atthe end (and preferably the 3’ end) of a coding sequence for the nonstructural proteins of said MMV.

[0048] An MMV stock produced according to any of the methods of making an MMV stock described herein is also contemplated. For example, the MMV stock may be made according to the method of making an MMV stock 100 described herein. The MMV stock may comprise an MMV titer of least 7 logi0TCID5o / mL, such as at least 7.5 logi0TCID5o / mL, at least 9 logic TCIDso / mL, 7-12 log10TCID50 / mL, 9-12 log TCIDso / mL, 9-12 logic TCID5o / mL, or 7-15 logic TCID5O / ITIL. The MMV stock may comprise less than 350pg / mL protein, and less than 1 pg / mL DNA, for example: less than 50pg / mL protein, and less than 0.1 pg / mL; or less than 40 pg / mL protein, and less than 0.05 pg / mL. The MMV stock may be provided, for example, in aliquots of 250 pL or 500 pL, in which each aliquot is suitable for a use. 10953-W001 -SEC

[0049] Methods of producing xMuLV stock

[0050] Described herein are methods of producing xenotropic murine leukemia virus (xMuLV) stock. FIG. 2 illustrates embodiments of methods of producing xMuLV stock 200. As shown in block 210 the method comprises expanding xMuLV-expressing mammalian cells to produce a seed culture. Byway of example, the xMuLV-expressing mammalian cells may comprise mink lung (Mv1 Lu) cells. Turning to block 220, the method further comprises introducing the seed culture to a single-chamber closed bioreactor. The single chamber comprises fibrous discs, and the single chamber has a volume of 3-5 L and a surface area of at least 150,000 cm2, such as 150,000 - 200,000 cm2, or 180,000 cm2. By way of example, the seed culture may be introduced to the single-chamber closed bioreactor at a seeding density of 1 - 5 x 109cells / cm2. Conventional methods of making xMuLV stocks, such as those using a stack bioreactor, typically require a large equipment footprint. Advantageously, the single-chamber closed bioreactor of the method of producing xMuLV stocks described herein has a smaller footprint, which saves lab space. As a further advantage, the single-chamber closed bioreactor can be harvested more easily than a conventional cell stack, and does not need to be moved during the method (in contrast to a conventional cell stack), which is ergonomically advantageous for the operator. Turning to block 230, the method further comprises incubating the seed culture in the single chamber of the bioreactor for at least 10 days to produce xMuLV therein. Turning to block 240, the method further comprises performing at least four harvests of xMuLV from the bioreactor within 10 days of introducing the seed culture to the single-chamber closed bioreactor 220. Each of these harvests collects virus at a titer of at least 7 LogioTCID50 / mL, such as 7-9 Log10TCID5o / mL, or 7-10 Log TCIDso / mL, and each of these harvests is on a different day. By way of example, the at least four harvests may comprise harvests on days 4, 6, 8, and 10 after introducing the seed culture to the single-chamber closed bioreactor 220. Conventional 10953-W001-SEC methods of making xMuLV stocks, such as those using a cell stack (typically a 10-layer chamber) typically only produce three harvests. Thus, the method of producingxMuLV stock describe herein advantageously produces an additional harvest from the same production run (compared to conventional “cell stack” methods). Additionally, in some conventional methods, the first harvest may not be used for producing viral stocks, but may be used, for example, for training or technical development. It will be appreciated that the at least four harvests of the method of producingxMuLV describe herein also permit, for example, use of the first harvest for purposes such as technical development or training, but still provide an additional harvest (compared to conventional methods) that may be used to produce xMuLV stock. Turning to block 250, the method further comprises purifying xMuLV from the harvests. As shown in block 260, thus, xMuLV stock is produced. As shown in block 252, in some implementations of the method, the purifying 250 comprises isolating, by centrifugation, clarification supernatant from the mammalian cells and debris thereof, in which the clarification supernatant comprises xMuLV. Optionally, the centrifugation is at 5,000 xg to 15000xg. Following block 252, as shown in block 254, in some implementations of the method, the purifying 250 comprises performing ultracentrifugation on the clarification supernatant, to produce a pellet comprising xMuLV and an ultracentrifugation supernatant. Optionally, the ultracentrifugation is at 25,000 xg to 75,000 xg for 4- 10 hours, more preferably at 50,000 xg to 75,000 xg for 6 - 8 hours. Following block 254, as shown in block 256, in some implementations of the method, the purifying 250 comprises replacing the ultracentrifugation supernatant with buffer, swelling the pelleted virus, and resuspending the swelled pelleted xMuLV. By way of example, the resuspending may be by pipetting. Optionally, the buffer comprises 10 mM Tris + 100 mM NaCl + 1 mM EDTA tetrasodium dihydrate (TNE). Optionally, the purifying further comprises filtering the resuspended virus, optionally with a 0.1 - 0.5 pm or 0.2 - 0.5 pm filter, preferably a 0.2 pm filter. The thus-produced MMV stock 260 may have a xMuLV titer of > 7.0 log TCID5o / mL, such as such as 7-10 LogioTCID50 / mL, or 7-9 Logi0TCID5o / mL. Optionally, the xMuLV stock produced may be subjected to quality control tests. Optionally, the xMuLV stock may be placed in containers such as vials and stored. 10953-W001 -SEC

[0051] Some methods of making xMuLV stock described herein further comprise performing a viral clearance study in a biologies manufacturing process. The viral clearance study may be performed after the xMuLV stock is produced 260. The viral clearance study comprises applying the viral stock to an upstream portion of a biologies manufacturing process and detecting a presence or amount of xMuLV is a downstream portion of the biologies manufacturing process. Byway of example, the biologies manufacturing process may be for manufacturing a therapeutic protein, such as a monoclonal antibody or derivative of a monoclonal antibody.

[0052] For any of the methods of making xMuLV stock described herein, the fibrous disks comprise a first layer of nonwoven polyester and polypropylene, and a second layer of nonwoven polyester and polypropylene. The first and second layer may be immobilized upon each, such as immobilization by sonication. For any of the methods of making xMuLV described herein, the fibrous disks may have an average diameter of 4-8 mm, such as 6 mm. By way of example, commercially available Fibra-Cel disks are suitable fibrous disks for methods of making xMuLV stock described herein.

[0053] An xMuLV stock produced according to any of the methods of making an xMuLV stock described herein is also contemplated. For example, the xMuLV stock may be made according to the method of making an xMuLV stock 200 described herein. The xMuLV stock may comprise an a xMuL titer of > 7.0 logi0TCID5o / mL, such as such as 7-9 Log TCIDso / mL, or 7-10 LogioTCID5o / mL.

[0054] GENERAL

[0055] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein. 10953-W001-SEC

[0056] The use of the terms “a” and “an” and “the” and similar referents in the context of describing the disclosure (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted.

[0057] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range and each endpoint, unless otherwise indicated herein, and each separate value and endpoint is incorporated into the specification as if it were individually recited herein.

[0058] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the disclosure and does not pose a limitation on the scope of the disclosure unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure.

[0059] Examples

[0060] Example 1 : Next-Generation MMV Production

[0061] Human newborn kidney cells (324K cells) were thawed and expanded as follows: A vial of liquid nitrogen (LN2)-frozen 324K cells was thawed by immersion of pre-warmed water. The contents of the vial were aseptically removed and transferred into a conical tube containing 324K maintenance media (the v:v ratio of cells to maintenance media was 1 :9). The conical tube containing cryopreserved cells and 324K maintenance media was then centrifuged at 200 xg for 10 minutes at room temperature to remove cryoprotectant. The media was aspirated and discarded. The cell pellet was resuspended in 324K maintenance media. A cell count was performed to verify that the initial viable cell density and viability has 10953-W001 -SEC been achieved. The entire contents were aseptically transferred to a sterile T-175 flask containing 324K maintenance media (the v:v ratio of cell-containing media to maintenance media was 1 :10). The flask was then placed in a temperature and %CO2controlled incubator and incubated for the culture duration. To passage the cells, a split ratio was used. The flask was emptied and then rinsed with 1 X Dulbecco’s Phosphate Buffered Saline (DPBS) without Ca2+or Mg2+. The DPBS was removed and 4 mL of Trypsin-EDTA, 0.05% (trypsin was put into the flask and incubated until the cells detach from the flask). After the cells detached (typically 4 minutes or more), 324K maintenance media was added to neutralize the trypsin. 3 to 4 passaging stages were performed to expand the culture from P0 (vial thaw) to a culture of sufficient cell count to inoculate a 10-layer cell stack to the target seed density of 2.0 X 104cells / cm2.

[0062] The seed culture was then seeded in a 10-layer stack as follows: The flasks were emptied and rinsed with 1 X Dulbecco’s Phosphate Buffered Saline (DPBS) without Ca2+or Mg2+. The DPBS was removed and Trypsin-EDTA, 0.05% (trypsin) was put into the flask and incubated until the cells detached from the flask. After the cells detached, 324K maintenance media was added to neutralize the trypsin. The cell suspension was then centrifuged at 200 x g for 10 minutes. The media was decanted from the cell pellet, and the cells were re-suspended in 324K Seed Media containing 1% fetal bovine serum (FBS) solution. Another cell count was performed. MMV was added to the cell suspension to a target multiplicity of infection (MOI) of 0.0001 to 0.01 . 324K Seed Media containing 1 % FBS was added to the cell stack so that the final volume within the 10-layer cell stack equaled 1 .5 L, accounting for the volume of cell suspension to be added. The appropriate quantity of cell suspension to seed the 10-layer stack at a seed density of 2.0 x 104cells / cm2infected with virus was added to the 10-layer cell stack, which was then placed in a temperature and %CO2controlled incubator for a duration of 10 days.

[0063] Cell stack culture and freeze-thaw were then performed as follows: A 10-day incubation period was performed in a temperature and %CO2controlled incubator. The cell stack was then removed and transferred from a -20°C to -70°C freezer to a temperature and 10953-W001 -SEC

[0064] %CO2controlled incubator until a total of three complete freeze-thaw cycles have been performed.

[0065] Clarification of the culture was then performed. The media from the cell stack was decanted into a sterile bottle and transferred into centrifuge bottles. The harvest was clarified from cellular debris by centrifugation at 9,000 x g for 30 minutes, producing clarification supernatant. The clarification supernatant was decanted and kept, while the pellet was discarded.

[0066] Endonuclease removal of nucleic acids was then performed. Benzonase is an endonuclease that degrades extracellular nucleic acid. Magnesium Chloride (MgCl2) was added to the clarified harvest material to a target concentration of 2mM to facilitate the activity of benzonase enzyme. Benzonase was added to the clarified harvest material to a target concentration of 25 units / mL. The clarified harvest was then placed in a temperature and %CO2controlled incubator for > 1 hour to facilitate benzonase activity. EDTA was added to the clarified harvest material to a final target concentration of 5mM to inhibit further benzonase activity.

[0067] To concentrate the clarification supernatant containing MMV, Tangential Flow Filtration (TFF) was then performed. TFF reduces process volume, which can alleviate time and resource requirements of the subsequent steps. A hollow-fiber filter with a molecular weight cut off (MWCO) of < 500 kDa was utilized to reduce the volume of the harvest material to a final concentration factor target of 8 x + / - 3 x. The filtration was performed on an Akta Flux 6 system. The Delta P (AP) of the filtration was set to <20 psi for the duration of the filtration, and the permeate line was left open without any induced back-pressure.

[0068] Ultrafiltration was then performed. Ultracentrifugation removes impurities, exchanges the buffer of the virus suspension, and concentrates the virus. The TFF- concentrated harvest was separated into appropriate ultracentrifugation tubes and subjected to 1 10,000 x g for 6 hours. Following ultracentrifugation, the ultracentrifugation supernatant was removed from the tubes as waste, while the virus pellets were kept and resuspended into TNE buffer (10 mM Tris + 100 mM NaCl + 1 mM EDTA tetrasodium dihydrate). 10953-W001-SEC

[0069] TNE swelling, sonication, and 0.22 m filtration were then performed. Swelling the virus pellets in TNE for 16 to 25 hours allows for the virus pellet to soften, enabling easier virus re-suspension. It is appreciated that sonication further breaks up larger virus aggregates into a homogenous virus solution. Virus particles that are not sufficiently resuspended are lost during subsequent 0.22 pm filtration steps. Afterthe ultracentrifugation supernatant waste was removed from the centrifuge tubes, TNE was split evenly among the virus pellets. The tubes were placed in a 2°C to 8°C refrigerator and allowed to swell in the TNE for 16 to 24 hours. Following incubation with TNE, the virus pellets are rigorously resuspended using pipettes. The suspensions from the centrifuge tubes are pooled and subjected to sonication in an aquasonic sonicator for 1 to 2 minutes. After sonication, the virus material was filtered through an appropriately-sized 0.22 pm filter.

[0070] Capto Core 400 purification was then performed. Capto Core 400 is a type of multimodal resin-based chromatography which functions to remove impurities based on both size and charge. It reduces the DNA and protein impurity levels in the virus material and is run in flow-through mode. Due to the small scale of the process volume and the flowthrough nature of the chromatography, it is executed in the Biosafety Cabinet (BSC) utilizing a simple syringe pump to control the flow rate. A Capto Core 400 column was loaded with filtered ultracentrifugation supernatant comprising virus material at a flow rate of 2mL / min. Following the load, 5mL of TNE buffer is loaded at 2 mL / min into the column to chase out the remaining load from the system and improve step yield. The output was collected directly from the column into a sterile container.

[0071] The output from the Capto Core 400 purification was subjected to a final 0.22 pm sterile filter and tested for sterility using a TSA media growth plate. Cryovials were labelled with identifying information, including virus type, unique lot number, storage condition, and expiration date. The virus was aliquoted into the cryovials which are then placed in a -70°C freezer for storage.

[0072] Two runs of next-generation MMV production as described were performed. An additional seven runs using a slightly different process were also successfully performed. 10953-W001-SEC

[0073] As shown in Table 1, each of the two next-generation MMV stock exhibited a yield of 9.6 - 10.2 Log TCIDso / mL, which was greater than conventional MMV stock, and also a superior nucleic acid impurity and protein impurity profile:

[0074] Table 1 : *Because the Next Generation MMV Stock Production has higher MMV concentration, it is contemplated that 250pl aliquots may also be used, yielding 200 vials of 250pL.

[0075] Example 2: Conventional MMV Stocks Can Cause Clogging of Filters in VC Studies, While Next-Generation MMV Stocks Can Improve Viral Filter Performance A series of viral clearance (VC) studies was performed on a Viresolve Pro (Millipore) viral filter operated in constant flow mode to a loading target of 250 L / m2. Four MMV stocks were compared: “Conventional MMV” (MMV stock produced accordingto a conventional inhouse method), “Stock 2” (an MMV stock produced by an alternate process that was generated during the course of development of Next-Generation MMV stocks), “TrueSpike” (a commercially available MMV stock), and “Next Generation MMV” (a next-generation MMV stock product produced accordingto Example 1 ). Four unique viral filter load materials were prepared by spiking a monoclonal antibody drug substance at 0.1 % (v / v) of the different MMV stocks of varying characteristics. Characteristics of these MMV stocks are summarized in Table 2, below. 10953-W001-SEC

[0076] Table 2

[0077] The results of filtration performance, as illustrated by Delta C (Psi) vs Loading (L / m2) are shown in FIG. 3. Delta C (Psi) indicates the pressure across the viral filter. Due to upper operating pressure limitations of viral filters, increasing Delta C (Psi) values across the Loading (L / m2) indicates undesirable filter clogging behavior. The MMV stock produced according to Example 1 (“Next Generation MMV” stock) out-performed in-house MMV Stocks (“Conventional MMV”), the MMV stock produced by an alternate method from the course of development (Stock 2), as well as the commercial TrueSpike stock. Protein impurity level differences between the stocks indicated that nucleic acid contaminants may be the lead contributor to clogging behavior on Viral Filters. It was appreciated that superior filterability indicated by the limited increase in Delt C (Psi) during the course of product loading (L / m2) of next-generation MMV stocks run indicated an increased potential for higher product loading targets and potentially permit viral filter (VF) re-use. This can yield significant efficiencies in the manufacture of biopharmaceutical molecules, as viral filters are raw materials that require extensive resources, which optimally should be loaded to the highest extent allowable by both the intrinsic biopharmaceutical molecule filterability and the ability of the Viral Clearance study to achieve loading targets prior to hitting the upper operating pressure limits. Accordingly, it is contemplated that the superior performance of the Next Generation MMV stock can optimize resource utilization, and reduce the potential cost of goods for the manufacture of biopharmaceutical molecules. 10953-W001 -SEC

[0078] Without being limited by theory, it is contemplated that VF filtration performance with MMV stock may be affected to different degrees for different biopharmaceutical molecules. However, for any biopharmaceutical molecule in which the MMV stock impacts VF performance high purity Next-Generation MMV stock as described herein is expected to improve VF performance. Furthermore, regardless of the degree to which MMV stock impacts VF performance for any molecules, the high titers of Next-Generation MMV stocks described herein are expected to improve the Logarithmic Reduction Value (LRV) of VF, which is the output of the VC study.

[0079] Example 3: Production of XMuLV

[0080] Mink cells were thawed and expanded as follows: Mink lung (Mv1 Lu) is an adherent cell line that is chronically infected (Inf.) with xMuLV. Mv1 Lu cells were removed from the liquid nitrogen (LN2) storage freezer tank and thawed by immersion of pre-warmed water. The contents of the vialwere aseptically removed and transferred into a conical tube containing Mv1 Lu maintenance media. The conical tube containing cryopreserved cells and Mv1 Lu maintenance media (v:v ratio of cells to maintenance media was 1 :9) was then centrifuged at 200 xg for 10 minutes at room temperature to remove cryoprotectant. The media was aspirated and discarded, and the cell pelletwas resuspended in Mv1 Lu maintenance media. A cell count was performed to verify that the initial viable cell density and viability has been achieved. The entire contents were aseptically transferred to a sterile T-175 flask containing Mv1 Lu maintenance media (v:v ratio of cell-containing media and maintenance media was 1 :10). The flask was then placed in a temperature and %CO2controlled incubator for the culture duration.

[0081] Cell passage was performed as follows: To passage the cells, a split ratio was used. The flask of cells was taken out of the incubator and the cell confluency is checked. The flask was emptied and then rinsed with 1 X Dulbecco’s Phosphate Buffered Saline (DPBS) without Ca2+or Mg2+, followed by incubation with Trypsin-EDTA, (0.05% trypsin) until the cells detach from the flask, at which point Mv1 Lu maintenance media was added to neutralize the 10953-W001 -SEC trypsin. A cell count was performed to determine the amount of cell suspension needed for the new flasks. There were typically 3 to 4 passages involved in expanding the culture from P0 (vial thaw) to a culture of sufficient density to inoculate the subsequent stage of the process.

[0082] Bioreactor preparation was performed as follows: A5p BioBLU Single-Use Bioreactor (Eppendorf) was prepared. The 5p BioBLU Bioreactor contains Fibra-Cel disks, which contain a layer of nonwoven polyester and polypropylene, and a layer of nonwoven polyester and polypropylene, which are attached by sonication. Per the manufacturer, Fibra-cel disks have an average diameter of about 6 mm. These disks may serve as a solid growth matrix for adherent cells and provide a shear-free environment for high-density cell cultivation. The 5p BioBLU Single-Use Bioreactor has a single impeller that is moved by a motor on top. The impeller has a low shear and pumps downward. The bioreactor was set up with accessories, included welding tubing connectors, standardizing probes [optical (opt) pH, Dissolved Oxygen (DO), Temperature], connecting heating blankets, agitator, gas connections, and base flask. The bioreactor was then filled with about 3L of pre-conditioned Mink Lung (Mv1 Lu) Seed Media. Through the BioCommand software, a batch and recipe was created. Once the setup is complete, the opt-pH and DO sensors were calibrated in the media, one day before inoculation. Once all these steps were complete, the 5p BioBLU was ready for inoculation.

[0083] Bioreactor inoculation and incubation were performed as follows: The bioreactor was operated in a batch process mode. It is appreciated that the cell culture typical undergoes four main phases: Lag, growth, stationary (when product secretion occurs), and death. Mv1 Lu cells were introduced into the bioreactor containing pre-conditioned media. The expanded Mv1 Lu cells in a number of 2.7 x 109were resuspended with 500 mL of Mv1 Lu seed media and transferred into a 2L transfer vessel. The 2L transfer vessel was welded to an addition port on the 5p BioBLU. An inoculum was added to media in the bioreactor, and the cells were left to mix and attach to the Fibra-Cel disks for a minimum of one hour. 10953-W001-SEC

[0084] Mv1 Lu cells were incubated in the Bioreactor. Table 3 outlines operation process parameters for the incubation:

[0085] Table 3 During incubation, various factors includin temperature, agitation, dissolved oxygen

[0086] (DO), pH, metabolites, overlay, and sparging gases were constantly monitored. The pH was controlled by sparging caron dioxide (CO2) gas and sterile 1 M sodium carbonate (Na2CO3) addition. Dissolved oxygen (DO) concentrations was controlled at 50 % by automatic gassing at a glow of 0.002 - 0.5 SLMP. Stock solutions of 50% Glucose (Glc) and 200mM glutamine (Gin) were supplemented through separate feeds.

[0087] The incubation took place for 10 days, and the media containing xMuLV was harvested on days 4, 6, 8, and 10, using a 5-liter (L) transfer vessel. Right after harvesting the cells, the 5p BioBLU Bioreactor was re-fed again with 3 - 3.5L of Mink Lung (Mv1 Lu) seed media on days 4, 6, and 8. The harvested culture medium was clarified by a low-speed centrifugation at 9000 xg for 30 minutes (min) at room temperature after the virus is harvested to product a clarification supernatant. This process removed insoluble particles like cells, cell debris, and large aggregates from the medium. Next, ultra-centrifugation was 10953-W001 -SEC performed (which may also be referred to as concentration). It is appreciated that the ultracentrifugation (concentration) lowers the volume of the culture medium orsupernatant, and removes impurities, while concentrating the target virus, thus preparing the virus for buffer exchange. The ultra-centrifugation was done at either 53,000 xgfor 6 hours (hrs) or 9,000 xg for 24 hrs at room temperate. The virus was then pelletized, and the supernatant discarded.

[0088] An amount of 100 mL TNE buffer was added to the virus pellet to help media change and swelling the virus helped resuspend the viral particles in liquid. It is appreciated that the swelling can allow the virus to mix and disperse well. The resuspended virus was then filtered, and a sterility check was done. Cryovials were labeled with the virus type, unique lot number, storage condition, and storage location.

[0089] Eleven runs of xMuLV production were performed as described in this Example. Titer summaries for the runs (xMuLVtiter are shown as logi0TCID5o / mL) are shown in Table 4A. For comparison, titer summaries for a conventional “cell stack” method of xMuLV production (which only included two harvests, at days 6 and 8) are shown in Table 4B. Table 4A 10953-W001-SEC

[0090] Table 4B

[0091] This example shows that the Next-Generation xMuLV production, achieved a virus titer comparable to conventional cell stack (10-layer chamber) production. Additionally, it yielded an extra production on day 10, resulting in three lots of xMuLV compared to the two lots produced in the conventional cell stack production process. Further, compared to the conventional cell stack process, the Nex-Generation xMuLV production process described in this Example utilizes a smaller footprint and a smaller piece of equipment that is ergonomically advantageous for the operator.

Claims

10953-W001-SECWhat is claimed:

1. A method of producing Minute Mouse Virus (MMV) stock, the method comprising: infecting mammalian cells with MMV; incubating the infected mammalian cells in a chamber for 5-15 days, 8-12 days, preferably 9-11 days, or, more preferably 10 days; isolating clarification supernatant from the mammalian cells, wherein the clarification supernatant comprises MMV; incubating the clarification supernatant with an endonuclease, thereby digesting nucleic acid in the clarification supernatant; and purifying MMV after said incubating with the endonuclease, thereby producing the MMV stock.

2. The method of claim 1 , wherein purifying the MMV comprises: performing ultracentrifugation to produce a pellet comprising MMV and an ultracentrifugation supernatant, optionally wherein the ultracentrifugation is at 75,000 xg to 150,000 xg for 4- 10 hours; and replacingthe ultracentrifugation supernatantwith buffer, swellingthe pelleted virus, and resuspending the swelled pelleted virus, optionally wherein the resuspending is by sonication, and optionally wherein the buffer comprises 10 mM Tris + 100 mM NaCl + 1 mM EDTA tetrasodium dihydrate (TNE).

3. The method of claim 1 or 2, wherein the purifying further comprises mixed modal chromatography, such as Capto Core 400 chromatography, after resuspending the swelled pelleted MMV.10953-W001 -SEC4. The method of any one of claims 1-3, further comprising after incubating with the endonuclease, and prior to the purifying, concentrating the MMV by tangential flow filtration (TFF).

5. The method of any one of claims 1 -4, wherein the MMV stock comprises an MMV titer of at least 7 log TCID50 / mL, such as at least 7.5 log10TCID5o / mL, at least 9 log10TCID5o / mL, or 7-12 logw TCID5o / mL, or 9-12 logw TCID5o / mL, or 7-15 logw TCID5o / mL or 9-12 log TCIDso / mL.

6. The method of any one of claims 1-5, wherein the MMV stock comprises less than 350pg / mL protein, and less than 1 pg / mL DNA.

7. The method of any one of claims 1 -6, wherein the MMV comprises a DNA genome, said DNA genome comprising a genetic barcode sequence specific to the MMV’s strain.

8. The method of claim 7, wherein the barcode sequence is positioned adjacent to a stop codon, wherein said stop codon is at an end of a coding sequence for the nonstructural proteins of said MMV, optionally wherein the end is a 3’ end.

9. The method of any one of claims 1-8, wherein the mammalian cells are an embryonic kidney cell line, preferably 324K cells.

10. The method of any one of claims 1-9, wherein the mammalian cells in the chamber are at a density of 1 -5 x 104cells / cm2, such as 2 x 104cells / cm2, prior to said infecting.

11. The method of any one of claims 1-10, wherein the mammalian cells are infected with MMV at a multiplicity of transfection (MOI) of 0.00001 - 0.1 , such as 0.0001 - 0.01.

12. The method of any one of claims 1 -1 1 , wherein the chamber is a multi-layer cell stack chamber, such as a 10-layer cell stack chamber.10953-W001 -SEC13. The method of any one of claims 1-12, wherein isolating the clarification supernatant comprises centrifugation to separate the mammalian cells and debris thereof from clarification supernatant comprising the MMV, such as centrifugation at 5,000 xg to 20,000 xg, or 5,000 xg to 10,000 x g.

14. The method of any one of claims 1-13, wherein the endonuclease comprises benzonase, or a combination of DNase and RNase.

15. The method of any one of claims 1-14, further comprising performing a viral clearance study in a biologies manufacturing process, the viral clearance study comprising applying the viral stock to an upstream portion of a biologies manufacturing process and detecting a presence or amount of MMV in a downstream portion of said biologies manufacturing process.

16. An MMV stock produced by the method of any one of claims 1-15.

17. A method of producing xenotropic murine leukemia virus (xMuLV) stock, the method comprising: expanding xMuLV-expressing mammalian cells to produce a seed culture; introducing the seed culture to a single-chamber closed bioreactor, said single chamber comprising fibrous discs, and said single chamber having a volume of 3-5 L and a surface area of at least 150,000 cm2, such as 150,000 - 200,000 cm2, or 180,000 cm2. incubating the seed culture in the single chamber of the bioreactor for at least10 days to produce xMuLV therein; and performing at least four harvests of xMuLV from the bioreactor within 10 days of said introducing, wherein each harvest collects virus at a titer of at least 7 Logic TCIDso / mL, such as 7-9 Logi0TCID5o / mL, or 7-10 Logw TCID50 / mL, and wherein each harvest is on a different day; and purifying xMuLV from the harvests, thereby producing xMuLV stock.10953-W001-SEC18. The method of claim 17, wherein the fibrous disks comprise a first layer of nonwoven polyester and polypropylene, and a second layer of nonwoven polyester and polypropylene, wherein the first and second layer are immobilized upon each, such as immobilization by sonication.

19. The method of claim 17 or 18, wherein the xMuLV-expressing mammalian cells comprise mink lung (Mv1 Lu) cells.

20. The method of any one of claims 17-19, wherein the seed culture is introduced to the single-chamber closed bioreactor at a seeding density of 1 - 5 x 109cells / cm221. The method of any one of claims 17-20, wherein the at least four harvests comprise harvests on days 4, 6, 8, and 10 after said introducing.

22. The method of any one of claims 17-21 , wherein purifying comprises: isolating, by centrifugation, clarification supernatant from the mammalian cells and debris thereof, wherein the clarification supernatant comprises xMuLV, optionally wherein the centrifugation is at 5,000 xg to 15000xg, performing ultracentrifugation on the clarification supernatant, to produce a pellet comprising xMuLV and an ultracentrifugation supernatant, optionally wherein the ultracentrifugation is at 25,000 xgto 75,000 xgfor4- 10 hours; and replacingthe ultracentrifugation supernatantwith buffer, swellingthe pelleted virus, and resuspending the swelled pelleted xMuLV, and optionally wherein the buffer comprises 10 mM Tris + 100 mM NaCl + 1 mM EDTA tetrasodium dihydrate (TNE).

23. The method of claim 22, wherein the ultracentrifugation is at 50,000 xg to 75,000 xg for 6 - 8 hours.

24. The method of claim 22 or 23, wherein the purifyingfurther comprises filtering the resuspended virus, optionally with a 0.1 - 0.5 pm, 0.2 - 0.5 pm, or 0.2 pm filter.10953-W001-SEC25. The method of any one of claims 17-24, wherein the xMuLV stock has a xMuLV titer of > 7.0 log o TCID5o / mL, such as such as 7-9 Logi0TCID5o / mL.

26. The method of any one of claims 15-25, further comprising performing a viral clearance study in a biologies manufacturing process, the viral clearance study comprising applying the viral stock to an upstream portion of a biologies manufacturing process and detecting a presence or amount of xMuLV is a downstream portion of said biologies manufacturing process.

27. An xMuLV stock produced by the method of any one of claims 17-26.

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