Bioreactor production of virus from adherent cells

Abstract

To provide a simple, high-yield, and scalable method for producing viruses from adherent cells.SOLUTION: Anchorage-dependent cells infected with a virus are used. The method comprises releasing virus from adherent host cells grown in a bioreactor and purifying the released virus by ultrafiltration and / or diafiltration. The method can be used to manufacture viruses, including for clinical use, at a lower cost compared to conventional virus manufacturing methods.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Applications The subject matter of this application is related to allowed U.S. Application No. 16 / 020,850, filed June 27, 2018, entitled "Bioreactor Production of Viruses from Adherent Cells," to inventors Joseph Cappello and Richard J. Aguilar. Where possible, the subject matter of the present invention is incorporated by reference in its entirety.

[0002] Technical Field Methods for producing viruses from adherent cells are provided. [Background technology]

[0003] background Viruses are used as therapeutic agents. Such applications include vaccines, gene therapy vectors, and viral therapeutic agents. The production of viruses for these purposes involves viral replication in suitable host cells and subsequent purification of the virus from the host cells. Host cells used for viral replication are grown in either adherent or suspension culture conditions in an anchorage-dependent manner. The advantage of suspension culture is that cells can be cultured in large quantities in a single bioreactor. However, certain viruses do not replicate well in suspension-cultured cells.

[0004] Anchorage-dependent cells in which viruses replicate well are generally cultured in roller bottles, requiring many bottles to obtain sufficient quantities. Therefore, these methods are difficult and costly to scale. Whether using adherent or suspension culture conditions, virus-infected cells are harvested from one or more culture tubes in a bioreactor or roller bottle and then lysed to release the virus. Viruses are purified from whole host cell components using many different steps, including one or more of the following: homogenization, sonication, centrifugation, filtration, affinity purification, chromatography, and density gradient ultracentrifugation. The culture conditions and purification steps associated with virus production add complexity and cost to the manufacturing process and can result in low yields. Therefore, a simple, scalable method for producing viruses is needed. Summary of the Invention

[0005] overview High-yield, simple, and scalable virus production is provided. The method uses anchorage-dependent cells infected with the virus. According to the method, the virus is released from the host cells while host cell components remain substantially attached to the culture surface. The virus is released into the cell culture medium while the host cell-derived components remain on the cells, and the virus is purified by a simple, cost-effective single-step process of filtration or diafiltration or a two-step process of ultrafiltration and diafiltration. The purification process is high-yield and can be performed in one day or less. The process is performed on cells grown in a bioreactor suitable for culturing anchorage-dependent cells, thereby combining a simplified purification method with the advantages of bioreactor scalability. Any virus that can infect and grow in cells that can be grown in an adherent form, particularly enveloped viruses, can be produced by these methods.

[0006] The method / process is for producing virus from adherent cells cultured in a bioreactor. The method includes releasing the virus from the adherent host cells in the bioreactor and then purifying the released virus by ultrafiltration and / or diafiltration. Various examples and embodiments of such methods are described herein.

[0007] A method (process) for producing a virus is provided. The method includes the steps of: a) culturing host cells containing the virus in a bioreactor, the bioreactor including a matrix for growing adherent or entrapped cells, the matrix being biocompatible; the matrix being entrapped and / or attached to the matrix; and the density of the matrix being such that the cells remain adherent under conditions in which the cells are lysed and treated to release the virus, and the flow of cell culture medium through the matrix is sufficient for cell growth; b) treating the cells to lyse and release the virus into the medium in the bioreactor; and c) purifying the released virus from the cell culture medium in only one or two steps without further processing. The first step is ultrafiltration or diafiltration; the second step is ultrafiltration and diafiltration. No other purification steps are used. Thus, after the virus is lysed and released from the cells, purification is achieved solely by ultrafiltration and / or diafiltration. The purification is performed solely by the first or second step. The purification can be accomplished in a day or less.

[0008] The bioreactor comprises a matrix or surface to which adherent cells can adhere and on which suspended cells can be trapped. The matrix (or macrocarrier, substrate, or surface) can be a non-fixed adhesive surface. The matrix or surface can be selected from, but is not limited to, macrocarrier beads in suspension, fibers, or a woven mesh. The matrix or surface can also be a fixed adhesive surface. The bioreactor can be, for example, a packed-bed bioreactor. Other configurations can be selected by those skilled in the art, as long as the bioreactor comprises a matrix or surface for retaining cells during growth and lysis.

[0009] The host cells are typically cells that adhere to or are entrapped in a matrix, including cells grown in suspension, and cannot be released by treating the cells and medium. Generally, the host cells are adherent cells. The cells can be primate cells or cell lines. The specific cells are cells suitable for propagating specific viruses. The cell lines are known cell lines, such as CV-1 cells, KB cells, Vero cells, and CHO cells. Cells include, but are not limited to, mammalian cells, including human and other primate cells, and human cells include, for example, human fibroblasts, epithelial cells, and endothelial cells.

[0010] As exemplified herein, the exemplified cells are CV-1 cells, and the virus is a vaccinia virus, e.g., a therapeutic vaccinia virus. Prior to purification, the cell-treated medium can be collected. If necessary, the medium can be stored. The method for producing the virus can include only steps a), b), and c) above, or can include additional steps, prior to purification, where the purification includes only one or two steps of ultrafiltration and / or diafiltration.

[0011] Virus release includes treatments to lyse cells, such as freezing / thawing, and / or treatment with a hypotonic medium, and / or treatment with a detergent. The virus is released from lysed cells into the medium by treatment with a protease and / or nuclease. The protease is generally a nonspecific protease, such as a digestive enzyme, for example, trypsin. The nuclease is a DNase or RNase, such as the endonuclease from Serratia marcescens, sold under the trade name Benzonase®. Nuclease treatment is optional. Lysis and enzyme treatment can be performed sequentially or together. Lysis must precede or be performed together with protease or nuclease treatment. The protease and nuclease treatments can be performed together or in any order.

[0012] The virus may be any virus of interest, such as a therapeutic virus (including oncolytic viruses), a vaccine, a gene therapy vector, or a virus for delivery of a gene product. Generally, the virus is an enveloped virus, since purification is performed on released viruses, and release may involve nuclease treatment. Viruses include, but are not limited to, poxviruses, such as vaccinia virus, myxoma virus, measles virus, reovirus, vesicular stomatitis virus (VSV), adenovirus, adeno-associated virus, poliovirus, herpes virus, Sindbis virus, and Seneca Valley virus, as well as derivatives thereof modified to contain nucleic acids encoding heterologous gene products. The virus may be an oncolytic virus, such as a vaccinia virus (e.g., GL-ONC1, Pexa-Vec, vvDD, JX-929, and WO-12), a vesicular stomatitis virus (e.g., VSV-IFNbeta-NIS, VSV-E6 / 7, VSV-GFP), a measles virus (e.g., MV-NIS, MV-Edm, MV-NPL), a Seneca Valley virus (e.g., SVV-001 and NTX-010), a reovirus (e.g., leolysin), an adenovirus (e.g., CGTG-102, Oncos-102, NG-348, NG-350, NG-347, NGaFAB, NG-aEpCAM, ONYX), or an HIV-1-associated virus (e.g., HIV-1-associated virus). The virus may be an avian virus (e.g., HIV-1, HIV-2, HIV-3, HIV-4, HIV-5, HIV-6, HIV-7, HIV-8, HIV-9, HIV-10, HIV-11, HIV-12, HIV-13, HIV-14, HIV-15, HIV-16, HIV-17, HIV-18, HIV-19, HIV-20, HIV-21, HIV-22, HIV-23, HIV-24, HIV-25, HIV-26, HIV-27, HIV-28, HIV-29, HIV-30, HIV-31, HIV-32, HIV-33, HIV-34, HIV-35, HIV-36, HIV-37, HIV-38, HIV-39, HIV-40, HIV-41, HIV-42, HIV-43, HIV-44, HIV-45, HIV-46, HIV-47, HIV-48, HIV-49, HIV-49, HIV-49, HIV-49, HIV-40, HIV-41, HIV-42, HIV-44, HIV-45, HIV-46, HIV-47, HIV-48, HIV-49, HIV-49, HIV-49, HIV-40, HIV-41, HIV-42, HIV-43 ...Oncolytic vaccinia viruses include, but are not limited to, Lister (e.g., the LIVP strain and its clonal strains), Western Reserve (WR), Copenhagen (Cop), Bern, Paris, Tashkent, Tiantan, Wyeth (DRYVAX), IHD-J, IHD-W, Brighton, Ankara, CVA382, Modified Vaccinia Ankara (MVA), Dalian I, LC16m8, LC16M0, LIVP, ACAM2000, WR65-16, Connaught, New York City Department of Health (NYCBH), EM-63, and NYVAC strains, as well as the modified Wyeth strain JX-594. An example of an oncolytic LIVP strain of virus is the strain designated GLV-1h68 (also designated GL-ONC1). Viruses include modified viruses encoding heterologous gene products, including therapeutic products and reporter genes and other detectable markers.

[0013] Viruses are purified by ultrafiltration and / or diafiltration. Those skilled in the art can select the appropriate membrane configuration and type of ultrafiltration. For example, ultrafiltration can be performed using a membrane with a nominal molecular weight cutoff of 300 to 750 kilodaltons or a nominal porosity of 0.05 to 0.2 μm. A membrane containing polyethersulfone can be used. The membrane may be a flat membrane or a hollow fiber membrane. The ultrafiltration can be performed in a tangential flow type, a cross-flow type, or another type selected by the skilled artisan. Ultrafiltration can be performed following diafiltration, or diafiltration can be performed without ultrafiltration. The recovery rate of the treated and released virus is 50% or more, and may be 90% or more, 95% or more.

[0014] This application contains at least one drawing in color. Copies of any patent or publication based on this application with color drawing(s) will be provided to the Patent Office upon request and payment of the necessary fee. [Brief explanation of the drawings]

[0015] [Figure 1]FIG. 1 shows an exemplary bioreactor, the ATMI iCellis® nanobioreactor and control / data management system. [Figure 2] FIG. 2 shows a microscopic image of a crystal violet stained carrier from iCellis® nanobioreactor experiment 6 below. [Figure 3] FIG. 3 shows fluorescence microscopy images of iCellis® nanobioreactor carriers before and after infection with the therapeutic LIVP strain of vaccinia virus designated GLV-1h68 (also designated GL-ONC1). [Figure 4] FIG. 4 shows the CV-1 cell (a well-known cell line that is widely available), e.g., ATCC® CCL-70 growth profile and viral amplification in Cellis® Bioreactor Experiment 1 and roller bottle controls. [Figure 5] FIG. 5 shows the CV-1 growth profile and viral amplification for iCellis® bioreactor experiment 2 and roller bottle control. [Figure 6] FIG. 6 shows the CV-1 growth profile and viral amplification for iCellis® bioreactor experiment 3 and roller bottle controls. [Figure 7] FIG. 7 shows the CV-1 growth profile and viral amplification for iCellis® bioreactor run 4 and roller bottle controls. [Figure 8] FIG. 8 shows the CV-1 growth profile and viral amplification for iCellis® bioreactor run 5 and roller bottle control. [Figure 9] FIG. 9 shows the CV-1 growth profile and viral amplification for iCellis® bioreactor run 6 and roller bottle controls. [Figure 10] FIG. 10 shows the growth curves for each of iCellis® Bioreactor Nano Experiments 2 to 6. [Figure 11]FIG. 11 shows proliferation data collected throughout the growth phase of CV-1 cells in iCellis® bioreactor experiments 2-6. [Figure 12] FIG. 12 shows proliferation data collected throughout 150 hours of the growth phase of CV-1 cells in iCellis® bioreactor experiments 2-6. [Figure 13] FIG. 13 shows cell density data collected throughout 300 hours of the growth phase of CV-1 cells in roller bottle controls from Experiments 1-6. [Figure 14] FIG. 14 shows cell density data collected throughout 150 hours of the growth phase of CV-1 cells in roller bottle controls from Experiments 1-6. [Figure 15] FIG. 15 shows the growth curves for Cellis® Bioreactor Nano experiments 1 and 3-6 after infection with GLV-1h68. [Figure 16] Figure 16 shows the viral amplification data from nano experiments 1 and 3-6. [Figure 17] FIG. 17 shows the CV-1 growth profile for iCellis® bioreactor runs 7, 8, and 9. [Figure 18] FIG. 18 shows the evaluation of virus extraction conditions from bioreactor carriers (Trial 4). [Figure 19] FIG. 19 shows evaluation of virus stability in TrypLE (Trial 8). [Figure 20]Figure 20 shows virus recovery during the harvest step from the bioreactor. The bioreactor is continuously processed by circulating various solutions and treatment media. Virus recovery is quantified by testing the collected circulating solution with a viral plaque assay. The results show that the majority of virus is released from the bioreactor after treatment with 1X TrypLE and subsequent washing with 10 mM Tris-Cl. Washing with PBS and 1 M NaCl results in virtually no further virus recovery. Extraction of the bioreactor macrocarrier (matrix) after harvest showed no residual virus in the matrix, demonstrating the efficiency of the harvest method.

[0016] The above figures are offered by way of illustration and not by way of limitation. DETAILED DESCRIPTION OF THE INVENTION

[0017] Overview A.Definition B. Overview C. Bioreactor and Matrix D. Cells and Growth E. Viruses and Dissemination F. Cell lysis and release of virus into treated media G. Diafiltration and Ultrafiltration of Processed Media to Purify Virus H. Working Example

[0018] Detailed Description A.Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All patents, patent applications, published applications and publications, Genbank® sequences, databases, websites, and other published documents referenced throughout this disclosure are incorporated by reference in their entirety unless otherwise indicated. In the event that multiple definitions of terms exist in this specification, those in this section prevail. When a URL or other such identification or address is referenced, it is understood that such identification may change and specific information on the Internet may vary, but that equivalent information may be found by searching the Internet. These references indicate the availability and public dissemination of such information.

[0019] As used herein, a bioreactor refers to a cell culture device. For use in the methods herein, a bioreactor includes a matrix for culturing adherent cells. The matrix retains the cells so that they are not released into the cell culture medium upon lysis. An exemplary bioreactor is sold under the trade name iCellis® bioreactor. This bioreactor was developed by ATMI Life Sciences and is available from PALL Biosciences.

[0020] As used herein, "carrier" or "substrate" means any solid substance that provides a biocompatible surface to which adherent cells adhere during culture.

[0021] As used herein, a matrix-type carrier comprises polyester fibers, which may be held within a (eg, polypropylene) cage to immobilize the fibers.

[0022] As used herein, processing medium refers to the medium in the bioreactor in which cells are cultured and virus is produced, and is used to process the cells and produce the virus.

[0023] As used herein, large-scale production is defined by a single patient dose and the required annual dose. Depending on the virus and treatment, each large-scale production provides about 1 / 6 to 1 / 12, e.g., about 1 / 10, of the annual dose. Thus, for example, for the vaccinia virus designated GLV-1h68 (GL-ONC1), a large-scale production provides about 6 x 10 per therapeutic dose. 9 Approximately 10,000 treatments or a total of 6x10 plaque-forming units (pfu) per year 13 Provides annual production of 8-12, e.g., 10, pfu. Assuming 8-12, e.g., 10, production batches per year, each large-scale batch would provide 1,000 therapeutic doses or approximately 10 11 ~10 13 , for example, about 5-6x10 12 pfu can be generated. Examples of pfu and doses for the exemplary virus GLV1h-68 (GLV-ONC1): [Table 1]

[0024] As used herein, "virus titer" or "virus titer" refers to the virus concentration, which is a predetermined number of infectious virus units per a given volume, such as plaque-forming units (pfu) / mL. Virus titer can be determined by serial dilution of a sample infected with target cells to quantify the number of infectious or active viruses in the sample. For example, virus titer can be determined using a plaque assay.

[0025] As used herein, "virus" or viral vector refers to any of a large group of infectious agents that cannot grow or replicate without host cells. Viruses typically contain a protein envelope surrounding an RNA or DNA core of genetic material, but do not contain a semipermeable membrane, and can only grow and multiply in living cells. As used herein, oncolytic viruses refer to viruses that selectively replicate in tumor cells in tumor-bearing subjects. Oncolytic viruses can kill tumor cells after infecting them. For example, oncolytic viruses can cause tumor cell death by lysing tumor cells or inducing cell death in tumor cells.

[0026] As used herein, therapeutic viruses, e.g., therapeutic oncolytic viruses, are viruses used to treat a disease or condition. Typically, they are not pathogenic or in a non-pathogenic state.

[0027] As used herein, the terms "vaccinia virus," "VACV," or "VV" refer to a large, complex, enveloped virus belonging to the poxvirus family. It has a linear, double-stranded DNA genome approximately 190 kbp long and encodes approximately 200 proteins. Vaccinia virus strains include, but are not limited to, Western Reserve (WR), Copenhagen, Tashkent, Tiantan, Lister, Wyeth, IHD-J, IHD-W, Brighton, Ankara, MVA, Dalian I, LIPV, LC16M8, LC16MO, LIVP, WR65-16, Connaught, and New York City Department of Health vaccinia virus strains, or strains derived therefrom or modified therefrom.

[0028] As used herein, Lister strain of the Institute of Viral Preparations (LIVP) or LIVP virus strain refers to a virus strain that is an attenuated Lister strain (ATCC catalog number VR-1549) produced by application to the skin of calves at the Institute of Viral Preparations in Moscow, Russia (Altshteyn et al. (1985) Dokl. Akad. Nauk USSR 285:696-699). The LIVP strain can be obtained, for example, from the Institute of Virus Production in Moscow, Russia (see, e.g., Kutinova et al. (1995) Vaccine 13:487-493); the FSRI SRC VB Vector Microbial Collection (Kozlova et al. (2010) Environ. Sci. Technol. 44:5121-5126); or the Moscow Ivanovsky Institute of Virology (C0355 K0602; Agranovski et al. (2006) Atmospheric Environment 40:3924-3929). It is also well known to those skilled in the art; it was a vaccine strain used for vaccination in the USSR, Asia, and India. Said strains are well known (e.g. Altshteyn et al. (1985) Dokl. Akad. Nauk USSR 285:696-699; Kutinova et al. (1994) Arch. Virol. 134:1-15; Kutinova et al. (1995) Vaccine 13:487-493; Shchelkunov et al. (1993) Virus Research 28:273-283; Sroller et al. (1998) Archives Virology 143:1311-1320; Zinoviev et al., (1994) Gene 147:209-214; and Chkheidze et al. (1993) FEBS 336:340-342).

[0029] As used herein, LIVP GLV-1h68 (also referred to as GL-ONC1; see, e.g., U.S. Patent No. 7,588,767 and U.S. Patent Publication No. US-2016-0339066-A1) is a LIVP virus containing ruc-gfp (a luciferase and green fluorescent protein fusion gene (see, e.g., U.S. Patent No. 5,976,796)), beta-galactosidase (LacZ), and beta-glucuronidase (gusA) reporter genes inserted into the F14.5L, J2R (thymidine kinase), and A56R (hemagglutinin) loci, respectively. The genome of GLV-1h68 has the nucleotide sequence set forth in SEQ ID NO:3 of co-pending published application US-2016-0339066-A1, or a nucleotide sequence having at least 97%, 98%, or 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO:3.

[0030] As used herein, the interchangeable terms "modified virus" or "recombinant virus" refer to a virus that has been altered compared to its parent strain. Typically, a modified virus has one or more truncations, mutations, insertions, or deletions in the viral genome. A modified virus may have one or more modified endogenous viral genes and / or one or more modified intergenic regions. A typical modified virus may have one or more heterologous nucleic acid sequences inserted into the viral genome. A modified virus may contain one or more heterologous nucleic acid sequences in the form of a gene expression cassette for expression of the heterologous gene.

[0031] As used herein, a modified LIVP virus strain refers to a LIVP virus having a genome not contained in the LIVP, but generated by modifying the genome of a strain derived from the LIVP. Typically, the viral genome is modified by nucleotide substitution (substitution), insertion (addition), or deletion (truncation). Modifications can be made by any method known to those skilled in the art, such as genetic modification and recombinant DNA techniques. Thus, a modified virus is a virus whose genome has been altered compared to the genome of the parent virus. A typical modified virus has one or more heterologous nucleic acid sequences inserted into the viral genome. Typically, the heterologous nucleic acid comprises an open reading frame encoding a heterologous protein. For example, a modified virus herein can comprise one or more heterologous nucleic acid sequences in the form of a gene expression cassette for the expression of a heterologous gene.

[0032] As used herein, when referring to doses such as plaque forming units (pfu) / kg based on kg weight of the subject, an average human subject would have a weight of about 70 kg to 75 kg, e.g., 70 kg.

[0033] For clarity of disclosure, and not by way of limitation, the detailed description is divided into the following subsections:

[0034] B. Overview A method / process is provided that includes two steps. The first step is a production step in which the virus is taken up into cells in which the virus can replicate, and the cells are cultured under conditions in which the virus is produced. The virus can be any suitable virus, including, but not limited to, enveloped viruses, such as poxviruses, e.g., vaccinia viruses. The cells are then lysed to release the virus, which can then be treated with one or more enzymes, such as nucleases and / or proteases. In the second step, the treated medium is collected, and the virus is purified solely by ultrafiltration and / or diafiltration. Thus, the second step is only a one- or two-step process that includes only ultrafiltration and / or diafiltration, resulting in purified virus.

[0035] Thus, the process involves growing adherent host cells, typically cell lines, in a bioreactor containing a packed biocompatible woven or fibrous matrix material, e.g., polyester, polyethylene terephthalate, which matrix is dense enough to retain cells and cell debris by adhesion and / or entrapment when the cells are lysed.

[0036] Host cells are cultured at an appropriate density, then inoculated with the virus and cultured to produce the virus. The cells are lysed, for example, by freezing and thawing, or by exposure to a low osmolarity medium, or both, followed by treatment with an enzyme, for example, a protease, particularly one that cleaves nonspecifically, such as trypsin. If necessary, the cells are treated with a nuclease before, together with, or after the protease. The lysed cells are held in place by a matrix material.

[0037] The virus is purified from the medium in only one or two steps, one of which is ultrafiltration or diafiltration. If two steps are used, they are ultrafiltration and diafiltration. No further purification steps other than ultrafiltration and / or diafiltration are used.

[0038] Thus, a scalable process (also referred to as a method) is provided for producing (also referred to as manufacturing) purified viruses, particularly therapeutic viruses such as oncolytic viruses, vaccines, and gene therapy vectors. The method can be performed in a bioreactor, and is therefore easily scaled up. Purification provides high yields of virus and can be accomplished in one day or less. The resulting virus is produced in high yields, with recovery rates of 95% or greater, typically at least 50%, 60%, 70%, 80%, or 90%.

[0039] C. Bioreactor and Matrix Bioreactors are vessels suitable for growing cells and contain a matrix substrate of woven or nonwoven fibers, fabric, or textiles, the fibers or fibrous mesh of which allows for cell attachment (or entrapment) and growth. The matrix can be a fixed or packed bed, or a fluidized bed.

[0040] Bioreactors include macrocarrier beads, woven or nonwoven fibers, fabrics, or fabric-filled matrices, the fibers or fibrous mesh of which allow cell attachment and proliferation. The matrix is such that cells cannot be detached or removed from the fabric by enzymatic digestion, e.g., trypsin digestion. The cells are such that cells cannot be detached or removed from the fabric by enzymatic digestion, e.g., trypsin. Hollow fiber bioreactors do not function because cells attached to the inner surface of the hollow fibers are not entrapped in the matrix and can be detached by enzymatic digestion, e.g., trypsin. Hollow fiber bioreactors are not contemplated because cells attached to the inner surface of the hollow fibers are not entrapped in the matrix and can be detached by enzymatic digestion, e.g., trypsin. According to the process herein, cells held by a matrix, such as anchorage-dependent ("attached") host cells, are grown in a bioreactor; the cells are infected with a virus and grown in the bioreactor on the matrix substrate.

[0041] An example of a bioreactor is the bioreactor sold under the trade name iCellis® (Pall Life Sciences). U.S. Patent Nos. 8,597,939 and 8,986,979 describe such bioreactors. The iCellis® bioreactor is exemplary of a bioreactor platform, including a scalable line of single-use, high-cell-density bioreactors that can be manufactured from small-scale (also referred to below as "nano") to large-scale scales. This bioreactor contains a pre-packed, fixed bed of pharmaceutical-grade polyester microfiber, which provides a large surface area for growth in a compact bioreactor volume. The primary bioreactor is equipped with a built-in magnetic drive impeller that circulates medium through the fixed bed from bottom to top, ensuring low shear stress and high cell viability. At the top of the fixed bed, the medium falls as a thin film onto an exterior wall that captures O2 to maintain dissolved oxygen levels in the bioreactor.

[0042] matrix material Examples of matrix materials are biocompatible adhesive materials that can be used to culture cells and to which cells can adhere, as described herein. Such materials include, but are not limited to, polyester, polypropylene, polyalkylene, polyfluorochloroethylene, polyvinyl chloride, polystyrene, polysulfone, cellulose acetate, glass fibers, ceramic particles, and inert metal fibers. Fibers of these materials can be used in the form of woven or nonwoven fabrics. For example, in the bioreactor commercially available under the trade name iCellis® and used in the examples herein, the matrix material is the polyester polyethylene terephthalate (PET). To improve its biocompatibility, cell adhesion, or cell retention, the matrix can be coated with a substance that promotes cell adhesion, such as Matrigel® cell culture substrate, extracellular matrix components (e.g., fibronectin, chondronectin, laminin, ProNectin® F), collagen, or poly-L-lactic acid.

[0043] The density of the matrix is sufficient to entrap cells, cell debris after lysis, but not so high as to prevent cell media flow through the matrix and bioreactor, which is necessary for cells to grow. The density of the matrix is such that cells remain entrapped within the matrix, but the density is not so high as to prevent fluid flow through the matrix.

[0044] The matrix may be a mesh, e.g., made of polyester fibers. The fiber diameter is typically about 10-40 micrometers. The mesh can be purchased from fiber manufacturers as a bulk fiber nonwoven mesh or as a woven fabric. Some manufacturers produce fibers specifically for pharmaceutical purposes and are biocompatible. This medical-grade mesh or fiber is particularly useful in the processes and methods herein because it is compatible with cell attachment and growth in bioreactors used in pharmaceutical manufacturing.

[0045] The packing density of such materials is a factor that affects the state of cell entrapment within the matrix. The higher the fiber density, the greater the filtration effect of the packing, and therefore the greater the likelihood that cells will remain entrapped within the matrix. However, the higher the packing density, the more restricted the medium flow and the greater the stirring force required to achieve the same medium flow. Example densities are approximately 80-160 g / L, e.g., 90-150 g / L, e.g., 96 g / L-144 g / L. This range retains cells; the higher the packing density, the more restricted the medium flow and the greater the stirring force required to achieve the same medium flow.

[0046] The iCellis® bioreactor system (see, e.g., FIG. 1) is a 500 ml 2 The projected virus (e.g., vaccinia virus) yields for scale-up using a fixed bed volume of 25 L are as follows: [Table 2]

[0047] In the iCellis® bioreactor, two PET packing densities are exemplified: 96 g / L and 144 g / L.

[0048] D. Cells and Growth Provided herein is a process for producing viruses from adherent host cells using a bioreactor. The method involves releasing viruses from adherent host cells in a bioreactor and then purifying the released viruses by one or two steps of ultrafiltration and / or diafiltration. The method can be used to produce viruses, including those for clinical use, at a lower cost than conventional virus production methods. The purification method requires only ultrafiltration and / or diafiltration, reducing the time required for purification and providing a higher virus yield. Purification can be achieved in one day or less.

[0049] Cells that are adherent cells or cells used to grow in or on a matrix support are cultured in a bioreactor. The host cells can be any cell suitable for growing viruses, and the choice of cell can depend on the specific virus. Generally, the host cells are adherent cells. Cells include, but are not limited to, mammalian cells of primate origin; transformed or immortalized cells; and cell lines. Typical examples of such cells are human fibroblasts; human epithelial cells; and human endothelial cells. Cell lines include, but are not limited to, CV-1 cells; Vero cells; and CHO cells. The cells may be recombinant and / or genetically modified.

[0050] The cells are seeded into the reactor in an appropriate amount. For example, such an amount may be about 3-6 x 10 3 cells / cm 2 , for example, about 4-5x10 3 cells / cm 2 , e.g., 4.5x10 3 cells / cm 2 The cells are grown for a sufficient time, generally 8 to 20 days depending on the growth conditions, to reach an optimal density for infection with the virus, for example, about 1 to 3 x 10 5 cells / cm 2 , for example, about 1.5x10 5 cells / cm 2 Those skilled in the art will know or be able to empirically determine the optimal density for growth and infection of particular cells.

[0051] In an exemplary method, cells are cultured in a reactor at 4.5E3 cells / cm. 2 were seeded at 1.5E5 cells / cm 2The cells are grown to an optimal infection density of 0.01% (MOI), which can take approximately 8-20 days depending on growth conditions. The cells are infected at a specific MOI (0.2-0.002, e.g., 0.02-0.1), and virus production proceeds for approximately 96 hours. Purification involves only a single step (ultrafiltration and / or diafiltration) and can be performed in one day, compared to traditional purification, which typically requires 5-7 steps over a longer period.

[0052] In certain embodiments, the bioreactor can be used for the growth of adherent cells; iCellis® bioreactors include components disclosed in U.S. Patent Nos. 8,597,939 and 8,986,979; include a matrix or surface to which adherent cells can adhere; include an unfixed adhesive surface such as macrocarrier beads, fibers, or woven mesh in suspension; and / or include an immobilized adhesive surface such as in a packed-bed bioreactor.

[0053] In certain embodiments, adherent host cells are cultured in a bioreactor under controlled conditions; in the presence of cell culture medium and medium additives that support the nutritional requirements of the cells; at a temperature suitable for optimal growth of the cells (e.g., for mammalian cells, 37±3°C); at a pH suitable for optimal growth of the cells (e.g., for mammalian cells, pH 7.3±3); with medium agitation or circulation or incubation suitable for optimal growth of the cells; in a fixed-bed reactor with a medium linear flow rate of about 0.5-5 cm / sec; at a dissolved oxygen level suitable for optimal growth of the cells (e.g., for mammalian cells, 50±25%); and / or at a high cell density (e.g., ≧2×10E6 cells / mL, or ≧1×10E7 cells / mL) relative to the medium volume of the bioreactor.

[0054] E. Viruses and Dissemination Viruses encompassed herein include, but are not limited to, therapeutic viruses, e.g., oncolytic viruses, viruses for vaccines, and viruses for any purpose, including recombinant production of encoded products. The viruses are generally enveloped viruses that can be released into cell culture medium.

[0055] The virus is taken up by cells and then cultured to allow the virus to replicate. The cells are infected at a specific multiplicity of infection (MOI) depending on the virus. In an exemplary embodiment, the virus is vaccinia virus, and the MOI is about 0.002. Virus production is allowed to proceed for a time that produces the maximum amount of virus, for example, about 72 to 120 hours, for example, 90 to 120 hours, for example, 96 hours, for a vaccinia virus such as GL-ONC1 (GLV1h-68).

[0056] The host cells can be infected with the virus before being introduced into the bioreactor, or the adherent host cells can be infected with the virus while growing in the bioreactor. The bioreactor medium and / or other culture conditions can be adjusted before or after introducing the infected or uninfected host cells to optimize viral infection efficiency and / or replication.

[0057] Viruses include, but are not limited to, poxviruses, herpesviruses, adenoviruses, adeno-associated viruses, lentiviruses, retroviruses, rhabdoviruses, papillomaviruses, vesicular stomatitis viruses, measles viruses, Newcastle disease viruses, picornaviruses, Sindbis viruses, papillomaviruses, parvoviruses, reoviruses, coxsackieviruses, influenza viruses, mumps viruses, polioviruses, and Semliki Forest viruses.

[0058] The virus may be endogenous, wild-type, recombinant, or genetically modified. The virus may be selected from Newcastle disease virus, parvovirus, vaccinia virus, myxoma virus, measles virus, reovirus, vesicular stomatitis virus (VSV), oncolytic adenovirus, adeno-associated virus, poliovirus, herpes virus, Sindbis virus, and Seneca Valley virus, or derivatives thereof that have been modified to contain a nucleic acid encoding a heterologous gene product. The virus may be an oncolytic virus. The oncolytic virus may be a vaccinia virus selected from the following strains: Lister, Western Reserve (WR), Copenhagen (Cop), Bern, Paris, Tashkent, Tiantan, Wyeth (DRYVAX), IHD-J, IHD-W, Brighton, Ankara, CVA382, Modified Vaccinia Ankara (MVA), Dalian I, LC16m8, LC16M0, LIVP, ACAM2000, WR65-16, Connaught, New York City Department of Health (NYCBH), EM-63, and NYVAC. The vaccinia virus may be derived from a Lister strain virus, a LIVP virus, or a clonal strain of a LIVP virus.

[0059] Virus can be vaccinia virus, for example, recombinant vaccinia virus.Representative examples of therapeutic vaccinia virus include modified LIVP strain virus, for example, those disclosed in U.S. Patent No. 7,588,767, 8,857,927, 9,005,602, 8,323,959 and 7,754,221, in particular the virus that is called GLV-1h68 (GL-ONC1) and the clone strain described in U.S. Publication No. US-2012-0308484-A1, and modified Wyeth strain vaccinia virus, for example, the virus that is called JX-594 (also called Pexa-Vec, Sillajen Biotherapeutics), which is a replication-competent Wyeth strain vaccinia virus that thymidine kinase gene is inactivated, and the virus is modified to encode and express human GM-CSF and LacZ gene.

[0060] The virus may be in a modified form containing a nucleic acid encoding a heterologous gene product, the heterologous gene product being a therapeutic or reporter gene product. The heterologous gene product may be selected from anti-cancer drugs, anti-metastatic drugs, anti-angiogenic drugs, immunomodulatory molecules, antigens, cell matrix degradation genes, genes for tissue regeneration and reprogramming human somatic cells to pluripotency, enzymes that modify substrates to produce detectable products or signals or are detectable by antibodies, proteins that can bind to contrast agents, genes for optical imaging or detection, genes for PET imaging, and genes for MRI imaging. The heterologous gene product may be a therapeutic agent selected from hormones, growth factors, cytokines, chemokines, costimulatory molecules, ribozymes, transporter proteins, single-chain antibodies, antisense RNA, prodrug-converting enzymes, siRNA, microRNA, toxins, antitumor oligopeptides, mitotic inhibitor proteins, mitotic inhibitor oligopeptides, anticancer polypeptide antibacterial drugs, angiogenesis inhibitors, tumor suppressors, cytotoxic proteins, cytostatic proteins, and tissue factors. Viruses can encode and optionally express heterologous gene products. Such products include therapeutic products that can be delivered by viruses. Representative examples of such products include anti-cancer drugs, anti-metastatic drugs, anti-angiogenic drugs, immunomodulatory molecules, and antigens. Other exemplary products include, but are not limited to, therapeutic agents selected from hormones, growth factors, cytokines, chemokines, costimulatory molecules, ribozymes, transporter proteins, single-chain antibodies, antisense RNA, prodrug-converting enzymes, siRNAs, microRNAs, toxins, anti-tumor oligopeptides, mitotic inhibitor proteins, mitotic inhibitor oligopeptides, anti-cancer polypeptides, antimicrobial agents, angiogenesis inhibitors, tumor suppressors, cytotoxic proteins, cytostatic proteins, and tissue factors. Viruses can also deliver nucleic acids encoding genes, such as cell matrix degradation genes, genes for tissue regeneration, and genes for reprogramming human somatic cells to pluripotency.The virus can encode a detectable reporter product, including, but not limited to, an enzyme that modifies a substrate or is detectable by an antibody to produce a detectable product or signal, a protein that can bind to an imaging agent, a gene for optical imaging or detection, a gene for PET imaging, and a gene for MRI imaging.

[0061] F. Cell lysis and release of virus into treated media In the production stage of the method, the cells in the bioreactor are lysed and treated to release the virus. According to the methods herein, the virus can be released from the host cells without substantial mechanical homogenization.

[0062] This can be achieved by a suitable method for lysing cells. The lysis method requires that the cells remain attached to or entrapped in the matrix. For example, cells can be lysed by freezing and thawing. If necessary, the release and recovery of virus can be enhanced by a hypotonic shock. This can be done, for example, by adding an aqueous solution with a total ionic strength of about 0.05M or less, for example, 0.01M or less, or for example, 0.001M to the frozen / thawed bioreactor.

[0063] Depending on the matrix selected, cells and lysed cell debris remain attached / trapped in the matrix and are not, or are not substantially, released into the cell culture medium along with the virus. Release of virus from trapped / attached cells is achieved by a suitable method for achieving lysis, such as freezing / thawing the bioreactor and / or exposure to a hypotonic medium, followed by enzymatic digestion with an enzyme, e.g., a protease, and optionally, a nuclease. Proteases include trypsin and other such proteases that typically detach cells but are not detached by the matrix. Examples of such proteases include trypsin (derived from porcine pancreas), TrypLE (a recombinant bacterial enzyme with trypsin-like enzyme activity), Accutase® solution (Sigma-Aldrich; a mixture of proteolytic and collagenolytic enzyme activities), proteinase K, papain and subtilisin, other such proteases with nonspecific cleavage sites that digest proteins, bromelain, ficain, etc.

[0064] Viruses can be released from host cells by a process involving freezing / thawing. For example, viruses can be released from cells in a fixed-bed reactor in a process in which the culture medium is removed, and the bioreactor is frozen at ≦10°C for ≧60 minutes, <−60°C for ≧15 minutes, <−120°C for ≧1 minute, or other suitable temperature combinations. The bioreactor can be frozen, for example, by placing it in a freezer, jacketed freezing, immersing it in dry ice, immersing it in liquid nitrogen, injecting liquid nitrogen gas, or other such methods. The bioreactor can be thawed by an appropriate method, for example, by exposure to room temperature air, adding liquid medium, such as phosphate-buffered saline (PBS), or other such methods known to those skilled in the art. For example, liquid medium can be added to the bioreactor at a temperature selected to maximize cell lysis while optimizing virus stability (e.g., ≦50°C or 37±3°C).

[0065] Optionally, when freeze / thaw is used, virus release and recovery can be enhanced by hypotonic shock. Hypotonic shock can be performed, for example, by adding an aqueous solution to the freeze / thawed bioreactor so that the total ionic strength is about 0.05 M or less, e.g., 0.01 M or less, or 0.001 M. Virus can be released from host cells by exposure to a hypotonic medium. In certain embodiments, the hypotonic medium can be water or a buffer solution with an ionic strength of ≦50 mM. Hypotonic conditions can be achieved by diluting the bioreactor medium with a hypotonic solution, such as water, to a final ionic strength of ≦50 mM. For example, in a fixed-bed bioreactor, the medium can be removed and replaced with a hypotonic medium with an ionic strength of ≦50 mM. In certain embodiments, the hypotonic medium can be agitated or circulated.

[0066] If necessary, virus release and recovery can be enhanced by treatment with an enzyme having DNase and / or RNase activity during or after freeze / thaw and / or hypotonic shock, or after treatment in an appropriate buffer. An example of a nuclease is the endonuclease sold under the trade name Benzonase® endonuclease, or other enzymes with DNase and / or RNase activity. Benzonase® nuclease (sold by Millipore; see, e.g., Franke et al., (1998) FEBS Letters 425: 517-522), which digests endogenous or heat-denatured DNA and RNA, is a genetically engineered endonuclease derived from Serratia marcescens. It is known to those skilled in the art as Serratia nuclease and is a protein dimer of 30 kDa subunits with two necessary disulfide bonds.

[0067] Conditions for releasing virus from host cells can be selected to maximize cell lysis while optimizing virus stability. For example, the temperature can be ≦50° C.; the pH can be ≧4 and ≦10; and / or the linear flow velocity through a packed-bed bioreactor can be ≧2 cm / sec.

[0068] Viruses can be released from host cells by exposure to a medium containing a detergent. The detergent can be ionic, cationic, or anionic. The concentration of the detergent can be, for example, ≦1%.

[0069] After freeze / thaw treatment, optional hypotonic medium treatment, and other such treatments, the cells are treated with a protease as described above. Viruses can be released from host cells by digestion with one or more enzymes. The cells are exposed to a digestion medium with an optimal ionic strength and pH for the selected digestive enzyme. An exemplary digestion medium is buffered at a neutral pH, e.g., pH 4, such as PBS, pH ≤ 8, e.g., 7-8. The digestion medium contains sodium salts and / or magnesium salts. The digestive enzyme can be a protease as described above, e.g., trypsin or recombinant trypsin (TrypLE). Protease treatment can be effective in combination with or in place of a nuclease, such as the endonuclease derived from Serratia marcescens sold under the trademark Benzonase®. Digestion can be performed using a nuclease and a protease sequentially or in combination. The digestion temperature can be set to optimize enzymatic digestion and virus stability (e.g., 37°C). The digestion time can be set to optimize enzymatic digestion and virus stability (e.g., ≥ 1 hour). The virus can be collected by removing the digestion medium and purifying the virus obtained therefrom.

[0070] The released virus can be rinsed with a medium selected to optimize virus recovery and / or virus stability. For example, the rinsing medium can be water; a low ionic strength buffer; a high ionic strength buffer; having a pH > 9; and / or 10 mM Tris-Cl, pH 9.0.

[0071] G. Diafiltration and Ultrafiltration of Processed Media to Purify Virus The purification process after release of the virus from the cells and enzymatic digestion is a single (or two) step of ultrafiltration or diafiltration, or both. No other purification steps are used or required. Purification can be performed in one day. Conventional prior art purification typically uses 5-7 steps carried out over an extended period of time. Not only do the purification methods provided herein reduce time and cost and provide higher yields, but the reduced steps and shorter time also reduce the degradation and inactivation of the virus that occurs over extended processing periods.

[0072] The virus released into the culture medium is purified by ultrafiltration or diafiltration or both. No other purification steps are used. The released virus can be purified by ultrafiltration. Those skilled in the art can select appropriate filtration membranes and protocols. In some embodiments, the filtration medium can be selected to optimize impurity removal and virus stability; it can be water; it can be a buffer solution; it can have high ionic strength; it can have low ionic strength; it can have physiological ionic strength; and / or it can be suitable for administration to animals or humans. The recovery of the released virus that is processed can be 50% or more; 70% or more; 80% or more; 90% or more; or 95% or more.

[0073] Ultrafiltration Viruses can be purified using large-porosity ultrafiltration membranes, which are commonly used to remove contaminating viruses from biological preparations. Such membranes typically have a porosity of 300,000 daltons or greater, allowing protein products to pass through the membrane while retaining viruses. By using these "virus clearance" membranes in the opposite mode (tangential flow or cross-flow filtration mode rather than single-pass mode), viruses can be retained and purified while proteins, nucleic acids, and other impurities are removed.

[0074] For example, the ultrafiltration membrane can be selected to minimize virus binding and maximize virus retention. The ultrafiltration membrane can have a porosity cutoff that prevents virus passage through the pores. For example, the ultrafiltration membrane can have a nominal molecular weight cutoff of 300 to 750 kilodaltons or a nominal porosity of 0.05 to 0.2 μm; the ultrafiltration membrane can include polyethersulfone (PES); the ultrafiltration membrane can be, for example, a flat membrane or a hollow fiber membrane; ultrafiltration can be performed in a tangential flow or crossflow mode; ultrafiltration can be performed under conditions that optimize virus retention and virus stability, and / or ultrafiltration can be performed at a temperature of ≦10°C.

[0075] Flat membrane filters and hollow fiber filters can be used for ultrafiltration. Membranes constructed from materials exhibiting low virus binding are suitable for high virus recovery. Examples of such membranes include, but are not limited to, Centramate T-series cassette flat membranes (300 kd mwco, PALL Life Sciences, Inc.) and MidiKros hollow fiber membrane cartridges (500 or 750 kd mwco, Spectrum Laboratories Inc.), all of which are constructed from polyethersulfone (PES).

[0076] diafiltration Ultrafiltration can be followed by diafiltration with high ionic strength, neutral pH and / or low ionic strength, high pH filtration solutions. As described herein, such methods can recover greater than 90% of the virus from lysed cells with high purity.

[0077] In some embodiments, the released virus can be purified by diafiltration. In some embodiments, diafiltration can be performed using a membrane selected to minimize virus binding and maximize virus retention; the diafiltration membrane can be the same as that used in ultrafiltration; diafiltration can be performed by adding filtration medium to the retentate of ultrafiltration; and / or diafiltration can be performed by adding a filtration volume of ≧5 that is the same as the volume of the retentate.

[0078] 95% of the virus can be recovered. The resulting virus can be any virus of interest, particularly vaccinia virus. The virus can be biologically active and can be used to infect cells, express homologous or heterologous genes or proteins, and / or induce an immune response.

[0079] Those skilled in the art will envision numerous modifications and other embodiments within the scope and spirit of the presently disclosed invention. Indeed, changes in the materials, methods, diagrams, and experimental examples described may be made by those skilled in the art without changing the fundamental aspects of the disclosed invention. Any of the disclosed embodiments may be used in combination with any of the other disclosed embodiments. [Example]

[0080] H. Working Example The following examples are not intended to limit the scope of the present disclosure or claims, and do not represent all or the only experiments performed. While efforts have been made to ensure accuracy with respect to the numbers used (e.g., amounts, temperatures, and other parameters), some experimental error and deviation should be accounted for. It should be understood that modifications in the methods described can be made without changing the basic aspects illustrated by the experiments and examples.

[0081] Example 1 General Method CV-1 cell culture All CV-1 cells used in these experiments were from the CV-1 Working Cell Bank (WuXi Apptec Acc#09-002346, Lot#090170885, 6.0 x 10 cells in 1.0 mL). 6 The cells were derived from a 100-well plate (cells / virus, frozen on December 9, 2009, passage 45). These cells were further expanded and cryopreserved at passage 55. An aliquot of these cells was used in iCellis® Bioreactor Nano experiments. CV-1 cells were cultured in DMEM (supplemented with 10% FBS) in T-flasks and roller bottles in a 5% CO2 incubator at 37°C.

[0082] Infection with GLV-1h68 iCellis® bioreactor nano experimental cultures were infected with GLV-1h68, a recombinant vaccinia virus derived from the LIVP strain. GLV-1h68 P3 2-28-14 was used for infection in nano experiments 1 through 6, and GLV-1h68 P3 8-28-14 was used for infection in nano experiments 7 through 11. The amount of inoculum virus used was calculated based on the cell number at the time of infection, the titer of the inoculum virus, and the specified MOI.

[0083] iCellis® Bioreactor Nanobioreactor Technology The iCellis® bioreactor nano bioreactor was set up and operated according to the manufacturer's recommended procedures ("Integrity iCellis® bioreactor Nano Bioreactor with Control System User Guide" - Document reference USG_nano_mycontrol_01, version 01, February 2014.).

[0084] analysis Viral plaque assays (VPAs) were performed in bioreactors, in which purified samples were infected with GLV-1h68. Reactor medium samples were removed and frozen at -20°C. Macrocarrier (matrix) samples from bioreactors containing one or two carriers were removed from the bioreactor, placed in microcentrifuge tubes containing 1 mL of fresh DMEM-10% FBS medium, and frozen at -20°C. Prior to testing, media and carrier samples were thawed at room temperature or 37°C, diluted with DMEM-2% FBS, and plated with CV-1 cells for plaque formation. Carrier trypsinization was performed by placing one carrier in 0.5 mL of 0.05% trypsin / 0.53 mM EDTA or 1x TrypLE in PBS and incubating at 37°C for up to 60 minutes with vortex mixing. Samples were diluted by adding 1.0 mL of DMEM-10% FBS. Purified samples were stored on ice and tested without freezing. VPA was performed using CV-1 cells in 24-well multiwell plates, and viral plaques were visualized by crystal violet staining.

[0085] β-Glucuronidase assays (GUS-A) were performed using samples of reactor medium and macrocarriers diluted and tested in DMEM-2% FBS. The fluorogenic substrate Cl-MUGlcU (stock solution: 36.5 mM in DMSO) was used in an assay buffer consisting of phosphate-buffered saline solution containing 2% FBS. Assays were performed in 96-well multiwell plates and read on a SpectraMax® M5 plate reader using SoftMaxPro v5.4.4 software.

[0086] Protein and DNA content were measured using the Quant-iT Protein Assay Kit (Invitrogen) and the Quant-iT dsDNA Assay Kit (High Sensitivity, Invitrogen), respectively. Assays were performed in 96-well multiwell plates and read on a SpectraMax® M5 plate reader using SoftMaxPro v5.4.4 software.

[0087] Example 2 Development of CV-1 Cell Culture Process in iCellis® Bioreactor Nanobioreactor The seeding density for all experiments was 4.5x10 4 cells / cm 2 This was consistent with the seeding density of CV-1 cells in roller bottles. The target cell density for infection was 1.0–2.0 x 10 5 cells / cm 2 It was.

[0088] The progress of the culture was monitored by aseptically opening the bioreactor and removing the carriers at different time points during the culture process. Cells attached to the carriers were lysed, and cell nuclei were counted using a hemocytometer. Cells were also visualized microscopically by crystal violet staining. Figure 2 shows microscopic images of stained carriers from Experiment 6. Before cell seeding, the carrier fibers appeared uniform in diameter and lacked appreciable staining. After carrier seeding, adherent staining material appeared to accumulate within the spaces between each fiber and fiber bundle. The staining material comprised cells attached to the fibers and to each other as the cells proliferated. After infection with GLV-1h68, the carriers were imaged with a microfluorescence microscope to assess the amount and distribution of GFP expression. Figure 3 shows carriers before infection, 24 hours after infection, and after harvesting. Strong green GFP fluorescence uniformly bound to the fibers was clearly visible 24 hours after infection. At higher magnification, individual fluorescent centers were observed on the fibers corresponding to the location of cells. The fluorescence significantly decreased.

[0089] During and after infection, virus titers were assessed to determine the amount of virus amplification and distribution of virus between cells and culture medium. Carriers were sampled from the reactor and freeze / thawed in fresh culture medium for assay by VPA. Culture medium from the reactor was simultaneously sampled and assayed directly by VPA. Virus load was calculated as PFU / cm of surface area. 2 , expressed as PFU / cell (cell number examined at time of infection), or total PFU in the reactor (either carrier-bound or in the medium).

[0090] Nano Experiment 1 Add 4.5x10 CV-1 cells to the reactor. 4 cells / cm 2 3.4x10 4 cells / cm 2The cells were allowed to attach to the bioreactor (76%) at 10°C. The agitation rate of the culture was set to create a linear flow velocity of 1.6 cm / sec during cell seeding and increased to 2.5 cm / sec over 10 days. The flow rate was decreased to 1.6 cm / sec on day 10, 1.0 cm / sec on day 14, and 0.5 cm / sec on day 16. The culture medium volume was increased from 600 mL after seeding to 762 mL and 2286 mL on day 3. The medium was changed on days 3, 6, 10, 14, and 16. On day 21, the medium was inoculated with GLV-1h68 at an MOI of 0.07 (1.12 x 10) in 600 mL of DMEM-2% FBS medium. 8 The cells were infected with 1000 sucrose (pfu). The flow rate was maintained at 0.5 cm / sec. The medium was collected 72 hours post-infection. Simultaneously, CV-1 cells were seeded into roller bottles, and cells were counted periodically. The roller bottles were cultured under standard laboratory conditions without changing the culture medium. Figure 6 shows the cell density profile of the culture.

[0091] The results showed that CV-1 cells seeded in iCellis® bioreactors expanded through a fairly comparable growth phase to roller bottle controls. CV-1 cultures in the reactors reached a target cell population of 1.6 x 10 cells. 5 cells / cm 2 (approximately 2 population cell doublings), indicating that the bioreactor was able to support cell growth up to this density. Changes in agitation speed or culture medium did not significantly affect the overall cell growth rate. During the growth phase, the culture medium was observed to become cloudy, indicating possible cell detachment from the reactor. This was reduced by reducing agitation.

[0092] Infection of the culture with GLV-1h68 on day 21 resulted in a rapid increase in virus titer, evident at day 1 postinfection, which reached a maximum at day 2. Throughout the 72-hour growth phase, virus adhered to the carrier (i.e., cells), with little free virus detected in the medium. Although there was a decrease in cell number on the carrier over a period of time postinfection, this decrease did not correspond to an increase in virus titer in the medium. At day 1 postinfection, the culture became acidic (pH 6.2), the medium became turbid, and cell density decreased. To counter this, sodium hydroxide solution was delivered to the bioreactor using an alkali pump. This was used in all subsequent bioreactor experiments.

[0093] Nano Experiment 2 Nano experiment 2 was performed by decreasing the flow rate immediately after cell seeding. The reactor was seeded with 4.5x10 CV-1 cells at passage 76 in 600 mL of medium at a linear flow rate of 1.6 cm / s. 4 cells / cm 2 After 1 hour, the volume of culture medium was increased to 1342 mL and the linear flow rate was increased to 2.5 cm / sec. After 24 hours, the adherent cell density was 3.5 x 10 4 cells / cm 2 On day 1, the volume of the culture medium was increased to 2286 mL and the flow rate was reduced to and maintained at 0.5 cm / sec. The culture medium was changed on day 5.

[0094] Day 10, 1.7x10 5 cells / cm 2 The cultures were cultured at an MOI of 0.1 (1.48x10 8Cells were infected with GLV-1h68 at 1000 pfu (pfu) and the flow rate was reduced to zero 3 days post-infection. Virus levels rapidly decreased below the initial infection level in the bioreactor, both on the carrier and in the medium. The linear flow rate was returned to a very low level (down to 0.3 cm / s) between days 4 and 8 post-infection to allow nutrients to circulate to the cells, but virus levels in the bioreactor remained low. Efficient virus infection and spread in the bioreactor was achieved by agitation caused by the circulation of the medium; therefore, subsequent experiments were performed at linear flow rates of 0.5 to 2.5 cm / s.

[0095] Nano Experiment 3 Nano experiment 3 was a repeat of experiment 2, except that the linear flow velocity was reduced to 0.44 cm / sec after day 1. The reactor was inoculated with 4.5 x 10 CV-1 cells at passage 78. 4 cells / cm 2 After 24 hours, 3.9x10 4 cells / cm 2 (87% seeding efficiency).

[0096] On day 10, the cell density was 1.7x10 5 cells / cm 2 On day 11, the culture was cultured at an MOI of 0.1 (1.74x10 8 The cells were infected with GLV-1h68 at 1000 pfu (pfu). The flow rate was increased to 0.5 cm / sec and maintained at that temperature. Virus rapidly increased on the carriers, but virus in the culture medium remained low. Virus amplification was intentionally extended until day 8 post-infection, and the entire amplification file was examined. Virus increased on day 2 post-infection, remained relatively stable until day 6, and then decreased significantly on days 7 and 8. This decrease did not correspond to an increase in virus in the culture medium.

[0097] Nano Experiment 4 For nano experiment 4, the reactor was inoculated with 4.5x10 CV-1 cells at passage 81. 4 cells / cm 2 The cells were seeded at a density of 4.5x10 for 24 hours. 4 cells / cm 2(100% seeding efficiency). After 24 hours, the flow rate was set to 0.44 cm / sec and the medium volume was increased to 2286 mL. One medium change was performed on day 7. On day 10, the flow rate was increased to 1.5 cm / sec and maintained.

[0098] In this experiment, cell density steadily increased in the bioreactor from days 8 to 10. On day 11, the linear flow rate was increased to 1.5 cm / sec, resuming cell growth and reaching 1.9 x 10 cells by day 12. 5 cells / cm 2 The maximum cell density was reached (more than two population cell doublings).

[0099] Infection of bioreactors on day 14 (MOI = 0.13, 1.72 x 10 8 pfu) resulted in maximum virus amplification on day 1 postinfection. Virus bound almost exclusively to the carrier. Virus decreased on the carrier on days 4 and 5 postinfection, corresponding to an increase in virus in the culture medium.

[0100] Nano Experiment 5 For nano experiment 5, CV-1 cells at passage 58 were used directly from cryopreservation. Cells were grown from the same working cell bank as the CV-1 cells used in the previous experiment.

[0101] In nano experiment 5, 4.5x10 4 cells / cm 2 4.7x10 4 cells / cm 2 The cells were allowed to attach (104% seeding efficiency). The volume of the culture medium was increased to 2286 mL, and the linear flow rate was set to 0.44 cm / s. The culture medium was changed once on day 4, and the flow rate was increased to 0.9 cm / s on day 9.

[0102] In this experiment, the cell density reached a maximum of 1.0x10 on day 7. 5 cells / cm 2 The growth rate reached approximately 1.5 population doublings and then decreased. Increasing the flow rate on day 9 did not appreciably increase the growth rate. The cultures were cultured at an MOI of 0.1 (8.26x10) on day 12.7 The cells were infected with GLV-1h68 at 1000 pfu (1000 ng / ml) and the flow rate was increased to 2.5 cm / sec. Virus accumulated on day 1 postinfection but subsequently decreased. Virus bound almost exclusively to the carrier, and no increase in virus was discernible in the culture medium.

[0103] Nano Experiment 6 Nano experiment 6 was performed with CV-1 cells at passage 62, continuing the passage of cells used in experiment 5. The method of experiment 5 was repeated, except that the flow rate was reduced to 0.44 cm / sec 24 hours after seeding and then remained unchanged during the growth phase, with no single change of culture medium until day 6. The reactor was charged with 4.5x10 4 cells / cm 2 5.3x10 4 cells / cm 2 were attached to the reactor (118% seeding efficiency).

[0104] In this experiment, 1.5x10 5 cells / cm 2 was achieved on day 11 (2 population doublings). Cultures were infected with GLV-1h68 on day 12 (MOI = 0.1, 1.40x10 8 pfu), slight virus amplification was detected by day 3 post-infection, with a further peak occurring at day 6 post-infection. Again, very little virus was detected in the culture medium.

[0105] Analysis of CV-1 cell proliferation phase in nano experiments 1-6 The proliferation of CV-1 cells was examined for nano experiments 2 to 6. The respective proliferation curves are shown in Figure 12.

[0106] The growth data from the five experiments were combined to generate an overall growth curve. Figure 13 shows the data fitted to an exponential equation with a time-dependent coefficient of 0.005. The population doubling time (PDT) calculated from the growth curve equation for CV-1 cells grown in the bioreactor was 140 hours.

[0107] Where proliferation data from experiments 2–6 cut off at 150 h after seeding, the PDT was 99 h.

[0108] Analysis of GLV-1h68 infection phase in nano experiments 1-6 Between 24 and 48 hours post-infection, cell density in the bioreactors decreased and continued to decrease throughout the infection period. Except for experiment 1, where a sudden oxidation of the culture occurred at 24 hours post-infection, no significant cell lysis was observed in any of the bioreactor experiments with properly controlled pH. Except for experiment 6, no decrease was observed at 24 hours post-infection.

[0109] Virus production in the iCellis® bioreactor was assessed by sampling the carriers and medium from the reactor every 24 hours post-infection. The virus titer in the medium was measured directly by viral plaque assay (VPA), while the carriers were subjected to freeze / thawing and then titrated in fresh medium. The results showed that virus accumulated in the bioreactor by 72 hours post-infection and then declined. Based on this analysis, the target time for virus harvest from the bioreactor was 72 hours post-infection.

[0110] GLV-1h68 production in iCellis® bioreactors Three sequential bioreactors were performed (nano experiments 7, 8, and 9). Experiments 7 and 8 were derived from CV-1 cells serially grown from the same working cell bank (WCB) stock vial, but at later linear passages. Experiment 9 was grown from the same WCB but from a different vial. Experiments 7, 8, and 9 also differed slightly in the linear flow rates during the growth phase (0.44, 0.56, and 0.67 cm / sec, respectively). Cell growth in experiments 7, 8, and 9 reached slightly different endpoints before infection (1.6x10 cells, respectively). 5 , 1.8x10 5 , 1.5x10 5 cells / cm 2 Experiments 7, 8, and 9 were performed with GLV-1h68 infection at an MOI of 0.2 (3.1x10 for experiments 7, 8, and 9, respectively). 8, 3.7x10 8 , and 3.1x10 8 pfu), and was grown for 72 hours post-infection, which differed from previous nano experiments 1-6. Finally, sampling of the carriers was minimized to minimize disturbance to the culture and reduce carrier loss from the bioreactor.

[0111] The growth characteristics of CV-1 cells in bioreactor runs 7, 8, and 9 are shown in Figure 28. The reactors were each filled with 4.5x10 4 cells / cm 2 The cells in experiments 7, 8, and 9 were at passages 65, 77, and 59, respectively. After 24 hours, experiment 7 had 4.6 x 10 4 cells / cm 2 (102% seeding efficiency), and experiment 8 was 5.8x10 4 cells / cm 2 (127% seeding efficiency), and experiment 9 was 4.4x10 4 cells / cm 2 The proliferation rate in Experiment 8 was higher than in Experiments 7 and 9. Experiment 8 had 1.8 x 10 cells on Day 8, before the planned medium change. 5 cells / cm 2 In Experiments 7 and 9, the infection density reached 1.6 x 10 on Day 13, and therefore the culture medium was not changed. The culture medium for Experiments 7 and 9 was changed on Days 10 and 12, respectively. In Experiments 7 and 9, the infection density reached 1.6 x 10 on Day 13, and therefore the culture medium was not changed. 5 cells / cm 2 and 1.5x10 on day 15 5 cells / cm 2 The calculated population doubling time (PDT) for Experiment 8 was 87 hours.

[0112] Example 3 Development of a method for extracting viruses from bioreactor carriers The collected results of various virus extraction methods showed that the method significantly affected the results (Figure 24). Varying the extraction method resulted in approximately 60-fold differences in virus recovery efficiency. The effects of time, temperature, and agitation on virus extraction from carriers were evaluated. When cells were lysed by hypotonic shock with 1 mM Tris (pH 9.0) and freeze / thawing, virus yields were approximately 3 PFU / cell. Microscopic examination of cells on carriers showed that the cells were lysed but virus was not released. Virus remained bound to the carriers either directly by binding to the carrier surface or indirectly by binding to cellular components. Microscopic examination of stained carriers after treatment showed that most cellular debris remained on the carriers.

[0113] Trypsin treatment was tested for virus extraction from bioreactor carriers (post-lysis carriers) that had undergone lysis by hypotonic shock. Trial 1 compared the effectiveness of extraction media: PBS, trypsin / EDTA (porcine trypsin), TrypLE (recombinant trypsin), or water. Additionally, the effects of freeze / thaw and sonication were evaluated. Results indicated that each extraction method was successful, with the most efficient extraction achieved with TrypLE, and that freeze / thaw alone or freeze / thaw and sonication were additionally beneficial. Virus extraction yielded 7.2 PFU / cell. Using freeze / thaw and sonication, 3.3 PFU / cell was extracted into PBS. Trial 2 repeated the comparison with TrypLE, with and without freeze / thaw and sonication. 7.0 PFU / cell was extracted with TrypLE and freeze / thaw and sonication.

[0114] In trial 3, trypsin / EDTA, TrypLE, and 1mM Tris (pH 9.0) were compared for extraction of bioreactor carriers without pre-lysis (pre-lysis carriers) with and without freeze / thaw and sonication. TrypLE and freeze / thaw and sonication extracted 9.1 PFU / cell.

[0115] The evaluation was repeated with carriers obtained from bioreactors at 24, 48, and 72 hours postinfection (hpi) that were not lysed but were frozen and thawed. Trial 4 compared extraction of 72 hpi carriers with PBS, trypsin / EDTA, and TrypLE with or without freeze / thaw and sonication (Figure 25). Trypsin / EDTA yielded 19 PFU / cell, and TrypLE yielded 60 PFU / cell. All of these results were obtained with freeze / thaw and sonication of the carriers after extraction. With freeze / thaw alone, PBS yielded 2.7 PFU / cell, trypsin / EDTA yielded 17.5 PFU / cell, and TrypLE yielded 40.4 PFU / cell. The carriers used in Trial 4 were extracted twice under the same conditions. In this case, TrypLE, TrypLE with freeze / thaw, and TrypLE with freeze / thaw and sonication each extracted less than 1.3 PFU / cell.

[0116] Extraction of carriers sampled at 48 hpi yielded less virus than carriers sampled at 72 hpi. TrypLE alone, TrypLE with freeze / thaw, and TrypLE with freeze / thaw and sonication extracted 12.2, 25.1, and 22.1 PFU / cell, respectively. For carriers sampled at 24 h, the three methods extracted 13.7, 18.3, and 19.8 PFU / cell, respectively.

[0117] Virus aliquots of known titers were incubated in TrypLE at 21°C or 37°C for 5, 10, 15, 30, 45, or 60 minutes. There was no significant decrease in virus titer at either temperature. In all cases, virus titer increased with incubation in TrypLE.

[0118] Example 4 Virus infection, amplification, and collection Increased contact of the reactor substrate with the harvest solution was achieved by increasing the time or volume.

[0119] In experiments 7, 8, and 9, GLV-1h68 was used to infect 600 mL of infection medium at an MOI of 0.2 to achieve rapid and efficient infection of cells. The growth phase was set to 72 hours for all three experiments. This was achieved as indicated by the rapid decrease in cell density immediately after infection (Figure 27). 72 hours after infection, the reactors were harvested by draining the medium and rinsing with PBS. The bioreactor in experiment 7 was harvested immediately. The bioreactors in experiments 8 and 9 were frozen and processed later. TrypLE solution was added to the reactor in PBS and circulated by agitation. The harvest volume was collected, and the reactor was rinsed with an additional volume. The harvested and rinsed volumes were sampled and analyzed for virus content by VPA.

[0120] In Experiment 7, the cells were treated with 300 mL of 1xTrypLE at 21°C for 45 minutes, followed by harvesting with nine successive rinses of 300 or 500 mL of 10 mM Tris-HCl (pH 9.0). The reactor was not frozen prior to harvesting. Analysis of the virus content of the harvested and rinsed fractions indicated that virus was continuously harvested from the bioreactor throughout the process. The third rinsed fraction contained the highest virus content, accounting for only 16% of the total virus collected. In fact, even the final rinsed fraction accounted for 4% of the total virus collected. 1.3x10 10 pfu of GLV-1h68 were recovered. Table 1: Virus collection for nano experiment 7 [Table 3]

[0121] Run 8 was harvested after a bioreactor freeze / thaw. It was treated with TrypLE (prepared by diluting 10x TrypLE in PBS to 300 mL) for 45 minutes at 21°C, followed by 10 consecutive rinses with 300 mL of 10 mM Tris-HCl (pH 9.0). The first five rinses were performed with continuous maximum agitation, while the remaining rinses used multiple intermittent bursts of agitation. Compared to Run 7, the second rinse fraction had the highest virus content, accounting for 20% of the total virus collected. Successive rinse fractions contained evaluable virus, with the final rinse fraction still accounting for 5% of the total virus collected. The total virus collected was 1.7x10 10 The pfu GLV-1h68. Table 2: Virus collection for nano experiment 8 [Table 4]

[0122] In experiment 9, harvesting was performed using 500 mL of TrypLE (prepared by diluting 10x TrypLE in PBS) after freeze / thawing for 60 minutes at 37°C, followed by two rinses with 500 mL each of 10 mM Tris-HCl (pH 9.0), two rinses with PBS, and finally two rinses with PBS containing 1 M NaCl. Maximum continuous agitation was used throughout. The second rinse with 10 mM Tris-HCl (pH 9.0) again contained the highest virus content, but the relative yield of the total harvest was higher (32%) than the previous harvest. The TrypLE harvest fraction and the first rinse fraction also contained significantly more virus (27 and 29%, respectively). Subsequent rinses with PBS contained lower virus content (9.4 and 1.9%), while the final rinse with PBS containing 1 M NaCl contained significantly less virus (0.5 and 0.2%). The total virus collected from experiment 9 was 7.3 x 10 10 It was PFU.

[0123] Reducing the total volume of virus harvest can facilitate downstream processing steps, so improving the efficiency of virus release at each step is beneficial.

[0124] Carriers were sampled from the bioreactor after harvest, extracted, and tested by VPA. 6 PFU / carrier was 4.5x10 for the whole bioreactor at harvest containing 914 carriers. 9 A residual post-harvest virus content of 6% of the total virus harvested or PFU was demonstrated. Thus, virus recovery during the harvest process was estimated to be at least 94%. 9 cells were contained in the bioreactor at the time of infection. Therefore, the specific viral productivity was [7.3x10 10 +(7.3x10 10 x0.06)]PFU / 1.5x10 9 cells = 52 PFU / cell Table 3: Virus collection for nano experiment 9 [Table 5]

[0125] Example 5 Purification of GLV-1h68 from iCellis® Bioreactor Harvest Five bioreactors (nano experiments 7, 8, 9, 10, and 11) were run to evaluate a virus purification process using tangential flow (i.e., cross-flow) ultrafiltration. The virus harvest from nano experiment 7 (3,500 mL total) was frozen in aliquots at -20°C. For each experiment, an aliquot of the virus harvest was thawed at 4°C and / or room temperature immediately prior to use. In other experiments, the virus harvest was directly purified by UF / DF without prior freezing.

[0126] Nanoexperiment 8 virus collection UF / DF purified using Centramate LV300 kilodalton cutoff (kdco) filters Virus was harvested from a 3.5 L Nano Experimental 8 bioreactor. Ultrafiltration was performed using a Centramate LV300kDCO filter at 9.1 L / min / mL until the retentate volume was reduced to 900 mL. 2 The retentate was stored overnight at -20°C. UF was resumed until the retentate volume was reduced to 500 mL. Diafiltration was performed by adding seven successive diavolumes of PBS. The final retentate and filter wash totaled 300 mL. The viral titer of the samples was determined by VPA, and the protein and DNA content was determined by protein and DNA assays.

[0127] The results showed that the virus recovery after ultrafiltration was 80% and after diafiltration was 36%. The protein content of the UF retentate was 21.3 mg / 10 9 pfu, and the DNA content was 455 μg / 10 9 After diafiltration, the protein content of the DF retentate was 9.0 mg / 10 pfu. 9 pfu (a 2.4-fold decrease), and the DNA content was 17 μg / 10 9 pfu (26-fold). Table 4: Nano Experiment 8 virus harvest UF / DF purified using Centramate LV300kdco filters [Table 6] Table 5: Protein and DNA analysis of Nano Experiment 8 virus harvest UF / DF purified using Centramate LV300kDCO filters [Table 7]

[0128] Nano Experiment 7 virus harvest purified by UF / DF using Centramate LV300kdco filter A 1000 mL aliquot of the virus harvest from the Nano Experiment 7 bioreactor was concentrated using a Centramate LV300kDCO filter. The UF retentate (200 mL) was then diafiltered first against 10 volumes (2000 mL) of PBS, followed by 10 volumes (2000 mL) of low ionic strength, high pH buffer (10 mM Tris-Cl, pH 9.0).

[0129] After initial concentration of the virus harvest from 1000 mL to 200 mL, virus recovery was 67%. After diafiltration against PBS, virus recovery was 16%. After subsequent diafiltration against 10 mM Tris-Cl (pH 9.0), virus recovery was 81%. Table 6: Nano Experiment 7 virus harvest UF / DF purified using Centramate LV300kdco filters [Table 8] 1 Not detected.

[0130] Nano Experiment 9 virus collection purified by UF / DF using Centramate LV300kdco filters Virus was collected from a 3.5 L Nano Experiment 9 bioreactor. Ultrafiltration was performed using a Centramate LV300kdco filter at an initial flow rate of 4.4 L / min / min. 2 Diafiltration was performed using PBS (200 mL each) until the retentate volume was reduced to 200 mL. Diafiltration was performed by adding five serial diafiltration volumes of PBS (200 mL each), followed by five serial diafiltration volumes of 10 mM Tris-Cl (pH 9.0) (200 mL each). Samples of the UF and DF retentate and permeate fractions were tested for viral titer by VPA and for protein and DNA content by protein and DNA assays.

[0131] The results show that the virus recovery in the retentate after ultrafiltration was 88% and after diafiltration against PBS was 67%. After diafiltration against 10 mM Tris-Cl (pH 9.0), the recovery in the retentate fraction was 107%. The protein content in the UF retentate was 1.3 mg / 10 9 pfu, and the DNA content was 20 μg / 10 9 After diafiltration, the protein content of the DF retentate was 0.6 mg / 10 pfu. 9 pfu (a 1.4-fold decrease), and the DNA content was 16 μg / 10 9 pfu (0.8-fold). Table 7: Nano Experiment 9 virus collection UF / DF purified using Centramate LV300kDCO filters [Table 9] Table 8: Protein and DNA analysis of Nano Experiment 9 virus UF / DF purified using Centramate LV300kDCO filters [Table 10]

[0132] Virus harvest and purification by UF / DF using MidiKros hollow fiber filter cartridges Centramate LV uses flat membrane filters. MidiKros filters are cartridges composed of hollow fibers with porosities of 500 kDa and 750 kDa. UF / DF purification of virus harvests was measured using these filter cartridges to examine the effects of increasing filter geometry and porosity. An aliquot of virus harvest from Nano Experiment 7, stored at -20°C, was thawed and used for UF / DF purification. A 500 mL virus harvest was concentrated to a volume of 100 mL using a MidiKros 500 kDa hollow fiber filter and then diafiltered against 10 diavolumes of 10 mM Tris-Cl (pH 9.0) (CFF12). Starting samples, UF and DF permeate and retentate fractions were tested for titer by VPA and protein and DNA content. Results showed a virus recovery of 79% after UF and 68% after DF. Virus was not detected in either the UF or DF permeate fractions. Similarly, 500 mL of virus harvest was purified by UF / DF using a MidiKros 750kDCO hollow fiber filter (CFF13). Virus recovery was 66% after UF and 43% after DF. The protein and DNA content of the starting material was 5.1 mg / 10, respectively. 9 pfu and 400 μg / 10 9 After UF / DF, the protein and DNA contents of the retentate of the MidiKros 500kDCO filter were 1.3 mg / 10 pfu, respectively. 9 pfu and 90 μg / 10 9 pfu (a 4.0-fold and 4.5-fold reduction, respectively). After UF / DF, the protein and DNA contents of the retentate of the MidiKros 750kDCO filter were 1.9 mg / 10 9 pfu and 63 μg / 10 9 pfu (2.7-fold and 6.4-fold reductions, respectively). Table 9: Nano Experiment 7 virus harvest UF / DF purified using MidiKros 500kDCO hollow fiber filters [Table 11] 1 Not detected. Table 10: Nano Experiment 7 virus harvest UF / DF purified using MidiKros 750kDCO hollow fiber filters [Table 12] 1 Not detected. Table 11: Protein and DNA analysis of Nano Experiment 7 virus harvest UF / DF purified on MidiKros 500kDCO and 750kDCO hollow fiber filters [Table 13]

[0133] Nanoexperiment 10 virus collection purified by UF / DF using MidiKros 750kdco hollow fiber filters The virus harvest from nano experiment 10 was purified by UF / DF using a MidiKros 750kDCO hollow fiber filter cartridge. 3.5 L of virus harvest was concentrated to 200 mL by ultrafiltration, followed by diafiltration against 10 diavolumes of 10 mM Tris-Cl, pH 9.0 (200 mL each). Samples of the permeate and retentate fractions were tested for virus titer by VPA and protein and DNA content. Virus recovery after UF concentration was 112% and after diafiltration was 97%. The protein content of the UF starting material was 4.0 mg / 10 9 pfu, and the DNA content was 166 μg / 10 9 After diafiltration, the retentate was 1.0 mg / 10 pfu. 9 pfu (a 4-fold reduction relative to the starting material) and the DNA content was 15 μg / 10 9 pfu (11.1-fold reduction relative to the starting material). Table 12: Nano Experiment 10 Virus Harvest Purified by UF / DF Using MidiKros 750kDCO Hollow Fiber Filters [Table 14] 1 Not detected. Table 13: Protein and DNA analysis of Nano Experiment 10 virus harvest purified by UF / DF using MidiKros 750kDCO hollow fiber filters [Table 15]

[0134] Nanoexperiment 11 virus collection purified by UF / DF using MidiKros 500kDCO hollow fiber filters The virus harvest from Nano experiment 11 was purified by UF / DF using a MidiKros 500kDCO hollow fiber filter cartridge. 2.5 L of virus harvest was concentrated to 200 mL by ultrafiltration, followed by diafiltration against 10 diavolumes of 10 mM Tris-Cl, pH 9.0 (200 mL each). Samples of the permeate and retentate fractions were tested for virus titer by VPA and protein and DNA content. Virus recovery after UF concentration was 147% and after diafiltration was 105%. The protein content of the UF starting material was 5.1 mg / 10 9 pfu, and the DNA content was 166 μg / 10 9 After ultrafiltration concentration, the retentate was 1.1 mg / 10 pfu. 9 pfu (4.9-fold reduction compared to the starting material) and the DNA content was 22 μg / 10 9 pfu (a 7.4-fold reduction relative to the starting material). After diafiltration, the retentate was 1.4 mg / 10 9 pfu (a 3.8-fold reduction relative to the starting material), and the DNA content was 25 μg / 10 9 pfu (a 6.5-fold reduction relative to the starting material). Table 14: Nano Experiment 11 Virus Harvest Purified by UF / DF Using MidiKros 500kDCO Hollow Fiber Filters [Table 16] 1 Not detected. Table 15: Protein and DNA analysis of Nano Experiment 11 virus harvest purified by UF / DF using MidiKros 500kDCO hollow fiber filters [Table 17]

[0135] Nanoexperiment 12 virus collection purified by UF / DF using MidiKros 500kDCO hollow fiber filters Virus was harvested from nano experiment 12 by thawing the frozen bioreactor by adding 500 mL of 10 mM Tris-HCl, 2 mM MgCl2 (pH 9.0). Benzonase® (recombinant Benzonase®, Speed BioSystems, Inc.) was added at 100 U / mL and incubated at 37°C for 60 minutes with agitation. The Benzonase® nuclease digestion medium was removed and replaced with 500 mL of PBS (containing 1x TrypLE) and further incubated at 37°C for 60 minutes with agitation. The TrypLE harvest medium was removed and the bioreactor was flushed twice with 500 mL of 10 mM Tris-HCl (pH 9.0) at 37°C for 10 minutes each with agitation. The TrypLE harvest and the two flushes were combined as the virus harvest (1.5 L). Analysis of virus titer by VPA showed that the TrypLE virus harvest fraction contained 80.1% of the virus, the first 10 mM Tris-HCl effluent contained 16.3%, and the second effluent contained 2.4%. Thus, 98.8% of the virus was released from the bioreactor and collected in the virus harvest. The Benzonase® nuclease digestion fraction contained 0.4% of the virus, and the post-harvest macrocarriers contained less than 0.1%.

[0136] The virus harvest was purified by UF / DF using MidiKros 500kDCO hollow fiber filter cartridges. 1.5 L of virus harvest was concentrated to 200 mL by ultrafiltration, followed by diafiltration against 10 diavolumes of 10 mM Tris-Cl, pH 9.0 (200 mL each). Samples of the permeate and retentate fractions were tested for virus titer by VPA and protein and DNA content. Virus recovery after UF concentration was 45% and after diafiltration was 123%. The protein content of the UF starting material was 4.3 mg / 10 9 pfu, and the DNA content was 38 μg / 10 9 After ultrafiltration concentration, the retentate was 4.0 mg / 10 pfu. 9 pfu (a 1.1-fold reduction relative to the starting material) and the DNA content was 5 μg / 10 9 pfu (8.7-fold reduction relative to the starting material). After diafiltration, the retentate was 1.6 mg / 10 9 pfu (a 2.7-fold reduction relative to the starting material) and the DNA content was 1 μg / 10 9 pfu (42-fold reduction relative to the starting material). Table 16: Nano Experiment 12 Virus Harvest Purified by UF / DF Using MidiKros 500kDCO Hollow Fiber Filters [Table 18] 1 Not detected. Table 17: Protein and DNA analysis of Nano Experiment 12 virus collection purified by UF / DF using MidiKros 500kDCO hollow fiber filters [Table 19]

[0137] Example 6 Comparison with conventional methods The virus was purified by several methods used in the art and the results were compared. One process, called the "CEF" process, produces virus from primary cells obtained from chicken eggs, grown in suspension culture, and infected with the virus. The cells are harvested by centrifugation, lysed by homogenization, purified by filtration, and then subjected to two rounds of sucrose gradient centrifugation, followed by formulation and packing / finalization.

[0138] "Process A" involves producing virus in the continuous cell line CV-1 by growing in roller bottles (adherent culture), infecting the cells, dissociating them from the substrate with trypsin, and harvesting them by centrifugation. Cells are lysed by freeze / thawing, and virus is produced from whole cells by affinity chromatography, then concentrated by centrifugation, resuspended in formulation buffer, and packed / finalized.

[0139] "Process B" is the process described herein. CV-1 cells are grown in a fixed-bed bioreactor (adherent culture), infected with virus, and the medium is drained from the reactor and frozen. The lysed adherent cells in the bioreactor are treated with Benzonase® nuclease followed by trypsin to release the virus and dissociate the cell debris adhered to the matrix in the bioreactor. The virus is produced, purified by ultrafiltration and diafiltration, formulated, and packaged / finalized.

[0140] The table below provides a step-by-step comparison of various conventional processes with the process described herein (referred to as "Process B"), which requires significantly fewer steps and results in a higher virus yield. Table 18 [Table 20] Table 19 (Comparing post-lysis steps of the process herein with prior art Process A) [Table 21] Table 20 (showing higher yields with the process herein) [Table 22]

[0141] Example 7 333m according to the method of this specification 2 Exemplary Large-Scale Process for Producing Vaccinia Virus in a Bioreactor Such as an iCellis® 500 Bioreactor Table 21: [Table 23]

[0142] *** Since modifications will be apparent to those of skill in this art, it is intended that this invention be limited only by the scope of the claims.

Claims

1. 1. A method for producing purified vaccinia virus, comprising: a) culturing host cells containing vaccinia virus in a bioreactor, said bioreactor comprising a matrix for growing adherent cells; the matrix is ​​biocompatible and has a cell-entrapping density or a cell-adherent density, such that cells are entrapped and / or attached to the matrix; the density of the matrix is ​​such that the cells remain trapped and / or the matrix remains attached under conditions in which the cells are lysed and treated to release the virus, and the flow of treated culture medium through the matrix is ​​sufficient for cell proliferation; b) after culturing, treating the cells entrapped and / or attached to the matrix to lyse them and release the virus into the treated culture medium in the bioreactor, the lysed cells remaining attached and / or entrapped to the matrix; and c) purifying the released viruses from said treated culture medium in only one or two steps, without any further treatment of said treated culture medium to remove cells or cell debris, the one step is ultrafiltration or diafiltration; and the two steps being ultrafiltration and diafiltration; A method comprising:

2. The method of claim 1 , wherein the host cell is an adherent cell.

3. 3. The method according to claim 1 or 2, wherein the medium in the bioreactor in step c) is removed from the bioreactor for purifying the virus; and then appropriately stored.

4. The method of any of claims 1 to 3, wherein the purification of step c) of the virus is achieved in one day or less.

5. The method according to any one of claims 1 to 4, comprising steps a), b), and c), wherein steps a) and b) are carried out in said bioreactor.

6. 5. The method of any one of claims 1 to 4, consisting essentially of steps a), b) and c), wherein the lysis step b) comprises treatment with proteases and / or nucleases to release the viruses from trapped and / or attached cells.

7. 5. The method of any one of claims 1 to 4, comprising steps a), b) and c), wherein the lysis step b) comprises treatment with a protease and / or a nuclease to release the virus from the trapped and / or attached cells.

8. The method of any one of claims 1 to 7, wherein the cells are cells of a single cell line.

9. The method according to any one of claims 1 to 8, wherein the cells are cells of a single cell line of adherent cells.

10. 10. The method of any one of claims 1 to 9, wherein the oncolytic vaccinia virus is selected from the group consisting of Lister, Western Reserve (WR), Copenhagen (Cop), Bern, Paris, Tashkent, Tiantan, Wyeth (DRYVAX), IHD-J, IHD-W, Brighton, Ankara, CVA382, JX-594, Modified Vaccinia Ankara (MVA), Dalian I, LC16m8, LC16m0, LIVP, ACAM2000, WR65-16, Connaught, New York City Department of Health (NYCBH), EM-63, and NYVAC strains.

11. The method according to any one of claims 1 to 11, wherein the vaccinia virus is a Lister strain virus.

12. 12. The method of claim 11, wherein the vaccinia virus is an LIVP virus or a clonal strain of an LIVP virus.

13. 13. The method of claim 12, wherein the virus is a LIVP virus designated GLV-1h68.

14. The method of any one of claims 1 to 13, wherein the virus is in a modified form that contains a nucleic acid encoding a heterologous gene product.

15. 13. The method of claim 12, wherein the heterologous gene product is a therapeutic or reporter gene product.

16. The method according to any one of claims 1 to 15, wherein the cells are mammalian cells.

17. The method of claim 16, wherein the mammalian cell is a CV-1 cell.

18. 17. The method of claim 16, wherein the mammalian cells are Vero cells.

19. 17. The method of claim 16, wherein the mammalian cell is a human cell.

20. 17. The method of claim 16, wherein the mammalian cells are human fibroblasts.

21. 17. The method of claim 16, wherein the mammalian cells are human epithelial cells.

22. 17. The method of claim 16, wherein the mammalian cells are human endothelial cells.

23. The method of any of claims 1 to 22, wherein the bioreactor comprises a matrix or surface to which adherent cells adhere.

24. 24. The method of claim 23, wherein the matrix in the bioreactor comprises a non-fixed adhesive surface.

25. 25. The method of claim 24, wherein the surface is a macrocarrier bead in suspension, a fiber, or a woven mesh.

26. 24. The method of claim 23, wherein the bioreactor comprises a fixed adhesive surface.

27. 27. The method of claim 26, wherein the bioreactor is a packed bed bioreactor.

28. 28. The method of any of claims 1-27, wherein treating the cells to lyse and release the virus comprises exposing the bioreactor to freeze / thaw.

29. 28. The method of any of claims 1-27, wherein treating the cells to lyse and release the virus comprises exposing the cells to a hypotonic medium.

30. 28. The method of any preceding claim, wherein treating the cells to lyse and release the virus comprises exposing the cells to a detergent.

31. 28. The method of any preceding claim, wherein treating the cells to lyse and release the virus comprises exposing the cells to an enzyme.

32. 32. The method of claim 31 , wherein the enzyme is a protease.

33. 32. The method of claim 31 , wherein the enzyme is a nuclease.

34. 32. The method of claim 31 , wherein treating the cells to lyse and release the virus comprises exposing the cells to a nuclease and a protease, either sequentially or in combination.

35. The method according to any one of claims 1 to 34, wherein the ultrafiltration uses a membrane having a nominal molecular weight cut-off of 300 to 750 kilodaltons or a nominal porosity of 0.05 to 0.2 µm.

36. The method of any one of claims 1 to 35, wherein the ultrafiltration uses a membrane comprising polyethersulfone.

37. The method according to any one of claims 1 to 36, wherein the ultrafiltration uses a flat membrane.

38. The method according to any one of claims 1 to 36, wherein the ultrafiltration uses a hollow fiber membrane.

39. The method according to any one of claims 1 to 38, wherein the ultrafiltration is carried out in a tangential flow mode.

40. The method according to any one of claims 1 to 38, wherein the ultrafiltration is carried out in a cross-flow manner.

41. The method of any one of claims 1 to 40, wherein ultrafiltration is performed subsequent to diafiltration.

42. 42. The method of any one of claims 1 to 41, wherein the recovery rate of the treated and released virus is 50% or more.

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