Patent Grant
10851350
Provided are methods for virus production from adherent cells.
Viruses are used as therapeutics. Among such uses are as vaccines, gene therapy vectors and virotherapy agents. The manufacture of viruses for these purposes involves the replication of virus in suitable host cells, and then purification of the virus from the host cells. Host cells used for replication of viruses are either grown in anchorage-dependent, adherent culture conditions or in suspension culture conditions. An advantage of the suspension culture is that the cells can be cultured in a single bioreactor in large volume. Some viruses, however, do not replicate well in suspension-cultured cells.
Anchorage-dependent cells, in which viruses replicate well, are commonly cultured in roller bottles, which require a large number of bottles to obtain sufficient amounts. These processes, thus, are difficult and expensive to scale. In either case, whether using adherent or suspension culture conditions, the virus infected cells are harvested from the culture vessel or vessels, either the bioreactor or the roller bottles, and lysed to release the virus. The virus is purified from the total host cell-derived components using numerous and various steps including several or more of homogenization, sonication, centrifugation, filtration, affinity purification, chromatography, and density gradient ultracentrifugation. The culture conditions and the purification steps involved in the manufacture of viruses add complication and cost to the manufacturing processes, and can result in low yields. Hence, simpler and scalable methods for manufacturing viruses are needed.
Provided are simplified scalable methods for the manufacture of viruses in high yield. The methods employ virus-infected anchorage-dependent cells. In accord with the methods, the virus is released from the host cells while the host cell components remain substantially attached to the culture surface. Host cell-derived components remain with cells remnants, while the virus is released into the cell culture medium, and the virus is purified by a simplified, cost-effective single step process of ultrafiltration or diafiltration or a two-step process of ultrafiltration and diafiltration. The purification process is high yield, and can be performed in a day or less. The process can be performed with cells grown in a bioreactor that is suitable for culturing anchorage-dependent cells, thereby combining the simplified purification methods with the advantages of bioreactor scale-up. Any virus, particularly enveloped viruses, which can infect and grow in cells that can be grown in an adherent format can be manufactured by these methods.
Among the methods/processes are those for producing viruses from adherent cells cultured in bioreactors. The methods involve releasing the virus from adherent host cells in a bioreactor, and purifying the released virus by ultrafiltration and/or diafiltration. Described herein are various examples and aspects of such methods.
Provided are methods (processes) for producing a virus. The methods include the steps of: a) culturing host cells, comprising a virus, in a bioreactor, wherein the bioreactor comprises a matrix for growing adherent cells or cells entrapped therein, wherein the matrix is biocompatible; the cells are entrapped in and/or adhere to the matrix; and the density of the matrix is such that the cells remain attached under conditions in which 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 them and release the virus into the medium in the bioreactor; and then c) without further treatment, and in only one or only two steps, purifying the released virus from the cell culture medium. The one step is ultrafiltration or diafiltration; and the two steps are ultrafiltration and diafiltration. No other purification steps are employed. Thus, purification after lysing and releasing the virus from the cells, is effected only by ultrafiltration and/or diafiltration. Purification is achieved in only the one or the two steps. The purification can be effected in 1 day or less.
The bioreactor contains a matrix or surface onto which adherent cells attach, and suspension cells can be entrapped. The matrix (or macrocarrier or substrate or surface) can be a non-fixed attachment surface. The matrix or surface can be selected from among, but not limited to microcarrier beads, fibers, or woven mesh in suspension. The matrix or surface can be a fixed attachment surface. The bioreactor can be, for example, a packed bed bioreactor. Other configurations can be selected by the skilled artisan, as long as the bioreactor contains a matrix or surface for retaining the cells during growth and lysis.
The host cells are cells that adhere to the matrix or that are entrapped in the matrix, including cells that normally grow in suspension, so that upon processing the cells and medium, the cells are not released. In general, the host cells are adherent cells. The cells can be primary cells, or cell lines. The particular cells are those suitable for growing a particular virus. Among the cell lines are known cell lines, such as CV-1 cells, KB cells, Vero cells, CHO cells and others. Cells include, but are not limited to, mammalian cells, including human and other primate cells; human cells include, for example, human fibroblast cells, epithelial cells and endothelial cells.
As exemplified herein, the exemplified cells are CV-1 cells, and the virus is a vaccinia virus, such as a therapeutic vaccinia virus. Prior to purification, the cell process medium can be harvested. Optionally, the medium can be stored. The methods for producing the virus can include only steps a), b) and c), above, or can include additional steps prior to purification, which only includes the one or two steps of ultrafiltration and/or diafiltration.
Releasing of the virus includes treatments, such as freeze/thaw and/or treatment with hypotonic medium and/or treatment with detergent, to lyse the cells. The virus is released into the medium from lysed cells by treatment with a protease and/or nuclease. The protease generally is a non-specific protease, such as a digestive enzyme, such as trypsin. The nuclease is a DNase or RNase, such as the endonuclease from Serratia marcescens, sold under the trademark Benzonase®. Treatment with nuclease is optional. The lysis and enzyme treatments can be performed sequentially or together. Lysis should precede or be conducted with the protease or nuclease treatment. The protease and nuclease treatments can be effected together or in any order.
The virus can be any virus of interest, such as therapeutic viruses, including oncolytic viruses, and vaccines, and gene therapy vectors, and viruses for delivery of gene products. Generally, since purification is effected with released virus, and release can include nuclease treatment, the viruses are enveloped viruses. Viruses include, but are not limited to, poxviruses, such as a vaccinia virus, myxoma virus, measles virus, reovirus, vesicular stomatitis virus (VSV), adenoviruses, adeno-associated virus, poliovirus, herpes viruses, Sindbis virus and Seneca Valley virus, ora derivative thereof that is modified to contain nucleic acid encoding a heterologous gene product. The virus can be an oncolytic virus, such as vaccinia viruses (e.g., GL-ONC1, Pexa-Vec, vvDD, JX-929, and WO-12), vesicular stomatitis viruses (e.g., VSV-IFNbeta-NIS, VSV-E6/7, VSV-GFP), measles viruses (e.g., MV-NIS, MV-Edm, MV-NPL), seneca valley viruses (e.g., SVV-001 and NTX-010), reovirus (e.g., Reolysin), adenoviruses (e.g., CGTG-102, Oncos-102, NG-348, NG-350, NG-347, NGaFAB, NG-aEpCAM, ONYX-015, CG7870, VCN-01, LOAd703, Ad5, Ad3/5, CRAd-CXCR4-5/3, OvAdl, dI1520), parvovirus (e.g., H1-PV), marabaviruses (e.g., MG1MA3, MG1-HPV, and MB1-Neoantigen), new castle disease virus (e.g., NDV-HUJ), retrovirus (e.g., Toca511), coxsackievirus (e.g., CAVATAK), and herpes viruses (e.g., HSV-1716, NV1020, Imlygic (T-Vec), ONCR-1/-2, and HSV-1716). The viruses can be attenuated and/or modified. Oncolytic vaccinia viruses include, but are not limited to, Lister, such as LIVP strains and clonal strains thereof, Western Reserve (WR), Copenhagen (Cop), Bern, Paris, Tashkent, Tian Tan, Wyeth (DRYVAX), IHD-J, IHD-W, Brighton, Ankara, CVA382, Modified Vaccinia Ankara (MVA), Dairen I, LC16m8, LC16M0, LIVP, ACAM2000, WR 65-16, Connaught, New York City Board of Health (NYCBH), EM-63 NYVAC strain, and the modified Wyeth strain, JX-594. Exemplary of oncolytic LIVP strains of virus is the strain designated GLV-1h68 (also referred to as GL-ONC1). Included among the viruses are modified viruses that encoded a heterologous gene product(s), including therapeutic products, and reporter genes and other detectable markers.
The virus is purified by ultrafiltration and/or diafiltration. The skilled person can select the appropriate form of membrane and mode of ultrafiltration. For example, ultrafiltration can employ, for example a membrane having a nominal molecular weight cut off of between 300 and 750 kilodaltons or a nominal porosity of between 0.05 and 0.2 μm. It can employ a membrane that contains polyethersulfone. The member can be flat, or can be a hollow fiber membrane. The ultrafiltration can be performed in a tangential flow mode, in a cross flow mode, or other mode selected by the skilled artisan. Ultrafiltration can be followed by diafiltration, or diafiltration can be performed without ultrafiltration. Recovery of the processed released virus is greater than 50%, and can be as high as 90%, 95% or more.
This application includes at least one drawing executed in color. Copies of any patent or any publication, based on this application, with color drawing(s), will be provided by the Office upon request and payment of the requisite fee.
The above-identified figures are provided by way of representation and not limitation.
Outline
A. Definitions
B. Overview. C. Bioreactor and matrix
D. Cells and growth
E. Virus and inoculation
F. Cell Lysis and release of virus into the process medium
G. Diafiltration and ultrafiltration of the process medium to purify virus
H. Examples
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the invention(s) belong. All patents, patent applications, published applications and publications, Genbank® sequences, databases, websites and other published materials referred to throughout the entire disclosure herein, unless noted otherwise, are incorporated by reference in their entirety. In the event that there are a plurality of definitions for terms herein, those in this section prevail. Where reference is made to a URL or other such identifier or address, it understood that such identifiers can change and particular information on the Internet can come and go, but equivalent information can be found by searching the internet. Reference thereto evidences the availability and public dissemination of such information.
As used herein, a bioreactor refers to a cell culture device. For use in the methods herein the bioreactor contains a matrix for culturing adherent cells. The matrix is one that retains the cells, such that they are not released into the cell culture medium when lysed. An exemplary bioreactor are those sold under the trademark iCellis® bioreactor. This bioreactor was developed by ATMI Life Sciences and is available from PALL Biosciences.
As used herein, “carrier” or alternatively “substrate” refers to any solid-state material that provides a biocompatible surface onto which adherent cells adhere in culture.
As used herein, matrix type carriers contain polyester fibers, optionally held within a cage (for example, of polypropylene) to immobilize the fibers.
As used herein, process medium refers to the medium in the bioreactor in which the cells are cultured, and virus produced, and the medium used in processing the cells and producing the virus.
As used herein, large-scale manufacture is defined by the single patient dose, and the annual demand for doses. It varies for the virus and treatment; but each large-scale preparation provides about ⅙ to 1/12, such as about 1/10 of the number yearly doses. Hence, for example, for the vaccinia virus, designated GLV-1h68 (GL-ONC1), large scale manufacturing provides an annual production of about 10,000 treatments at a dose of about 6×109 plaque forming units (pfu) per treatment dose or 6×1013 total annual pfu. Assuming 8-12, such as 10 production batches per year, each large scale batch should produce 1,000 treatment doses or about 1011 to 1013, such as about 5-6×1012 pfu. Exemplary pfu and doses of the exemplary virus GLV1h-68 (GLV-ONC1):
As used herein, “virus titer” or “viral titer” refers to the concentration of a virus and is a given number of infectious virus units per volume, such as plaque forming units (pfu)/mL. Virus titer can be determined by serial dilution of a sample for infection of target cells in order to quantify the number of infectious or active virus in a sample. For example, virus titer can be determined using a plaque assay.
As used herein, a “virus” or virus vector refers to any of a large group of infectious entities that cannot grow or replicate without a host cell. Viruses typically contain a protein coat surrounding an RNA or DNA core of genetic material, but no semipermeable membrane, and are capable of growth and multiplication only in living cells. As used herein, oncolytic viruses refer to viruses that replicate selectively in tumor cells in tumorous subjects. Some oncolytic viruses can kill a tumor cell following infection of the tumor cell. For example, an oncolytic virus can cause death of the tumor cell by lysing the tumor cell or inducing cell death of the tumor cell.
As used herein, a therapeutic virus, such as a therapeutic oncolytic virus, is a virus that is used to treat disease or condition. Typically they are not pathogenic or have been rendered non-pathogenic.
As used herein the term “vaccinia virus” or “VACV” or “VV” denotes a large, complex, enveloped virus belonging to the poxvirus family. It has a linear, double-stranded DNA genome approximately 190 kb pairs in length, and which encodes approximately 200 proteins. Vaccinia virus strains include, but are not limited to, strains of, derived from, or modified forms of Western Reserve (WR), Copenhagen, Tashkent, Tian Tan, Lister, Wyeth, IHD-J, and IHD-W, Brighton, Ankara, MVA, Dairen I, LIPV, LC16M8, LC16MO, LIVP, WR 65-16, Connaught, and New York City Board of Health vaccinia virus strains.
As used herein, Lister Strain of the Institute of Viral Preparations (LIVP) or LIVP virus strain refers to a virus strain that is the attenuated Lister strain (ATCC® Catalog No. VR-1549™) that was produced by adaption to calf skin at the Institute of Viral Preparations, 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 Viral Preparations, Moscow, Russia (see. e.g., Kutinova et al. (1995) Vaccine 13:487-493); the Microorganism Collection of FSRI SRC VB Vector (Kozlova et al. (2010) Environ. Sci. Technol. 44:5121-5126); or can be obtained from the Moscow Ivanovsky Institute of Virology (C0355 K0602; Agranovski et al. (2006) Atmospheric Environment 40:3924-3929). It also is well known to those of skill in the art; it was the vaccine strain used for vaccination in the USSR and throughout Asia and India. The strain is well known (see 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).
As used herein, LIVP GLV-1h68 (also designated GL-ONC1; see, e.g., U.S. Pat. No. 7,588,767, and US Patent Publication No. US-2016-0339066-A1) is an LIVP virus that contains ruc-gfp (a luciferase and green fluorescent protein fusion gene (see, e.g., U.S. Pat. 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 a sequence of nucleotides set forth in SEQ ID NO: 3 or a sequence of nucleotides that has at least 97%, 98% or 99% sequence identity to the sequence of nucleotides set forth in SEQ ID NO: 3 of copending published application US-2016-0339066-A1.
As used herein, the term “modified virus” or “recombinant virus,” used interchangeably, refers to a virus that is altered compared to a parental strain of the virus. Typically modified viruses have one or more truncations, mutations, insertions or deletions in the genome of virus. A modified virus can have one or more endogenous viral genes modified and/or one or more intergenic regions modified. Exemplary modified viruses can have one or more heterologous nucleic acid sequences inserted into the genome of the virus. Modified viruses can contain one or more heterologous nucleic acid sequences in the form of a gene expression cassette for the expression of a heterologous gene.
As used herein, a modified LIVP virus strain refers to an LIVP virus that has a genome that is not contained in LIVP, but is a virus that is produced by modification of a genome of a strain derived from LIVP. Typically, the genome of the virus is modified by substitution (replacement), insertion (addition) or deletion (truncation) of nucleotides. Modifications can be made using any method known to one of skill in the art such as genetic engineering and recombinant DNA methods. Hence, a modified virus is a virus that is altered in its genome compared to the genome of a parental virus. Exemplary modified viruses have one or more heterologous nucleic acid sequences inserted into the genome of the virus. Typically, the heterologous nucleic acid contains an open reading frame encoding a heterologous protein. For example, modified viruses herein can contain one or more heterologous nucleic acid sequences in the form of a gene expression cassette for the expression of a heterologous gene.
As used herein, when referencing dosage, such as plaque forming units (pfu)/kg, based on mass kg of the subject, an average human subject is considered to have a mass of about 70 kg-75 kg, such as 70 kg.
For clarity of disclosure, and not by way of limitation, the detailed description is divided into the subsections that follow.
Provided are methods/processes that include two stages: a production stage in which the virus is introduced into cells in which it can replicate, and the cells are cultured under conditions in which the virus is produced. The viruses can be enveloped viruses, including but not limited to, poxviruses, such as vaccinia virus. The cells are then lysed to release the virus, and can be treated with an enzyme or enzymes that are nucleases and/or proteases. In the second stage, the process medium is harvested, and the virus is purified only by ultrafiltration or diafiltration or both. Thus, the second stage is only a one or two step process involving only ultrafiltration and/or diafiltration to produce the purified virus.
The processes, thus, include the steps of growing adherent host cells, generally a cell line, in a bioreactor that contains packed biocompatible woven or fibrous matrix material, such as polyester, polyethylene terephthalate. The matrix is of sufficient density to retain by adherence and/or entrapment the cells and also, cellular debris when the cells are lysed.
The host cells are cultured to an appropriate density, and then inoculated with the virus, and cultured to produce the virus. The cells are lysed, such as by freezing and thawing or exposure to hypotonic medium or both, followed by treatment with an enzyme, such as protease, particularly one that cleaves non-specifically, such as trypsin. The cells optionally are treated with a nuclease before, with or after the protease. The lysed cells are retained by the matrix material.
The virus is purified from the medium in only one or two steps, where one step is the ultrafiltration or diafiltration. If two steps are used, they are ultrafiltration and diafiltration. No additional purification steps, other than ultrafiltration and/or diafiltration are employed.
Hence, provided are scalable processes (also referred to as methods) for producing (also referred to as manufacturing) purified viruses, particularly therapeutic viruses, such as oncolytic viruses, vaccines, and gene therapy vectors. Because the methods can be practiced in a bioreactor, the methods are readily scaled up. Purification, which provides high yields of virus, can be effected in one day or less. The resulting virus is produced in high yield, with as much as 95% or more, generally at least 50%, 60%, 70%, 80%, 90% recovery.
A bioreactor is a vessel suitable for growing cells and contains a packed matrix substrate of woven or non-woven fibers, fabric or strips of fabric whose fibers or fibrous mesh allow cell attachment (or entrapment) and growth. The matrix can be in a fixed or packed bed or in a fluidized bed.
The bioreactor contains a packed matrix of microcarrier beads, woven or non-woven fibers, fabric or strips of fabric whose fibers or fibrous mesh allow cell attachment and growth. The matrix is such that the cells are not detached or removed from the fabric by enzymatic digestion, such as trypsin digestion. The cells are not detached or removed from the fabric by enzymatic digestion such as trypsin. A hollow fiber bioreactor would not work because cells adherent to the inner surface of the hollow fibers would not be trapped in the matrix and the cells would be able to be detached by enzymatic digestion, such as trypsin. A hollow fiber bioreactor is not contemplated because cells adherent to the inner surface of the hollow fibers would not be trapped in the matrix and the cells can be detached by enzymatic digestion, such as trypsin. In accord with the processes herein, cells that are retained by the matrix, such as anchorage-dependent (“adherent”) host cells are grown in the bioreactor; the cells are infected with the virus and grown in the bioreactor on the matrix substrate.
Exemplary of bioreactors, are the bioreactors sold under the trademark iCELLis® (Pall Life Sciences). U.S. Pat. Nos. 8,597,939 and 8,986,979 describe such bioreactors. The iCellis® bioreactor is exemplary of a bioreactor platform that is a scalable line of single-use high cell density bioreactors, allowing small-scale (referred as the “nano” below) to large-scale manufacturing. This bioreactor contains a pre-packed, fixed bed of medical grade polyester microfibers providing a large surface area for growth in a compact bioreactor volume. The main bioreactor is equipped with a built-in magnetic drive impeller that circulates the medium through the fixed bed from the bottom to the top, ensuring low shear stress and high cell viability. At the top of the fixed bed, the medium falls as a thin film down the outer wall where it takes up 02 to maintain dissolved oxygen levels in the bioreactor.
Matrix Materials
Exemplary of matrix materials, are material to which cells can adhere, adherent material, that are biocompatible so that they can be used to culture cells as described herein. Such materials, include, but are not limited to, a polyester, a polypropylene, a polyalkylene, a polyfluorochloroethylene, a polyvinyl chloride, a polystyrene, a polysulfone, a cellulose acetate, a glass fiber, a ceramic particle, and an inert metal fiber. Fibers of these materials can be used, in woven or non-woven form. For example in the bioreactor sold under the trademark iCellis®, and used in the working examples herein, the matrix material is polyethylene terephthalate (PET), a polyester. The matrix can be coated with materials that promote cell attachment such as a Matrigel® cell culture substrate, an extra cellular matrix component (e.g., fibronectin, chondronectin, laminin, ProNectin® F), a collagen, or a poly L lactic acid in order to improve its biocompatibility, its cell adherence or its cell retention.
The density of the matrix is sufficient so that the cells and, after lysis, cell debris remain trapped, but is not too high to impede cell medium flow through the matrix and bioreactor, which is necessary for cells to grow. The density of the matrix is such that the cells remain entrapped in the matrix, but the density is not too high to prevent fluid flow throughout the matrix.
The matrix can be a mesh composed of polyester fiber. The diameter of the fiber is typically about 10-40 micrometers. The mesh can be purchased from a textile manufacturer either as bulk fiber, non-woven mesh, or as woven fabric. Some manufacturers produce the fiber specifically for medical purposes and it is qualified for biocompatibility. This medical grade mesh or fabric is of particular utility in that it is compatible with cell attachment and growth in a bioreactor that would be used for biopharmaceutical production.
The density of the packing of such materials is a factor that influences cells remaining trapped in the matrix. The more the fiber density, the greater the filtration effect of the packing, therefore the cells are more likely to remain entrapped in the matrix. The greater packing density, however, is more restrictive to media flow and requires more agitation force to achieve the same media flow. Exemplary densities are about 80-160 g/L, such as 90-150 g/L, such as 96 g/L-144 g/L. This range retains cells, but the greater packing density is more restrictive to media flow and requires more agitation force to achieve the same media flow.
The iCELLis® bioreactor system (see, e.g.,
For the iCellis® bioreactors, two packing densities of PET: 96 g/L and 144 g/L, are exemplified.
Provided herein are processes (methods) for producing viruses from adherent host cells using the bioreactors. The methods involve releasing the virus from adherent host cells in a bioreactor, and purifying the released virus by one or two steps of ultrafiltration and/or diafiltration. The methods are used to manufacture viruses, including for clinical use, at reduced cost relative to conventional virus manufacturing methods. The purification method, which requires only ultrafiltration and/or diafiltration reduces the time for purification, and provides higher yields of the virus. Purification can be effected in a day or less.
The cells, which are adherent cells, or cells adapted to grow in or on a matrix support, are cultured in a bioreactor. The host cells can be any cells suitable for growing a virus; selection of the cell can depend on the particular virus. Generally, the host cells are adherent cells. The cells include, but are not limited to, mammalian cells of primary origin; transformed or otherwise immortalized cells and cell lines. Exemplary of such cells are: human fibroblast cells; 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 can be recombinant, and/or genetically modified.
Cells are seeded into the reactor in an appropriate amount. For example, such amount can be about 3-6×103 cells/cm2, such as about 4-5×103 cells/cm2, such as 4.5×103 cells/cm2. The cells are grown for sufficient time, generally 8 days to 20 days depending on growth conditions, to reach an optimal density for infection with the virus, such as, for example, about 1-3×105 cells/cm2, such as about 1.5×105 cells/cm2. The skilled person knows or can empirically determine optimal densities for growth and infection of particular cells.
In an exemplary process, cells are seeded into the reactor at 4.5E3 cells/cm2, and grown to an optimal infection density of 1.5E5 cells/cm2, which can take about 8 days to 20 days depending on growth conditions. The cells are infected at a specific MOI (0.2 to 0.002, such as 0.02 to 0.1), and virus production proceeds for about 96 hours. Because the purification is only a single step (ultrafiltration and/or diafiltration), it can be conducted in a single day compared to conventional purifications which are typically 5-7 steps conducted over a number of days.
In some embodiments, the bioreactor can be adapted for growth of adherent cells; the iCellis® bioreactor; include elements as disclosed in U.S. Pat. Nos. 8,597,939 and 8,986,979; contain a matrix or surface onto which adherent cells may attach; contain a non-fixed attachment surface such as microcarrier beads, fibers, or woven mesh in suspension; and/or contain a fixed attachment surface as in a packed bed bioreactor.
In some embodiments adherent host cells are cultured in the 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 optimum growth of the cells (e.g., 37±3° C. for mammalian cells); at a pH suitable for optimal growth of the cells (e.g., pH 7.3±3 for mammalian cells); with agitation or circulation of the medium or culture suitable for optimal growth of the cells; in a fixed bed reactor at a linear flow velocity of medium at about 0.5 to 5 cm/second; at a dissolved oxygen level suitable for optimal growth of the cells (e.g., 50±25% for mammalian cells); and/or to high cell density relative to the medium volume of the bioreactor (e.g., ≥2×10E6 cells/mL, or 1×10E7 cells/mL).
The viruses contemplated herein, include, but are not limited to, therapeutic viruses, such as oncolytic viruses, viruses for vaccines, and viruses for any purpose, including for recombinant production of an encoded product. The viruses generally are enveloped viruses that can be released to the cell culture medium.
The virus is introduced into the cells, which then are cultured so that the virus replicates. The cells are infected at a specific multiplicity of infection (MOI), which depends on the virus. In exemplary embodiments, the virus is a vaccinia virus, and the MOI is about 0.002. Virus production proceeds for a time to result in maximum amount of virus, such as, for vaccinia, such as the virus designated GL-ONC1 (GLV1h-68), about 72-120, such as 90-120, such as 96, hours.
The host cells can be infected with the virus before being introduced to the bioreactor, or the adherent host cells can be infected with the virus while growing in the bioreactor. Bioreactor medium and/or other culture conditions can be adjusted before or after introduction of infected or uninfected host cells to optimize infection efficiency and/or replication of the virus.
Viruses include, but are not limited to, poxviruses, herpesviruses, adenoviruses, adeno-associated viruses, lentiviruses, retroviruses, rhabdoviruses, papillomaviruses, vesicular stomatitis virus, measles virus, Newcastle disease virus, picornavirus, Sindbis virus, papillomavirus, parvovirus, reovirus, coxsackievirus, influenza virus, mumps virus, poliovirus, and semliki forest virus.
The virus can be native; wild-type; recombinant; or genetically modified. The virus can selected from among a Newcastle Disease virus, parvovirus, vaccinia virus, myxoma virus, measles virus, reovirus, vesicular stomatitis virus (VSV), oncolytic adenoviruses, adeno-associated virus, poliovirus, herpes viruses, Sindbis virus and Seneca Valley virus, or a derivative thereof that is modified to contain a nucleic acid encoding a heterologous gene product. The virus can be an oncolytic virus. The oncolytic virus can be a vaccinia virus, wherein the vaccinia virus is selected from among Lister, Western Reserve (WR), Copenhagen (Cop), Bern, Paris, Tashkent, Tian Tan, Wyeth (DRYVAX), IHD-J, IHD-W, Brighton, Ankara, CVA382, Modified Vaccinia Ankara (MVA), Dairen I, LC16m8, LC16M0, LIVP, ACAM2000, WR 65-16, Connaught, New York City Board of Health (NYCBH), EM-63, and NYVAC strains. The vaccinia virus can be derived from a Lister strain virus: an LIVP virus or a clonal strain of an LIVP virus.
The virus can be a vaccinia virus, such as a recombinant vaccinia virus. Exemplary of therapeutic vaccinia viruses are the modified LIVP strain viruses, such as those described in U.S. Pat. Nos. 7,588,767, 8,857,927, 9,005,602, 8,323,959 and 7,754,221, particularly, the virus designated GLV-1h68 (GL-ONC1), and the clonal strains described in U.S. Publication No. US-2012-0308484-A1, and modified Wyeth strain vaccinia virus, such as the virus designated JX-594 (also referred to as Pexa-Vec, Sillajen Biotherapeutics), which is a replication-competent Wyeth strain vaccinia virus that is modified so that the thymidine kinase gene is inactivated, and the virus encodes and expresses the human GM-CSF and LacZ genes.
The virus can be a modified form containing a nucleic acid encoding a heterologous gene product, wherein the heterologous gene product is a therapeutic or reporter gene product. The heterologous gene product is selected from among an anticancer agent, an antimetastatic agent, an antiangiogenic agent, an immunomodulatory molecule, an antigen, a cell matrix degradative gene, genes for tissue regeneration and reprogramming human somatic cells to pluripotency, enzymes that modify a substrate to produce a detectable product or signal or are detectable by antibodies, proteins that can bind a contrasting agent, genes for optical imaging or detection, genes for PET imaging and genes for MRI imaging. The heterologous gene product can be a therapeutic agent selected from among a hormone, a growth factor, cytokine, a chemokine, a costimulatory molecule, ribozymes, a transporter protein, a single chain antibody, an antisense RNA, a prodrug converting enzyme, an siRNA, a microRNA, a toxin, an antitumor oligopeptide, a mitosis inhibitor protein, an antimitotic oligopeptide, an anti-cancer polypeptide antibiotic, an angiogenesis inhibitor, a tumor suppressor, a cytotoxic protein, a cytostatic protein and a tissue factor. The viruses can encode and, if desired, express heterologous gene products. The products include therapeutic products that can be delivered by the virus. Exemplary of such products is an anticancer agent, an antimetastatic agent, an antiangiogenic agent, an immunomodulatory molecule, and an antigen. Other exemplary products, include, but are not limited to, a therapeutic agent selected from among a hormone, a growth factor, cytokine, a chemokine, a costimulatory molecule, ribozymes, a transporter protein, a single chain antibody, an antisense RNA, a prodrug converting enzyme, an siRNA, a microRNA, a toxin, an antitumor oligopeptide, a mitosis inhibitor protein, an antimitotic oligopeptide, an anti-cancer polypeptide antibiotic, an angiogenesis inhibitor, a tumor suppressor, a cytotoxic protein, a cytostatic protein and a tissue factor. Viruses also can deliver nucleic acids encoding genes, such a cell matrix degradative gene, genes for tissue regeneration and reprogramming human somatic cells to pluripotency. The viruses can encode detectable reporter products, such as, but are not limited to, enzymes that modify a substrate to produce a detectable product or signal or are detectable by antibodies, proteins that can bind a contrasting agent, genes for optical imaging or detection, genes for PET imaging and genes for MRI imaging.
In the production stage of the process, the cells in the bioreactor are lysed, and treated to release the virus. In accord with the methods herein, the virus can be released from host cells without substantial mechanical homogenization.
This can be effected by any suitable method for lysing cells. The lysis method should be such that the cells remain adhered to or are entrapped in the matrix. For example, the cells are lysed by freezing and thawing. Optionally, release and recovery of the virus can be enhanced by hypotonic shock. This can be effected, for example, by, addition of aqueous solutions with total ionic strength less than approximately 0.05 M, such as 0.01 M or less, or such as 0.001 M to the freeze/thawed bioreactor.
By virtue of the selected matrix, the cells and lysed cell debris remains attached/entrapped in the matrix and is not released, or is substantially not released, into the cell culture medium with the virus. Release of the virus from the entrapped/adhered cells is effected by suitable methods to effect lysis, such as freeze/thaw of the bioreactor and/or exposure to hypotonic medium, and, then, enzymatic digestion with enzymes, such as proteases and, optionally, nucleases. Proteases include trypsin or other such proteases normally used to detach cells, but that by virtue of the matrix do not detach the cells. Exemplary of such proteases, are trypsin (porcine pancreatic source), TrypLE (recombinant bacterially-produced enzyme with trypsin-like enzymatic activity), Accutase® solution (Sigma Aldrich; a mixture of proteolytic and collagenolytic enzyme activities), proteinase-K, papain and subtilisin, other such protease with non-specific cleavage sites to digest proteins, bromelain, ficain, and others.
The virus can be released from host cells by a process involving freeze/thaw. For example, the virus can be released from cells in a fixed bed reactor in a process where the culture medium is removed and the bioreactor is frozen at about ≤10° C. for ≥60 minutes, or at <−60° C. for ≥15 minutes, or at ≤−120° C. for ≥1 minute, or other suitable combination of temperature. The bioreactor can be frozen, for example, by placing it into a freezer, by jacketed refrigeration, by immersion in dry ice, by immersion in liquid nitrogen, by infusion of liquid nitrogen vapor, or other such methods. The bioreactor can be thawed by suitable methods, such as exposure to room temperature air, by addition of liquid medium, such as phosphate buffered saline (PBS), or by other such methods known to those of skill in the art. For example, liquid medium can be added to the bioreactor at a temperature selected to maximize cell lysis while optimizing the stability of the virus (e.g., ≤50° C. or 37±3° C.).
Optionally, if freeze/thaw is employed, release and recovery of the virus can be enhanced by hypotonic shock. Hypotonic shock can be effected, for example, by the addition of aqueous solutions so that the total ionic strength is less than approximately 0.05 M, such as 0.01 M or less, or 0.001 M to the freeze/thawed bioreactor. The virus can be released from host cells by exposure to hypotonic medium. In some embodiments, the hypotonic medium can be water or aqueous buffer with ionic strength ≤50 mM. Hypotonic condition can be accomplished by dilution of the bioreactor medium with hypotonic solution such as water to achieve a final ionic strength ≤50 mM. For example, in a fixed bed bioreactor, the medium can be removed and replaced with hypotonic medium of ionic strength ≤50 mM. In some embodiments, the hypotonic medium can be agitated or circulated.
Optionally, release and recovery of the virus can be enhanced by treatment with an enzyme with DNase and/or RNase activity either during or after the freeze/thaw and/or hypotonic shock or after in a suitable buffer. Exemplary of a nuclease is the endonuclease sold under the trademark Benzonase® endonuclease or other enzyme 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 native or heat-denatured DNA and RNA, is a genetically engineered endonuclease from Serratia marcescens. Known to those of skill in the art as Serratia nuclease, it is a protein dimer of 30 kDa subunits with two essential disulfide bonds.
Conditions for releasing the virus from host cells can be selected to maximize cell lysis while optimizing the stability of the virus. 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.
The virus can be released from host cells by exposure to medium containing detergent. The detergent can be ionic, cationic or anionic. The concentration of detergent can be, for example, ≤1%.
After treatment with freeze/thaw and optional hypotonic medium and other such treatments, the cells are treated with proteases, as discussed above. The virus can be released from host cells by digestion with one or more enzymes. The cells are exposed to a digestion medium, which has an ionic strength and pH optimal for the selected digestive enzyme(s). Exemplary digestion medium can be buffered at pH ≤8, such as neutral pH between 7 and 8, such as pH 7.4, such as can be PBS. Digestion medium contains a sodium salt; and/or contains a magnesium salt. The digestive enzyme can be a protease, as discussed above, such as trypsin, or recombinant trypsin (TrypLE). The protease treatment can be effective combined with or replaced with a nuclease such as the endonuclease from Serratia marcescens sold as Benzonase®. Digestion can employ the nuclease and the protease sequentially or in combination. Digestion temperature can be set to optimize enzymatic digestion and optimize stability of the virus (e.g., 37° C.). The digestion time can be set to optimize enzymatic digestion and optimize stability of the virus (e.g., ≥1 hour). The virus can be harvested by removing the digestion medium, and purifying the virus therefrom.
The released virus can be rinsed with medium selected to optimize virus recovery and/or virus stability. For example, the rinsing medium can be water; can be a buffer of low ionic strength; can be a buffer of high ionic strength; has a pH ≥9; and/or can be 10 mM Tris-Cl, pH 9.0.
The purification process, following release of the virus from the cells and enzyme digestion is a single (or dual) step of ultrafiltration or diafiltration or both. No other purification steps are employed or needed. Purification can be conducted in a single day. Conventional prior art purifications typically employ 5-7 steps conducted over a number of days. Not only is there a reduction in time and a cost savings and higher yield by virtue of the purification method provided herein, the fewer steps and shorter time reduces degradation and inactivation of the virus that occurs over time during processing.
The released virus in the culture medium is purified by ultrafiltration or diafiltration, or both. No other purification steps are employed. The released virus can be purified by ultrafiltration. The skilled person can select appropriate filtration membranes and protocols. In some embodiments the filtration medium can be chosen to optimize removal of impurities and optimize stability of the virus; can be water; can be an aqueous buffer; can be high ionic strength; can be low ionic strength; can be physiological ionic strength; and/or can be suitable for administration to animals or humans. The recovery of processed released virus can be greater than 50%; greater than 70%; greater than 80%; greater than 90%; or greater than 95%.
Ultrafiltration
The virus can be purified using large porosity ultrafiltration membranes typically used to remove contaminating viruses from biopharmaceutical preparations. Such membranes typically have porosities of greater than or equal to 300,000 daltons molecular weight cut off such that protein products pass through the membrane while viruses are retained. Using these “virus clearance” membranes in the opposite mode (tangential flow or cross flow filtration mode rather than a single pass-through mode) viruses can be retained and purified while proteins, nucleic acids and other impurities are removed.
For example, the ultrafiltration membrane can be chosen to minimize virus binding and maximize virus retention. The ultrafiltration membrane can be one that has a porosity with a permeability cut off to prevent virus passage through the pores. For example, the ultrafiltration membrane has a nominal molecular weight cut off of between 300 and 750 kilo-daltons or a nominal porosity between 0.05 and 0.2 μm; the ultrafiltration membrane includes 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 cross flow mode; ultrafiltration can be performed under conditions to optimize virus retention and optimize virus stability and/or ultrafiltration can be performed at ≤10° C.
Flat membrane filters and hollow fiber filters can be used for ultrafiltration. Membranes composed of materials that have low binding of virus 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 composed of polyethersulfone (PES).
Diafiltration
Ultrafiltration can be followed by diafiltration with high ionic strength, neutral pH and/or low ionic strength, high pH filtration solutions. As disclosed herein, such methods are capable of recovering greater than 90% of the virus from the lysed cells at high purity.
In some embodiments, the released virus can be purified by diafiltration. In some embodiments, diafiltration can be performed with a membrane chosen to minimize virus binding and maximize virus retention; the diafiltration membrane can be the same one used for ultrafiltration; diafiltration can be performed by addition of filtration medium to the ultrafiltration retentate; and/or diafiltration can be conducted by addition of ≥5 filtration volumes, each equivalent to the retentate volume.
As much as 95% of virus can be recovered. The resulting virus is any virus of interest, particularly vaccinia virus. The virus can be biologically active, can be used to infect cells, can be used to express homologous or heterologous genes or proteins, and/or can be used to elicit an immunological response.
Those skilled in the art can devise many modifications and other embodiments within the scope and spirit of the presently disclosed inventions. Indeed, variations in the materials, methods, drawings, experiments examples and embodiments described may be made by skilled artisans without changing the fundamental aspects of the disclosed inventions. Any of the disclosed embodiments can be used in combination with any other disclosed embodiment.
The following examples are not intended to limit the scope of the disclosure or the claims nor to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature and other parameters) but some experimental errors and deviations should be taken into account. It should be understood that variations in the methods as described can be made without changing the fundamental aspects that the experiments and examples illustrate.
General Methods
CV-1 Cell Cultivation.
All CV-1 cells used in these experiments were derived from CV-1 Working Cell Bank (WuXi Apptec Acc #09-002346, Lot #090170885, 6.0×106 cells/vial in 1.0 mL, Freeze date 9 Dec. 2009, Passage 45). These cells were further expanded and cryopreserved at passage 55. Aliquots of these cells were used in the iCellis® bioreactor Nano experiments. CV-1 cells were cultivated in DMEM supplemented with 10% FBS in T-flasks and roller bottles in 37° C., 5% CO2 incubators.
Infection with GLV-1h68.
iCellis® bioreactor Nano experiment 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 of Nano Experiments 1-6 and GLV-1h68 P3 8-28-14 for Nano Experiments 7-11. The amount of seed virus used was calculated based on the cell number at time of infection, the titer of the seed virus, and the specified MOI.
iCellis® Bioreactor Nano Bioreactor Procedures.
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.)
Analyses.
Viral Plaque Assay (VPA) was conducted on bioreactor and purification samples infected with GLV-1h68. Samples of reactor medium were removed and frozen at −20° C. Samples of macrocarrier (the matrix) in the bioreactor containing 1 or 2 carriers were removed from the bioreactor and placed in a microcentrifuge tube containing 1 mL of fresh DMEM-10% FBS medium and frozen at −20° C. Prior to testing, medium and carrier samples were thawed at room temperature or 37° C., diluted in DMEM-2% FBS and plated on CV-1 cells for plaque formation. Trypsinization of carriers was conducted by placing one carrier into 0.5 mL of 0.05% Trypsin/0.53 mM EDTA or 1×TrypLE in PBS and incubating at 37° C. for up to 60 minutes with intermittent vortex mixing. Samples were diluted by addition of 1.0 mL of DMEM-10% FBS. Purification samples were stored on ice and tested without prior freezing. VPA was conducted using CV-1 cells according in 24-well multi-well plates. Viral plaques were visualized by crystal violet staining.
ß-Glucuronidase Assay (GUS-A) was conducted on samples of reactor medium and macrocarriers, diluted in DMEM-2% FBS and tested. The fluorogenic substrate Cl-MUGlcU (stock solution: 36.5 mM in DMSO) was used in assay buffer consisting of phosphate buffered saline solution containing 2% FBS. Assays were carried out in 96-well multi-well plates and read in the SpectraMax® M5 plate reader using SoftMaxPro v5.4.4 software.
Protein and DNA content were measured using Quant-iT Protein Assay Kit (Invitrogen®) and Quant-iT dsDNA Assay Kit (high sensitivity, Invitrogen®), respectively. Assays were carried out in 96-well multi-well plates and read in the SpectraMax® M5 plate reader using SoftMaxPro v5.4.4 software.
CV-1 Cell Culture Process Development in the iCellis® Bioreactor Nano Bioreactor
The seeding density for all experiments was 4.5×104 cells/cm2, matching the seeding density of CV-1 cells in roller bottles. The target cell density for infection was 1.0 to 2.0×105 cells/cm2.
Culture progress was assessed by aseptically opening the bioreactor and removing carriers at various times during the culture process. The cells attached to the carriers were lysed and cell nuclei were counted using a hemocytometer. Cells were also visualized microscopically on the carriers by crystal violet staining. Micrographic images of the stained carriers of Experiment 6 are shown in
During and after infection, viral titers were assessed to determine the amount of virus amplification and the distribution of the virus between the cells and the culture medium. Carriers were sampled from the reactor and underwent freeze-thaw in fresh culture medium for assay by VPA. The culture medium from the reactor was sampled at the same time and assayed directly by VPA. Virus amount was either expressed as PFU/cm2 of surface area, PFU/cell (cell number determined at time of infection), or as total PFU in the reactor (either carrier-associated or in the medium).
The reactor was seeded with CV-1 cells of passage 71 at 4.5×104 cells/cm2 and at 24 hours, 3.4×104 cells/cm2 were attached to the bioreactor (76%). The agitation rate of the culture was set to create a linear flow velocity of 1.6 cm/sec during cell seeding and raised to 2.5 cm/sec through Day 10. The flow velocity was reduced to 1.6 cm/sec on Day 10, 1.0 cm/sec on Day 14, and 0.5 cm/sec on Day 16. The volume culture medium was increased from 600 mL to 762 mL after seeding and to 2286 mL on Day 3. The medium was exchanged on Days 3, 6, 10, 14, and 16. On Day 21, the culture was infected with GLV-1h68 at an MOI of 0.07 (1.12×108 pfu) in 600 mL of DMEM-2% FBS medium. The flow velocity was maintained at 0.5 cm/sec. The culture was harvested 72 hours post-infection. Concurrently, CV-1 cells were seeded into roller bottles and cell counts were performed periodically. Roller bottles were cultured under standard laboratory conditions without changes of culture medium.
The results showed that CV-1 cells seeded into the iCellis@ bioreactor grew throughout the growth period fairly comparably to roller bottle controls. The CV-1 culture in the reactor reached the target 1.6×105 cells/cm2 (approximately 2 population cell doublings), indicating that the bioreactor could support the growth of cells to this density. Neither the changes in agitation rate nor the culture media changes significantly affected the overall growth rate of the cells. During the growth phase, it was observed that the culture medium became cloudy, which is indicative of the possible detachment of cells from the reactor. Lower agitation reduced this.
Infection of the culture with GLV-1h68 on Day 21 caused an immediate increase in the virus titer evident on Day 1 post-infection and reaching a maximum at Day 2. Throughout the 72 hour amplification period, the virus was associated with the carriers (i.e. the cells) and very little was detected free in the medium. There was a decrease in cell number on the carriers over the post-infection period, although this decrease did not correspond with increased virus titer in the medium. On Day 1 post-infection, the culture had acidified to pH 6.2, the medium became cloudy and cell density dropped. To counteract this, an alkali pump was used to deliver a sodium hydroxide solution to the bioreactor. This was used in all subsequent bioreactor experiments.
Nano Experiment 2 was performed by reducing the flow velocity immediately after cell seeding. The reactor was seeded with CV-1 cells of passage 76 at 4.5×104 cells/cm2 in 600 mL of medium at a linear flow velocity of 1.6 cm/sec. After 1 hour the volume of culture medium was increased to 1342 mL, and the linear velocity was increased to 2.5 cm/sec. After 24 hours the attached cell density was 3.5×104 cells/cm2 (78%). On Day 1, the volume the culture medium was increased to 2286 mL, and the flow velocity was reduced and maintained at 0.5 cm/sec. The culture medium was changed on Day 5.
On Day 10, 1.7×105 cells/cm2 was achieved (more than 2 population cell doublings). The culture was infected with GLV-1h68 at an MOI of 0.1 (1.48×108 pfu) and the flow velocity was reduced to zero through Day 3 post-infection. Virus levels decreased rapidly in the bioreactor, both on the carriers and in the medium, below the level of the initial infection. Linear velocity was restored on Days 4-8 post-infection but only to very low levels (up to 0.3 cm/sec) in order to circulate nutrients to the cells, but virus levels in the bioreactor remained very low. Efficient virus infection and spreading in the bioreactor was achieved with agitation via circulation of the medium, therefore subsequent experiments were conducted at linear velocities between 0.5-2.5 cm/sec.
Nano Experiment 3 was a repeat of Experiment 2 except that the linear flow velocity after Day 1 was further reduced to 0.44 cm/sec. The reactor was seeded with CV-1 cells of passage 78 at 4.5×104 cells/cm2 and after 24 hours, 3.9×104 cells/cm2 were attached (87% seeding efficiency).
On Day 10, the cell density had achieved 1.7×105 cells/cm2. On Day 11, the culture was infected with GLV-1h68 at an MOI of 0.1 (1.74×108 pfu). The flow velocity was increased and maintained at 0.5 cm/sec. The virus increased immediately on the carriers, whereas the virus in the culture medium remained low. Virus amplification was intentionally extended to Day 8 post-infection to examine the full amplification profile. The virus increased through Day 2 post-infection, then remained relatively stable through Day 6, before decreasing significantly at Days 7 and 8. The decrease did not correspond with an increase of virus in the culture medium.
In Nano Experiment 4 the reactor was seeded with CV-1 cells of passage 81 at 4.5×104 cells/cm2 and at 24 hours, the cell density was 4.5×104 cells/cm2 (100% seeding efficiency). After 24 hours, the flow velocity was set at 0.44 cm/sec and the media volume was increased to 2286 mL. There was a single media change on Day 7. On Day 10, the flow velocity was increased and maintained at 1.5 cm/sec.
In this experiment, the cell density increased in the bioreactor steadily until Day 8-10. On Day 11, the linear velocity was increased to 1.5 cm/sec and cell growth resumed, reaching a maximum cell density of 1.9×105 cells/cm2 on Day 12 (more than 2 population cell doublings).
Infection of the bioreactor on Day 14 (MOI=0.13, 1.72×108 pfu) produced a maximum virus amplification on Day 1 post-infection. The virus was associated almost exclusively with the carriers. The virus declined on the carriers on Days 4 and 5 post-infection with a corresponding rise in the virus in the culture medium.
In Nano Experiment 5 CV-1 cells at passage 58 were used directly from cryopreservation. The cells were propagated from the same working cell bank as the CV-1 cells used in the previous experiments.
Nano Experiment 5 was seeded at 4.5×104 cells/cm2 and at 24 hours, 4.7×104 cells/cm2 were attached (104% seeding efficiency). The volume of the culture medium was increased to 2286 mL, and the linear flow velocity was set at 0.44 cm/sec. A single change of culture medium occurred on Day 4, and on Day 9 the flow velocity was increased to 0.9 cm/sec.
In this experiment, cell density reached a maximum 1.0×105 cells/cm2 (approximately 1.5 population cell doublings) on Day 7 and declined thereafter. The increase in flow velocity on Day 9 did not appreciably increase the growth rate. The culture was infected with GLV-1h68 on Day 12 at an MOI of 0.1 (8.26×107 pfu) and the flow velocity was increased to 2.5 cm/sec. Virus accumulated on Day 1 post-infection, but then declined thereafter. The virus was associated almost exclusively with the carriers, and no appreciable increase of virus in the culture medium occurred.
Nano Experiment 6 was conducted with CV-1 cells of passage 62, continued passage of the cells used in Experiment 5. The procedure repeated that of Experiment 5, except that no change in the flow velocity occurred during the growth phase after its reduction to 0.44 cm/sec at 24 hours post-seeding and the single change of culture medium did not occur until Day 6. The reactor was seeded with 4.5×104 cells/cm2 and at 24 hours, 5.3×104 cells/cm2 were attached to the reactor (118% seeding efficiency).
In this experiment, 1.5×105 cells/cm2 was achieved on Day 11 (approximately 2 population cell doublings). The culture was infected with GLV-1h68 on Day 12 (M01=0.1, 1.40×108 pfu), little virus amplification was detected until Day 3 post-infection, and an additional peak occurred on Day 6 post-infection. Again, very little virus was detected in the culture medium.
Analysis of CV-1 Cell Growth Phase in Nano Experiments 1-6
CV-1 cell growth was examined for Nano Experiments 2-6. The individual growth curves are displayed in
The growth data for the 5 experiments were combined, and an overall growth curve was generated.
When the growth data from Experiments 2-6 was truncated at 150 hours post-seeding, the PDT was 99 hours.
Analysis of GLV-1h68 Infection Phase in Nano Experiments 1-6
Within 24 to 48 hours after infection, cell density in the bioreactor declined and continued to decline throughout the infection period. Except for Experiment 1, in which sudden acidification of the culture occurred at 24 hours post-infection, there was no evidence of extensive cell lysis in any of the bioreactor experiments in which pH was adequately controlled. Except for Experiment 6, the decline was not evident at 24 hours post-infection.
Virus production in the iCellis® bioreactor was assessed by sampling carriers and the medium from the reactor every 24 hours after infection. The viral titer of the medium was assayed directly by viral plaque assay (VPA), whereas the carriers were subjected to freeze/thaw in fresh medium and then titered. The results revealed that the virus accumulated in the bioreactor up to 72 hours post-infection and then declined. Based on this analysis, the targeted time for virus harvest from the bioreactor was 72 hours post-infection.
Production of GLV-1h68 in the iCellis® Bioreactor
Three consecutive bioreactor runs were conducted (Nano Experiments 7, 8 and 9). Experiments 7 and 8 were derived from CV-1 cells continuously expanded from the same working cell bank (WCB) stock vial, but of subsequent linear passages. Experiment 9 was expanded from a different vial, but of the same WCB stock. Experiments 7, 8 and 9 also varied slightly in linear flow velocity during the growth phase (0.44, 0.56 and 0.67 cm/sec, respectively). Cell growth of Experiments 7, 8 and 9 proceeded to slightly different endpoints prior to infection (1.6×105, 1.8×105, 1.5×105 cells/cm2, respectively). Experiments 7, 8 and 9 varied from the previous Nano Experiments 1-6 in that GLV-1h68 infection was conducted at MOI 0.2 (3.1×108, 3.7×108 and 3.1×108 pfu for Nano Experiment 7, 8 and 9, respectively) and was amplified for 72 hours post-infection. Finally, minimum sampling of the carriers was done to minimize disturbance of the culture and reduce the depletion of carriers from the bioreactor.
The growth characteristics of the CV-1 cells in bioreactor Experiments 7, 8 and 9 are shown in
Method Development for Extraction of Virus from Bioreactor Carriers
The compiled results of the various virus extraction methods illustrated that the methodology greatly impacted the results (
Trypsin treatment was investigated for extraction of the virus from bioreactor carriers that had undergone lysis by hypo osmotic shock (post-lysis carriers). Trial 1 compared the effects of the extraction medium, either PBS, Trypsin/EDTA (porcine trypsin), TrypLE (recombinant trypsin) or water. Additionally, the effects of freeze/thaw and sonication were evaluated. The results showed that each extraction method was successful, with the most effective extraction being obtained with TrypLE with added benefit from freeze/thaw alone or freeze/thaw and sonication. The virus extracted was 7.2 PFU/cell. With freeze/thaw and sonication, 3.3 PFU/cell were extracted in the PBS. Trial 2 repeated the comparison of TrypLE with and without freeze/thaw and sonication. The TrypLE with freeze/thaw and sonication extracted 7.0 PFU/cell.
Trial 3 compared Trypsin/EDTA, TrypLE, and 1 mm Tris pH 9.0 with and without freeze/thaw and sonication for extraction of bioreactor carriers without prior lysis (pre-lysis carriers). TrypLE with freeze/thaw and sonication extracted 9.1 PFU/cell.
The evaluation was repeated with carriers obtained from the bioreactor at 24, 48 and 72 hours post-infection (hpi) that had been frozen and thawed, but not previously lysed. Trial 4 compared the extraction of 72 hpi carriers with PBS, Trypsin/EDTA and TrypLE with and without freeze/thaw and sonication (
Extraction of the carriers sampled at 48 hpi yielded less virus than the 72 hpi carriers. TrypLE alone, TrypLE with freeze/thaw, and TrypLE with freeze/thaw and sonication extracted 12.2, 25.1 and 22.1 PFU/cell, respectively. The carriers sampled at 24 hours extracted 13.7, 18.3 and 19.8 PFU/cell under the three methods, respectively.
Virus aliquots of known titer were incubated in TrypLE at either 21° C. or 37° C. for 5, 10, 15, 30, 45 or 60 minutes. There was no substantial reduction in viral titer at either temperature. If anything, the viral titer increased with incubation with TrypLE.
Virus Infection, Amplification and Harvest
Increasing the contact of the reactor carriers with the harvest solution was accomplished by either increasing time or increasing volume.
Experiments 7, 8 and 9 were infected with GLV-1h68 in 600 mL of infection medium at MOI of 0.2 in order to achieve rapid and efficient infection of the cells. The amplification period was set at 72 hours for all three experiments. The result was achieved as evident by the rapid decline in cell density immediately after infection (
Experiment 7 was harvested with 300 mL of 1×TrypLE at 21° C. for 45 minutes, followed by 9 consecutive rinses with 300 or 500 mL of 10 mM Tris-HCL pH 9.0. The reactor was not frozen prior to harvest. Analysis of the virus content of the harvest and rinse fractions indicated that the virus was harvested from the bioreactor continuously throughout the process. The 3rd rinse fraction, with the maximum virus content, was only 16% of the total virus harvested. In fact, the last rinse fraction still had 4% of the total virus harvested. 1.3×1010 pfu of GLV-1h68 was recovered.
Experiment 8 was harvested after freeze/thaw of the bioreactor. TrypLE (prepared by dilution of 10×TrypLE to 300 mL in PBS) at 21° C. for 45 minutes, followed by 10 consecutive rinses with 300 mL each of 10 mM Tris-HCL pH 9.0. The first 5 rinses were carried out with continuous maximum agitation and the final rinses used multiple intermittent burst agitations. Compared to Experiment 7, the 2nd rinse fraction had the maximum virus content, with 20% of the total virus harvested. Successive rinse fractions had appreciable virus and the last rinse fraction still had 5% of the total virus harvested. The total virus harvested was 1.7×1010 pfu of GLV-1h68.
In Experiment 9 harvesting occurred after freeze/thaw with 500 mL of TrypLE (prepared by dilution of 10×TrypLE in PBS) at 37° C. for 60 minutes, followed by 2 rinses with 500 mL each of 10 mM Tris-HCL pH 9.0, 2 rinses with PBS, and then lastly with 2 rinses with PBS containing 1 M NaCl. Maximum continuous agitation was used throughout. The 2nd rinse fraction with 10 mM Tris-HCl pH 9.0 again contained the maximum virus content; however the relative proportion of the total harvest was greater than previous harvests (32%). Additionally, the TrypLE harvest fraction and the 1st rinse contained significant fractions of the virus (27 and 29%, respectively). Subsequent rinses with PBS had diminishing virus content (9.4 and 1.9%), and the last rinses with PBS containing 1 M NaCl had significantly reduced virus content (0.5 and 0.2%). The total virus harvested from Experiment 9 was 7.3×1010 PFU.
Reducing the total volume of virus harvest could facilitate downstream processing steps. Therefore, improving the efficiency of virus release at each step is advantageous.
Carriers were sampled from the bioreactor after harvest, extracted and tested by VPA. The carriers contained 4.9×106 PFU/carrier, which for the entire bioreactor at time of harvest containing 914 carriers indicated a residual post-harvest virus content of 4.5×109 PFU or 6% of the total virus harvested. Therefore, the recovery of virus during the harvest procedure was estimated to be at least 94%. There were 1.5×109 cells contained in the bioreactor at time of infection. Therefore, the specific virus productivity was [7.3×1010+(7.3×1010×0.06)] PFU/1.5×109 cells=52 PFU/cell.
Purification of GLV-1h68 from the iCellis® Bioreactor harvest
Five bioreactor runs (Nano Experiments 7, 8, 9, 10 and 11) were used to evaluate the virus purification process using ultrafiltration in a tangential flow (i.e. cross flow) mode. The virus harvest of Nano Experiment 7 (3,500 mL in total) was frozen at −20° C. in aliquots. For each experiment, an aliquot of the virus harvest was thawed at 4° C. and/or at room temperature just prior to use. For other experiments, the virus harvest was purified directly by UF/DF without prior freezing.
Nano Experiment 8 Virus Harvest UF/DF Purified Using Centramate LV 300 Kilodaltons Cut-Off (Kdco) Filter
The virus was harvested from the Nano Experiment 8 bioreactor in 3.5 L. Ultrafiltration was performed using a Centramate LV 300 kdco filter at an initial flow rate of 9.1 L/min/M2 until the retentate volume was reduced to 900 mL. The retentate was stored at −20° C. overnight. UF was resumed until the retentate volume was reduced to 500 mL. Diafiltration was conducted by adding seven successive diavolumes of PBS. The final retentate and filter washes were combined for a total volume of 300 mL. The viral titer of samples was determined by VPA and the protein and DNA contents were determined by protein and DNA assays.
The results showed that virus recovery after ultrafiltration was 80% and after diafiltration was 36%. The protein content of the UF retentate was 21.3 mg/109 pfu and the DNA content was 455 μg/109 pfu. After diafiltration, the protein content of the DF retentate was reduced to 9.0 mg/109 pfu (2.4 fold reduction) and the DNA content was reduced to 17 μg/109 pfu (26 fold).
Nano Experiment 7 Virus Harvest Purified Using Centramate LV 300 Kdco Filter UF/DF
An aliquot of the virus harvest of the Nano Experiment 7 bioreactor (1000 mL) was concentrated using the Centramate LV 300 kdco filter. The UF retentate (200 mL) was then diafiltered first against 10 diavolumes (2000 mL) of PBS followed by 10 diavolumes (2000 mL) of low ionic strength and high pH buffer (10 mM Tris-CL pH 9.0).
Virus recovery after initial concentration of the virus harvest from 1000 ml to 200 mL was 67%. After diafiltration against PBS, the virus recovery was 16%. After subsequent diafiltration against 10 mM Tris-CL, pH 9.0, the virus recovery returned to 81%.
1None detected.
Nano Experiment 9 Virus Harvest Purified Using Centramate LV 300 Kdco Filter UF/DF
The virus was harvested from the Nano Experiment 9 bioreactor in 3.5 L. Ultrafiltration was performed using a Centramate LV 300 kdco filter at an initial flow rate of 4.4 L/min/M2 until the retentate volume was reduced to 200 mL Diafiltration was conducted by adding five successive diavolumes of PBS (200 mL each) followed by adding five successive diavolumes 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 protein and DNA contents by protein and DNA assays.
The results show that 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 of the UF retentate was 1.3 mg/109 pfu and the DNA content was 20 μg/109 pfu. After diafiltration, the protein content of the DF retentate was reduced to 0.6 mg/109 pfu (1.4 fold reduction) and the DNA content was reduced to 16 μg/109 pfu (0.8 fold).
Virus Harvest Purification by UF/DF Using MidiKros Hollow-Fiber Filter Cartridges.
The Centramate LV uses flat membrane filters. The MidiKros filters are cartridges assembled from hollow fibers with porosities of 500 kdco and 750 kdco. UF/DF purification of the virus harvest was evaluated with these filter cartridges to determine the effect of filter geometry and increased porosity. Aliquots of the virus harvest from Nano Experiment 7 stored at −20 C were thawed and used in UF/DF purification. The MidiKros 500 kdco hollow-fiber filter was used to concentrate 500 mL of the virus harvest to a volume of 100 mL, followed by diafiltration against 10 diavolumes of 10 mM Tris-Cl, pH 9.0 (CFF 12). Samples of the starting material, UF and DF permeate and retentate fractions were tested for titer by VPA and protein and DNA content. The results showed a virus recovery of 79% after UF and 68% after DF. No virus was detected in the permeate fractions either during UF or DF. Similarly, 500 mL of the virus harvest was purified by UF/DF using the MidiKros 750 kdco hollow-fiber filter (CFF 13). The virus recovery was 66% after UF and 43% after DF. The protein and DNA content of the starting material was 5.1 mg/109 pfu and 400 μg/109 pfu, respectively. After UF/DF, the protein and DNA content of the retentate of the MidiKros 500 kdco filter was 1.3 mg/109 pfu and 90 μg/109 pfu, respectively (4.0 and 4.5 fold reductions, respectively). After UF/DF, the protein and DNA content of the retentate of the MidiKros 750 kdco filter was 1.9 mg/109 pfu and 63 μg/109 pfu, respectively (2.7 and 6.4 fold reductions, respectively).
1None detected.
1None detected.
The virus harvest from Nano Experiment 10 was purified by UF/DF using a MidiKros 750 kdco hollow-fiber filter cartridge. 3.5 L of the virus harvest was concentrated by ultrafiltration to 200 mL 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/109 pfu and the DNA content was 166 μg/109 pfu. After diafiltration, the retentate was 1.0 mg/109 pfu (4 fold reduction over the starting material) and the DNA content was 15 μg/109 pfu (11.1 fold reduction over the starting material).
1None detected.
The virus harvest from Nano Experiment 11 was purified by UF/DF using a MidiKros 500 kdco hollow-fiber filter cartridge. 2.5 L of the virus harvest was concentrated by ultrafiltration to 200 mL 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/109 pfu and the DNA content was 166 μg/109 pfu. After ultrafiltration concentration, the retentate was 1.1 mg/109 pfu (4.9 fold reduction over the starting material) and the DNA content was 22 μg/109 pfu (7.4 fold reduction over the starting material). After diafiltration, the retentate was 1.4 mg/109 pfu (3.8 fold reduction over the starting material) and the DNA content was 25 μg/109 pfu (6.5 fold reduction over the starting material).
1None detected.
The virus was harvested from Nano Experiment 12 by thawing the frozen bioreactor by addition of 500 mL 10 mM Tris-HCl, 2 mM MgCl2, pH 9.0. Benzonase® (Recombinant Benzonase®, Speed BioSystems, Inc.) was added to 100 U/mL and incubated with agitation at 37° C. for 60 minutes. The Benzonase® nuclease digestion medium was removed and replaced with 500 mL of PBS containing 1×TrypLE and further incubated with agitation at 37° C. for 60 minutes. The TrypLE harvest medium was removed and the bioreactor was flushed twice with 500 mL of 10 mM Tris-HCl, pH 9.0 with agitation at 37° C. for 10 minutes each. The TrypLE harvest and both flush volumes were combined as the virus harvest (1.5 L). Viral titer analysis by VPA indicated that the TrypLE virus harvest fraction contained 80.1% of the virus, the first 10 mM Tris-HCl flush contained 16.3%, and the second flush contained 2.4%. Therefore, 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 macrocarriers after harvest contained less than 0.1%.
The virus harvest was purified by UF/DF using a MidiKros 500 kdco hollow-fiber filter cartridge. 1.5 L of the virus harvest was concentrated by ultrafiltration to 200 mL 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/109 pfu and the DNA content was 38 μg/109 pfu. After ultrafiltration concentration, the retentate was 4.0 mg/109 pfu (1.1 fold reduction over the starting material) and the DNA content was 5 μg/109 pfu (8.7 fold reduction over the starting material). After diafiltration, the retentate was 1.6 mg/109 pfu (2.7 fold reduction over the starting material) and the DNA content was 1 μg/109 pfu (42 fold reduction over the starting material).
1None detected.
The virus was produced by several processes employed in the art, and the results compared. One process referred to as the “CEF” process is the production of a 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, followed by two sucrose gradient centrifugations, followed by formulation and fill/finish.
“Process A” is the production of a virus in a continuous cell line, CV-1, grown in roller bottles (adherent culture), infected with the virus, the cells dissociated from the substrate with trypsin and collected by centrifugation. The cells are lysed by freeze thaw and the virus purified from the total cell lysate by affinity chromatography, followed by concentration by centrifugation, resuspension in formulation buffer and fill/finish.
“Process B,” is a process as described herein. CV-1 cells are grown in a fixed-bed bioreactor (adherent culture), infected with the virus, the reactor drained of medium and frozen. The lysed adherent cells in the bioreactor are treated with Benzonase® nuclease followed by trypsin to release the virus, leaving the cell debris adhered to the matrix in the bioreactor. The virus is purified and formulated ultrafiltration and diafiltration, and filled/finished.
The tables below provide step-by-step comparisons of the various prior art processes, and the process herein (referred to a “Process B”). The process provided herein requires far fewer steps, and results in higher yield of virus.
Exemplary large-scale process for producing vaccinia virus in a bioreactor, such as the 333 m2 iCellis® 500 Bioreactor by the methods herein.
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 appended claims.
Benefit of priority is claimed to U.S. provisional application Ser. No. 62/525,734, filed Jun. 27, 2017, inventors Joseph Cappello and Richard J. Aguilar, entitled “Bioreactor Production of Virus from Adherent Cells.” The subject matter of this application is incorporated by reference in its entirety.
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