A method for optimizing VERO cell growth

Abstract

The invention discloses an upstream process for producing a virus. The process includes cell expansion of adherent cell cultures grown on cell culture containers and on microcarriers in bioreactors. In particular, a scalable process for the upstream manufacture of a virus is described, which includes optimization of the amount of cell detachment agent used per surface area of a microcarrier to enhance cell growth in production bioreactors.

Description

A METHOD FOR OPTIMIZING VERO CELL GROWTHCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 678,076 filed August 1, 2024, the entire contents of which are incorporated by reference herein.FIELD OF THE INVENTION

[0002] The invention relates to a scalable process for the upstream manufacture of a virus.BACKGROUND OF THE INVENTION

[0003] The manufacture of virus and virus vaccines is well known in the art. Viral vaccines have been manufactured for human use since the 18th century’ with continuous improvements to vaccine manufacturing made during the last 300 years (Plotkin. S. Proc. Natl. Acad. Sci.U.S.A. August 26, 2014 111 (34) 12283-1228). During this time, the state of the manufacturing art has moved from the use of primary' cell cultures to continuous cell lines, the most common and most accepted of which is the Vero cell line (P Noel Barrett, Wolfgang Mundt, Otfried Kistner & M Keith Howard (2009) Expert Review of Vaccines. 8:5, 607-618).

[0004] Certain viral vaccine production processes use anchorage-dependent Vero cells as the substrate for viral production. One issue associated with utilizing Vero cells is that the initial culture of Vero cells occurs in static vessels or static cell culture containers, such as CellSTACKs® (Coming Incorporated, Coming, N.Y.). Culturing Vero cells at the N-l stage requires a large number of vessels to obtain the amount of cells necessary’ to provide adequate titers in a large scale production bioreactor. These vessels include microcarriers as a solid substrate for adherent cell growth. A cell detachment agent is required to remove the cells from the microcarriers prior to planting in a bioreactor.

[0005] With their spherical shape, microcarriers offer a higher surface area to volume ratio compared to static vessels. Accordingly, microcarriers have been successfully used for large- scale production involving anchorage dependent cells. Microcarrier-based systems allow for culturing higher cell density and harvesting more cells for cell train development. Unfortunately, Vero cells do not grow well in production bioreactors and cell growth decreases during each passage. The lower the yield, the less cells obtained that can be expanded and infected with a virus. Without a significant yield, the process is not scalable up to production. Accordingly, a need exists for an optimization of cell detachment agent to provide improved yield and growth.There is a need in the art to improve cell harvesting to enhance growth of cells in the production bioreactor.SUMMARY OF THE INVENTION

[0006] Herein, a scalable process for the upstream manufacture of a virus is described, which includes optimization of the amount of cell detachment agent used per surface area of a microcarrier to enhance cell grow th in production bioreactors. The invention provides a manufacturing process for the production of a virus comprising: expanding a population of Vero cells in one or more static cell culture containers (a first adherent cell expansion), wherein the first adherent cell expansion comprises one or more cell passages; adding a cell detachment agent to the one or more static cell culture containers in an amount of about 0.005-0.025 mL / cm2per container; harvesting the Vero cells from the one or more static cell culture containers; planting the Vero cells harvested from the one or more static cell culture containers in a first bioreactor, wherein the first bioreactor contains microcarriers and medium to support the growth of the Vero cells; expanding the Vero cells in the first bioreactor (a second adherent cell expansion), wherein the second adherent cell expansion comprises one or more cell passages; adding a cell detachment agent to the first bioreactor in an amount of about 0.005-0.025 mL / cm2; harvesting the Vero cells from the first bioreactor; planting the Vero cells harvested from the first bioreactor in a second bioreactor, wherein the second bioreactor contains microcarriers and medium to support the growth of the Vero cells; expanding the Vero cells in the second bioreactor (a final adherent cell expansion); adding a virus to the second bioreactor and allowing the vims to infect the Vero cells; amplifying the vims in the second bioreactor; and optionally harvesting the virus from the second bioreactor.BRIEF DESCRIPTION OF THE DRAWINGS

[0007] FIG. 1 is a graphical representation of the effect of reduced cell detachment agent on cell growth at the N-l stage, as described in Example 4.

[0008] FIG. 2 is a graphical representation of the improvement of cell growth at the production phase based on reduced cell detachment agent at N-l stage, as described in Example 4.DETAILED DESCRIPTION OF THE INVENTION

[0009] It is challenging to develop a cell train process with planar cell culture vessels for large- scale production because of the limited available cell quantities. Use of a microcarrier-based system allows for culturing higher cell densify7and harvesting more cells for cell train development. The cells required for this process are typically supplied by an N-l bioreactorwhere the cells are grown on microcarriers to confluency. and subsequently dissociated using a cell detachment agent / enz me.

[0010] The disclosure is directed to the production of a viral vaccine that uses anchorage dependent cells grown on microcarriers. The invention focuses on the optimization of the amount of cell detachment agent used per surface area of the static cell culture containers (e.g..CellSTACKs® (Coming) and microcarriers in the N-2 and N-l stage respectively. It was found that optimization of N-l cells in the harvest process improved cell grow th in the production bioreactor. By reducing the amount of enzyme used for cell detachment in the N-l bioreactor, N- stage cells improved in growth. The summary of the technology described above is non-limiting and other features and advantages of the technology will be apparent from the follow ing detailed description, and from the claims.Definitions

[0011] Listed below are definitions of various terms used herein. These definitions apply to the terms as they are used throughout this specification and claims, unless otherwise limited in specific instances, either individually or as part of a larger group.

[0012] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, and peptide chemistry are those well-known and commonly employed in the art.

[0013] As used herein, the articles “a” and “an” refer to one or to more than one (i.e., to at least one) of the grammatical object of the article, unless the context clearly dictates otherwise. By w ay of example, “an element” means one element or more than one element. Furthermore, use of the term “including” as well as other forms, such as “include,” “includes,” and “included,” is not limiting.

[0014] Reference to “or” indicates either or both possibilities unless the context clearly dictates one of the indicated possibilities. In some cases, “and / or” was employed to highlight either or both possibilities.

[0015] The term “about”, when modifying the quantity (e.g., mM, or M) of a substance or composition, the percentage (v / v or w / v) of a formulation component, the pH of a solution / formulation, or the value of a parameter characterizing a step in a method, or the like refers to variation in the numerical quantity that can occur, for example, through typical measuring, handling and sampling procedures involved in the preparation, characterization and / or use of the substance or composition; through instrumental error in these procedures;through differences in the manufacture, source, or purity of the ingredients employed to make or use the compositions or carry out the procedures; and the like. In certain embodiments, “about” means a variation of ± 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, or 10%. In one embodiment, the term “about” means ± 10% of the value it modifies.

[0016] All ranges disclosed herein are inclusive of the recited endpoint and independently combinable (for example, the range of “from 50 mg to 500 mg” is inclusive of the endpoints, 50 mg and 500 mg, and all the intermediate values). The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value; they are sufficiently imprecise to include values approximating these ranges and / or values.

[0017] As used herein, the term “comprising” may include the embodiments “consisting of’ and “consisting essentially of.” The terms “comprise(s),” “include(s),” “having,” “has,” “may,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that require the presence of the named ingredients / steps and permit the presence of other ingredients / steps. However, such description should be construed as also describing compositions or processes as “consisting of’ and “consisting essentially of’ the enumerated components, which allows the presence of only the named components or compounds, along with any acceptable carriers or fluids, and excludes other components or compounds.

[0018] “Adherent cells” means cells that must be attached to a surface to grow (including, for example, containers, closed-system containers, open-system containers, and microcarriers). Examples of adherent cells include African green monkey kidney cell(s), i.e., Vero cells.

[0019] “Bioreactor” means a multi- or single-use bioreactor. In particular embodiments, “bioreactor” means a single-use bioreactor. The bioreactor may be a 50L volume single-use bioreactor, a 250L volume single-use bioreactor, a 2000L volume single-use bioreactor, or any volume in between 50L and 2000L. In another embodiment, a “bioreactor” refers to a Thermo Scientific Single-Use Bioreactor (S.U.B. or “SUB”).

[0020] “Cell expansion”, or “CE”. or “amplification” means a series of consecutive cell growth and passage steps undertaken to generate the required number of cells for the final infection and viral production steps. “Expanding” and “amplifying” have similar meanings.

[0021] “CellSTACK®” (Coming Incorporated, Coming, NY) refers to a static cell culture container. In an embodiment, “CellSTACK®” refers to Coming® CellSTACK® Culture Chambers comprising 2 (CCS-2; or a 2-layer static cell culture container), 10 (CCS-10; or a 10- layer static cell culture container) or 40 (CCS-40; or a 40-layer static cell culture container) layers for cell grow th as specified in each embodiment. A “closed-system CellSTACK®” meansa CellSTACK® that has been modified to allow sterile transfer or manipulation of cells or the processing of cells, for example, allowing the processing of cell culture materials such as cells and medium through sterile weldable tubing. Specifically, the “closed-system CellSTACK®” is modified to replace vent caps with double filters and weldable tubing to allow for closed-system processing.

[0022] “Container” means a cell culture vessel including CellSTACKs®, T-flasks and bioreactors. A “closed-system container” means a container or vessel that has been modified to allow sterile transfer or manipulation of cells or the processing of cells, for example, allow the processing of cell culture materials such as cells and medium through sterile weldable tubing.

[0023] “Infection” refers to the addition of virus to a cell culture for the purpose of virus production. Infection occurs in a bioreactor. As an example, “infection” refers to the addition of at least one strain of a virus.

[0024] “Inoculation” or “plant” refers to the act of adding cells to anew cell culture vessel during a cell expansion process. For example adding cells to a new CellSTACK® or adding cells to the bioreactor.

[0025] “Media” or “medium” means any serum-free media. In an embodiment, “media” means OptiPro™ (Thermo Fisher Scientific, Waltham, MA) serum-free media.

[0026] “Media exchange” or “medium exchange” or “ME” refers to replacing some or all of the spent cell culture media in a vessel or container with new, unused cell culture media.

[0027] “Microcarrier” refers to support matrices composed of microscopic beads that bind to adherent cells and allow for the adherent cell (i.e., Vero cell) grow th in bioreactors. In an embodiment, “microcarrier(s)” refers to a dextran microcarrier(s). In an embodiment, “microcarrier(s)” refers to a Cytodex® 1 gamma irradiated microcarrier(s) (Cytiva Sweden AB. Uppsala, Sweden).

[0028] “MOI” refers to the multiplicity' of infection, which is the number of infectious virons per cell during infection. In an embodiment, the target MOI is O.OOl-O.OlPFU / vc.

[0029] “PFU” refers to the plaque forming units or number of virus particles capable of forming plaques per unit volume.

[0030] “Scalable” refers to the increase in virus production through increasing the volume of the cell culture container or vessel (i.e., bioreactor) rather than increasing the number of cell culture containers or vessels.

[0031] “Serum-free” means, without animal serum.

[0032] “Trypsinization” refers to the process of dissociating adherent cells from a cell culture vessel using a proteolytic enzyme such as tr psin or TrypLE™ (Thermo Fisher Scientific,Waltham, MA). A proteolytic enzyme such as trypsin or TrypLE™ may also be referred to as a “cell-detachment agent”.

[0033] “Upstream” means the manufacturing process steps that include cell expansion, infection, virus production and harvest.

[0034] “vc” refers to viable cells.

[0035] “Virus harvest” or “viral harvest” refers to the act of harvesting virus-containing cell culture supernatant for the purpose of isolating virus. “

[0036] “Vims production” or viral production” refers to vims amplification over a period of time from vims culture addition (i.e., infection) through virus harvest. As an example, the period of time is from 1-20 days, or from 2 5 day, or from 3-10 days, or from 5-10 days.

[0037] In order to achieve large-scale production of vims through cell culture, it is preferred in the art to have cells capable of growing in suspension or microcarriers. A microcarrier is a support matrix allowing for the growth of adherent cells in spinner flasks or bioreactors (such as stirred bioreactors, rotating wall microgravity bioreactors and fluidized bed bioreactors). Microcarriers are typically 125 - 250 mM spheres with a density that allows them to be maintained in suspension with gentle stirring. Microcarriers can be made from a number of different materials including, but not limited to, DEAE-dextran, glass, polystyrene plastic, acrylamide, and collagen. The microcarriers can have different surface chemistries including, but not limited to, extracellular matrix proteins, recombinant proteins, peptides and charged molecules. In one embodiment, the microcarriers are Dry Gamma Irradiated Cytodex-1 microcarriers (Fisher Scientific), which are based on cross-linked dextran matrices.

[0038] In one embodiment, the invention provides a manufacturing process for the production of virus comprising: a) expanding a population of Vero cells in one or more static cell culture containers (a first adherent cell expansion), wherein the first adherent cell expansion comprises one or more cell passages; b) adding a cell detachment agent to the one or more static cell culture containers in an amount of about 0.005-0.025 mL / cm2per container; c) harvesting the Vero cells from the one or more static cell culture containers; d) planting the Vero cells harvested from the one or more static cell culture containers in a first bioreactor, wherein the first bioreactor contains microcarriers and medium to support the growth of the Vero cells; e) expanding the Vero cells in the first bioreactor (a second adherent cell expansion), wherein the second adherent cell expansion comprises one or more cell passages;f) adding a cell detachment agent to the first bioreactor in an amount of about 0.005-0.025 rnL / cm2; g) harvesting the Vero cells from the first bioreactor; h) planting the Vero cells harvested from the first bioreactor in a second bioreactor, wherein the second bioreactor contains microcarriers and medium to support the growth of the Vero cells; i) expanding the Vero cells in the second bioreactor (a final adherent cell expansion); j) adding a virus to the second bioreactor and allowing the virus to infect the Vero cells; k) amplifying the virus in the second bioreactor; and l) optionally, harvesting the virus from the second bioreactor.

[0039] In some embodiments, the Vero cells are grown in serum-free medium.

[0040] In an embodiment, the static cell culture containers are CellSTACK® containers. In another embodiment, the static cell culture containers are closed-system CellSTACK® containers. In another embodiment, the static cell culture containers are open-system CellSTACK® containers.

[0041] In an embodiment, the cell detachment agent is a proteolytic enzyme such as try psin or TrypLE™. In an embodiment, the cell detachment agent is try psin. In an embodiment, the cell detachment agent is TrypLE™. In an embodiment, about 0.010 to about 0.020 mL / cm2cell detachment agent is added to each static cell culture container. In an embodiment, about 0.010 to about 0.020 mL / cm2cell detachment agent is added to the first bioreactor. In an embodiment, about 0.011 to about 0.015 mL / cm2cell detachment agent is added to each static cell culture container. In an embodiment, about 0.011 to about 0.015 mL / cm2cell detachment agent is added to the first bioreactor. In an embodiment, about 0.013 mL / cm2cell detachment agent is added to each static cell culture container. In an embodiment, about 0.013 mL / cm2cell detachment agent is added to the first bioreactor.

[0042] In an embodiment, bioreactors used in the process are single-use bioreactors. In an embodiment, bioreactors used in the process are multiple-use bioreactor. In an embodiment, the single-use bioreactor is a 50L bioreactor. In an embodiment, the single-use bioreactor is a 250L bioreactor. In an embodiment, the single-use bioreactor is a 2000L bioreactor. In an embodiment, the bioreactor is a closed-system bioreactor. In an embodiment, the bioreactor is an open-system bioreactor.

[0043] In another embodiment, the first bioreactor is a single-use bioreactor. In an embodiment, the first bioreactor is a multiple-use bioreactor. In an embodiment, the first bioreactor is a 50L single-use bioreactor. In an embodiment, the first bioreactor is a 250L single-use bioreactor. Inan embodiment, the first bioreactor is a 500L single-use bioreactor. In an embodiment, the first bioreactor is a closed-system bioreactor. In an embodiment, the first bioreactor is an open-system bioreactor. In an embodiment, the first bioreactor is a closed-system single-use bioreactor. In an embodiment, the first bioreactor is an open-system single-use bioreactor. In an embodiment, the first bioreactor is Thermo Scientific Hyperforma Single-Use Bioreactor.

[0044] In another embodiment, the second bioreactor is a single-use bioreactor. In an embodiment, the second bioreactor is a multiple-use bioreactor. In an embodiment, the second bioreactor is a 50L single-use bioreactor. In an embodiment, the second bioreactor is a 250L single-use bioreactor. In an embodiment, the first bioreactor is a 2000L single-use bioreactor. In an embodiment, the second bioreactor is a closed-system bioreactor. In an embodiment, the second bioreactor is an open-system bioreactor. In an embodiment, the second bioreactor is a closed-system single-use bioreactor. In an embodiment, the second bioreactor is an open-system single-use bioreactor. In an embodiment, the second bioreactor is Thermo Scientific Hyperforma Single-Use Bioreactor.

[0045] In another embodiment, the first adherent cell expansion occurs over 2-20 days, or 3-15 days, or 2-10 days, or 2-5 days, or 2-4 days, or 2-3 days. In another embodiment, the second adherent cell expansion occurs over 2-20 days, or 3-15 days, or 2-10 days, or 2-5 days, or 2-4 days, or 2-3 days. In another embodiment, the final adherent cell expansion occurs over 2-20 days, or 3-15 days, or 2-10 days, or 2-5 days, or 2-4 days, or 2-3 days.

[0046] In another embodiment, the first adherent cell expansion occurs at a temperature of 37 ± 1°C and at 5% ± 1% CO2. In another embodiment, the second adherent cell expansion occurs at a temperature of 37 ± 1°C and at 5% ± 1% CO2. In another embodiment, the final adherent cell expansion occurs at a temperature of 37 ± 1°C and at 5% ± 1% CO2.

[0047] In another embodiment, the microcarriers are Cytodex® 1 Gamma microcarriers.

[0048] In another embodiment, the medium is serum-free medium. In another embodiment, the medium is supplemented with Polaxamer 188. In an embodiment, the medium is ThermoScientific’s VP-SFM.

[0049] In another embodiment, the final adherent cell expansion in the closed-system bioreactor occurs over 120 ± 12 hours.

[0050] In another embodiment, the final adherent cell expansion in the closed-system bioreactor occurs at a pH of 7.3 ± 0.05.

[0051] In another embodiment, the final adherent cell expansion in the closed-system bioreactor occurs at a temperature of 37 ± 1°C.

[0052] In an embodiment, a virus is added to the second bioreactor in an MOI of 0.001-0.01 PFU / vc. In an embodiment, the MOI is 0.001 PFU / vc.

[0053] In another embodiment, virus production occurs at a pH of 7.0 ± 0.05.

[0054] In another embodiment, virus production occurs at a temperature of 34 ± 1°C. In another embodiment, the virus harvest occurs at a temperature of 5 ± 3°C.

[0055] In another embodiment, the virus harvest occurs in a closed-system environment. In another embodiment, the virus harvest occurs in an open-system environment.

[0056] In an embodiment, the harvest yield of virus is at least 1E7 PFU / mL. In an embodiment, the harvest yield of virus is at least 2E7 PFU / mL.

[0057] In an embodiment, the cell detachment agent is TrypLE™.

[0058] In a specific embodiment, the invention provides an upstream manufacturing process for the production of virus comprising: a) incubating Vero cells in one or more system containers for about 120 ± 12 hours at 37 ± 1°C at 5% ± 1% CO2; b) performing a first medium exchange and incubating the Vero cells in the one or more system containers for about 48 ± 12 hours at 37 ± 1°C at 5% ± 1% CO2 after the first medium exchange; c) adding a cell detachment agent to the first bioreactor in an amount of about 0.011-0.015 mL / cm2; d) harvesting the Vero cells from the first bioreactor, planting the Vero cells into one or more system containers and incubating the Vero cells for about 120 ± 12 hours at 37 ± 1°C at 5% ± 1% CO2 ; e) performing a second medium exchange and incubating the Vero cells in the one or more containers for about 48 ± 12 hours at 37 ± 1°C at 5% ± 1% CO2 after the second medium exchange;1) harvesting the Vero cells, and planting the Vero cells into one or more system containers and incubating the Vero cells for about 120 ± 12 hours at 37 ± 1°C at 5% ± 1% CO2 ; g) performing a second medium exchange and incubating the Vero cells in the one or more containers for about 24 ± 12 hours at 37 ± 1°C at 5% ± 1% CO2 after the second medium exchange; h) adding a cell detachment agent to the first bioreactor in an amount of about 0.011-0.015 mL / cm2;i) harvesting the Vero cells from the first bioreactor, planting the Vero cells into one or more system containers and incubating the Vero cells for about 120 ± 12 hours at 37 ± 1°C at 5% ± 1% CO2; j) performing a third medium exchange and incubating the Vero cells in the one or more system containers for about 24 ± 12 hours at 37 ± 1°C at 5% ± 1% CO2; k) harvesting the Vero cells harvest and plant of the Vero cells into one or more system bioreactors, each bioreactor containing microcarriers and medium, for about 120 ± 12 hours for continued incubation and grow th of Vero cells; j) performing a fourth medium exchange; k) adding a virus to the one or more bioreactors and allowing the virus to infect the Vero cells; l) allowing the virus infected Vero cells to incubate in the bioreactor; and m) optionally harvesting the virus.

[0059] In an embodiment, the cell detachment agent is TrypLE. In an embodiment, about 0.010 to about 0.020 mL / cm2cell detachment agent is added to each static cell culture container. In an embodiment, about 0.010 to about 0.020 mL / cm2cell detachment agent is added to the first bioreactor. In an embodiment, about 0.011 to about 0.015 mL / cm2cell detachment agent is added to each static cell culture container. In an embodiment, about 0.011 to about 0.015 mL / cm2cell detachment agent is added to the first bioreactor. In an embodiment, about 0.013 mL / cm2cell detachment agent is added to each static cell culture container. In an embodiment, about 0.013 mL / cm2cell detachment agent is added to the first bioreactor.

[0060] In a specific embodiment, the invention provides an upstream manufacturing process for the production of virus comprising: a) incubating Vero cells in one or more system CCS-2 CellSTACKs® for about 120 ± 12 hours at 37 ± 1°C at 5% ± 1% CO2; b) performing a first medium exchange and continuing to incubate the Vero cells in the one or more system CCS2 CellSTACKs® for about 48 ± 12 hours at 37 ± 1°C at 5% ± 1% CO2; c) harvesting the Vero cells and planting the Vero cells into one or more system CCS10 CellSTACKs® for about 120 ± 12 hours at 37 ± 1°C at 5% ± 1% CO2 for continued incubation and growth of Vero cells; d) performing a second medium exchange and continuing to incubate the Vero cells in the one or more system CCS10 CellSTACKs® for about 48 ± 12 hours at 37 ± 1°C at 5% ± 1% CO2;e) harvesting the Vero cells and planting the Vero cells into one or more system CCS10 CellSTACKs® for about 120 ± 12 hours at 37 ± 1°C at 5% ± 1% CO2 for continued incubation and grow th of Vero cells; f) performing a third medium exchange and continuing to incubate the Vero cells in the one or more system CCS 10 CellSTACKs® for about 24 ± 12 hours at 37 ± 1°C at 5% ± 1% CO2; g) harvesting the Vero cells and planting the Vero cells into one or more system CCS10 CellSTACKs® for about 120 ± 12 hours at 37 ± 1°C at 5% ± 1% CO2 for continued incubation and growth of Vero cells; h) performing a fourth medium exchange and continuing to incubate the Vero cells in the one or more system CCS10 CellSTACKs® for about 24 ± 12 hours at 37 ± 1°C at 5% ± 1% CO2; i) harvesting the Vero cells and planting the Vero cells into one or more system bioreactors, each system bioreactor containing microcarriers and medium, for about 120 ± 12 hours for continued incubation and growth; j) performing a fifth medium exchange; k) adding a virus to the one or more bioreactors and allowing the virus to infect the Vero cells; l) allowing the virus infected Vero cells to incubate in the bioreactor; and m) harvesting the virus.

[0061] In an embodiment, the Vero cell harvest occurs via try psinization. In an embodiment, the Vero cell harvest utilizes a cell detachment agent, about 0.010 to about 0.020 mL / cm2cell detachment agent is added to each static cell culture container. In an embodiment, about 0.010 to about 0.020 mL / cm2cell detachment agent is added to the first bioreactor. In an embodiment, about 0.011 to about 0.015 mL / cm2cell detachment agent is added to each static cell culture container. In an embodiment, about 0.011 to about 0.015 mL / cm2cell detachment agent is added to the first bioreactor. In an embodiment, about 0.013 mL / cm2cell detachment agent is added to each static cell culture container. In an embodiment, about 0.013 mL / cm2cell detachment agent is added to the first bioreactor.

[0062] In a specific embodiment, the invention provides a process for the production of virus comprising: a) incubating Vero cells in one CCS2 CellSTACK® for about 120 ± 12 hours at 37 ± 1°C at 5% ± 1% CO2;b) performing a medium exchange and continuing to incubate the Vero cells in the system CCS2 CellSTACKs® for about 48 ± 12 hours at 37 ± 1°C at 5% ± 1% CO2; c) han esting the Vero cells and planting the Vero cells into one or two CCS 10 CellSTACKs® for about 120 ± 12 hours at 37 ± 1°C at 5% ± 1% CO2 for continued incubation and growth; d) performing a medium exchange and continuing to incubate the Vero cells in the one or two system CCS 10 CellSTACKs® for about 48 ± 12 hours at 37 ± 1°C at 5% ± 1% CO2; e) harvesting the Vero cells and planting the Vero cells into two CCS 10 CellSTACKs® for about 120 ± 12 hours at 37 ± 1°C at 5% ± 1% CO2 for continued incubation and grow th; f) performing a medium exchange and continuing to incubate the Vero cells in the two system CCS10 CellSTACKs® for about 24 ± 12 hours at 37 ± 1°C at 5% ± 1% CO2; g) harvesting the Vero cells and planting the Vero cells into 4-6 CCS 10 CellSTACKs® for about 120 ± 12 hours at 37 ± 1°C at 5% ± 1% CO2 for continued incubation and growth: h) performing a medium exchange and continuing to incubate the Vero cells in the 4-6 CCS10 CellSTACKs® for about 24 ± 12 hours at 37 ± 1°C at 5% ± 1% CO2; i) harvesting the Vero cells and planting the Vero cells into a bioreactor, the bioreactor containing microcarriers and medium, for about 120 ± 12 hours for continued incubation and growth; j) performing a medium exchange; k) adding a virus to the system bioreactor and allowing the virus to infect the Vero cells; l) allowing the virus infected Vero cells to incubate in the bioreactor; and m) harvesting the virus.

[0063] In an embodiment, the Vero cell harvest occurs via trypsinization. In an embodiment, the Vero cell harvest utilizes a cell detachment agent, about 0.010 to about 0.020 mL / cm2cell detachment agent is added to each static cell culture container. In an embodiment, about 0.010 to about 0.020 mL / cm2cell detachment agent is added to the first bioreactor. In an embodiment, about 0.011 to about 0.015 mL / cm2cell detachment agent is added to each static cell culture container. In an embodiment, about 0.011 to about 0.015 mL / cm2cell detachment agent is added to the first bioreactor. In an embodiment, about 0.013 mL / cm2cell detachment agent is added to each static cell culture container. In an embodiment, about 0.013 mL / cm2cell detachment agent is added to the first bioreactor.

[0064] In an aspect of the various embodiments, step i) occurs at a pH between 7.0 - 7.4 and at a temperature between 36.0 - 38°C. In a further embodiment, the pH is 7.3 and the temperature is 37 °C.

[0065] The process of the invention may be used in large-scale production of any viruses that can be grown on Vero cells using microcarriers. For example, a viral vaccine utilizing this process may be adapted to incorporate vesicular stomatitis virus, yellow fever, enterovirus, measles, dengue, rabies, or polio virus.Example 1 : Static Cell Growth

[0066] Frozen vials of Vero Working Cell Bank were thawed in a 37°C dry heat block and used to inoculate one 2-layer Coming CellSTACK® (CCS-2) at a cell density of approximately 2.4*104 viable cells / cm2(vc / cm2). The Vero cells were maintained in CellSTACKs® using OptiPRO supplemented with 4 mM L-glutamine at 37°C and 5% CO2 in an incubator. Cells were passaged 4 times using TrypLE™ for cell detachment and Soybean Trypsin Inhibitor (STI: 1 g / L) for protease inactivation. Cell concentration and viability were determined using the Vi- CELL™ XR cell counter.Example 2: N-l Cell Grow th

[0067] At least two days prior to plant, the 250 L Single Use Bioreactor (SUB) was batched to -90% of the working volume with 225 L of N-l Growth Medium (VP-SFM + 6mM L-Glutamine + 0.1 % P-188) and a dry addition of 520 g of Dry Gamma Irradiated Cytodex-1 microcarriers (each having a surface area of 4400 cm2 / g) to reach a concentration of 2g / L in the 250 L SUB.

[0068] Using the harvested cell suspension from the previous passage, the 250 L SUB was then planted with a target plant cell density of 1.3 x 104viable cells / cm2(vc / cm2), which is approximately equivalent to 13 cells per bead or 1.14 x 105viable cell / mL (vc / mL). The surface area of Cytodex-1 is 4400 cm2 / g, so a plant density of 1.3 x 104viable cells / cm2(vc / cm2) is approximately equivalent to 13 cells per bead or 1.14 x io5viable cell / mL (vc / mL). Once planted, the 250L SUB was controlled at 37.0°C, pH 7.3 + 0.05 (two-sided pH control, with CO2 and sodium carbonate), Dissolved Oxygen (DO) > 50% and 7.5 standard liter per minute (slpm) air overlay for approximately 4 days (96 ± 12 hours). Agitation was controlled at 40 rotation per minute (rpm) for one day post-plant, then increased to 45 rpm for the remaining duration of the growth period. Three days post-plant, a -80% medium exchange was performed.

[0069] Four days post-plant, the 250 L SUB was washed with Formulated Phosphate Buffered Saline solution (PBS + 0.1% Pl 88) and harvested enzymatically via addition of 3.47 L of Formulated 10X TrypLE Select + 15.85 mM EDTA. Upon completion of trypsinization, 33.6 L of Quench Medium (VP-SFM + 6 mM L-Glutamine + Img / mL Soybean Try psin Inhibitor (STI))was added to the 250 L SUB. Cells were separated from the microcarriers via two 12 L Harvestainers. Once the harvested cell suspension was transferred into the DS300 mixing bag, 5 L of Chase Medium (VP-SFM + 6 mM L-Glutamine) was added to the Harvestainer to recover the remaining cells. The Harvestainers were rocked back and forth; and contents pumped into the DS300 Mixer. This process was repeated with 10L of chase medium to rinse the harvestainers. Once all contents were collected in a DS300 Mixer, cell counts were performed. Using the harvested cell suspension from the 250L SUB, the N-stage 2000L Thermo SUB was planted. Example 3: Production Bioreactor Cell Growth

[0070] Immediately following the N-l step harvest, cells were inoculated in the N-stage 2000L Thermo SUB production bioreactor with a seeding cell density of 2.2 x 104 vc / cm2Temperature was maintained at 37°C and pH w as controlled at 7.3 via CO2 or sodium carbonate addition. DO was maintained at > 50% saturation. Agitation w as controlled at 40 rpm for one day post-plant, then increased to 45 rpm for the remaining duration of the growth period.Example 4: Results

[0071] Slow growth was observed in the N-l 250L SUB when cells were sourced from N-2 CCS-40s compared to CCS-lOs. The CCS-lOs used TrypLE™ volumes to match a volume to surface area ratio (Vol:SA) of 0.013 rnL / cm2at the time of harvest. Using CCS-lOs as an N-2 source to plant the N-l 250 L SUB was not deemed as a path forward for final manufacturing and further optimization was required to implement CCS-40s at this step. The initial CCS-40 process used a Vol:SA for TrypLE™ that matched 0.051mL / cm2which was 4 times more than that used in CCS-lOs. To optimize for cell growth at the N-l step, the Vol:SA for TrypLE™ at the N-2 step in CCS-40s was adjusted to 0.013ml / cm2 to match that of CCS-lOs. Once implemented, a significant improvement in cell growth of approximately 50% was observed at the N-l stage with cells sourced from the optimized CCS-40 process.

[0072] Surprisingly, and as shown in Figure 1 and Table 1, reducing Vol:SA of Try pLE™ used to harvest cells at N-2 step improved cell growth at N-l step. Specifically, controls XL6 Ageis, XL5 Aegis, and XL5 CX showed significantly less cell growth than the optimized MCE9, MCE12, and MCE13. The improvement from the standard (XL5 and XL6) process to the optimized (MCE9, MCE12, and MCE13) process is shown below' in Table 1: Table 1:

[0073] The improvement from the standard (x) process to the optimized (circles) process is shown in Figure 2. Also shown in the cell grow th when using CCS-10 (+) where the volume per surface area of cell detachment agent is lower. Similarly, the same strategy was applied to the N- 1 step to harvest cells from microcarriers.

[0074] As shown in Figure 2, a reduction in cell detachment agent also showed an increase in growth at the N-stage. In particular, optimizing Vol:SA for TrypLE™ to 0.0013 mL / cm2for cells harvest at the N-l microcarriers SUB step improved cell growth in the 250 L production SUB.Table 2

[0075] Upon successful cell growth at the production (N) stage post modification of dissociation reagent amount, the cells were infected with Measles virus at an MOI of 0.001 pfu / vc. The virus and cells were allowed to incubate for 2 days and the bioreactor was then harvested. The harvest yielded 2E7 PFU / rnL of virus on average, which was significantly higher than the 1E7 PFU / mL target.

[0076] The disclosed subject matter is not to be limited in scope by the specific embodiments and examples described herein. Indeed, various modifications of the disclosure in addition to those described will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

[0077] All references (e.g., publications or patents or patent applications) cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual reference (e.g., publication or patent or patent application) was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Other embodiments are within the following claims.

Claims

CLAIMSWhat is claimed is:

1. A manufacturing process for the production of a vims comprising: a. expanding a population of Vero cells in one or more static cell culture containers (a first adherent cell expansion), wherein the first adherent cell expansion comprises one or more cell passages; b. adding a cell detachment agent to the one or more static cell culture containers in an amount of about 0.005-0.025 mL / cm2per container; c. harvesting the Vero cells from the one or more static cell culture containers; d. planting the Vero cells harvested from the one or more static cell culture containers in a first bioreactor, wherein the first bioreactor contains microcarriers and medium to support the grow th of the Vero cells; e. expanding the Vero cells in the first bioreactor (a second adherent cell expansion), wherein the second adherent cell expansion comprises one or more cell passages; f. adding a cell detachment agent to the first bioreactor in an amount of about 0.005-0.025 mL / cm2; g. harvesting the Vero cells from the first bioreactor; h. planting the Vero cells harvested from the first bioreactor in a second bioreactor, wherein the second bioreactor contains microcarriers and medium to support the growth of the Vero cells; i. expanding the Vero cells in the second bioreactor (a final adherent cell expansion); j. adding a vims to the second bioreactor and allowing the virus to infect the Vero cells; k. amplifying the virus in the second bioreactor; and l. harvesting the vims from the second bioreactor.

2. The process of claim 1, wherein each adherent cell expansion of the Vero cells comprises at least 4 cell passages.

3. The process according to any of claims 1-2, wherein the Vero cells are grown in serum-free medium.

4. The process according to any one of claims 1-3, wherein the static cell culture containers are closed-system static cell culture containers.

5. The process according to any one of claims 1-4, wherein the static cell culture containers are open-system static cell culture containers.

6. The process according to any one of claims 1-5, wherein each adherent cell expansion occurs over 2-20 days.

7. The process according to any one of claims 1-6, wherein each adherent cell expansion occurs at a temperature of 37 ± 1°C and at 5% ± 1% CO2.

8. The process according to any of claims 1-7, wherein the microcarriers are Cytodex-1 microcarriers.

9. The process according to any of claims 1-8. wherein about 0.010 to about 0.020 mL / cm2cell detachment agent is added to each static cell culture container.

10. The process according to any of claims 1-9, wherein about 0.010 to about 0.020 mL / cm2cell detachment agent is added to the first bioreactor.

11. The process according to any of claims 1-10, wherein about 0.011 to about 0.015 mL / cm2cell detachment agent is added to each static cell culture container.

12. The process according to any of claims 1-11, wherein about 0.011 to about 0.015 mL / cm2cell detachment agent is added to the first bioreactor.

13. The process according to any of claims 1-12, wherein about 0.013 mL / cm2cell detachment agent is added to each static cell culture container.

14. The process according to any of claims 1-13, wherein about 0.013 mL / cm2cell detachment agent is added to the first bioreactor.

15. The process according to any of claims 1-9, wherein the final cell expansion in the second bioreactor occurs over 120 ± 12 hours.

16. The process according to any of claims 1-10, wherein the final cell expansion in the second bioreactor occurs at a pH range of 7.05 to 7.55.

17. The process according to any of claims 1-11, wherein the final cell expansion in the second bioreactor occurs at a temperature of 37 ± 1°C.

18. The process according to any one of claims 1-12, wherein the virus is selected from the group consisting of vesicular stomatitis virus, yellow fever, enterovirus, measles, dengue, rabies and polio virus.

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