Methods for infecting cells with viruses

Abstract

To provide a method for infecting cells with a virus. [Solution] The present invention relates to a method for increasing the yield of viruses, viral particles, or viral vectors from host cells in a bioreactor. The present invention provides a reproducible and robust method and system for determining and controlling the optimal time for host cell infection using correlations of process air parameters, including airflow, O2 flow, and their respective tendencies, which result in increased viral yield. The present invention relates to a method for growing viruses and viral vectors for vaccine and viral vector production. More specifically, the present invention relates to a specific method for initiating host cell infection resulting in increased viral yield from host cells in a fixed-bed bioreactor.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the interests of U.S. Provisional Application No. 63 / 104,803, filed on 23 October 2020, which is incorporated herein by reference in its entirety, including any numerical values, tables, nucleic acid sequences, amino acid sequences, or drawings.

[0002] Field of Invention The present invention relates to a method for growing viruses and viral vectors for vaccine and viral vector production. More specifically, the present invention relates to a specific method for initiating host cell infection resulting in an increased viral yield from host cells in a fixed-bed bioreactor. [Background technology]

[0003] Background of the Invention To meet the ever-increasing demand for vaccines and other therapeutics, robust technologies that enable the reproducible and robust production of viruses and viral vectors are essential. Furthermore, technologies that improve viral yield from host cells also play a crucial role in accelerating the development of vaccine processes and production, for the development of versatile host cell technology platforms such as Vero cells and other mammalian cell platforms, avian cell platforms, and insect cell technology platforms. This invention satisfies the need to improve methods for virus generation. [Overview of the Initiative] [Means for solving the problem]

[0004] Embodiments of the present invention provide systems and methods for utilizing airflow and O2 flow parameters to determine the optimal time for infection of host cells growing in fixed-bed and other bioreactor systems where cell count sampling is difficult, impractical, or impossible.

[0005] Known methods that use metabolite data to determine the optimal time of infection in a fixed-bed reactor with respect to cell density require sampling and a wide infection window. Through the observation of process air parameters, including comparing the total air flow to the O2 flow within the reactor, the inventors have identified factors, parameters, and trends of interest, including a tendency for the volume of air flow to the fixed-bed reactor to decrease and cross the increasing trend of the volume of O2 flow to the reactor. The present invention provides a reproducible and robust process for determining and controlling the optimal time of infection of host cells using the correlation of process air parameters, including air flow, O2 flow, and their respective trends, that results in an increase in virus yield. The data disclosed herein supports the trends and correlations of air / O2 with respect to cell density within the reactor.

[0006] The patent or application file contains at least one drawing created in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Patent and Trademark Office upon request and payment of the required fee.

Brief Description of the Drawings

[0007] [Figure 1] Figure 1 compares the air flow and oxygen (O2) flow of two representative runs according to an embodiment of the present invention.

[0008] [Figure 2] Figure 2 shows the air - O2 difference vs. UNVC viable cell density according to an embodiment of the present invention.

[0009] [Figure 3] Figure 3 shows the hourly average air - O2 difference over time vs. viable cell density according to an embodiment of the present invention.

[0010] [Figure 4] Figure 4 shows the air - O2 difference model applied to representative run 2 of Figure 1 according to an embodiment of the present invention.

[0011] [Figure 5A] Figure 5A shows microcarrier strips used in a fixed-bed bioreactor, with 13 microcarrier strips, each with a three-dimensional area of ​​approximately 11.2 cm², in 5 mL of culture medium.

[0012] [Figure 5B] Figure 5B shows a cross-section of a bioreactor that could be used to hold up to 3,500 strips per floor.

[0013] [Figure 5C] Figure 5C shows different parts of the bioreactor in a cross-sectional view. [Modes for carrying out the invention]

[0014] Detailed description of the invention The present invention provides a system and method for a reproducible and robust process that determines and controls the optimal time for host cell infection using correlations of process air parameters, including airflow, O2 flow, and their respective tendencies, which result in increased viral yield.

[0015] In one embodiment, the present invention provides a method for infecting host cells with a virus, which does not require a step of counting host cells, and includes the steps of: culturing host cells in a bioreactor; observing a set of bioreactor process air parameters; identifying a first time marker based on the bioreactor process air parameters; calculating an optimal time for an infection window based on the first time marker; and infecting the host cells during the calculated optimal time for an infection window.

[0016] Equipment compliant with Current Good Manufacturing Practice (CGMP) does not need to be equipped for cell counting. The advantage of the present invention is to provide a process that allows process monitoring to initiate the optimal time for infection based on the correlation between airflow and oxygen flow, instead of or in addition to time, cell count, or a cell count substitute (such as biomass).

[0017] In certain embodiments, the host cells may include adherent cells. In certain embodiments, the bioreactor may be a fixed-bed bioreactor. In certain embodiments, the step of observing bioreactor process air parameters includes measuring the velocity of the total airflow into the bioreactor, measuring the velocity of the O2 flow into the bioreactor at multiple time points, and creating a set of current measurements at each respective time point. In certain embodiments, the step of identifying a first time marker includes determining the time at which the velocity of the total airflow into the bioreactor decreases and crosses an increasing trend in the velocity of the O2 flow into the bioreactor. In certain embodiments, the step of identifying a first time marker includes calculating a value of 1 or greater from the current set of measurements to predict a future time at which the velocity of the total airflow into the bioreactor decreases and crosses an expected increasing trend in the velocity of the O2 flow into the bioreactor.

[0018] If necessary, once an infection window is established for a specific cell line and virus, the established infection window can be applied and utilized together with other viruses. However, monitoring the rate of total airflow to the bioreactor and the rate of O2 flow to the bioreactor may still be desirable for active monitoring and real-time process control to collect data.

[0019] The type of host cell used for culturing the virus in the present invention may be natural or genetically modified (e.g., recombinant cells, cell lines, etc.) and may be any eukaryotic cell suitable for producing viral antigens, viral vectors, or virus production. In some embodiments, the host cell is a Vero cell and the virus is a viral vector such as recombinant vesicular stomatitis virus (rVSV).

[0020] In some embodiments, determining the optimal infection window time is based on the correlation of process air parameters, including, but not limited to, airflow, O2 flow, and their convergence, the relationship between them (e.g., the ratio between them), or the tendencies of each, resulting in an increase in virus yield. In some embodiments, the optimal infection window time is the convergence of airflow and O2 flow. In some embodiments, the infection window is a time interval in which airflow decreases and O2 flow increases so that they are within ±30% of each other, or within ±20% of each other, or within ±10% of each other, or within ±5% of each other.

[0021] Embodiments of the present invention include a method for producing a virus in a bioreactor, comprising the steps of: providing host cells in a bioreactor; growing the host cells in a bioreactor; observing a set of bioreactor process air parameters; calculating an optimal infection window time based on the set of bioreactor process air parameters; infecting the host cells with at least one virus or viral particle during the optimal infection window time; incubating the host cells infected with the virus or viral particle to propagate the virus; and optionally recovering the virus.

[0022] In some embodiments, the host cells are adherent cells. In some embodiments, the bioreactor is a flatbed bioreactor, and the process of growing the host cells is carried out at a constant initial dissolved oxygen (dO2) level, pH, and temperature. In certain embodiments, the bioreactor is a disposable flatbed bioreactor.

[0023] In certain embodiments, calculating the optimal infection window time, either alone or in combination with any of the aforementioned embodiments, involves determining the time at which the velocity of the total airflow to the bioreactor decreases and intersects with the increasing trend in the velocity of the O2 flow to the bioreactor. In alternative embodiments, calculating the optimal infection window time, either alone or in combination with any of the aforementioned embodiments, involves determining the time at which the air-O2 difference in the bioreactor approaches or is zero. In other embodiments, calculating the optimal infection window time, either alone or in combination with any of the aforementioned embodiments, involves determining the time at which the air-O2 difference in the bioreactor is below a predetermined threshold or is expected to fall below a predetermined threshold. In some embodiments, the optimal infection window time is the convergence of the airflow and O2 flow. In some embodiments, the infection window is a time interval at which the airflow decreases and the O2 flow increases so that they are within ±30% of each other, or within ±20% of each other, or within ±10% of each other, or within ±5% of each other.

[0024] Considering convergence, and referring to a representative run 1 in Figure 1 as a non-restrictive example, a convergence threshold of + / - 30 units (e.g., mL / min) may be selected for this run. If the airflow is 80 (e.g., mL / min) and the O2 flow is 20 (e.g., mL / min), then 80-20=60, convergence is not achieved, and the infection window is not filled. If the airflow is 66 and the O2 flow is 38, then 65-38=27, convergence is achieved, and the infection window is filled (in this example, this is the first point in the dashed box near the time of the infection window). If the airflow is 60 and the O2 flow is 40, then 60-40=20, convergence is achieved, and the infection window is filled (in this example, this is the second point in the dashed box near the time of the infection window). In this run, this is when infection occurs. It should be noted that in certain embodiments, additional parameters may apply, such as a minimum convergence time, a narrower convergence window (lower threshold), or the need for convergence along with a different value before infection.

[0025] Referring to a typical run 2 in Figure 1, as a non-restrictive example, a convergence threshold of + / - 30 units (e.g., mL / min) may be selected for this run. If the airflow is 80 and the O2 flow is 20, then 80-20=60, convergence is not achieved, and the infection window is not filled. If the airflow is 60 and the O2 flow is 30, then 60-30=30, convergence is achieved, and the infection window is filled (this is the first point in the dashed box around the time of the infection window). If the airflow is 50 and the O2 flow is 50, then 50-50=0, convergence (which is also the intersection in this case) is achieved, and the infection window is filled (this is the intersection of the dashed boxes around the time of the infection window). If the airflow is 40 and the O2 flow is 60, then 40-60=-20, convergence is achieved, and the infection window is filled (this is the second point in the dashed box around the time of the infection window). In this run, this is when infection occurs. It should be noted that in certain embodiments, additional parameters may be applied, such as a minimum convergence time, a narrower convergence window (lower threshold), or the need for convergence with an infection of another value (such as a specific absolute value for either airflow or O2 flow).

[0026] Advantageously, this method of infecting host cells based on the rates of airflow and O2 flow allows for controllable and reproducible virus production based on process monitoring and enables the use of a closed system without the need for cell counting. As a closed system, the bioreactor is a self-contained environment that is closed (e.g., hermetically sealed) during operation, enabling continuous culture without the need for cell counting or access ports for cell counting.

[0027] Alternatively, host cell infection may be based on time derived from historical data using the present invention, but without active monitoring.

[0028] In some embodiments, the host cells provided (e.g., seeded) in the bioreactor are not counted before the infection step. In some embodiments, the method further includes recovering the host cells or host cell products after infection. In some embodiments, the host cells in the bioreactor are not counted before recovery.

[0029] In some embodiments, the bioreactor does not include and is not connected to a cell counting device (e.g., an instrument such as a counting chamber, an automated cell counter, a Coulter counter, or a flow cytometer). The cell counting device can utilize various techniques such as image-based counting (e.g., bright-field or fluorescence) or non-image-based counting (e.g., current exclusion). In some embodiments, the bioreactor does not have an access port for cell counting. In some embodiments, the bioreactor is a closed, sealed (e.g., hermetically sealed) system.

[0030] In certain embodiments, the infection of host cells by the virus has an infection multiplicity (MOI) of approximately 0.1 to 0.05. In certain embodiments, the infection of host cells by the virus has an MOI of 0.05. In certain embodiments, the step of incubating host cells infected with the virus or viral particles to grow the virus includes incubating the host cells at a certain final dO2 level, pH, and temperature, which is different from a certain initial dO2 level, pH, and temperature.

[0031] The virus may be any naturally occurring virus or a genetically modified virus (e.g., a recombinant or engineered virus). In certain embodiments, the virus is selected from the group consisting of naturally occurring VSV or genetically modified VSV, adenovirus, influenza virus, Ross River virus, hepatitis A virus, vaccinia virus, herpes simplex virus, Japanese encephalitis virus, herpes simplex virus, West Nile virus, yellow fever virus, rhinovirus, reovirus, Ebola-Zaire virus, Ebola-Sudan virus, Ebola-Marburg virus, Nipah virus, or any of the aforementioned chimeras. In some embodiments, the virus is a viral vector. In certain embodiments, the virus is a VSV vector. In certain embodiments, the virus is a modified viral vector, such as a VSV, containing a glycoprotein derived from another virus of interest.

[0032] The type of host cell used for culturing the virus in the present invention may be natural or genetically modified (e.g., recombinant cells, cell lines, etc.) and may be any eukaryotic cell suitable for producing viral antigens, viral vectors, or viruses. In some embodiments, the host cell is selected from naturally occurring or genetically modified mammalian cells (e.g., human cells and mouse cells), avian cells (e.g., chicken cells and quail cells), and insect cells.

[0033] In some embodiments, the host cells are selected from the group consisting of Vero cells, MBCK cells, MDBK cells, MRC-5 cells, BSC-1 cells, LLC-MK cells, CV-1 cells, CHO cells, COS cells, HeLa cells, HEK 293 cells, MDOK cells, CRFK cells, RAF cells, TCMK cells, LLC-PK cells, PK 15 cells, W1-38 cells, T-FLY cells, BHK cells, SP2 / 0 cells, NSO cells, PerC6 cells, COR cells, and QOR cells.

[0034] In certain embodiments, the host cells are Vero cells or HEK 293 cells.

[0035] If necessary, the method of the present invention includes steps beyond the expansion, proliferation, infection, and optional recovery of host cells. For example, in some embodiments, the method may further include the steps of determining viral titer by a plaque assay; purifying and / or characterizing the virus; or producing a vaccine, a viral vector for gene delivery, or an immunotherapy composition using cells, or a cell-derived product such as a virus, or a portion of a virus. The present invention includes compositions such as vaccines and immunotherapy compositions produced by the method, which can be formulated for administration to human or animal subjects via any suitable route of administration.

[0036] For example, to produce compositions such as vaccines, vectors, or immunotherapy compositions, recovered cells or cell-derived products (e.g., viruses or parts thereof, or other biomolecules) may be combined with one or more excipients, diluents (such as water, phosphate-buffered saline, or saline), carriers, adjuvants, or any combination of two or more of the aforementioned. The adjuvants may be any class suitable for the intended use of the vaccine or composition, such as alum salts and other mineral adjuvants, bacterial products or bacterial-derived adjuvants, tensoactive agents (e.g., saponins), oil-in-water (o / w) and water-in-oil (w / o) emulsions, liposomal adjuvants, cytokines (e.g., IL-2, GM-CSF, IL-12, and IFN-γ), and α-galactosylceramide analogs. Some non-exclusive examples of adjuvants include montanide emulsion, QS21, Freund's complete or incomplete adjuvant, aluminum phosphate, aluminum hydroxide, Bacillus Calmette-Guerin (BCG), and alum.

[0037] If desired, the method may further include recovering cells and recovering cell-derived products from infected host cells using methods known in the art. Various biomolecules produced by naturally occurring or non-genetically modified cells produced using the methods of the invention can be recovered for various uses such as the manufacture of drugs or biologics and for pharmacological research (e.g., isolated from biomolecule-producing cells). Thus, the invention can be used to utilize cells as biological “factories” for providing products of the cells such as biomolecules. The term “biomolecule” refers to molecules (plural) that can be produced by cells (cell-derived products). Such biomolecules include, but are not limited to, proteins, peptides, amino acids, lipids, carbohydrates, nucleic acids, nucleotides, viruses, parts of viruses (e.g., virus particles), and other substances. Biomolecules can be, for example, intracellular, transmembrane, or secreted by cells and can be purified or isolated using methods known in the art.

[0038] The volume of the bioreactor can be any that meets the production objectives for, e.g., screening, laboratory research and development, clinical research, and commercial production. Various types, sizes, and models of suitable bioreactors are commercially available. Examples of commercially available bioreactors that can be used in the present invention include, but are not limited to, disposable fixed-bed bioreactors produced by Univercells Technologies and iCELLis.

[0039] In some embodiments, the bioreactor is 1 m 2 ~600 m 2 (as surface area), or 10 m 2 ~30 m 2 , or 30 m 2 ~200 m 2 , or 200 m 2 ~600 m 2 , or 600 m 2 ~2400 m 2 , or 2.4 m 2~2400m 2 It has a capacity in the range of . In some embodiments, the bioreactor has a volume capacity of 700-800 mL (e.g., iCELLis nano). In some embodiments, the bioreactor has a volume capacity of 25 L-50 L (e.g., iCELLis 500). In some embodiments, the bioreactor has a volume capacity of 10 m 2 ~30m 2 It has a capacity of 1.5L to 3.5L (e.g., UNVC carbo). In some embodiments, the bioreactor is 200m 2 ~600m 2 It has a capacity of , or a volumetric capacity of 30L to 100L (e.g., UNVC nitro). In some embodiments, the bioreactor is approximately 2,400m 2 It has a volume of 350L to 400L (e.g., UNVC oxo) or more.

[0040] The culture medium can be any medium suitable for the host cells and production purpose, such as a serum-free, chemically defined medium.

[0041] Embodiments of the method of the present invention may include the steps of: providing host cells in a bioreactor; growing the host cells at a constant initial dO2 level, pH, and temperature until confluence; calculating an optimal infection window time based on a set of bioreactor process air parameters; infecting the host cells with at least one virus or viral particle during the optimal infection window time; incubating the host cells infected with the virus or viral particle to grow the virus; and, if necessary, recovering the virus.

[0042] Embodiments may advantageously utilize the correlation between factors, including airflow and O2 flow, and cell density within the bioreactor. This correlated trend can be used to better determine the optimal time for infection in fixed-bed reactors (e.g., reactors that do not allow cell counting sampling or where cell counting is impractical).

[0043] Referring to the drawings, Figure 1 compares airflow and oxygen (O2) flow in two representative implementations of a fixed-bed bioreactor system according to an embodiment of the present invention.

[0044] Figure 2 shows the air-O2 difference versus Univercells (UNVC) live cell density according to an embodiment of the present invention. The embodiment allows for evaluation of the difference between airflow velocity and O2 flow velocity. In this case, a positive difference indicates that the airflow volume is greater than the O2 flow volume, and a negative difference indicates the opposite, i.e., that the airflow volume is less than the O2 flow volume. If the airflow volume is equal to the O2 flow volume, the difference is 0.

[0045] In this chart, as the difference (d) approaches 0, the viable cell density (VCD, measured here in viable cells per square centimeter) is 1 × 10⁻⁶. 5 cells / cm 2 It approaches this value. Around d=20, VCD is 1×10 5 cells / cm 2 It is within half the logarithm of and is known as the “infection trigger point”. Embodiments may use airflow and O2 flow trends to estimate the time required for infection. In this example, using d, approximately 42% of the variability of VCD is retained, and R 2 = 0.4224, and the difference: VCD correlation coefficient = -0.64.

[0046] Figure 3 is a graph showing the average hourly air-O2 difference versus viable cell density over time according to an embodiment of the present invention, with the right axis representing the air-O2 difference versus the left axis representing VCD (cells / cm²) against time (time after inoculation, or hpi) on the x-axis. 2 ) compare. Up to 64hpi, past VCDs were 5×10 4 ~1 × 10 5 cells / cm 2 It has been shown that within a window of 84-96 hpi, the VCD is 1 x 10 5 cells / cm 2This is the closest trend. Within this window, the trend lines for air and O2 begin to converge. Embodiments of the present invention can advantageously provide a more reliable estimate of the optimal time for infection.

[0047] Figure 4 shows the air-O2 difference model retrospectively applied to data collected from representative run 2 (shown in Figure 1) according to an embodiment of the present invention. Extrapolating the analysis to representative run #2 suggests that the actual time of infection occurred later than the optimal time of infection. In this embodiment, the cells were 1 × 10⁶ 5 cells / cm 2 A cell density of 1.3 × 10⁻¹⁰ is higher than the target density. 5 live cells / cm 2 Infection occurred when the density was such that it was high.

[0048] Figure 5A shows the microcarrier strips used in a fixed-bed bioreactor, with approximately 11.2 cm per strip. 2 Thirteen microcarrier strips with a three-dimensional area are shown in 5 mL of culture medium.

[0049] Figure 5B shows a cross-section of a bioreactor that could be used to hold up to 3,500 strips per floor.

[0050] Figure 5C shows different parts of the bioreactor in a cross-sectional view.

[0051] The present invention provides a method for infecting host cells with a virus without the need for the step of counting host cells. In particular, the present invention provides a reproducible and robust process for determining and controlling the optimal time for host cell infection that results in increased viral yield. In one embodiment, the method includes 1) providing (e.g., seeding) host cells to a bioreactor in an environment in which system parameters such as total airflow, O2 flow, dissolved oxygen (dO2), pH, and temperature can be measured and controlled; 2) growing the host cells with a first set of pre-infection system parameters; 3) monitoring the system parameters; 4) infecting the host cells with at least one virus at a time determined at least partially by changes in two or more of the measured system parameters; incubating the host cells with the virus with a second set of post-infection system parameters; and optionally, 5) recovering the virus. In some embodiments, the host cells are adherent cells that are scaffold-dependent and require a flat surface, microcarrier, and / or fixation bed for fixation. In certain embodiments, Vero cells or HEK 293 cells are used as host cells in a fixed-bed bioreactor.

[0052] In some embodiments, the method includes: 1) providing (e.g., seeding) host cells to a bioreactor at a constant initial level of 80-100% dO2, pH of 7.2-7.4, and temperature of 36°C+2°C; 2) reducing the dO2 level to a maximum of 50% of the initial dO2 level while maintaining constant pH and temperature; 3) infecting the host cells with at least one virus during an infection window defined as when the airflow decreases and the oxygen flow increases, so that they are within ±20% of each other; 4) incubating the host cells with the virus at a dO2 level of 20-50%, pH of 7.2-7.4, and temperature of 36°C±2°C; and optionally, 5) recovering the virus.

[0053] In specific embodiments of the present invention, dO2 is reduced by at least 50% 12 hours before infecting host cells with the virus. In one embodiment, the host cells are infected with the virus after they have grown to their highest cell density. In yet another embodiment of the present invention, dO2 is reduced after the host cells have reached their highest cell density.

[0054] The amount of virus produced by this method is significantly higher and more reproducible than that produced by conventional methods where all parameters, including dO2, are kept constant throughout the entire process.

[0055] The following applies to the section of the detailed description of this application.

[0056] When an indefinite or definite article, such as "a," "an," or "the," is used to refer to a singular noun, unless otherwise specified, it includes the plural form of that noun.

[0057] In the context of this invention, the terms “about” or “approximate” indicate an interval of precision that a person skilled in the art would understand to still guarantee the technical effect of the feature in question. When used in conjunction with a numerical value, the term typically indicates a deviation of ±10%, preferably ±5%, from the given numerical value.

[0058] As used herein, the term “bioreactor” refers to a device that provides a biologically active environment in which biological processes, such as the growth of viruses and vectors, can take place under controlled conditions. Bioreactors may be designed for small-scale cultures, such as those used in laboratories, as well as large-scale bioreactors equipped with containers or vats for the production and recovery of biological macromolecules, such as vaccine viruses, antigens, and vectors, in pilot plants or on a commercial scale. Both suspension cells and adherent cells can be grown using bioreactors. A bioreactor is a controlled environment in which the levels of oxygen / dO2, nitrogen, carbon dioxide, and pH can be adjusted.

[0059] A "fixed-bed bioreactor" refers to a type of bioreactor that includes a fixed bed of packing material that promotes cell adhesion and growth. Fixed-bed bioreactors have been used to produce viral vaccine products, both on a small and large scale, due to their ability to perfuse high cell densities with low shear forces. Any configuration or platform of a fixed-bed bioreactor can be used in conjunction with the present invention.

[0060] The fixed-bed bioreactor may be a disposable bioreactor such as the commercially available iCELLis system (Pall Corporation) or the scale-X® system (Univercells), described in whole herein by reference by Berrie DM et al., Vaccine, 2020, 38:3639-3645. The iCELLis system platform provides a novel fixed-bed technology in a robust, single, closed system that does not require aseptic handling, containing a carrier composed of woven medical-grade polyethylene terephthalate (PET) fibers. Furthermore, the system incorporates rapid gas exchange using a “waterfall” technique with controlled temperature, O2, pH, carbon dioxide (CO2), and nitrogen (N2), and further by the use of a magnetic impeller that generates low cell shear stress and uniformly distributed medium circulation. For most viruses, the titer produced from the iCELLis system is significantly increased compared to classical adherent cell flatstock flasks. The iCELLis technology is suitable for growth areas of 0.5–4 m². 2 In small-scale applications such as iCELLis Nano, and with a growing area of ​​66-500 m² 2 It can be used in manufacturing scales such as iCELLis 500, which falls within this range. Processes developed in small-scale systems can be scaled up to manufacturing scale processes.

[0061] Univercells' scale-X™ bioreactor system is a series of growth surface scale-X "hydro" (<3m 2) "carbo" (10~30m 2 ) and "nitro" (200~600m 2 ) provides. This range offers scalable process and clinical lot production capabilities. Within the Univercells product line, the height of the bioreactor increases, but the diameter remains constant. For example, carbo 10m 2 The bioreactor is 30m 2 It is 1 / 3 the height of the bioreactor. However, scaling up between different lines is achieved by keeping the fixed bed height constant and increasing the diameter, similar to scaling up in chromatography systems. For example, 200m 2 The bioreactor is 10m 2 While having the same height as other bioreactors, the diameter is different. The scale-X carbo system is a disposable bioreactor coupled with inline product concentration operated by a bench-scale automated process controller (pH, DO, T, agitation, liquid flow rate), enabling the generation and simultaneous concentration of viral products (this is novel and a feature that distinguishes this type of fixed-bed bioreactor from others on the market). The fixed bed in the scale-X carbo bioreactor has a total container volume of 1.6 to 3.2 L, depending on the surface area, and is 10 m³ 2 ~30m 2 It provides surface area for cell growth. This results in a volume for high cell density per unit volume and a compact footprint that allows for integration into standard biosafety cabinets. While many commercially available fixed-bed bioreactors use randomly filled discs or cloth strips as substrate for cell adhesion, the scale-X bioreactor utilizes a fixed bed composed of a mesh layer that provides uniformity and consistency between containers for cell growth. The fixed-bed bioreactor may have sensors to measure and monitor biomass indicating pH, temperature, dissolved oxygen, and adherent cell density. The fixed-bed bioreactor may also have different ports for adding oxygen or nitrogen, a medium exchange port, sodium hydroxide (NaOH) and / or CO2. 2It may have a port for adjusting the pH by adding [something]. The dO2 of the culture medium is O 2 or N 2 It can be modified by the addition of [a certain substance]. Preferably, the dO2 level can be depleted in a controlled manner by injecting Na into the headspace of the bioreactor while simultaneously stirring and monitoring the dO2.

[0062] The host cells used in the present invention may be anchorage-dependent cells or may be adapted to be anchorage-dependent cell lines. The host cells of the disclosed method may be cultured on microcarriers and may be suspended in a bioreactor or microcarrier strip. In some embodiments, the host cells are cultured on a microcarrier strip in a fixed bed of a fixed bed bioreactor. In some embodiments, the fixed bed bioreactor is a commercially available iCELLIS Nano (Pall Corporation), iCELLis 500 bioreactor (Pall Corporation), or Univercells fixed bed bioreactor (Univercells SA). In some embodiments, the fixed bed is up to 40,000 cm³ in an 800 mL fixed bed bioreactor such as the iCELLis Nano. 2 In 25L fixed-bed bioreactors such as the iCELLis 500 (Figures 5A-C; Table 1), the maximum volume is 5,000,000 cm³. 2 This can be provided. The height of the fixed bed can range from 20 mm to 10 mm, and in an 800 mL fixed bed bioreactor, it can reach 5300 cm². 2 ~40,000cm 2 From 660,000 cm³ in a 25L fixed-bed bioreactor 2 ~5,000,000cm 2 It provides a growth area. [Table 1]

[0063] The host cell population is 2,000 to 20,000 cells / cm³. 2Culturing can be achieved by using seeding densities within a range. Seeding density can be adjusted based on the host cell type, bioreactor volume, and fixed bed height in a fixed bed bioreactor. Selecting the optimal seeding density for the process is within the scope of the knowledge of those skilled in the art. Growth of metabolically active cells can be monitored by correlations of process air parameters, including but not limited to airflow, O2 flow, and their convergence, the relationship between them (e.g., the ratio between them), or the tendencies of each, resulting in increased viral yield.

[0064] The present invention may include measuring further parameters, such as biomass, using a biomass sensor within the fixed bed of a bioreactor. Biomass, which indicates the mass of adherent cells via conductivity, can be used to monitor the overall growth of host cells and the decrease in cell mass due to viral replication after infection. Higher biomass, indicated by higher conductivity as monitored by the biomass sensor, indicates a higher growth rate of cells. However, reliance on biomass measurement has drawbacks. Biomass does not represent metabolic activity or peak production. For example, host cells may enter senescence and still record high biomass. Furthermore, biomass measurement in some systems requires access to the bioreactor, which may present challenges in clinical and commercial processes, including CGMP compliance.

[0065] As used herein, “culture medium” or “culture medium” refers to the liquid used to culture host cells in a bioreactor. The culture medium used in the procedures of this disclosure may contain, but is not limited to, a variety of components that support the growth of host cells, including amino acids, vitamins, organic and inorganic salts, and carbohydrates. The culture medium may be a serum-free medium, which is a medium formulated without any animal serum. When used, serum-free media may be selected from DMEM, DMEM / F12, Medium 199, MEM, RPMI, OptiPRO SFM, VP-SFM, VP-SFM AGT, HyQ PF-Vero, MP-Vero, etc. The culture medium may be an animal-free medium, i.e., it does not contain any products of animal origin. The culture medium may be a protein-free medium, i.e., the medium is formulated without protein. Serum-free or protein-free media can be formulated without serum or protein, but may contain cellular proteins derived from host cells and, if necessary, proteins specifically added to the serum-free or protein-free media.

[0066] The pH for culture may be, for example, 6.5 to 7.5, depending on the pH stability of the host cells. Preferably, cells are cultured at a pH of 7.4. Host cells can be cultured at a temperature between 20 and 40°C, specifically between 30 and 40°C, preferably 36°C ± 1°C for mammalian cells.

[0067] The host cells or host cell lines used for culturing the virus in the methods of this disclosure may be any eukaryotic cells suitable for viral antigen production, viral vector production, or virus production. Preferably, the host cells may be “adherent cells” or “scaffold-dependent cells.” Adherent cells are cells that adhere to a surface under culture conditions and may require a scaffold to grow; they are sometimes called scaffold-dependent cells.

[0068] The host cells used in the present invention may be naturally occurring or genetically modified (e.g., recombinant cells, cell lines, etc.) and may be any eukaryotic cell suitable for viral antigen production, viral vector production, or virus production. In some embodiments, the host cells are selected from naturally occurring or genetically modified mammalian cells (e.g., human cells and mouse cells), avian cells (e.g., chicken cells and quail cells), and insect cells.

[0069] In some embodiments, the host cells are selected from Vero cells, MBCK cells, MDBK cells, MRC-5 cells, BSC-1 cells, LLC-MK cells, CV-1 cells, CHO cells, COS cells, HeLa cells, HEK 293 cells, MDOK cells, CRFK cells, RAF cells, TCMK cells, LLC-PK cells, PK 15 cells, W1-38 cells, T-FLY cells, BHK cells, SP2 / 0 cells, NSO cells, PerC6 cells, COR cells, and QOR cells.

[0070] In certain embodiments, the host cells are Vero cells or HEK 293 cells.

[0071] Preferred adherent cells are anchorage-dependent cells that can be grown on a carrier such as a PET strip, but suspension cells that can be adapted to grow as adherent cells may also be used. In some embodiments, the anchorage-dependent cells used in the present invention are Vero cells. Selecting an adherent host cell suitable for use in the process of the present invention is within the knowledge of those skilled in the art.

[0072] The virus may be any naturally occurring virus or a genetically modified virus (e.g., a recombinant or engineered virus). In certain embodiments, the virus is selected from the group consisting of naturally occurring VSV or genetically modified VSV, adenovirus, influenza virus, Ross River virus, hepatitis A virus, vaccinia virus, herpes simplex virus, Japanese encephalitis virus, herpes simplex virus, West Nile virus, yellow fever virus, rhinovirus, reovirus, Ebola-Zaire virus, Ebola-Sudan virus, Ebola-Marburg virus, Nipah virus, or any of the aforementioned chimeras. In some embodiments, the virus is a viral vector. In certain embodiments, the virus is a VSV vector. In certain embodiments, the virus is a modified viral vector, such as a VSV, containing a glycoprotein derived from another virus of interest.

[0073] In one embodiment of the present invention, the virus is a viral vector. The viral vector is a virus that can be used to transfer a passenger nucleic acid sequence into a target cell. The viral vector may be a viral expression vector that can be used to induce recombinant proteins. Preferably, the viral vector may be a modified vaccinia virus Ankara (MVA), VSV, adeno-associated virus (AAV), lentivirus, retrovirus, or adenovirus. More preferably, the viral vector of the present invention is a VSV vector. The recombinant protein expressed by the viral vector may be a viral protein, a bacterial protein, a therapeutic recombinant protein, or a combination thereof. More preferably, the recombinant protein produced by the viral vector is a viral protein.

[0074] In some embodiments, the virus of the present invention is a VSV vector. VSV, a member of the Rhabdoviridae family, is an enveloped virus with a negative-strand RNA genome that causes self-limiting diseases in livestock. Attenuated VSV is a desirable viral vector because it is nonpathogenic in humans, nearly non-toxic in animals, exhibits robust growth in target serial mammalian cell lines, lacks DNA intermediates during replication, induces strong cellular and humoral immune responses, and induces a genomic structure that allows for transgene insertion at multiple sites (Humphreys and Sebastian, Immunology, 2018, 153:1-9; Clarke et al., Vaccine. 2016 34:6597-6609).

[0075] As used herein, “infection” or “viral infection” refers to the entry of a virus into a host cell and the subsequent replication of the virus within the cell. Host cell infection in the methods of this disclosure may be performed when the optimal window of infection, determined by the correlation of process air parameters including, for example, airflow, O2 flow, and their respective tendencies, results in an increase in viral yield.

[0076] The host cells of the method disclosed herein can be cultured with an initial dO2 of 100%. The dO2 may be reduced from a level of 90% to a lower level of 20% before infection. The dO2 may be reduced from about 80% to about 60%, from about 70% to about 40%, and from about 50% to about 15%. Preferably, the dO2 may be reduced from about 50% to about 20% before infection. More preferably, this level is reduced to about 20% before infection.

[0077] dO2 can be decreased starting in the range of 2 to 24 hours before infection and can be maintained at this level throughout the infection process and virus recovery. dO2 is decreased starting approximately 2 to 10 hours, 5 to 15 hours, 10 to 20 hours, and 18 to 24 hours before infection. Preferably, dO2 is decreased starting in the range of 8 to approximately 12 hours before infection.

[0078] As used herein, “recovery” refers to the collection of cell-derived products, such as cells and viruses, by collecting unclarified culture media and / or host cells from a bioreactor. Virus recovery may be performed, for example, 2–5 days after infection, or 3–6 days after the decrease in dO2. In some embodiments, virus recovery may be performed 2 days after infection. Some viruses may require a further step of host cell lysis before recovery.

[0079] The viruses of this disclosure may be quantified by methods including, but not limited to, plaque assays, endpoint dilution assays, hemagglutination assays, bicinchoninate assays, or electron microscopy. Preferably, the viruses may be quantified by plaque assays. As used herein, a plaque assay is a method for determining the number of infectious viral particles based on the measurement of plaque-forming units (PFUs). In a plaque assay, a cell monolayer is infected with serial dilutions of a viral stock solution, and an agarose overlay is used to restrict viral flow. Infected cells release progeny viruses, which then infect adjacent cells. The cells are lysed to produce clear regions surrounded by uninfected cells called plaques, which are visualized using a dye. The higher the viral titer of the sample, the greater the number of plaques.

[0080] Embodiments of the present invention provide novel and advantageous systems and methods for infecting host cells with a virus without the necessary step of counting host cells. In one embodiment, the system comprises a bioreactor configured and adapted for cell culture, viral infection of cells, viral replication, and viral recovery; a cell culture medium within the bioreactor; an air space above the cell culture medium within the bioreactor; an air inlet to the bioreactor; an airflow sensor for measuring the airflow to the bioreactor; an O2 inlet to the bioreactor; an O2 flow sensor for measuring the O2 flow to the bioreactor; a data acquisition module configured and adapted to collect the following values: current airflow to the bioreactor, current trend of airflow to the bioreactor, current O2 flow to the bioreactor, and current trend of O2 flow to the bioreactor; and an indicator unit configured and adapted to indicate a decreasing trend in airflow to the bioreactor, an increasing trend in O2 flow to the bioreactor, and when convergence occurs between the current values ​​of airflow to the bioreactor and O2 flow to the bioreactor, respectively.

[0081] In one embodiment, the data acquisition unit comprises at least one first processor operably communicating with an airflow sensor and an O2 flow sensor, and at least one first machine-readable medium operably communicating with the at least one first processor, having instructions stored thereon that, when executed by the at least one first processor, perform the steps of: recording a reading from the airflow sensor to generate a current airflow value to the bioreactor; comparing the current airflow value to at least one previous airflow value to generate a current trend of the airflow to the bioreactor; recording a reading from the O2 flow sensor to generate a current O2 flow value to the bioreactor; and comparing the current O2 flow value to at least one previous O2 flow value to generate a current trend of the O2 flow to the bioreactor.

[0082] In one embodiment, the instruction unit comprises at least one second processor operably communicating with a data acquisition unit and a bioreactor, and at least one second machine-readable medium operably communicating with the at least one second processor, having instructions stored thereon, which, when executed by the at least one second processor, perform the steps of indicating a time window for viral infection of cells, where a current trend of airflow to the bioreactor is decreasing, a current trend of O2 flow to the bioreactor is increasing, and a convergence exists between the respective current values ​​of airflow to the bioreactor and O2 flow to the bioreactor, and only in that case.

[0083] The increasing or decreasing trend may be defined by comparing a single pair of data points (e.g., flow at time T0 versus flow at time T1), comparing multiple data points (e.g., the average of all flow values ​​collected, observed, or recorded over a first period versus the average of all flow values ​​collected, observed, or recorded over a second period), or by other methods (e.g., statistical analysis, machine learning, or artificial intelligence methods). The criteria, methods, or thresholds for determining the increasing or decreasing trend in one or more parameters may be the same or different.

[0084] Convergence is defined as the point at which two values ​​(e.g., air flow rate and O2 flow rate) become significantly the same over time. Convergence can be calculated at a single point in time or at two neighboring but separate point in time by comparing one flow rate measurement with another. Flow rates may be averaged, sampled, or otherwise processed before comparison. A convergence threshold may be set to determine or find convergence. The convergence threshold may be established in units of flow (e.g., mL / min) or on a percentage basis (e.g., the smaller value is within 30% of the larger value). Alternatively, flow values ​​may be converted to relative percentages and compared using percentage values ​​(e.g., a + / - 30% flow threshold applied to a flow of 62% air and 38% O2 may have convergence). Alternatively, the two values ​​can be converted to absolute percentages of the total flow and compared using the percentage values ​​(for example, a + / -10% flow threshold applied to a flow that is 42% air, 12% N2, and 46% O2 would have convergence between the airflow and the O2flow).

[0085] The embodiment may further comprise a decision-making unit configured and adapted to initiate viral infection of cells when a time window for viral infection of cells is indicated. Each of the data acquisition unit, instruction unit, and decision-making unit (to be employed individually or in any combination) may be a digital computer, an embedded component of a controller (e.g., a module or application in a commercially available or manufactured bioreactor control unit), a mechanical system, a pneumatic or hydraulic system, an analog electrical or electronic system, a standard operating procedure, a person, or any combination of the foregoing, may constitute any of them, may comprise any of them, or may be connected to any of them.

[0086] In one embodiment, at least one first processor and at least one second processor are the same processor.

[0087] In one embodiment, at least one first machine-readable medium and at least one second machine-readable medium are the same machine-readable medium.

[0088] In one embodiment, the bioreactor is a sealed bioreactor.

[0089] In one embodiment, the convergence threshold is selected as + / -20%, and convergence is found when the value of the O2 flow percentage to the bioreactor is within + / -20% of the value of the air flow percentage to the bioreactor.

[0090] In one embodiment, the data acquisition module includes a mechanical sensor or an analog electrical sensor.

[0091] In one embodiment, the indicator unit comprises one or more audible, visual, or tactile indicators.

[0092] One embodiment provides a method for infecting host cells with a virus without the need for the step of counting host cells. The method may include providing a bioreactor configured and adapted for cell culture, viral infection of cells, viral replication, and viral recovery; providing a cell culture medium within the bioreactor; providing an air space above the cell culture medium within the bioreactor; providing an air inlet to the bioreactor; providing an airflow sensor for measuring the airflow to the bioreactor; providing an O2 inlet to the bioreactor; providing an O2 flow sensor for measuring the O2 flow to the bioreactor; collecting the current airflow to the bioreactor, the current trend of the airflow to the bioreactor, the current O2 flow to the bioreactor, and the current trend of the O2 flow to the bioreactor by a data acquisition module; and indicating by an indicator unit a decreasing trend in the airflow to the bioreactor, an increasing trend in the O2 flow to the bioreactor, and when convergence occurs between the current values ​​of the airflow to the bioreactor and the O2 flow to the bioreactor, respectively.

[0093] In one embodiment, the data acquisition unit comprises at least one first processor operably communicating with an airflow sensor and an O2 flow sensor, and at least one first machine-readable medium operably communicating with the at least one first processor, having instructions stored thereon that, when executed by the at least one first processor, perform the steps of: recording a reading from the airflow sensor to generate a current airflow value to the bioreactor; comparing the current airflow value to at least one previous airflow value to generate a current trend of the airflow to the bioreactor; recording a reading from the O2 flow sensor to generate a current O2 flow value to the bioreactor; and comparing the current O2 flow value to at least one previous O2 flow value to generate a current trend of the O2 flow to the bioreactor.

[0094] In one embodiment, the instruction unit comprises at least one second processor operably communicating with a data acquisition unit and a bioreactor, and at least one second machine-readable medium operably communicating with the at least one second processor, having instructions stored thereon, which, when executed by the at least one second processor, perform the steps of indicating a time window for viral infection of cells, where a current trend of airflow to the bioreactor is decreasing, a current trend of O2 flow to the bioreactor is increasing, and a convergence exists between the respective current values ​​of airflow to the bioreactor and O2 flow to the bioreactor, and only in that case.

[0095] In one embodiment, the method may further include initiating viral infection of cells when a time window for viral infection of cells is indicated.

[0096] In one embodiment, at least one first processor and at least one second processor are the same processor.

[0097] In one embodiment, at least one first machine-readable medium and at least one second machine-readable medium are the same machine-readable medium.

[0098] In one embodiment, the bioreactor is a sealed bioreactor.

[0099] In one embodiment, the value of the O2 flow to the bioreactor is within + / - 20% of the value of the air flow to the bioreactor.

[0100] In one embodiment, the data acquisition module includes a mechanical sensor or an analog electrical sensor.

[0101] In one embodiment, the indicator unit comprises one or more audible, visual, or tactile indicators.

[0102] The methods and processes described herein can be embodied as code and / or data. The software code and data described herein can be stored in one or more machine-readable media (e.g., computer-readable media), which may include any device or medium capable of storing code and / or data for use by a computer system. When a computer system and / or processor reads and executes the code and / or data stored in the computer-readable media, the computer system and / or processor implements the methods and processes embodied as data structures and code stored in the computer-readable storage medium.

[0103] Those skilled in the art will understand that computer-readable media include removable and non-removable structures / devices that can be used to store information such as computer-readable instructions, data structures, program modules, and other data used by computing systems / environments. Computer-readable media include, but are not limited to, volatile memory such as random access memory (RAM, DRAM, SRAM); non-volatile memory such as flash memory, various read-only memories (ROM, PROM, EPROM, EEPROM), magnetic memory and ferromagnetic / ferroelectric memory (MRAM, FeRAM), and magnetic and optical storage devices (hard drives, magnetic tapes, CDs, DVDs); network devices; or other media currently known or to be developed that can store computer-readable information / data. Computer-readable media should not be considered or interpreted as including any propagating signals. The computer-readable media of the present invention may be, for example, a compact disc (CD), a digital video disc (DVD), a flash memory device, volatile memory, or a hard disk drive (HDD) such as an external HDD or a hard disk drive of a computing device, but the embodiments are not limited to these. The computing device may be, for example, a laptop computer, a desktop computer, a server, a mobile phone, or a tablet, but the embodiments are not limited to these.

[0104] All patents, patent applications, provisional applications, and publications mentioned or cited herein are incorporated by reference in their entirety, including all figures and tables, unless they conflict with the express teachings herein. [Examples]

[0105] material and method Examples 1-4 used the iCELLis Nano fixed-bed bioreactor system. The iCELLis Nano bioreactor can hold approximately 800 mL, which corresponds to a total surface growth area of ​​approximately 5,300-40,000 with a fixed-bed height of 20 mm-10 mm. The growth area corresponds to 35-267 T-150 flasks that can be used for layered growth (see Figures 5A, 5B, and Table 1). Runs with different parameters were performed using iCELLis.

[0106] Univercells' scale-X® Carbo fixed-bed bioreactor system may be used in predicted examples 5–7 (Berrie DM et al., Vaccine, 2020, 38:3639–3645). Depending on the surface area, the scale-X® Carbo bioreactor can be used in a total container volume of 1.6–3.2 L, up to 10 m³. 2 ~30m 2 It has a surface area for cell growth within a certain range. Using the scale-X® Carbo bioreactor, executions with different parameters can be performed.

[0107] The following are examples illustrating the procedure for carrying out the present invention. These examples should not be construed as limitations. Unless otherwise specified, all percentages are by weight, and all solvent mixture proportions are by volume.

[0108] Example 1. VSV production from a campaign where the parameters served as a baseline for virus production.

[0109] Vero cells were grown in an iCELLis bioreactor at approximately 100% dOi, a temperature of 37°C, and a pH of 7.4. Cell growth was monitored throughout the Vero cell culture period using the bioreactor's biomass sensor, and the Vero cells were infected with VSV at a conductivity (measure of cell growth) of 55 mS / cm. rVSV-LASV was provided by NIAID under mass transfer agreement LAB-18-P_LV-22.

[0110] The system reached peak conductivity (peak cell growth) of approximately 75 mS / cm about 12–24 hours after infection. Infection was at 0.05 MOI. Virus was recovered two days after infection. Viral production increased by more than 1 log / mL compared to titers from the same cells growing in flat stocks (Table 2).

[0111] Example 2. VSV production from a campaign in which dO2 is reduced for approximately 12 hours prior to infection and maintained and recovered throughout the infection.

[0112] Vero cells were cultured at approximately 100% dO2, 37°C, and pH 7.4. Cell growth was monitored using a biomass sensor throughout the Vero cell culture period, and cells were infected with a conductivity of 80 mS / cm (near maximum conductivity) (i.e., Vero cells were infected when cell growth reached its maximum). Approximately 12 hours before infection, the dO2 level was reduced to 45-50% and maintained at this reduced level throughout the infection and recovery period. The temperature was maintained at 37°C and the pH at 7.4. The virus was recovered approximately 2 days after infection. When dO2 was reduced 12 hours before infection and Vero cells were infected with VSV after they had achieved maximum cell growth, the VSV titer increased by more than 2 log compared to the VSV titer from flat stock (Table 2), and the viral titer increased 5.9 times compared to Example 1 (no dO2 reduction). rVSV-LASV was provided by NIAID under the material transfer agreement LAB-18-P_LV-22.

[0113] Example 3. VSV production from campaign with maximum conductivity, in addition to dO2, pH, and temperature remaining constant.

[0114] Vero cells were cultured in a bioreactor at approximately 100% dO2, 37°C, and pH 7.4. Cell growth was monitored using a biomass sensor throughout the Vero cell culture period, and cells were infected with a conductivity of 110 mS / cm (near maximum conductivity, and therefore maximum cell growth was achieved). Infection was 0.05 MOI. No adjustments were made to the dO2 level. The temperature was maintained at 37°C, and the pH was maintained at 7.4 throughout the culture and infection period. Viruses were recovered approximately 2 days after infection. The VSV titer from this experiment was similar to that of Example 1, and the higher yield observed in Example 2 was due to the modification of dO2, not to infection of Vero cells at peak cell growth, which likely increased the overall titer due to more cells being infected (Table 2). [Table 2]

[0115] Table 2 compares proliferation data between different runs. Proliferation data was compared between: 1. VSV grown from Vero cells in a flat stock flask, 2. VSV grown from Vero cells in an iCELLis system (run 1) with a dO2% of 90% during infection, 3. VSV grown from Vero cells in an iCELLis system (run 2) with a dO2% of 40% during infection, and 4. VSV grown from Vero cells in an iCELLis system (run 3) with a dO2% of 20% during infection. The data in Table 2 show significant gradual increases in VSV titer and total virus production from CS10 flask stock, run 1, run 2, and run 3, respectively. This indicates that using the iCELLis flatbed bioreactor to grow VSV resulted in a 1-2 log increase in virus production per mL compared to the virus produced from flat stock. More significantly, the gradual decrease in dO2 during infection resulted in a gradual and significant increase in VSV titer and total viral production. rVSV-LASV was provided by NIAID under material transfer agreement LAB-18-P_LV-22.

[0116] Example 4. VSV production at different dO2 levels during infection.

[0117] VSV was grown in Vero cells at approximately 100% dO2, a temperature of 37°C, and a pH of 7.4. Approximately 12 hours before infection, dO2 levels were reduced to 90%, 40%, and 20%, and maintained at this reduced level throughout the infection. The temperature was maintained at 37°C and the pH at 7.4. The virus was collected approximately 2 days after infection. The results show a gradual increase in viral yield with the level of dO2 reduction during infection, as shown in Table 2. rVSV-LASV was provided by NIAID under material transfer agreement LAB-18-P_LV-22.

[0118] Prediction Example 5 - VSV production from a campaign where parameters can serve as a baseline for future virus production.

[0119] Vero cells can be grown in a Univercells scale-X™ Carbo bioreactor at approximately 100% dO2, 37°C, and pH 7.4 using a suitable chemically defined serum-free medium. Process air parameters can be monitored using O2 and airflow sensors throughout the Vero cell culture period. When a decreasing trend in total airflow to the bioreactor, an increasing trend in total O2 flow to the bioreactor, and convergence of the current values ​​of total airflow and total O2 flow to the bioreactor (e.g., + / - 20%) (an indicator of metabolically active cell density) are observed, the Vero cells can be infected with VSV. The virus can then be recovered 2–5 days after infection. Viral production can be expected to increase by more than 1 log / mL compared to the titer from the same cells grown in flat stock (similar to the actual results shown in Table 2).

[0120] Prediction Example 6 - Influenza production from a campaign where parameters can serve as a baseline for future virus production.

[0121] Quail cells can be grown in a Univercells scale-X™ Carbo bioreactor at approximately 100% dO2, 37°C, and pH 7.4 using a suitable chemically defined serum-free medium. Process air parameters can be monitored using O2 and airflow sensors throughout the quail cell culture period. Quail cells can be infected with VSV when a decreasing trend in total airflow to the bioreactor, an increasing trend in total O2 flow to the bioreactor, and convergence (an indicator of metabolically active cell density) (e.g., current values ​​of total airflow and total O2 flow to the bioreactor) are observed. The virus can then be recovered 2–5 days after infection. Viral production can be expected to increase by more than 1 log / mL compared to titers from the same cells grown in flat stock (similar to actual results shown in Table 2).

[0122] Prediction Example 7 - LVV production from a campaign where parameters can serve as a baseline for future virus production.

[0123] HEK 293 cells can be grown in a Univercells Scale-X® Carbo bioreactor at approximately 100% dO2, 37°C, and pH 7.4 using a suitable chemically defined serum-free medium. Process air parameters can be monitored using O2 and airflow sensors throughout the culture period of HEK 293 cells. When a decreasing trend in total airflow to the bioreactor, an increasing trend in total O2 flow to the bioreactor, and convergence (an indicator of metabolically active cell density) (e.g., current values ​​of total airflow and total O2 flow to the bioreactor) are observed, the HEK 293 cells can be infected with VSV. The virus can then be recovered 2–5 days after infection. Viral production can be expected to increase by more than 1 log / mL compared to titers from the same cells grown in flat stock (similar to actual results shown in Table 2).

[0124] The examples and embodiments described herein are for illustrative purposes only, and various modifications or changes will be suggested in light thereof to those skilled in the art, and it should be understood that these should be included in the spirit and scope of this application and the appended claims. Furthermore, any element or limitation of any invention or embodiment disclosed herein may be combined (individually or in any combination) with any and / or all other elements or limitations, or with any other invention or embodiment disclosed herein, and all such combinations are intended to be within the scope of the present invention without limitation. In certain embodiments, for example, the following items are provided: (Item 1) A method for infecting host cells with a virus without requiring a step of counting host cells, The process of culturing host cells in a bioreactor, A process for observing a set of bioreactor process air parameters, A step of identifying a first time marker based on the bioreactor process air parameters, A step of calculating the optimal time for the infection window based on the first time marker, and The process of infecting the host cells during the calculated optimal time of the infection window. Methods that include... (Item 2) The method according to item 1, wherein the host cells are adherent cells. (Item 3) The method according to item 2, wherein the bioreactor is a fixed-bed bioreactor. (Item 4) The method according to item 3, wherein the step of observing the bioreactor process air parameters includes measuring the velocity of the airflow into the bioreactor, measuring the velocity of the O2 flow into the bioreactor at multiple time points, and creating a set of current measurements for each respective time point. (Item 5) The method according to item 4, wherein the step of identifying a first time marker includes determining the time at which the velocity of the airflow to the bioreactor decreases and intersects with an increasing trend in the velocity of the O2 flow to the bioreactor. (Item 6) The method of item 5, wherein the step of identifying a first time marker includes calculating a value of 1 or greater from a current set of measurements to predict a future time when the velocity of the airflow to the bioreactor is expected to decrease and intersect with an expected increasing trend in the velocity of the O2 flow to the bioreactor. (Item 7) The method according to item 1, wherein the host cell is a naturally occurring cell. (Item 8) The method according to item 1, wherein the host cell is a genetically modified cell. (Item 9) The method according to item 1, wherein the host cell is a naturally occurring or genetically modified mammalian cell, avian cell, or insect cell. (Item 10) The method according to item 1, wherein the host cells are selected from Vero cells, MBCK cells, MDBK cells, MRC-5 cells, BSC-1 cells, LLC-MK cells, CV-1 cells, CHO cells, COS cells, HeLa cells, HEK 293 cells, MDOK cells, CRFK cells, RAF cells, TCMK cells, LLC-PK cells, PK 15 cells, W1-38 cells, T-FLY cells, BHK cells, SP2 / 0 cells, NSO cells, PerC6 cells, COR cells, and QOR cells. (Item 11) The method according to item 1, wherein the host cell is a Vero cell. (Item 12) The method according to item 1, wherein the aforementioned virus is a naturally occurring virus. (Item 13) The method according to item 1, wherein the aforementioned virus is a genetically modified virus. (Item 14) The method according to item 1, wherein the virus is selected from naturally occurring VSV or genetically modified VSV, adenovirus, influenza virus, Ross River virus, hepatitis A virus, vaccinia virus, herpes simplex virus, Japanese encephalitis virus, herpes simplex virus, West Nile virus, yellow fever virus, rhinovirus, reovirus, Ebola-Zaire virus, Ebola-Sudan virus, Ebola-Marburg virus, Nipah virus, or any of the aforementioned chimeras. (Item 15) The method according to item 1, wherein the virus is a viral vector. (Item 16) The method according to item 1, wherein the virus is a modified viral vector containing a glycoprotein derived from a virus of another purpose. (Item 17) The method according to item 15, wherein the viral vector is recombinant VSV (rVSV). (Item 18) The method according to item 1, wherein the optimal time for the infection window includes the time at which the host cells reach the optimal viable cell density. (Item 19) The method according to any one of items 1 to 18, wherein the step of calculating the optimal time for the infection window includes determining the time at which the velocity of the airflow to the bioreactor decreases and intersects with an increasing trend in the velocity of the O2 flow to the bioreactor. (Item 20) The method according to any one of items 1 to 18, wherein the step of calculating the optimal time for the infection window includes determining the time at which the air-O2 difference in the bioreactor is below a predetermined threshold or is expected to fall below the predetermined threshold. (Item 21) The method according to any one of items 1 to 18, wherein the optimal time for the infection window is the time interval at which the decreasing trend in the velocity of the airflow to the bioreactor and the increasing trend in the velocity of the O2 flow to the bioreactor converge to within ±30% of each other, or within ±20% of each other, or within ±10% of each other, or within ±5% of each other. (Item 22) The bioreactor has a surface area of ​​1 m². 2 ~600m 2 A method described in any one of items 1 to 18, having a capacity in the range of [specified range]. (Item 23) The bioreactor has a surface area of ​​600 m². 2 ~2400m 2 A method described in any one of items 1 to 18, having a capacity in the range of [specified range]. (Item 24) The method according to any one of items 1 to 18, wherein the host cells in the bioreactor are not counted before the infection step. (Item 25) The method according to any one of items 1 to 18, further comprising the step of recovering the host cells or products of the host cells after the infection step. (Item 26) The method according to item 25, wherein the host cells in the bioreactor are not counted before the recovery step. (Item 27) The method according to any one of items 1 to 18, wherein the bioreactor does not include a cell counting device and is not connected to a cell counting device. (Item 28) The method according to any one of items 1 to 18, wherein the bioreactor does not have an access port for cell counting. (Item 29) The method according to any one of items 1 to 18, wherein the bioreactor is a closed, sealed (e.g., hermetically sealed) system. (Item 30) A composition comprising cells or cell-derived products produced by the method described in item 1. (Item 31) A method for producing viruses in a bioreactor, a) A step of providing host cells into the bioreactor, b) A step of growing the host cells in the bioreactor, c) A step of observing a set of bioreactor process air parameters, d) A step of calculating the optimal time for the infection window based on the set of bioreactor process air parameters, e) A step of infecting the host cell with at least one virus or viral particle during the optimal time of the infection window, f) A step of incubating the host cells infected with the virus or viral particles to grow the virus, and, if necessary, g) A method comprising the step of recovering the virus. (Item 32) The method according to item 31, wherein the host cells are adherent cells. (Item 33) The method according to item 32, wherein the bioreactor is a flatbed type bioreactor, and the step is to grow the host cells at a constant initial dO2 level, pH, and temperature. (Item 34) The method according to item 31, wherein the bioreactor is a disposable flatbed bioreactor. (Item 35) The method according to items 31-34, wherein the step of calculating the optimal time for the infection window includes determining the time at which the velocity of the airflow to the bioreactor decreases and intersects with an increasing trend in the velocity of the O2 flow to the bioreactor. (Item 36) The method according to any one of items 31 to 34, wherein the step of calculating the optimal time for the infection window includes determining the time at which the air-O2 difference in the bioreactor is below a predetermined threshold or is expected to fall below the predetermined threshold. (Item 37) The method according to any one of items 31 to 34, wherein the optimal time for the infection window is the time interval at which the decreasing trend in the velocity of the airflow to the bioreactor and the increasing trend in the velocity of the O2 flow to the bioreactor converge to within ±30% of each other, or within ±20% of each other, or within ±10% of each other, or within ±5% of each other. (Item 38) The method according to item 31, wherein the infection of the host cell by the virus has a multiple of infection (MOI) of approximately 0.1 to 0.05. (Item 39) The method according to item 338, wherein the infection of the host cell by the virus has an MOI of 0.05. (Item 40) The method according to item 39, wherein the step of incubating the host cells infected with the virus or viral particles to grow the virus comprises incubating the host cells at a certain final dO2 level, pH, and temperature different from the certain initial dO2 level, pH, and temperature. (Item 41) The method according to any one of items 31 to 34, wherein the host cell is a naturally occurring cell. (Item 42) The method according to any one of items 31 to 34, wherein the host cell is a genetically modified cell. (Item 43) The method according to any one of items 31 to 34, wherein the host cell is a naturally occurring or genetically modified mammalian cell, avian cell, or insect cell. (Item 44) The method according to any one of items 31 to 34, wherein the host cells are selected from Vero cells, MBCK cells, MDBK cells, MRC-5 cells, BSC-1 cells, LLC-MK cells, CV-1 cells, CHO cells, COS cells, HeLa cells, HEK 293 cells, MDOK cells, CRFK cells, RAF cells, TCMK cells, LLC-PK cells, PK 15 cells, W1-38 cells, T-FLY cells, BHK cells, SP2 / 0 cells, NSO cells, PerC6 cells, COR cells, and QOR cells. (Item 45) The method according to any one of items 31 to 34, wherein the host cell is a Vero cell. (Item 46) The method described in any one of items 31 to 34, wherein the virus is a naturally occurring virus. (Item 47) The method according to any one of items 31 to 34, wherein the aforementioned virus is a genetically modified virus. (Item 48) The method according to any one of items 31 to 34, wherein the virus is selected from naturally occurring VSV or genetically modified VSV, adenovirus, influenza virus, Ross River virus, hepatitis A virus, vaccinia virus, herpes simplex virus, Japanese encephalitis virus, herpes simplex virus, West Nile virus, yellow fever virus, rhinovirus, reovirus, Ebola-Zaire virus, Ebola-Sudan virus, Ebola-Marburg virus, Nipah virus, or any of the chimeras mentioned above. (Item 49) The method according to any one of items 31 to 34, wherein the virus is a viral vector. (Item 50) The method according to any one of items 31 to 34, wherein the virus is a modified viral vector containing a glycoprotein derived from a virus of another purpose. (Item 51) The method according to item 49, wherein the viral vector is recombinant VSV (rVSV). (Item 52) The bioreactor has a surface area of ​​1 m². 2 ~600m 2 The method described in any one of items 31 to 34, having a capacity in the range of [specified range]. (Item 53) The bioreactor has a surface area of ​​600 m². 2 ~2400m 2 The method described in any one of items 31 to 34, having a capacity in the range of [specified range]. (Item 54) The method according to any one of items 31 to 34, wherein the host cells in the bioreactor are not counted before the infection step. (Item 55) The method according to any one of items 31 to 34, further comprising the step of recovering the host cells or products of the host cells after the infection step. (Item 56) The method according to any one of items 31 to 34, wherein the host cells in the bioreactor are not counted before the recovery step. (Item 57) The method according to any one of items 31 to 34, wherein the bioreactor does not include a cell counting device and is not connected to a cell counting device. (Item 58) The method according to any one of items 31 to 34, wherein the bioreactor does not have an access port for cell counting. (Item 59) The method according to any one of items 31 to 34, wherein the bioreactor is a closed, sealed (e.g., hermetically sealed) system. (Item 60) The method according to any one of items 31 to 34, further comprising the step of determining the viral titer by a plaque assay. (Item 61) The method according to item 60, further comprising the step of purifying and / or characterizing the virus. (Item 62) The method according to any one of items 31 to 34, further comprising the step of manufacturing a vaccine using the aforementioned virus. (Item 63) The method according to any one of items 31 to 34, wherein the bioreactor comprises a chemically defined culture medium. (Item 64) A composition comprising cells or cell-derived products produced by the method described in item 31. (Item 65) A method for producing viruses in a bioreactor, a) A step of providing host cells into the bioreactor, b) A step of growing host cells at a constant initial dO2 level, pH, and temperature until confluence, c) A step of calculating the optimal infection window time based on a set of bioreactor process air parameters. d) The step of infecting the host cell with at least one virus or viral particle during the optimal time of the infection window, e) A step of incubating the host cells infected with the virus or viral particles to grow the virus, and, if necessary, f) Steps to recover the virus Methods that include... (Item 66) A system for infecting host cells with a virus without requiring a step of counting host cells, A bioreactor configured and adapted for cell culture, viral infection of cells, viral replication, and viral recovery, The cell culture medium in the bioreactor, The air space above the cell culture medium in the bioreactor, The air inlet to the bioreactor, An airflow sensor for measuring the airflow to the bioreactor, The O2 inlet to the bioreactor, An O2 flow sensor for measuring the O2 flow to the bioreactor, A data collection module, The current airflow to the bioreactor, Current trends in airflow to the bioreactor, The current O2 flow to the bioreactor, and Current trends in O2 flow to the bioreactor A data acquisition module configured and adapted to collect values ​​representing, It is an instruction unit, The decreasing trend of airflow to the bioreactor, The increasing trend of O2 flow to the bioreactor, and Convergence between the current values ​​of the airflow to the bioreactor and the O2 flow to the bioreactor An indicator unit configured and adapted to indicate when an event occurs. A system that includes the following. (Item 67) The aforementioned data acquisition unit, At least one first processor that is operably communicating with the airflow sensor and the O2 flow sensor, At least one first machine-readable medium that is operably communicating with the at least one first processor, when executed by the at least one first processor, A step of recording readings from the airflow sensor in order to generate the current airflow value to the bioreactor, A step of generating the current trend of the airflow to the bioreactor by comparing the current airflow value with at least one previous airflow value, A step of recording readings from the O2 flow sensor in order to generate the current O2 flow value to the bioreactor, and The system according to item 66, comprising: at least one first machine-readable medium having instructions stored in the at least one first machine-readable medium, which performs the step of: comparing the current value of the O2 flow with at least one previous value of the O2 flow to generate a current trend of the O2 flow to the bioreactor. (Item 68) The instruction unit, At least one second processor that is operationally communicating with the data acquisition unit, At least one second machine-readable medium that is operably communicating with the at least one second processor, when executed by the at least one second processor, A set of instructional conditions The current trend of airflow to the bioreactor is decreasing. The current trend of O2 flow to the bioreactor is increasing, and When the set of indicative conditions is met, including the existence of convergence between the current values ​​of the airflow to the bioreactor and the O2 flow to the bioreactor, A step of indicating a time window for the virus to infect the cells, comprising at least one second machine-readable medium having instructions stored in the at least one second machine-readable medium, The system described in item 67, comprising: (Item 69) The system according to item 68, further comprising a decision-making unit configured and adapted to initiate infection of the cells by the virus when the time window for infection of the cells by the virus is indicated. (Item 70) The system according to item 69, wherein the at least one first processor and the at least one second processor are the same processor. (Item 71) The system according to item 70, wherein the at least one first machine-readable medium and the at least one second machine-readable medium are the same machine-readable medium. (Item 72) The system according to item 71, wherein the bioreactor is a sealed bioreactor. (Item 73) The system according to item 72, wherein the value of the O2 flow to the bioreactor is within + / - 20% of the value of the air flow to the bioreactor. (Item 74) The system according to item 66, wherein the data acquisition module comprises a mechanical sensor or an analog electrical sensor. (Item 75) The system according to item 66, wherein the indicator unit comprises one or more audible indicators, visual indicators, or tactile indicators. (Item 76) A method for infecting host cells with a virus without requiring a step of counting host cells, To provide a bioreactor configured and adapted for cell culture, viral infection of cells, viral replication, and viral recovery, To provide cell culture medium in the bioreactor, To provide an air space above the cell culture medium in the bioreactor, To provide an air inlet to the bioreactor, To provide an airflow sensor for measuring the airflow to the bioreactor, To provide an O2 inlet to the bioreactor, To provide an O2 flow sensor for measuring the O2 flow to the bioreactor, The data collection module, The current airflow to the bioreactor, Current trends in airflow to the bioreactor, The current O2 flow to the bioreactor, and Current trends in O2 flow to the bioreactor To collect and According to the instruction unit, The decreasing trend of airflow to the bioreactor, The increasing trend of O2 flow to the bioreactor, and Convergence between the current values ​​of the airflow to the bioreactor and the O2 flow to the bioreactor A method that includes indicating when something occurs. (Item 77) The aforementioned data acquisition unit, At least one first processor that is operably communicating with the airflow sensor and the O2 flow sensor, At least one first machine-readable medium that is operably communicating with the at least one first processor, when executed by the at least one first processor, A step of recording readings from the airflow sensor in order to generate the current airflow value to the bioreactor, A step of generating the current trend of the airflow to the bioreactor by comparing the current airflow value with at least one previous airflow value, A step of recording readings from the O2 flow sensor in order to generate the current O2 flow value to the bioreactor, and The method according to item 76, comprising: at least one first machine-readable medium having instructions stored in the at least one first machine-readable medium, which performs the step of: comparing the current value of the O2 flow with at least one previous value of the O2 flow to generate a current trend of the O2 flow to the bioreactor. (Item 78) The instruction unit, The data acquisition unit and at least one second processor that is operably communicating with the bioreactor, At least one second machine-readable medium that is operably communicating with the at least one second processor, when executed by the at least one second processor, The current trend of airflow to the bioreactor is decreasing. The current trend of O2 flow to the bioreactor is increasing, and If there is a convergence between the current values ​​of the airflow to the bioreactor and the O2 flow to the bioreactor, and only if so, A step of indicating a time window for the virus to infect the cells, comprising at least one second machine-readable medium having instructions stored in the at least one second machine-readable medium, The method described in item 77, comprising: (Item 79) The method of item 78, further comprising initiating infection of the cells by the virus when the time window for infection of the cells by the virus is indicated. (Item 80) The method according to item 78, wherein the at least one first processor and the at least one second processor are the same processor. (Item 81) The method according to item 80, wherein the at least one first machine-readable medium and the at least one second machine-readable medium are the same machine-readable medium. (Item 82) The method according to item 81, wherein the bioreactor is a sealed bioreactor. (Item 83) The method according to item 82, wherein the value of the O2 flow to the bioreactor is within + / - 20% of the value of the air flow to the bioreactor. (Item 84) The method according to item 76, wherein the data acquisition module comprises a mechanical sensor or an analog electrical sensor. (Item 85) The method according to item 76, wherein the indicator unit comprises one or more audible indicators, visual indicators, or tactile indicators.

Claims

[Claim 1] The invention described in the specification.

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