Virus recovery method

Abstract

To provide a virus recovery method with a high recovery rate.SOLUTION: The purpose of the invention is to provide a virus recovery method including reducing viability of cells in a culture solution and filtering, through a hollow fiber membrane, the culture solution containing a virus produced by the cells to recover the virus. The hollow fiber membrane has a gradient structure in which an average pore size becomes smaller from an upstream side to a downstream side in a membrane thickness direction.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a filtration method. [Background technology]

[0002] Cell culture technology is an essential technology for the production of various biopharmaceuticals, such as viruses including viral vectors, antibodies, growth hormones, and insulin, and has made a significant contribution to the advancement of medicine in recent years. Among biopharmaceuticals, viral preparations and viral vaccines in particular have attracted attention. Highly efficient and stable production of viral preparations and viral vaccines by culturing virus-producing cells is one of the industrially important themes (see, for example, Patent Documents 1 to 4).

[0003] When producing viruses for use in biopharmaceuticals, it is necessary to culture virus-producing cells, remove them from the cell culture solution, and purify the virus. Conventional methods for removing cells from cell culture solutions include gel filtration, centrifugation, adsorption separation, precipitation, and membrane filtration. However, gel filtration has problems such as dilution of the target substance by the solvent used in gel filtration and its unsuitability for large-scale processing, making it difficult to use industrially. Centrifugation can only be applied when the solution has a low viscosity, making it difficult to install large-scale equipment. Sedimentation has the problem that it cannot completely remove cells by itself. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Publication No. 2018-076291 [Patent Document 2] Patent No. 6530171 [Patent Document 3] International Publication No. 2010 / 035793 [Patent Document 4] International Publication No. 2020 / 023612 Summary of the Invention [Problem to be solved by the invention]

[0005] Membrane filtration using microfiltration membranes or ultrafiltration membranes is suitable for industrial use because it is easy to remove cells and can continuously process large amounts of solution. However, conventional membrane filtration methods have the problem that a concentrated layer of cells and cell debris forms on the membrane surface, clogging the membrane surface, increasing the filtration pressure, and decreasing the filtration rate over time. In particular, when the cell viability in the cell culture medium is low and the cell culture medium contains many dead cells, a large amount of cell debris is present in the cell culture medium, and the cell debris accumulates on the membrane surface and inside the membrane, preventing the permeation of the target product and reducing the recovery rate of the target product.

[0006] Furthermore, according to the findings of the present inventors, the viability of cells decreases with the passage of culture time. However, the amount of substances produced by cells increases with the passage of culture time. Furthermore, the amount of substances produced by cells may increase at the same time as the viability of the cells decreases. Furthermore, according to the findings of the present inventors, when the substances produced accumulate inside the cells, for example, prolonged culture can decrease the cell viability and allow the substances produced to be released outside the cells. However, according to the findings of the present inventors, there is a problem in that a cell culture solution containing cells with low viability has a lower filtration throughput, i.e., a lower filtration efficiency, compared to a cell culture solution containing cells with high viability.

[0007] Furthermore, conventional filtration membranes have the problem that their permeability to the target substance decreases with continued use. When the permeability of the target substance through the filtration membrane decreases, the recovery rate of the target substance decreases and the proportion of impurities such as protein aggregates can increase. A decrease in the recovery rate of the target substance can lead to an increase in the manufacturing costs of pharmaceuticals that use cell-produced substances as raw materials, as well as an increase in medical expenses.

[0008] Therefore, an object of the present invention is to provide a method for recovering viruses with a high recovery rate. For example, an object of the present invention is to provide a method for filtering a culture solution containing viruses with a good virus permeability. [Means for solving the problem]

[0009] A virus recovery method according to an embodiment of the present invention includes reducing the viability of cells in a culture solution, filtering the culture solution containing the virus produced by the cells using a hollow fiber membrane, and recovering the virus, wherein the hollow fiber membrane has a gradient structure in which the average pore size decreases from the primary side to the secondary side in the membrane thickness direction. The filtration may be tangential flow filtration.

[0010] In the above virus recovery method, cells may be removed by filtration.

[0011] In the above virus recovery method, cellular debris may be removed by filtration.

[0012] In the above-mentioned virus recovery method, the pore size on the primary side of the hollow fiber membrane may be 20 μm or more and 100 μm or less.

[0013] In the above-mentioned virus recovery method, the blocking pore size of the pores in the hollow fiber membrane may be 0.05 μm or more and 20 μm or less.

[0014] In the above-mentioned virus recovery method, the hollow fiber membrane may be made of a synthetic polymer.

[0015] In the above-mentioned virus recovery method, the synthetic polymer may be polysulfone.

[0016] In the above-mentioned virus recovery method, the hollow fiber membrane may have a coarse layer and a dense layer.

[0017] In the above-mentioned virus recovery method, the cell viability may be reduced by chemical or physical treatment.

[0018] In the above-mentioned virus recovery method, the cells may be contacted with a chemical substance to reduce the cell viability.

[0019] In the above-described virus recovery method, the chemical substance may be a surfactant, an acidic substance, or a basic substance.

[0020] In the above-mentioned virus recovery method, cells may be lysed with a surfactant.

[0021] In the above-mentioned virus recovery method, the surfactant may be a nonionic surfactant or a zwitterionic surfactant.

[0022] In the above-mentioned virus recovery method, the concentration of the nonionic surfactant in the culture medium may be 0.005% or more, 0.01% or more, 0.02% or more, 0.03% or more, 0.04% or more, 0.05% or more, 0.06% or more, 0.07% or more, 0.08% or more, 0.09% or more, or 0.1% or more.

[0023] In the above-mentioned virus recovery method, the concentration of the nonionic surfactant in the culture medium may be 1% or less, 0.9% or less, 0.8% or less, 0.7% or less, 0.6% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, or 0.1% or less.

[0024] In the above-mentioned virus recovery method, the concentration of the zwitterionic surfactant in the culture medium may be 0.01% or more, 0.02% or more, 0.04% or more, 0.06% or more, 0.08% or more, 0.1% or more, 0.2% or more, 0.3% or more, 0.4% or more, or 0.5% or more.

[0025] In the above-mentioned virus recovery method, the concentration of the zwitterionic surfactant in the culture medium may be 2.0% or less, 1.8% or less, 1.6% or less, 1.4% or less, 1.2% or less, 1.0% or less, 0.8% or less, 0.6% or less, or 0.5% or less.

[0026] In the above virus recovery method, the cells may be transfected with a chemical to reduce the cell viability.

[0027] In the above-mentioned virus recovery method, the physical treatment may include disrupting the cells.

[0028] In the above-mentioned method for recovering a virus, the cell viability after reducing the cell viability may be 60% or less.

[0029] In the above-mentioned method for recovering a virus, the cell viability after reducing the cell viability may be 30% or less.

[0030] In the above virus recovery method, the cell density in the culture medium before reducing the cell viability is 1.0 × 10 5 cells / mL or more, 2.0×10 5 cells / mL or more, 4.0×10 5 cells / mL or more, 6.0×10 5 cells / mL or more, 8.0×10 5 cells / mL or more, or 1.0 × 10 6 cells / mL or more.

[0031] In the above virus recovery method, the cell density in the culture medium before reducing the cell viability is 1.0 × 10 8 cells / mL or less, 8.0×10 7 cells / mL or less, 6.0×10 7 cells / mL or less, 4.0×10 7 cells / mL or less, 2.0×10 7 cells / mL or less, 1.0×107 cells / mL or less, 8.0×10 6 cells / mL or less, 6.0×10 6 cells / mL or less, 4.0×10 6 cells / mL or less, or 2.0 x 10 6 It may be less than cells / mL.

[0032] In the above-mentioned method for recovering a virus, the cells may be genetically modified cells.

[0033] In the above-mentioned method for recovering a virus, the virus may be a non-enveloped virus.

[0034] In the above-mentioned method for recovering a virus, the virus may be a parvovirus.

[0035] In the above-mentioned method for recovering a virus, the virus may be an adeno-associated virus.

[0036] In the above-mentioned method for recovering a virus, the virus may be an enveloped virus.

[0037] In the above-mentioned method for recovering a virus, the virus may be a retrovirus.

[0038] In the above-mentioned method for recovering a virus, the virus may be a lentivirus.

[0039] The above virus recovery method may be substantially free of removing cells and / or cell debris with a precipitant prior to filtration. [Effects of the Invention]

[0040] According to the present invention, it is possible to provide a method for recovering viruses with a high recovery rate. [Brief explanation of the drawings]

[0041] [Figure 1]1 is a graph showing AAV permeability according to an example. [Figure 2] 1 is a graph showing HCP permeability according to an example. DETAILED DESCRIPTION OF THE INVENTION

[0042] The following provides a detailed description of the present invention (hereinafter, sometimes abbreviated as "embodiments" in this specification). Note that the following embodiments are merely examples of methods for embodying the technical ideas of the present invention, and the present invention is not limited to these examples.

[0043] The virus recovery method according to the embodiment includes reducing the viability of cells in a culture medium, filtering the culture medium containing the virus produced by the cells using a hollow fiber membrane, and recovering the virus, wherein the hollow fiber membrane has a gradient structure in which the average pore size decreases in the membrane thickness direction from the primary side to the secondary side.

[0044] The step of reducing cell viability is not particularly limited as long as it is a step of reducing the viability of cells in a cell culture medium by an active action, and may be, for example, a step of reducing cell viability by chemical or physical treatment. For example, simply culturing cells for a long period of time does not involve chemical or physical treatment and is not an active action that reduces cell viability. On the other hand, for example, transfection is an active step of reducing cell viability by causing cells to produce substances that may be cytotoxic, and is exemplified as one aspect of a step of reducing cell viability by chemical treatment. Cell disruption is a chemical or physical treatment that can damage cell membranes. The method of reducing viability may be either physical or chemical, but chemical treatment is preferred as it is easy to scale up.

[0045] The chemical treatment according to the embodiment includes the addition of a chemical substance. The chemical substance is not particularly limited as long as it reduces the viability. Examples of the chemical substance include at least one of a factor such as a nucleic acid that produces a useful substance, a vector, and a transfection reagent, which are introduced into cells during transfection. Other examples of the chemical substance include a surfactant, an acidic substance, or a basic substance. An acidic substance may be dissolved in a solvent (e.g., water or alcohol) to form an acidic solution. A basic substance may be dissolved in a solvent (e.g., water or alcohol) to form a basic solution. Examples of the acidic substance include hydrochloric acid, sulfuric acid, nitric acid, and acetic acid. Examples of the basic substance include sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, and ammonia.

[0046] The cells may be derived from animals, including humans, or from microorganisms. Examples of animals include mammals, reptiles, birds, amphibians, fish, and insects. Examples of the cells include, but are not limited to, cells that produce useful substances. The cells may be genetically modified cells. Examples of the cells include, but are not limited to, HEK293 cells, HEK911 cells, PER.C6 cells, Sf9 cells, and CHO (Chinese Hamster Ovary) cells.

[0047] Viruses include virus-like particles (VLPs). Viruses include viral vectors. Viruses include oncolytic viruses. Viruses include viral vaccines. Viral vaccines include bioengineered vaccines. Viruses include viruses that are released from cells into cell culture medium and viruses that accumulate inside cells.

[0048] The virus may be a non-enveloped virus or an enveloped virus. Examples of non-enveloped viruses include, but are not limited to, adenovirus, adeno-associated virus (AAV), and parvovirus. AAV has a weaker charge than adenovirus and tends to be more easily adsorbed to hollow fiber membranes. Examples of enveloped viruses include, but are not limited to, retrovirus, lentivirus, Sendai virus, rabies virus, Sindbis virus, and herpes simplex virus.

[0049] The cells may be transfected, for example. Transfection introduces factors such as nucleic acids that produce viruses into the cells. Transfection reduces cell viability. Factors such as nucleic acids are chemical substances. Factor introduction by transfection includes, for example, factor introduction by electroporation, factor introduction by lipofection, and factor introduction by a viral vector.

[0050] Cells may be cultured by either adherent culture, in which cells adhere to the inner surface of a culture vessel, or suspension culture, in which cells are suspended in a cell culture medium.

[0051] In a method of culturing cells by suspension culture, a stirring mechanism is installed in a culture vessel such as a spinner flask to suspend the cells. The stirring mechanism may be a magnetic stirrer or a mechanically driven shaft-shaped impeller. A method of culturing cells by suspension culture, in which fresh medium is supplied to the culture vessel while old cell culture medium containing impurities such as growth inhibitors is discharged from the culture vessel, is called continuous culture.

[0052] The culture method may be a batch culture method, a fed-batch culture method, or a continuous culture method. A batch culture method is a culture method in which new cell culture medium is not supplied to the culture tank. A fed-batch culture method is a culture method in which new cell culture medium is supplied to the culture tank. A continuous culture method is a culture method in which new cell culture medium is supplied to the culture tank and old cell culture medium is discharged from the culture tank while maintaining a constant amount of cell culture medium in the culture tank. The step of reducing the viability may be performed after the culture or during the culture. The filtration operation may be performed after the culture or continuously during the culture.

[0053] When recovering viruses from cells, the cells may be disrupted. Disrupting the cells allows the viruses inside the cells to diffuse into the cell culture medium. Disrupting the cells reduces the cell viability. Chemical treatment to lyse the cells may be performed by adding chemicals such as surfactants, acidic substances, and basic substances to the cells. However, enveloped viruses are produced extracellularly, so cell lysis is not required. Furthermore, non-enveloped viruses are generally produced intracellularly, but adeno-associated viruses (AAVs), such as AAV8 and AAV9, are produced extracellularly depending on their serotypes. In this case, cell lysis is not required.

[0054] The surfactant may be a nonionic surfactant or a zwitterionic surfactant. A nonionic surfactant and a zwitterionic surfactant may be mixed at an appropriate concentration. The surfactant may be present in an appropriate solvent. Nonionic and zwitterionic surfactants generally tend to denature proteins less than ionic surfactants.

[0055] Examples of non-ionic surfactants include, but are not limited to, Triton X-100, Triton X-114, NP-40, Brij-35, Brij-58, Tween-20, Tween-80, Octyl Glucoside, and Octylthio Glucoside.

[0056] Examples of zwitterionic detergents include, but are not limited to, CHAPS (3-[(3-Cholamidopropyl)dimethylammonio]propanesulfonate) and CHAPSO (3[(3-Cholamidopropyl)dimethylammonio]-2-hydroxypropanesulfonic acid).

[0057] The cell density in the cell culture medium before cell disruption is, for example, 1.0 × 10 5 cells / mL or more, 2.0×10 5 cells / mL or more, 4.0×10 5 cells / mL or more, 6.0×10 5 cells / mL or more, 8.0×10 5 cells / mL or more, or 1.0 × 10 6 cells / mL or more and 1.0 x 10 8 cells / mL or less, 8.0×10 7 cells / mL or less, 6.0×10 7 cells / mL or less, 4.0×10 7 cells / mL or less, 2.0×10 7 cells / mL or less, 1.0×10 7 cells / mL or less, 8.0×10 6 cells / mL or less, 6.0×10 6 cells / mL or less, 4.0×10 6 cells / mL or less, or 2.0 x 10 6 The cell density in the cell culture medium can be measured using an automatic cell counter (CYTORECON, manufactured by GE Healthcare).

[0058] When the cell density in the cell culture medium is as described above, the concentration of the nonionic surfactant in the cell culture medium is, for example, preferably 0.005% or more, and from the viewpoint of releasing intracellular substances, more preferably 0.01% or more, 0.02% or more, 0.03% or more, 0.04% or more, 0.05% or more, 0.06% or more, 0.07% or more, 0.08% or more, 0.09% or more, or 0.1% or more. Furthermore, the concentration is preferably 1% or less, and from the viewpoint of being able to remove the surfactant used in the downstream purification step, more preferably 0.9% or less, 0.8% or less, 0.7% or less, 0.6% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, or 0.1% or less.

[0059] When the cell density in the cell culture medium is as described above, the concentration of the zwitterionic surfactant in the cell culture medium is, for example, preferably 0.01% or more, and from the viewpoint of releasing intracellular substances, more preferably 0.02% or more, 0.04% or more, 0.06% or more, 0.08% or more, 0.1% or more, 0.2% or more, 0.3% or more, 0.4% or more, or 0.5% or more. Furthermore, the concentration is preferably 2.0% or less, and from the viewpoint of removing the surfactant used in the downstream purification step, more preferably 1.8% or less, 1.6% or less, 1.4% or less, 1.2% or less, 1.0% or less, 0.8% or less, 0.6% or less, or 0.5% or less.

[0060] The method for disrupting cells is not limited to chemical treatment. For example, cells may be disrupted using enzymes. Alternatively, cells may be disrupted by mechanical or physical treatment that applies pressure to the cells. Cells may be disrupted using machines such as high-pressure homogenizers or mills. The pressure may be ultrasonic, cavitation, or osmotic pressure. Alternatively, cells may be disrupted by heat treatment that applies heat to the cells. Alternatively, cells may be disrupted by applying a heat shock to the cells using a freeze-thaw method. After the cells are disrupted, they may be maintained in a batch culture without supplying new cell culture medium to the culture vessel.

[0061] In one embodiment, the production of a cell-produced substance (virus) can be carried out in the following order: cell culture, transfection, cell culture, detergent treatment of the cells, and batch filtration of the cell culture medium. In another embodiment, the production of a cell-produced substance can be carried out in the following order: cell culture, transfection, cell culture, and batch filtration of the cell culture medium. In yet another embodiment, the production of a cell-produced substance can be carried out in the following order: cell culture, transfection, and continuous culture.

[0062] Cell viability refers to the ratio of the number of living cells to the total number of cells in a solution. In some embodiments, the cell viability before a step of reducing cell viability is, for example, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more. The cell viability after a step of reducing cell viability and immediately before filtration is, for example, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less. Here, a step of reducing cell viability is, for example, but not limited to, the transfection described above.

[0063] In one embodiment, the cell viability before a step of reducing cell viability is, for example, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, or 70% or more, and the cell viability after a step of reducing cell viability and immediately before filtration is, for example, 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less. Here, the step of reducing cell viability is, for example, but not limited to, the above-mentioned cell disruption treatment, which is a step subsequent to transfection.

[0064] In some embodiments, the cell viability before a step of reducing cell viability is, for example, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more, and the cell viability after a step of reducing cell viability and immediately before filtration is, for example, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less. Here, a step of reducing cell viability is, for example, but not limited to, the above-mentioned transfection combined with continuous culture.

[0065] In addition, when there are multiple steps of reducing cell viability, the above cell viability refers to the cell viability before and after one step of reducing cell viability that is closest to the step of filtering the cell culture solution using a hollow fiber membrane.

[0066] The cell viability can be measured using an automatic cell counter (Vi-CELL XR, manufactured by Beckman Coulter).

[0067] The culture vessel containing the cell culture solution containing the cells to be filtered may be provided with an outlet for sending the cell culture solution to the primary surface of the hollow fiber membrane, and an inlet for returning the cell culture solution that has passed through the primary surface of the hollow fiber membrane without permeating the interior of the hollow fiber membrane to the culture vessel. The outlet and the inlet may be the same or different.

[0068] The surface of the hollow fiber membrane where the cell culture fluid to be filtered is supplied is called the primary side, and the surface where the permeate that has permeated the hollow fiber membrane flows out is called the secondary side.

[0069] In an embodiment in which the cell culture medium to be filtered is supplied to the inner peripheral surface, the inner peripheral surface of the hollow fiber membrane is the primary side, and the outer peripheral surface of the hollow fiber membrane is the secondary side. In an embodiment in which the cell culture medium to be filtered is supplied to the outer peripheral surface, the outer peripheral surface of the hollow fiber membrane is the primary side, and the inner peripheral surface of the hollow fiber membrane is the secondary side.

[0070] The plurality of hollow fiber membranes may be included in, for example, a filtration module. The filtration module may be provided with a cell culture medium inlet that is an inlet for the inflow of cell culture medium delivered from the culture tank and that is connected to the primary surface of the hollow fiber membrane, and a cell culture medium outlet that returns the cell culture medium that has passed through the primary surface of the hollow fiber membrane without permeating the interior of the hollow fiber membrane to the culture tank. The cell culture medium inlet and the cell culture medium outlet may be the same or different. The filtration module may also be provided with a permeate outlet that allows the virus-containing permeate that has permeated the interior of the hollow fiber membrane and flowed out from the secondary surface of the hollow fiber membrane to flow out.

[0071] The culture tank and the filtration module are connected by a liquid transfer mechanism including, for example, a flow path and a pump. The liquid transfer mechanism may include a pressure gauge and a weight gauge. Examples of the pump include, but are not limited to, a diaphragm pump, a tube pump, and a rotary pump.

[0072] By filtering a cell culture solution containing cultured cells using a hollow fiber membrane, the cells in the cell culture solution are removed, and the virus in the cell culture solution passes through the hollow fiber membrane and is purified.

[0073] The filtration may be tangential flow filtration. Tangential flow filtration (TFF) is a filtration method in which a cell culture solution is flowed in a direction parallel to the primary surface of a hollow fiber membrane on the primary surface of the hollow fiber membrane. Tangential flow filtration (TFF) includes alternating tangential flow filtration (ATF). In this embodiment, the term "tangential flow filtration (TFF)" may refer to a filtration method in which a cell culture solution is flowed in one direction in the hollow portion of a hollow fiber membrane. Alternating tangential flow filtration (ATF) refers to a filtration method in which a cell culture solution is flowed back and forth in the hollow portion of a hollow fiber membrane.

[0074] The porous structure of a hollow fiber membrane has a gradient structure as a whole. In a hollow fiber membrane with a gradient structure, the average pore size on the primary surface is larger than the average pore size on the secondary surface, and the pore size decreases from the primary surface toward the minimum pore size layer. Note that there may be a portion between the primary surface and the secondary surface where the pore size does not change. Therefore, in a hollow fiber membrane with a gradient structure, the pore size distribution is asymmetric in the membrane thickness direction. The layer with a relatively large pore size near the primary surface of the hollow fiber membrane is called the coarse layer. The layer with a relatively small pore size near the secondary surface of the hollow fiber membrane is called the dense layer. The minimum pore size layer, where the pore size is smallest, is included in the dense layer.

[0075] The average pore size of the primary surface of a hollow fiber membrane having a gradient structure is, for example, 1 μm or more, 10 μm or more, 20 μm or more, or 30 μm or more, and 100 μm or less, 90 μm or less, or 80 μm or less. A pore size of 1 μm or more on the primary surface tends to facilitate depth filtration, which retains removed materials inside the membrane, and tends to reduce clogging of membrane pores due to accumulation of removed materials on the membrane surface. Furthermore, a pore size of 100 μm or less on the primary surface tends to facilitate maintaining the strength of the hollow fiber membrane.

[0076] The average pore size of the secondary surface of the hollow fiber membrane having a gradient structure is, for example, 0.1 μm to 20 μm, 0.2 μm to 15 μm, or 0.3 μm to 10 μm.

[0077] To determine the average pore size on the surface of a hollow fiber membrane, the hollow fiber membrane is freeze-dried and 10 or more pores are observed in one field of view using an electron microscope (Keyence Corporation, VE-9800). Each of the 10 observed pores is approximated to a circle, and the average diameter calculated from the area of ​​the 10 circularly approximated pores is calculated as the average pore size.

[0078] The blocking pore size of the hollow fiber membrane having a gradient structure is, for example, 0.05 μm or more, 0.1 μm or more, 0.2 μm or more, or 0.3 μm or more. The blocking pore size of the hollow fiber membrane having a gradient structure is, for example, 20 μm or less, 10 μm or less, 5 μm or less, 3 μm or less, 1 μm or less, 0.8 μm or less, or 0.5 μm or less. A blocking pore size of 0.05 μm or more tends to reduce permeation resistance, reduce the pressure required for filtration, and prevent clogging of the membrane surface due to destruction and deformation of microbial particles, as well as a decrease in filtration efficiency. A blocking pore size of 20 μm or less tends to provide sufficient fractionation.

[0079] The blocking pore size is exemplified as the pore size of particles at which a 90% permeation rejection rate is achieved when a particle dispersion containing particles of a certain pore size is filtered using a porous hollow fiber membrane. It is also sometimes referred to as the minimum pore size. To determine the blocking pore size, polystyrene latex particles (Size Standard Particulates, manufactured by JSR Corporation) are dispersed in a 0.5% by mass aqueous solution of sodium dodecyl sulfate (manufactured by Wako Pure Chemical Industries, Ltd.) to a particle concentration of 0.01% by mass to prepare a latex particle dispersion. The latex particle dispersion is filtered using a porous hollow fiber membrane, and the change in latex particle concentration before and after filtration is measured. This measurement is performed while varying the latex particle size in approximately 0.1 μm increments from 0.1 μm, and a blocking curve for the latex particles is created. From this blocking curve, the particle size that blocks 90% permeation is read and used as the blocking pore size.

[0080] The hollow fiber membrane having a gradient structure may include a minimum pore size layer near the secondary surface, where the pore size is smallest. The minimum pore size in the hollow fiber membrane is approximately the same as the blocking pore size.

[0081] The inner diameter of the hollow portion of the hollow fiber membrane is, for example, 1000 μm to 2000 μm, 1000 μm to 1500 μm, or 1100 μm to 1400 μm. When the inner diameter of the hollow portion is 1000 μm or more, the entrance of the hollow portion tends to be less likely to be blocked by cells. When the inner diameter of the hollow portion is 2000 μm or less, the number of hollow fiber membranes constituting the filtration module increases, the effective cross-sectional area per filtration module increases, and filtration performance tends to be excellent.

[0082] The membrane thickness of the hollow fiber membrane is, for example, 300 μm to 1000 μm, 350 μm to 800 μm, or 350 μm to 500 μm. A membrane thickness of 300 μm or more can retain the removed material inside the membrane, and tends to more easily demonstrate the effect of depth filtration. Furthermore, it tends to be easier to maintain an appropriate filtration rate. Furthermore, a membrane thickness of 1000 μm or less increases the number of hollow fiber membranes constituting the filtration module, increasing the effective cross-sectional area per filtration module and tending to improve filtration performance.

[0083] The inner diameter (μm) and outer diameter (μm) of the hollow fiber membrane can be measured by cutting the hollow fiber membrane into a thin cylindrical shape and observing the slice under an optical microscope (Keyence Corporation, VH6100). I and outer diameter D O From the above, the membrane thickness T of the hollow fiber membrane is calculated using the following formula: h (μm) can be calculated. T h =(D O -D I ) / 2

[0084] The virus permeability can be measured by quantitatively comparing the virus concentration in the permeate after the filtration step with the virus concentration in the cell culture medium or cell lysate before the filtration step using quantitative PCR. For example, the virus permeability can be calculated using the following formula: Permeability X = (virus concentration in the permeate) / (virus concentration in the cell culture medium or cell lysate before filtration) x 100

[0085] The virus concentration is determined by measuring the amount of virus. The amount of virus can be measured by real-time PCR. When the virus is AAV, the AAVpro Titration Kit (Takara Bio) can be used as a reagent to measure the amount of AAV. The cell culture medium and cell lysate before filtration are centrifuged at 300 × g for 2 minutes, and the supernatant is sampled.

[0086] The antibody concentration is measured by HPLC using the following method. (1) Detector: ultraviolet absorption photometer (measurement wavelength: 280 nm) (2) Column: POROS G 20 μm Column, 4.6 × 50 mm, 0.8 mL (ThermoFisher) (3) Column temperature: room temperature (4) Mobile phase Mobile phase A: Dissolve 7.098 g of disodium hydrogen phosphate (anhydrous) and 8.766 g of sodium chloride in 800 mL of water, add 1 mol / L hydrochloric acid to adjust the pH to 7.0, and then add water to make 1000 mL. Mobile phase B: Dissolve 12 mL of 1 mol / L hydrochloric acid and 8.766 g of sodium chloride in water to make 1000 mL. (5) Delivery of mobile phase The flow rate is 2 mL / min, and the ratio of mobile phase A to mobile phase B is changed as shown in the table below. [Table 1]

[0087] The cell culture medium and cell lysate were centrifuged at 300 × g for 2 minutes, and the supernatant was sampled. Using the above procedure, nine serial dilutions of commercially available human immunoglobulin G (Japan Blood Products Organization, Donated Blood Venoglobulin IH 5% Intravenous Injection 2.5 g / 50 mL), as well as the permeate, cell culture medium supernatant, and cell lysate supernatant, were prepared. A standard curve was created using the nine peak areas of human immunoglobulin G, and the antibody concentration in each solution was calculated from the standard curve and the peak areas of the samples.

[0088] The hollow fiber membrane is made of, for example, a synthetic polymer membrane. The synthetic polymer is, for example, hydrophobic. An example of the synthetic polymer is polysulfone, but it is not limited to this. Generally, impurities such as cells and debris contained in a cell culture solution are hydrophobic. The impurities are captured by the hollow fiber membrane through hydrophobic interactions. This makes it possible to purify the cell culture solution containing useful substances. The hollow fiber membrane according to the embodiment can be manufactured by referring to, for example, the method described in International Publication No. 2010 / 035793.

[0089] The virus recovery method according to this embodiment may include removing cells and cell debris before filtering, but because the virus recovery method according to this embodiment can recover viruses with high efficiency, it may not substantially include removing cells and cell debris before filtering. Examples of methods for removing cells and cell debris include removing cells and cell debris using a precipitant and removing cells and cell debris by centrifugation. When removing cells and cell debris, impurity proteins and DNA may also be removed.

[0090] The turbidity in the solution can be measured using a portable turbidity meter (Fuji Tecom Portable Turbidity Meter 210Q).

[0091] Without being bound by theory, reducing the likelihood of clogging of hollow fiber membranes can suppress a decrease in the throughput of filtration through hollow fiber membranes. For example, the turbidity of the cell culture solution and the particle size of particles present in the cell culture solution may contribute to clogging of the hollow fiber membranes. In one embodiment, reducing the turbidity of the cell culture solution before filtration can suppress a decrease in the throughput of filtration through hollow fiber membranes. In another embodiment, reducing the particle size of particles present in the cell culture solution before filtration can suppress a decrease in the throughput of filtration through hollow fiber membranes. [Example]

[0092] The present invention will be described in detail below with reference to reference examples, reference comparative examples, experimental examples, examples, and comparative examples, but the present invention is not limited to the following examples, comparative examples, and experimental examples. Test methods for the reference examples, reference comparative examples, experimental examples, examples, and comparative examples are as follows. Reference Example 1 A hollow fiber microfilter (manufactured by Asahi Kasei Medical Co., Ltd., BioOptimal (registered trademark) MF-SL) with a blocking pore size of 0.4 μm, made of polysulfone, and having a gradient structure was prepared. 2 A BioOptimal MF-SL mini-module was produced so that the following was true: In addition, similarly to Patent Document 1 (JP 2018-076291 A), the pore diameter, inner diameter, outer diameter, and membrane thickness on the primary side of the BioOptimal MF-SL hollow fiber membrane were measured, and the average pore diameter on the primary side surface was 30 μm or more and 80 μm or less, the inner diameter was 1.4 mm, the outer diameter was 2.3 mm, and the membrane thickness was 0.45 mm.

[0093] A serum-free medium (Irvine Scientific, IS CHO-CD medium) in which monoclonal antibody-producing Chinese hamster ovary (CHO) cells were cultured was prepared as a cell culture medium for producing raw materials for pharmaceutical substances. The cell culture medium was cultured at a density of approximately 1.7 × 10 6 The cells were contained at 100 cells / mL, and the cell viability was approximately 83%.

[0094] Triton X-100 (Merck) at a final concentration of 0.05%, Tween 20 (Promega, Molecular Biology Grade) at a final concentration of 0.2%, and CHAPS (Fujifilm Wako) at a final concentration of 0.5% were added to the cell culture medium, which was then incubated at 37°C for 1 hour to lyse the cells and prepare a cell culture medium with reduced cell viability.

[0095] Water was pumped into a BioOptimal MF-SL mini-module using a peristaltic pump at a shear rate of 3000 / s (48.5 mL / min), and the transmembrane pressure was adjusted to 20 kPa using a valve at the primary outlet of the hollow fiber membrane. While maintaining the valve position and the transmembrane pressure, water was removed from the tubing and module. Then, cell culture medium and cell culture medium with lysed cells and reduced viability were pumped into the mini-module. After 90 minutes or more, antibody permeability was measured. Antibody concentration was measured by HPLC (Shimadzu Prominence) using a Protein G column (Thermo Scientific PORPS G 20 μm). The results are shown in Table 2. High antibody permeability was observed in both cases. [Table 2]

[0096] Reference Example 2 The same BioOptimal MF-SL mini-module as in Reference Example 1 was prepared.

[0097] A serum-free medium (IS CHO-CD medium, Irvine Scientific) in which antibody-producing Chinese hamster ovary (CHO) cells were cultured was prepared as a cell culture medium for producing raw materials for pharmaceutical substances. The cell culture medium was cultured at a density of approximately 2.0 × 10 6 The cells were contained at 100 cells / mL, and the cell viability was approximately 57%.

[0098] Triton X-100 (Merck) was added to the cell culture medium to a final concentration of 0.05%, and the mixture was incubated at 37°C for 1 hour to lyse the cells and prepare a cell culture medium with reduced cell viability.

[0099] As in Reference Example 1, the cell culture medium in which the cells had been lysed and the cell viability had been reduced was sent to the mini-module, and the antibody permeability was measured after 100 minutes or more had elapsed. The results are shown in Table 3. It was shown that the antibody permeability was high. [Table 3]

[0100] Reference Example 3 The same BioOptimal MF-SL mini-module as in Reference Example 1 was prepared.

[0101] A serum-free medium (IS CHO-CD medium, Irvine Scientific) in which antibody-producing Chinese hamster ovary (CHO) cells were cultured was prepared as a cell culture medium for producing raw materials for pharmaceutical substances. The cell culture medium was cultured at a density of approximately 2.0 × 10 6 The cells were contained at 100 cells / mL, and the cell viability was approximately 42%.

[0102] Triton X-100 (Merck) was added to the cell culture medium to a final concentration of 0.05%, and the mixture was incubated at 37°C for 1 hour to lyse the cells and prepare a cell culture medium with reduced cell viability.

[0103] As in Reference Example 1, the cell culture medium in which the cells had been lysed and the cell viability had been reduced was sent to the mini-module, and the antibody permeability was measured after 100 minutes or more had elapsed. The results are shown in Table 4. It was shown that the antibody permeability was high. [Table 4]

[0104] (Reference Comparative Example 1) A hollow fiber microfilter (MICROZA (registered trademark) UMP, manufactured by Asahi Kasei Corporation) with a blocking pore size of 0.2 μm, made of polyvinylidene fluoride (PVDF), and having a uniform structure was prepared. 2 A mini-module of MICROZA UMP was fabricated so that the pore size of the hollow fiber membrane having a uniform structure is substantially uniform throughout the hollow fiber membrane and in the thickness direction of the hollow fiber membrane.

[0105] A hollow fiber microfilter (MICROZA (registered trademark) UJP, manufactured by Asahi Kasei Corporation) with a blocking pore size of 0.65 μm, made of polyvinylidene fluoride (PVDF), and having a uniform structure was prepared. 2 A MICROZA UJP mini module was created to achieve the following.

[0106] As in Reference Examples 2 and 3, a cell culture medium and a cell culture medium in which cells had been lysed and the cell viability had been reduced were prepared.

[0107] Except for using a MICROZA UMP mini-module and a MICROZA UJP mini-module, the cell culture medium in which the cells had been lysed and cell viability had been reduced was pumped into each mini-module in the same manner as in Reference Examples 2 and 3, and the antibody permeability was measured after 100 minutes or more. The results are shown in Table 5. [Table 5]

[0108] Reference Example 4 The same BioOptimal MF-SL mini-module as in Reference Example 1 was prepared.

[0109] A serum-free medium (IS CHO-CD medium, Irvine Scientific) in which antibody-producing Chinese hamster ovary (CHO) cells were cultured was prepared as a cell culture medium for producing raw materials for pharmaceutical substances. The cell culture medium was cultured at a density of approximately 2.2 × 10 6 The cells were contained at 100 cells / mL, and the cell viability was approximately 84%.

[0110] Triton X-100 (Merck) was added to the cell culture medium to a final concentration of 0.05%, and the mixture was incubated at 37°C for 1 hour to lyse the cells and prepare a cell culture medium with reduced cell viability.

[0111] While feeding 60 mL of biopharmaceutical culture solution into the module at a shear rate of 3000 / s (48.5 mL / min), the transmembrane pressure was adjusted to 20 kPa using the valve at the primary outlet. While maintaining the transmembrane pressure, the antibody permeability was measured after dead-end filtration. The results are shown in Table 6.

[0112] (Reference Comparative Example 2) A mini-module of the same MICROZA UMP and MICROZA UJP as in Comparative Example 1 was fabricated.

[0113] As in Reference Example 4, a cell culture medium and a cell culture medium in which cells had been lysed and the cell viability had been reduced were prepared.

[0114] Except for using a MICROZA UMP mini-module and a MICROZA UJP mini-module, the cell culture medium in which the cells had been lysed and the cell viability had been reduced was pumped into each mini-module in the same manner as in Reference Example 4, and the antibody permeability was measured after 250 minutes or more. The results are shown in Table 6. [Table 6]

[0115] Reference Example 5 The same BioOptimal MF-SL mini-module as in Reference Example 1 was prepared.

[0116] A serum-free medium (IS CHO-CD medium, Irvine Scientific) in which antibody-producing Chinese hamster ovary (CHO) cells were cultured was prepared as a cell culture medium for producing raw materials for pharmaceutical substances. The cell culture medium was cultured at a density of approximately 2.2 × 10 6 The cells were cultured at a density of approximately 2.0 × 10 cells / mL, and the cell viability was approximately 26%. 6 The cells were contained at 0.575 cells / mL, and the cell viability was approximately 13%.

[0117] Triton X-100 (Merck) was added to each cell culture medium to a final concentration of 0.05%, and the mixture was incubated at 37°C for 1 hour to lyse the cells and prepare cell culture medium with reduced cell viability.

[0118] While the biopharmaceutical culture solution was pumped through the module at a shear rate of 3000 / s (48.5 mL / min), the transmembrane pressure was adjusted to 20 kPa using the valve at the primary outlet. The antibody permeability was measured after filtration for 100 minutes or more while maintaining the transmembrane pressure. The results are shown in Table 7.

[0119] (Reference Comparative Example 3) A mini-module of the same MICROZA UMP and MICROZA UJP as in Comparative Example 1 was fabricated.

[0120] As in Reference Example 5, a cell culture medium and a cell culture medium in which cells had been lysed and the cell viability had been reduced were prepared.

[0121] Except for using a MICROZA UMP mini-module and a MICROZA UJP mini-module, the cell culture medium in which cells had been lysed and cell viability had been reduced was pumped into each mini-module in the same manner as in Reference Example 5, and the antibody permeability was measured after 100 minutes or more. The results are shown in Table 7. [Table 7]

[0122] Reference Example 6 The same BioOptimal MF-SL mini-module as in Reference Example 1 was prepared.

[0123] A serum-free medium (IS CHO-CD medium, Irvine Scientific) in which Chinese hamster ovary (CHO) cells were cultured was prepared as a cell culture medium for producing raw materials for pharmaceutical substances. The cell culture medium was cultured at a density of approximately 2.3 × 10 6 The cells were contained at 100 cells / mL, and the cell viability was approximately 69%.

[0124] Triton X-100 (Merck) at final concentrations of 0.05% and 0.1%, and CHAPS (Fujifilm Wako) at final concentrations of 0.5% and 1%, were added to the cell culture medium and incubated at 37°C for 1 hour to lyse the cells and prepare a cell culture medium in which the cell viability was reduced to 15% or less.

[0125] While the biopharmaceutical culture solution was pumped through the module at a shear rate of 3000 / s (48.5 mL / min), the transmembrane pressure was adjusted to 20 kPa using the valve at the primary outlet, and the antibody permeability was measured after 150 minutes or more while maintaining the transmembrane pressure. The results are shown in Table 8. [Table 8]

[0126] (Experimental Example 1) A BioOptimal MF-SL mini-module similar to that in Reference Example 1 and a MICROZA UMP and MICROZA UJP mini-module similar to that in Reference Comparative Example 1 are prepared.

[0127] A culture medium in which human embryonic kidney (HEK) 293 cells are cultured is prepared as a cell culture medium for producing raw materials for pharmaceutical substances.

[0128] The cells are transfected with plasmid DNA for AAV production and cultured for several days. After that, Triton X-100 and CHAPS are added and the cells are incubated at 37°C for 1 hour to lyse the cells and prepare a cell culture medium with reduced cell viability.

[0129] While the biopharmaceutical culture solution is pumped into each module at a shear rate of 3000 / s, the transmembrane pressure is adjusted to 20 kPa using the valve at the primary outlet, and the AAV permeability and solution throughput are measured after dead-end filtration or after filtration for 100 minutes or more while maintaining the transmembrane pressure.

[0130] (Experimental Example 2) A BioOptimal MF-SL mini-module similar to that in Reference Example 1 and a MICROZA UMP and MICROZA UJP mini-module similar to that in Reference Comparative Example 1 are prepared.

[0131] A culture medium in which human embryonic kidney (HEK) 293 cells are cultured is prepared as a cell culture medium for producing raw materials for pharmaceutical substances.

[0132] Cells are transfected with plasmid DNA for producing AAV, and the cells are cultured for several days to prepare a cell culture medium with reduced cell viability.

[0133] While the biopharmaceutical culture solution is pumped into each module at a shear rate of 3000 / s, the transmembrane pressure is adjusted to 20 kPa using the valve at the primary outlet, and the AAV permeability and solution throughput are measured after dead-end filtration or after filtration for 100 minutes or more while maintaining the transmembrane pressure.

[0134] (Experimental Example 3) A BioOptimal MF-SL mini-module similar to that in Reference Example 1 and a MICROZA UMP and MICROZA UJP mini-module similar to that in Reference Comparative Example 1 are prepared.

[0135] A culture medium in which human embryonic kidney (HEK) 293 cells are cultured is prepared as a cell culture medium for producing raw materials for pharmaceutical substances.

[0136] The cells are transfected with a plasmid DNA for producing AAV, and after culturing for several days, an acidic substance is added and the cells are incubated at 37°C for 1 hour to lyse the cells and prepare a cell culture solution with reduced cell viability.

[0137] While the biopharmaceutical culture solution is pumped into each module at a shear rate of 3000 / s, the transmembrane pressure is adjusted to 20 kPa using the valve at the primary outlet, and the AAV permeability and solution throughput are measured after dead-end filtration or after filtration for 100 minutes or more while maintaining the transmembrane pressure.

[0138] Example 1 A BioOptimal MF-SL mini-module similar to that in Reference Example 1, and a MICROZA UMP and MICROZA UJP mini-module similar to that in Reference Comparative Example 1 were prepared.

[0139] HEK293EB cells stably expressing the adenovirus E1 gene region and Bcl-xL gene were prepared. They were cultured in DMEM medium (Wako) supplemented with 10% FBS and 1% penicillin-streptomycin solution using a Corning HyperFlask. After 4 days of culture, the cell culture medium was discarded and the HEK293EB cells were transfected with plasmid DNA for producing AAV5 and the fluorescent protein ZsGreen. For transfection, 66.5 μg of helper plasmid, 33.3 μg of Rep / Cap plasmid, and 33.3 μg of GOI plasmid were prepared. These were mixed with 399 μg of polyethyleneimine and allowed to stand for 15 minutes to prepare the transfection reagent. Plasmid DNA was transfected into a HyperFlask containing DMEM medium supplemented with 1% penicillin-streptomycin solution, 1% GlutaMax, 0.1% NaHCO3, and 0.1% D-glucose, along with a transfection reagent. After transfection, HEK293EB cells were cultured for 5 days. The viability of HEK293EB cells in culture decreased to 35%.

[0140] Each module has 4.8 x 10 5 300 mL of HEK293EB cell culture medium (cells / mL) was prepared for the MF-SL system, and 150 mL for the UMP and UJP systems. The medium was filtered while maintaining the transmembrane pressure at 20 kPa using the valve at the primary outlet while pumping at a shear rate of 3000 / s. The filtered volume of the medium, the concentration rate after filtration, and the AAV permeability during filtration were measured. The concentration rate is the ratio of the volume of solution remaining after filtration to the volume of solution before filtration, as shown in the following formula: The higher the concentration rate, the more concentrated the solution. Concentration rate = (amount of solution before filtration) ÷ (amount of solution remaining after filtration)

[0141] As a result, as shown in Table 9, the throughput of culture medium was highest when MF-SL was used, the concentration rate was highest when MF-SL was used, and the AAV penetration rate was highest when MF-SL was used. In addition, under conditions where the cell viability was low at 35%, the cell concentration was 4.8 × 10 5 A solution with a high concentration of 485 L / m 2 The throughput was 96% and the AAV was filtered at a cell concentration of 1 × 10 7 It was suggested that filtration with high permeability was possible even at concentrations as high as about particles / mL. [Table 9]

[0142] Example 2 The same BioOptimal MF-SL mini-module as in Reference Example 1 was prepared.

[0143] HEK293EB cells were cultured and transfected with AAV1 and plasmid DNA for producing the fluorescent protein ZsGreen in the same manner as in Example 1. After transfection, HEK293EB cells were cultured for 5 days. The viability of HEK293EB cells in the culture medium decreased to 10%.

[0144] To a portion of the HEK293EB cell culture medium, 0.1 M disodium EDTA (2NA (EDTA·2Na), DOJINDO) and 0.05 (w / v)% Triton X-100 (SIGMA) were added, and the cells were lysed for 1 hour. After lysis, the viability of HEK293EB cells in the medium decreased to 6%.

[0145] The module was treated with 8.4 × 10 EDTA at a final concentration of 0.1 M and 0.05 (w / v)% Triton without Triton. 5A culture solution of HEK293EB cells (cells / mL) was pumped at a shear rate of 3000 / s and a flow rate of 48.5 mL / min. The transmembrane pressure was adjusted to 20 kPa using the valve at the primary outlet, and the culture solution was filtered while maintaining the transmembrane pressure. The filtration throughput, concentration rate, and AAV permeability during filtration were measured.

[0146] The module was filled with 1.3 × 10 cells containing 0.1 M EDTA and 0.05% (w / v) Triton. 6 A culture solution of HEK293EB cells (cells / mL) was pumped at a shear rate of 3000 / sec and a flow rate of 48.5 mL / min. The transmembrane pressure was adjusted to 20 kPa using the valve at the primary outlet, and the culture solution was filtered while maintaining the transmembrane pressure. The filtration throughput, concentration rate, and AAV permeability during filtration were measured. The addition of EDTA caused cells that had adhered to the culture vessel to float in the culture solution, increasing the cell count in the culture solution.

[0147] As a result, as shown in Table 10, the AAV permeability was higher when cells were lysed with a surfactant before filtration. Although the amount of culture medium processed was lower when a surfactant was added, AAV was released from the cells when a surfactant was added, resulting in a higher AAV concentration in the culture medium. Therefore, it is thought that the amount of AAV that permeated through filtration was greater when a surfactant was added. Furthermore, under conditions where the cell viability was low at 10%, the cell concentration was 8.4 × 10 5 A solution with a high concentration of 374 L / m 2 The throughput was 1×10 and the AAV was filtered with 80% permeability. 7 It was suggested that filtration with high permeability was possible even when the cell concentration was as high as about 1.3 × 10 cells / mL. 6 High solution of 280L / m 2 The AAV was filtered at a throughput of 117% and a cell concentration of 1 × 10 7 It was suggested that filtration with high permeability was possible even at concentrations as high as about particles / mL. [Table 10]

[0148] Example 3 The same BioOptimal MF-SL mini-module as in Reference Example 1 was prepared.

[0149] HEK293EB cells were cultured and transfected with AAV1 and plasmid DNA for producing the fluorescent protein ZsGreen in the same manner as in Example 1. After transfection, HEK293EB cells were cultured for 5 days. The viability of HEK293EB cells in the culture medium decreased to 39%.

[0150] A portion of the HEK293EB cell culture medium was lysed by adding 0.1M EDTA and 0.5% (w / v) CHAPS to a final concentration for 1 hour. After lysis with 0.5% (w / v) CHAPS, the viability of HEK293EB cells in the medium decreased to 13%.

[0151] A portion of the HEK293EB cell culture medium was lysed by adding 0.1 M EDTA and 0.05% (w / v) Triton for 1 hour. After lysis with 0.05% (w / v) Triton, the viability of HEK293EB cells in the culture medium decreased to 19%.

[0152] The module contained 8.1 x 10 cells without EDTA and CHAPS or Triton. 5 A culture solution of HEK293EB cells (cells / mL) was pumped at a shear rate of 3000 / s and a flow rate of 48.5 mL / min. The transmembrane pressure was adjusted to 20 kPa using the valve at the primary outlet, and the culture solution was filtered while maintaining the transmembrane pressure. The filtration throughput, concentration rate, and AAV permeability during filtration were measured.

[0153] The module was filled with 1.3 × 10 cells containing 0.1 M EDTA and 0.5% (w / v) CHAPS. 6A culture solution of HEK293EB cells (cells / mL) was pumped at a shear rate of 3000 / s and a flow rate of 48.5 mL / min. The transmembrane pressure was adjusted to 20 kPa using the valve at the primary outlet, and the culture solution was filtered while maintaining the transmembrane pressure. The filtration throughput, concentration rate, and AAV permeability during filtration were measured.

[0154] The module was filled with 1.4 × 10 EDTA and 0.05 (w / v)% Triton. 6 A culture solution of HEK293EB cells (cells / mL) was pumped at a shear rate of 3000 / s and a flow rate of 48.5 mL / min. The transmembrane pressure was adjusted to 20 kPa using the valve at the primary outlet, and the culture solution was filtered while maintaining the transmembrane pressure. The filtration throughput, concentration rate, and AAV permeability during filtration were measured.

[0155] The results are shown in Table 11. Because AAV was released from the cells when surfactant was added, the AAV concentration in the medium was higher when surfactant was added, and the amount of AAV that permeated through filtration was thought to be greater when surfactant was added. Furthermore, under conditions where the cell viability was low at 19%, the cell concentration was 1.4 × 10 6 A solution with a high concentration of 217 L / m 2 The throughput was 90% and the AAV was filtered at a cell concentration of 1 × 10 7 It was suggested that filtration with high permeability was possible even at concentrations as high as about particles / mL. [Table 11]

[0156] Example 4 A BioOptimal MF-SL mini-module similar to that in Reference Example 1, and a MICROZA UMP and MICROZA UJP mini-module similar to that in Reference Comparative Example 1 were prepared.

[0157] HEK293EB cells were cultured and transfected with AAV2 and plasmid DNA for producing the fluorescent protein ZsGreen in the same manner as in Example 1. After transfection, the HEK293EB cells were cultured for 5 days. 0.05 (w / v)% Triton was added to the culture medium of the HEK293EB cells, and the cells were lysed for 1 hour.

[0158] While feeding HEK293EB cell culture medium into each module at a shear rate of 5000 / s, the transmembrane pressure was adjusted to 20 kPa using the valve at the primary outlet, and the culture medium was filtered while maintaining the transmembrane pressure. The filtration throughput, concentration rate, and AAV permeability during filtration were measured.

[0159] As a result, as shown in Table 12, the culture medium throughput was highest when MF-SL was used, the concentration rate was highest when MF-SL was used, and the AAV permeability rate was highest when MF-SL was used. [Table 12]

[0160] Example 5 The same BioOptimal MF-SL mini-module as in Reference Example 1 was prepared.

[0161] A first depth filter and a second depth filter were prepared. The first depth filter had a pore size of 2 μm to 20 μm, was made of cellulose, diatomaceous earth, etc., and had a membrane area of ​​0.002 m. 2 The second depth filter has a pore size of 0.4 μm to 1.0 μm, is made of cellulose, and has a membrane area of ​​0.002 m. 2 is.

[0162] HEK293EB cells stably expressing the adenovirus E1 gene region and Bcl-xL gene were prepared, cultured, and transfected with AAV1 and plasmid DNA for producing the fluorescent protein ZsGreen in the same manner as in Example 1. After transfection, the HEK293EB cells were cultured for 5 days.

[0163] While the HEK293EB cell culture medium was pumped through the MF-SL mini module at a shear rate of 3000 / s and a flow rate of 48.5 mL / min, the transmembrane pressure was adjusted to 20 kPa using the valve at the primary outlet, and the culture medium was filtered while maintaining the transmembrane pressure.

[0164] The culture medium of HEK293EB cells was passed through the combination of the first and second depth filters at a rate of 3.7 mL / min, and the culture medium was batch filtered.

[0165] The results are shown in Table 13. The initial volume is the volume of culture solution before filtration. The treatment time is the time of filtration. The filtration volume is the volume of culture solution filtered. The turbidity of the filtrate was 1 NTU or less in all cases. [Table 13]

[0166] As shown in Figure 1, filtration with the MF-SL mini-module resulted in a higher AAV permeability than filtration with a depth membrane. Also, as shown in Figure 2, filtration with the MF-SL mini-module resulted in a lower permeability of the impurity host cell protein (HCP) than filtration with a depth membrane. This suggests that hollow fiber membranes may be able to remove impurities better than depth membranes, allowing only the target substance to pass through.

[0167] Example 6 HEK293EB cells were cultured and transfected with AAV1 and plasmid DNA for producing the fluorescent protein ZsGreen in the same manner as in Example 1. After transfection, the HEK293EB cells were cultured for 5 days. After the culture, the viability of HEK293EB cells in the culture medium had decreased to 35%.

[0168] Hollow fiber microfilter (Asahi Kasei Medical Corporation, BioOptimal® MF-SL0005 (50 cm 2 )) to 7.4 × 10 5 Two liters of HEK293EB cell culture medium (2 cells / mL) was pumped at a flow rate of 100 mL / min while adjusting the transmembrane pressure to 10-50 kPa using the valve at the primary outlet, and the culture medium was filtered. After dead-end filtration, AAV remaining in the hollow fibers and tubing was recovered by extrusion washing with 200 mL of buffer (50 mM HEPES, 150 mM NaCl, 1 mM MgCl2). The filtered volume of the culture medium, the concentration rate by filtration, the AAV permeability during filtration, and the AAV recovery rate, including extrusion washing, were measured. The recovery rate is expressed by the following formula:

[0169] Recovery rate X = {(virus concentration in permeate) × volume of permeate} / {(virus concentration in cell culture medium before filtration) × volume of culture medium} × 100 As a result, as shown in Table 14, the recovery rate was 100%. By performing extrusion washing, the AAV remaining in the tube and membrane was recovered. This means that AAV does not adsorb to MF-SL and a good recovery rate can be achieved, suggesting that this method is highly valuable for industrial use. [Table 14]

[0170] (Experimental Example 4) A BioOptimal MF-SL mini-module identical to that in Reference Example 1 is prepared.

[0171] HEK293EB cells are cultured and transfected with AAV2 and plasmid DNA for producing the fluorescent protein ZsGreen in the same manner as in Example 1. After transfection, HEK293EB cells are cultured for 5 days.

[0172] HEK293EB cells were detached by adding 0.1M EDTA disodium salt (2NA (EDTA·2Na), DOJINDO) to the culture vessel. The resulting cell solution was centrifuged to pellet the cells, and Triton X-100 (SIGMA) was added to a final concentration of 0.05 (w / v)% to lyse the cells for 1 hour. After lysis, the viability of HEK293EB cells in the culture medium dropped to less than 10%.

[0173] The module was filled with 2.0 × 10 cells containing 0.1 M EDTA and 0.05% (w / v) Triton. 7 A culture solution of HEK293EB cells (cells / mL) was pumped at a shear rate of 3000 / sec and a flow rate of 48.5 mL / min. The transmembrane pressure was adjusted to 20 kPa using the valve at the primary outlet, and the culture solution was filtered while maintaining the transmembrane pressure. The filtered volume of the culture solution, the concentration rate by filtration, and the AAV permeability during filtration were measured.

[0174] (Reference Experimental Example 1) A hollow fiber microfilter (manufactured by Asahi Kasei Medical Co., Ltd., BioOptimal (registered trademark) MF-SL) with a blocking pore size of 0.4 μm, made of polysulfone, and having a gradient structure was prepared. 2 A BioOptimal MF-SL mini-module was created to achieve the following:

[0175] A serum-free medium (IS CHO-CD medium, Irvine Scientific) in which Chinese hamster ovary (CHO) cells were cultured was prepared as a cell culture medium for producing raw materials for pharmaceutical substances. The cell culture medium was cultured at a density of approximately 1.7 × 10 6 The cells were contained at 100 cells / mL, and the cell viability was approximately 83%.

[0176] Triton X-100 (Merck) at a final concentration of 0.05%, Tween 20 (Promega, Molecular Biology Grade) at a final concentration of 0.2%, and CHAPS (Fujifilm Wako) at a final concentration of 0.5% were added to the cell culture medium, which was then incubated at 37°C for 1 hour to lyse the cells and prepare a cell culture medium with reduced cell viability.

[0177] Water was pumped into a BioOptimal MF-SL mini-module using a peristaltic pump at a shear rate of 3000 / s (48.5 mL / min), and the transmembrane pressure was adjusted to 20 kPa using the valve at the primary outlet of the hollow fiber membrane. Water was removed from the tubing and module while maintaining the valve position and transmembrane pressure. Then, the cell culture medium and the cell culture medium with reduced cell viability were pumped into the mini-module. The permeate flow rate after 100 minutes of filtration was used to calculate the solution throughput per membrane area. The permeate volume was measured at regular intervals using a gravimeter. The turbidity of the solution was measured using a portable turbidity meter (Fuji Tecom Portable Turbidity Meter 210Q). The results are shown in Table 15. The throughput did not decrease significantly even when cell viability was reduced. [Table 15]

[0178] (Reference example 1) Density approximately 11×10 6 cells / mL to 18.5 × 10 6 Cell culture medium containing cells at 1000 cells / mL was cultured in BioOptimal MF-SL0.005m 2 If batch filtration is performed at a shear rate of 3000 / s without controlling the transmembrane pressure with a valve or other device, the throughput will be approximately 300 L / m for cell culture fluid with a cell viability of 99.5%. 2 In contrast, the cell culture medium, which reduced the cell viability to 26% by prolonging the culture time, had a processing volume of 60 L / m, about one-sixth of that. 2As described above, in Reference Example 1, when the viability of the cell culture medium was low due to long-term culture, the filterability was significantly reduced. The results are shown in Table 16. However, when the viability of the cell culture medium was approximately 1 × 10 7 cells / mL to approximately 2 × 10 7 The permeation of cells at a concentration of approximately 1 × 10 cells / mL was possible under conditions where the cell viability was as low as 26%. 7 cells / mL to approximately 2 × 10 7 A solution with high cell counts per mL was used at 60 L / m 2 The cell concentration was 1×10 7 It was suggested that MF-SL can filter even particles up to about 1 / mL. [Table 16]

[0179] (Reference Experimental Example 2) The same BioOptimal MF-SL mini-module as in Reference Experimental Example 1 was fabricated.

[0180] A hollow fiber microfilter (MICROZA (registered trademark) UJP, manufactured by Asahi Kasei Corporation) with a blocking pore size of 0.65 μm, made of polyvinylidene fluoride (PVDF), and having a uniform structure was prepared. 2 A MICROZA UJP mini module was created to achieve the following.

[0181] A serum-free medium (IS CHO-CD medium, Irvine Scientific) in which Chinese hamster ovary (CHO) cells were cultured was prepared as a cell culture medium for producing raw materials for pharmaceutical substances. The cell culture medium was cultured at a density of approximately 2.0 × 10 6 The cells were contained at 100 cells / mL, and the cell viability was approximately 57%.

[0182] Triton X-100 (Merck) was added to the cell culture medium to a final concentration of 0.05%, and the mixture was incubated at 37°C for 1 hour to lyse the cells, thereby preparing a cell culture medium in which the cell viability was reduced to 10%.

[0183] Water was pumped through each module at a shear rate of 3000 / sec (MF-SL: 48.5 mL / min, UJP: 23.5 mL / min), and the transmembrane pressure was adjusted to 20 kPa using the valve at the primary outlet of the hollow fiber membrane. After removing water from the tubing and module while maintaining the valve position and transmembrane pressure, each cell culture solution with reduced cell viability was pumped through the mini-module. The permeate flow rate after 100 minutes of filtration was used to measure the solution throughput per membrane area. The results are shown in Table 17. [Table 17]

[0184] (Reference Experimental Example 3) A serum-free medium (IS CHO-CD medium, Irvine Scientific) in which Chinese hamster ovary (CHO) cells were cultured was prepared as a cell culture medium for producing raw materials for pharmaceutical substances. The cell culture medium was cultured at a density of approximately 2.0 × 10 6 The cells were contained at 100 cells / mL, and the cell viability was approximately 42%.

[0185] Triton X-100 (Merck) was added to the cell culture medium to a final concentration of 0.05%, and the mixture was incubated at 37°C for 1 hour to lyse the cells, thereby preparing a cell culture medium in which the cell viability was reduced to 7%.

[0186] The solution throughput per membrane area was measured in the same manner as in Reference Experimental Example 2. The results are shown in Table 18. [Table 18]

[0187] (Reference Experimental Example 4) The same BioOptimal MF-SL mini-module as in Reference Experimental Example 1 was fabricated.

[0188] A serum-free medium (IS CHO-CD medium, Irvine Scientific) in which Chinese hamster ovary (CHO) cells were cultured was prepared as a cell culture medium for producing raw materials for pharmaceutical substances. The cell culture medium was cultured at a density of approximately 1.3 × 10 6 The cells were contained at 100 cells / mL, and the cell viability was approximately 26%.

[0189] Triton X-100 (Merck) was added to the cell culture medium to a final concentration of 0.05%, and the mixture was incubated at 37°C for 1 hour to lyse the cells, thereby preparing a cell culture medium in which the cell viability was reduced to 5%.

[0190] While the biopharmaceutical culture solution was pumped through the module at a shear rate of 3000 / s (48.5 mL / min), the transmembrane pressure was adjusted to 20 kPa using the valve at the primary outlet. While maintaining the transmembrane pressure, the solution throughput per membrane area was calculated from the permeation flow rate after 100 minutes of filtration. The results are shown in Table 19.

[0191] (Reference Experimental Example 5) A serum-free medium (IS CHO-CD medium, Irvine Scientific) in which Chinese hamster ovary (CHO) cells were cultured was prepared as a cell culture medium for producing raw materials for pharmaceutical substances. The cell culture medium was cultured at a density of approximately 1.3 × 10 6 The membrane contained cells at 0.5 cells / mL, and the cell viability was approximately 13%. The amount of solution processed per membrane area was measured in the same manner as in Reference Experimental Example 4. The results are shown in Table 19. [Table 19]

[0192] (Reference Experimental Example 6) The same BioOptimal MF-SL mini-module as in Reference Experimental Example 1 was fabricated.

[0193] A serum-free medium (IS CHO-CD medium, Irvine Scientific) in which Chinese hamster ovary (CHO) cells were cultured was prepared as a cell culture medium for producing raw materials for pharmaceutical substances. The cell culture medium was cultured at a density of approximately 2.3 × 10 6 The cells were contained at 100 cells / mL, and the cell viability was approximately 69%.

[0194] Triton X-100 (Merck) at final concentrations of 0.05% and 0.1%, and CHAPS (Fujifilm Wako) at final concentrations of 0.5% and 1%, were added to the cell culture medium and incubated at 37°C for 1 hour to lyse the cells and prepare a cell culture medium in which the cell viability was reduced to 15% or less.

[0195] While the biopharmaceutical culture solution was pumped through the module at a shear rate of 3000 / s (48.5 mL / min), the transmembrane pressure was adjusted to 20 kPa using the valve at the primary outlet. While maintaining the transmembrane pressure, the solution throughput per membrane area was calculated from the permeation flow rate after 150 minutes of filtration. The results are shown in Table 20. [Table 20]

[0196] (Reference Experimental Example 7) A BioOptimal MF-SL mini-module was fabricated, the same as in Reference Experimental Example 1. Also, a MICROZA UJP mini-module was fabricated, the same as in Reference Experimental Example 2.

[0197] A serum-free medium (IS CHO-CD medium, Irvine Scientific) in which Chinese hamster ovary (CHO) cells were cultured was prepared as a cell culture medium for producing raw materials for pharmaceutical substances. The cell culture medium was cultured at a density of approximately 1.3 × 10 6 The cells were contained at 100 cells / mL, and the cell viability was approximately 13%.

[0198] Triton X-100 (Merck) was added to the cell culture medium to a final concentration of 0.05%, and the mixture was incubated at 37°C for 1 hour to lyse the cells, thereby preparing a cell culture medium in which the cell viability was reduced to 10%.

[0199] Each module was filled with 60 mL of cell culture medium with reduced cell viability at a shear rate of 3000 / sec (MF-SL: 48.5 mL / min, UJP: 23.5 mL / min), with the transmembrane pressure adjusted to 20 kPa using the valve at the primary outlet of the hollow fiber membrane. While maintaining the valve position and transmembrane pressure, each cell culture medium with reduced cell viability was pumped through the mini-module. The permeate flow rate during dead-end filtration or 100-minute filtration was measured to determine the solution throughput per membrane area. The results are shown in Table 21. [Table 21]

[0200] (Reference Experimental Example 8) A BioOptimal MF-SL mini-module was fabricated, the same as in Reference Experimental Example 1. Also, a MICROZA UJP mini-module was fabricated, the same as in Reference Experimental Example 2.

[0201] A cell culture medium for culturing human embryonic kidney (HEK) 293 cells (293AAV, Cell Biolabs) was prepared as a cell culture medium for producing raw materials for pharmaceutical substances. The cell culture medium was cultured at a density of approximately 2.0 × 10 6 cells / mL.

[0202] Triton X-100 (Sigma-Aldrich) was added to the cell culture medium to a final concentration of 0.05%, and the mixture was incubated at 37°C for 1 hour to lyse the cells and prepare a cell culture medium with reduced cell viability.

[0203] A KrosFlo KR2i TFF System (Repligen) was used for each module, with a shear rate of 3000 / sec (MF-SL: 48.5 mL / min, UJP: 23.5 mL / min) and the transmembrane pressure was adjusted to 20 kPa using a valve at the primary outlet of the hollow fiber membrane. The valve opening and closing was automatically adjusted to maintain the transmembrane pressure at 20 kPa. Approximately 100 mL of each cell culture solution with reduced cell viability was pumped through a mini-module, and the solution throughput per membrane area was calculated from the permeate flow rate during dead-end filtration or 120-minute filtration. The throughput was 340 L / m for the MF-SL. 2 , 140L / m for UJP 2 It was.

Claims

1. 1. A method for recovering a virus, comprising: reducing the viability of cells in culture; filtering the culture solution containing the virus produced by the cells using a hollow fiber membrane to recover the virus; Including, The hollow fiber membrane has a gradient structure in which the average pore size decreases from the primary side to the secondary side in the membrane thickness direction. Virus recovery methods.

2. 2. The method for recovering viruses according to claim 1, wherein the pore size on the primary side of the hollow fiber membrane is 20 μm or more and 100 μm or less.

3. 2. The method for recovering viruses according to claim 1, wherein the blocking pore size of the pores in the hollow fiber membrane is 0.05 μm or more and 20 μm or less.

4. The method for recovering viruses according to claim 1, wherein the hollow fiber membrane is made of a synthetic polymer.

5. The method for recovering a virus according to claim 4, wherein the synthetic polymer is polysulfone.

6. The method for recovering viruses according to claim 1 , wherein the hollow fiber membrane has a coarse layer and a dense layer.

7. The method for recovering a virus according to claim 1, wherein the viability of the cells is reduced by chemical or physical treatment.

8. The method for recovering a virus according to claim 1, wherein the cells are contacted with a chemical substance to reduce the viability of the cells.

9. The method for recovering a virus according to claim 8 , wherein the chemical substance is a surfactant, an acidic substance, or a basic substance.

10. The method for recovering a virus according to claim 9 , wherein the cells are lysed by the surfactant.

11. The method for recovering a virus according to claim 9, wherein the surfactant is a nonionic surfactant or a zwitterionic surfactant.

12. The method for recovering a virus according to claim 1, wherein the cells are transfected in a manner that reduces the viability of the cells.

13. The method for recovering a virus according to claim 1, wherein the viability of the cells after reducing the viability of the cells is 60% or less.

14. The method for recovering a virus according to claim 1, wherein the viability of the cells after reducing the viability of the cells is 30% or less.

15. 2. The method for recovering a virus according to claim 1, which is substantially free of removing the cells and / or cell debris with a precipitant prior to said filtering.

16. The method for recovering a virus according to claim 1, wherein the virus is an adeno-associated virus.

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