JP2023006419A - Filtering method for cell culture medium with high impurity removability - Google Patents

Abstract

To provide a filtering method for a cell culture medium having a high rate of recovery of a purification target material.SOLUTION: A filtering method for a cell culture medium includes the step of filtering a cell culture medium in which cells have been cultured, using a hollow fiber membrane, by tangential flow filtration, in which before the filtration, a chemical substance is added to the cell culture medium, the hollow fiber membrane having its average pore diameter being uniform in the thickness direction.SELECTED DRAWING: None

Description

The present invention relates to a method for filtering cell culture fluid.

Cell culture technology is an essential technology in the production of various biopharmaceuticals such as viruses including viral vectors, antibodies, growth hormones, and insulin, and has greatly contributed to recent medical progress. Among biopharmaceuticals, viral preparations and viral vaccines are particularly attracting attention. Highly efficient and stable production of virus preparations and virus vaccines by culturing virus-producing cells is one of the important industrial themes (see, for example, Patent Documents 1 to 4).

When producing a raw material for a biopharmaceutical, it is necessary to culture cells that produce the raw material, remove the cells that produce the raw material from the cell culture medium, and purify the raw material. Conventional methods for removing cells from cell culture media include gel filtration, centrifugation, adsorption separation, sedimentation, and membrane filtration. However, the gel filtration method has problems such as dilution of the substance to be purified by the solvent used for the gel filtration and unsuitability for large-scale processing, making it difficult to use it industrially. The centrifugal separation method can only be applied when the viscosity of the solution is low, and it is difficult to introduce large-scale equipment. The sedimentation method alone has the problem that the cells cannot be completely removed.

JP 2018-076291 A Japanese Patent No. 6530171 WO2010/035793 WO2020/023612

Membrane filtration using a microfiltration membrane or an ultrafiltration membrane is suitable for industrial use because cells can be easily removed and a large amount of solution can be processed continuously. However, in the conventional membrane filtration method, there is a problem that a concentrated layer of cells and cell debris is formed on the membrane surface, clogging the membrane surface, causing an increase in filtration pressure and a decrease in filtration rate over time. . In particular, when the viability of cells contained in the cell culture medium is low and the cell culture medium contains many dead cells, there is a large amount of cell debris in the cell culture medium, and the cell debris accumulates on the membrane surface and inside the membrane. As a result, there is a problem that the permeation of the object to be purified is hindered, and the recovery rate of the object to be purified is lowered.

In addition, the conventional filtration membrane has a problem that its permeability to the substance to be purified decreases with continued use. When the permeability of the purification target to the filtration membrane decreases, the recovery rate of the purification target decreases and the proportion of impurities such as protein aggregates may increase. Impurities such as protein aggregates make it difficult to further purify the substance to be purified in subsequent steps, and reduce the yield of the substance to be purified in subsequent steps.

A decrease in the recovery rate of the object to be purified can lead to soaring manufacturing costs of pharmaceuticals made from cell-produced substances and soaring medical expenses. When impurities such as protein aggregates are administered to the human body, they can induce neutralizing antibodies and cause side effects.

Accordingly, one of the objects of the present invention is to provide a method for filtering a cell culture medium that is highly capable of removing impurities. For example, one of the objects of the present invention is to provide a method for filtering a cell culture solution that is highly capable of removing host cell proteins (HCPs).

A method for filtering a cell culture solution according to an aspect of the present invention includes filtering a cell culture solution in which cells are cultured by tangential flow filtration using a hollow fiber membrane, and before filtering, the cell culture solution is chemically filtered. Including the step of adding a substance, the hollow fiber membrane has a uniform average pore size in the film thickness direction.

In the method for filtering a cell culture medium described above, cells may be removed in the step of filtering the cell culture medium.

In the method for filtering a cell culture medium described above, cell debris may be removed in the step of filtering the cell culture medium.

In the method for filtering a cell culture solution described above, substances produced by the cells may be purified in the step of filtering the cell culture solution.

In the method for filtering a cell culture medium described above, the step of reducing the viability of cells in the cell culture medium may be a step of reducing the viability of cells by chemical treatment or physical treatment.

In the method for filtering a cell culture medium described above, the step of adding a chemical substance to the cell culture medium may be a step of transfecting the cells.

In the method for filtering a cell culture solution described above, the step of adding a chemical substance to the cell culture solution may be a step of adding a surfactant, an acidic substance, or a basic substance to the cell culture solution.

In the method for filtering a cell culture medium described above, the cell viability in the cell culture medium before filtration may be 60% or less.

In the method for filtering a cell culture medium described above, the cell viability in the cell culture medium before filtration may be 30% or less.

In the method for filtering a cell culture solution described above, the surfactant may be a nonionic surfactant or an amphoteric surfactant.

In the method for filtering a cell culture solution described above, the blocking pore size of the pores of the hollow fiber membrane may be 0.05 μm or more and 20 μm or less.

In the method for filtering a cell culture solution described above, the hollow fiber membrane may be made of a synthetic polymer membrane.

In the method for filtering a cell culture solution described above, the synthetic polymer may be polyvinylidene fluoride.

Although there is no particular limitation as long as intracellular substances can be released from the cells, in the above method for filtering cell culture medium, the concentration of the nonionic surfactant in the cell culture medium is 0.005% or more. 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, 0.05% or more, 06% or more, 0.07% or more, 0.08% or more, 0.09% or more, or 0.1% or more. Furthermore, although it is not particularly limited as long as the surfactant used in the downstream purification step can be removed, it is preferably 1% or less, and more preferably 0.9% or less and 0.8% from the viewpoint of removing the surfactant. % 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 . However, the appropriate concentration of surfactant may vary depending on the type of surfactant and the relationship between cells.

Although there is no particular limitation as long as intracellular substances can be released from the cells, in the above method for filtering cell culture medium, the concentration of the amphoteric surfactant in the cell culture medium is 0.01% or more. From the viewpoint of releasing intracellular substances, it is more preferably 0.02% or more, 0.04% or more, 0.06% or more, 0.08% or more, 0.1% or more, 0.1% or more. It may be 2% or more, 0.3% or more, 0.4% or more, or 0.5% or more. Furthermore, although it is not particularly limited as long as the surfactant used in the downstream purification step can be removed, it is preferably 2% or less, and more preferably 1.8% or less and 1.6% from the viewpoint of removing the surfactant. % 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. However, the appropriate concentration of surfactant may vary depending on the type of surfactant and the relationship between cells.

There is no particular limitation as long as the cell density is sufficient to produce a substance from the cells, but in the above cell culture filtration method, the cell density in the cell culture before adding the chemical substance is the lower limit. 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 as upper limits, 1.0×10 ⁸ cells/mL or less, 8.0×10 ⁷ cells/mL or less, 6.0×10 ⁷ cells 6./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; It may be 0×10 ⁶ cells/mL or less, 4.0×10 ⁶ cells/mL or less, or 2.0×10 ⁶ cells/mL or less.

In the method for filtering a cell culture solution described above, the cells may be genetically modified cells.

In the method for filtering a cell culture solution described above, the cells may be cells that produce a virus as a useful substance. By useful substance is meant a useful cell product intended to be obtained by cell culture.

In the method for filtering a cell culture solution described above, the cells may be cells that produce a protein as a useful substance.

In the cell culture medium filtering method described above, the cell culture medium may contain a virus.

In the method for filtering a cell culture solution described above, the virus may permeate the hollow fiber membrane during the filtration.

In the cell culture medium filtration method described above, the virus may be a non-enveloped virus.

In the method for filtering a cell culture solution described above, the virus may be a parvovirus.

In the method for filtering a cell culture solution described above, the virus particles may be adeno-associated viruses.

In the method for filtering a cell culture solution described above, the virus particles may be enveloped viruses.

In the method for filtering a cell culture solution described above, the virus particles may be retroviruses.

In the method for filtering a cell culture solution described above, the virus particles may be lentiviruses.

ADVANTAGE OF THE INVENTION According to this invention, the filtration method of the cell culture solution with high impurity removal property can be provided.

EMBODIMENT OF THE INVENTION Hereinafter, the form (it may be abbreviate|abbreviated as "embodiment" hereafter in this specification.) of this invention is demonstrated concretely. It should be noted that the embodiments shown below are intended to exemplify methods and the like for embodying the technical idea of the present invention, and are not limited to these exemplifications.

A method for filtering a cell culture medium according to an embodiment includes filtering a cell culture medium in which cells are cultured by tangential flow filtration using a hollow fiber membrane, and adding a chemical substance to the cell culture medium before filtration. Including the step of adding, the hollow fiber membrane has a uniform average pore size in the film thickness direction.

A method for filtering a cell culture medium according to an embodiment may include a step of reducing cell viability. The step of reducing the viability of cells is not particularly limited as long as it is a step of reducing the viability of cells in a cell culture medium by a positive action. It is a process to lower. For example, mere long-term culturing of cells does not involve chemical or physical treatments and does not constitute a positive action to reduce cell viability. On the other hand, for example, transfection is an active cell viability-reducing process that reduces cell viability by causing the cells to produce substances that may be cytotoxic, and chemical treatment reduces cell viability. It is exemplified as one aspect of the step of causing. Cell disruption treatments are chemical or physical treatments that can damage cell membranes.

Chemical treatment according to embodiments includes adding chemicals. The chemical substance is not particularly limited as long as it reduces the survival rate. Examples of chemical substances include at least one of factors such as nucleic acids that produce useful substances, vectors, and transfection reagents, which are introduced into cells during transfection. Another aspect of the chemical substance is a surfactant, an acidic substance, or a basic substance. The acidic substance may be dissolved in a solvent (water or alcohol is exemplified) to form an acidic solution. Also, the basic substance may be dissolved in a solvent (water or alcohol is exemplified) to form a basic solution. Examples of acidic substances include hydrochloric acid, sulfuric acid, nitric acid, acetic acid, and the like. Examples of basic substances include sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, and ammonia.

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

Useful substances are, for example, substances used as pharmaceuticals, but are not limited thereto. Examples of useful substances include, but are not limited to, viruses, proteins, hormones, cytokine growth factors, enzymes, and plasma proteins. Viruses include virus-like particles (VLPs). Viruses include viral vectors. Viruses include oncolytic viruses. Viruses include viral vaccines. Viral vaccines include bioengineered vaccines. Examples of proteins include, but are not limited to, antibodies. Antibodies are, for example, monoclonal antibodies. Useful substances include useful substances released from cells into cell culture medium and useful substances accumulated inside cells.

The virus may be a non-enveloped virus or an enveloped virus. Examples of non-enveloped viruses include, but are not limited to, adenoviruses, adeno-associated viruses, and parvoviruses. Examples of enveloped viruses include, but are not limited to, retroviruses, lentiviruses, Sendai virus, rabies virus, Sindbis virus, and herpes simplex virus.

Cells may be transfected, for example. Factors such as nucleic acids that produce useful substances are introduced into cells by transfection. Cell viability is reduced upon transfection. Agents such as nucleic acids are chemical entities. Factor introduction by transfection includes, for example, factor introduction by electroporation, factor introduction by lipofection, and factor introduction by viral vectors.

Cells may be cultured by an adherent culture method or by a suspension culture method (floating culture method). In the adherent culture method, cells adhere to the inner surface of the culture vessel. In suspension culture methods, cells are suspended in a cell culture medium.

In the method of culturing cells by suspension culture, for example, a stirring mechanism is provided in a culture vessel such as a spinner flask to float the cells. As the stirring mechanism, a magnetic stirrer, a mechanically driven shaft-shaped impeller, or the like is used. During suspension culture, a method of culturing cells while supplying fresh medium into a culture tank and discharging old cell culture medium containing impurities such as growth inhibitors to the outside of the culture tank is called continuous culture.

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 no new cell culture solution is supplied into the culture tank. The fed-batch culture method is a culture method in which fresh cell culture medium is supplied into the culture vessel. The continuous culture method is a culture method in which a new cell culture solution is supplied into the culture tank and an old cell culture solution is discharged from the culture tank while keeping the amount of the cell culture solution in the culture tank constant. The step of decreasing viability may be performed after culturing or during culturing. The tangential flow filtration operation may be performed after culturing, or may be performed continuously during culturing.

When recovering useful substances such as viruses from cells, the cells may be disrupted. By crushing the cells, useful substances inside the cells diffuse into the cell culture medium. Cell viability is reduced when the cells are disrupted. Cells may be chemically treated by adding chemicals such as surfactants, acidic substances, and basic substances to lyse the cells. However, since enveloped viruses are produced extracellularly, the cells do not need to be lysed. Non-enveloped viruses are generally produced intracellularly, but adeno-associated viruses (AAV), such as AAV8 and AAV9, are produced extracellularly depending on the difference in serotypes. In this case, the cells may not be lysed.

Examples of impurities in cell culture media include host cell protein (HCP) and host cell nucleic acid (genomic DNA, RNA), which are host cell-derived impurities, plasmid DNA and medium components, which are process-derived impurities, and target product-derived impurities. Examples of useful substances are protein aggregates and virus aggregates. HCP and DNA are examples of impurities that can be effectively removed in this embodiment.

Surfactants may be nonionic surfactants or zwitterionic surfactants. Nonionic surfactants and amphoteric surfactants may be mixed at appropriate concentrations. A surfactant may be present in a suitable solvent. Nonionic and zwitterionic detergents generally tend to denature proteins less than ionic detergents.

Examples of nonionic surfactants include Triton X-100, Triton X-114, NP-40, Brij-35, Brij-58, Tween-20, Tween-80, Octyl Glucoside, and Octylthio Glucoside. but not limited to these.

Examples of zwitterionic surfactants include CHAPS (3-[(3-Cholamidopropyl)dimethylammonio]propanesulfonate) and CHAPSO (3[(3-Cholamidopropyl)dimethylammonio]-2-hydroxypropanesulfonic acid), which include Not limited.

The density of cells in the cell culture medium before crushing the cells 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 1.0×10 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 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 2.0×10 ⁶ cells/mL or less. The cell density in the cell culture solution can be measured by an automatic cell counting device (CYTORECON manufactured by GE Healthcare).

When the density of cells in the cell culture medium is the above, the concentration of the nonionic surfactant in the cell culture medium is preferably, for example, 0.005% or more, and more preferably from the viewpoint of releasing intracellular substances. 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, 0.08% or more 09% or more, or 0.1% or more. Furthermore, it is preferably 1% or less, and more preferably 0.9% or less, 0.8% or less, 0.7% or less, 0.6% from the viewpoint that the surfactant used in the downstream purification step can be removed. 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, or 0.1% or less.

When the cell density in the cell culture medium is as described above, the concentration of the amphoteric surfactant in the cell culture medium is preferably, for example, 0.01% or more, and more preferably from the viewpoint of releasing intracellular substances. 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 It may be 0.5% or more. Furthermore, it is preferably 2% or less, and more preferably 1.8% or less, 1.6% or less, 1.4% or less, 1.2% from the viewpoint of removing the surfactant used in the downstream purification step. 1.0% or less, 0.8% or less, 0.6% or less, or 0.5% or less.

Methods for disrupting cells are not limited to chemical treatments. For example, cells may be disrupted by enzymes. Alternatively, cells may be disrupted by mechanical or physical treatment that applies pressure to the cells. Machines such as high-pressure homogenizers and mills may be used to disrupt cells. The pressure may be ultrasonic, cavitational, or osmotic pressure. Alternatively, the cells may be disrupted by heat treatment in which heat is applied to the cells. Alternatively, the cells may be disrupted by subjecting the cells to heat shock by the freeze-thaw method. After disrupting the cells, the cells may be retained by batch culture without supplying new cell culture medium into the culture vessel.

In one aspect, the production of cell products can be performed in the order of cell culture, transfection, cell culture, detergent treatment of cells, and batch filtration of cell culture fluid. In another form, the production of cell products can also be carried out in the order of cell culture, transfection, cell culture, batch filtration of the cell culture fluid. Furthermore, in another form, production of cell products can be performed in the order of cell culture, transfection, and continuous culture.

In some embodiments, cell viability prior to a step of decreasing 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, and the cell viability immediately before tangential flow filtration after one step of decreasing the cell viability 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, one step that reduces cell viability is, for example, but not limited to, transfection as described above.

In some embodiments, cell viability prior to a step of decreasing 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 immediately before tangential flow filtration after one step of decreasing cell viability is, for example, 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less. Here, one step for reducing cell viability is, for example, the above-described cell disruption treatment and a step after transfection, but is not limited thereto.

In some embodiments, the cell viability prior to a step of reducing cell viability is, for example, 80% or greater, 85% or greater, 90% or greater, 95% or greater, or 99% or greater; The viability of the cells immediately before tangential flow filtration after one step of reducing the viability of 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, one step that reduces cell viability is for example, but not limited to, transfection as described above in combination with continuous culture.

In addition, when there are multiple steps that reduce the cell viability, the above cell viability is the closest to the step of filtering the cell culture solution by tangential flow filtration using a hollow fiber membrane. Refers to cell viability before and after one step of decreasing rate.

The viability of cells can be measured by an automatic cell counting device (Vi-CELL XR manufactured by Beckman Coulter).

The culture tank containing the cell culture solution containing the cells with reduced viability has an outlet for sending the cell culture solution to the primary side surface of the hollow fiber membrane and a and an inlet for returning the cell culture solution that has passed through the primary side surface of the hollow fiber membrane to the culture tank. The outlet and inlet may be the same or different.

The surface to which the filtered cell culture medium is supplied is called the primary side of the hollow fiber membrane. Further, the surface from which the permeated liquid that has permeated the hollow fiber membrane flows is called the secondary side of the hollow fiber membrane.

In an embodiment in which the cell culture solution 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 solution 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.

A plurality of hollow fiber membranes are included in, for example, a filtration module. The filtration module has an inflow port for inflowing the cell culture solution sent from the culture tank, and is a cell culture solution inflow port that communicates with the primary side surface of the hollow fiber membrane, and the inside of the hollow fiber membrane and a cell culture medium outlet for returning the cell culture medium that has passed through the primary side surface of the hollow fiber membrane to the culture vessel without permeating the hollow fiber membrane. The cell culture medium inlet and the cell culture medium outlet may be the same or different. In addition, the filtration module may be provided with a permeated liquid outlet port for discharging the permeated liquid containing useful substances, which has permeated the inside of the hollow fiber membrane and flowed out from the secondary side surface of the hollow fiber membrane.

The culture tank and the filtration module are connected by, for example, a liquid-sending mechanism including a channel and a pump. The liquid delivery mechanism may include a pressure gauge and a weighing scale. Examples of pumps include, but are not limited to, diaphragm pumps, tube pumps, and rotary pumps.

By filtering the cell culture medium in which cells are cultured by tangential flow filtration using a hollow fiber membrane, cells in the cell culture medium are removed and useful substances in the cell culture medium permeate the hollow fiber membrane. , refined.

The tangential flow filtration (TFF) method is a filtration method in which a cell culture solution is flowed in a direction parallel to the primary side surface of the hollow fiber membrane on the primary side surface of the hollow fiber membrane. Tangential flow filtration (TFF) methods include alternating tangential flow filtration (ATF) methods. In the present embodiment, the tangential flow filtration (TFF) method may refer to a filtration method in which a cell culture solution flows in one direction in the hollow portion of the hollow fiber membrane. The alternating tangential flow filtration (ATF) method refers to a filtration method in which a cell culture solution is reciprocated in the hollow portion of a hollow fiber membrane.

The porous structure of the hollow fiber membrane may have a uniform structure. In a hollow fiber membrane having a uniform structure, the average pore size is substantially uniform throughout the hollow fiber membrane and substantially uniform in the thickness direction of the hollow fiber membrane. Therefore, in a hollow fiber membrane having a uniform structure, the pore size distribution is symmetrical in the film thickness direction. The blocking pore size of the hollow fiber membrane having a uniform structure is, for example, 0.05 μm or more and 20 μm or less, 0.1 μm or more and 10 μm or less, 0.1 μm or more and 5 μm or less, 0.1 μm or more and 3 μm or less, or 0.1 μm or more and 1 μm or less. be. When the blocking pore size is 0.05 μm or more, the permeation resistance is suppressed, the pressure required for filtration is suppressed, and there is a tendency to be able to suppress membrane clogging due to destruction and deformation of microbial particles, a decrease in filtration efficiency, and the like. Moreover, when the blocking pore size is 20 μm or less, there is a tendency that sufficient fractionation is obtained.

The blocking pore size is exemplified as the pore size of the particles when the permeation blocking rate of the particles is 90% when a particle dispersion in which particles with a certain pore size are dispersed is filtered using a porous hollow fiber membrane. . Sometimes called the minimum pore size. When determining the blocking pore size, polystyrene latex particles (SIZE STANDARD PARTICLES manufactured by JSR Corporation) were added to a 0.5% by mass sodium dodecyl sulfate aqueous solution (manufactured by Wako Pure Chemical Industries, Ltd.), and the particle concentration was 0.5%. 01% by mass to prepare a latex particle dispersion. A latex particle dispersion liquid is filtered using a porous hollow fiber membrane, and the concentration change of latex particles before and after filtration is measured. This measurement is performed while varying the latex particle size in steps of 0.1 μm to about 0.1 μm to generate an inhibition curve for the latex particles. From this blocking curve, the particle diameter that can block 90% permeation is read, and the obtained diameter is taken as the blocking pore size.

When determining the average pore size of the hollow fiber membrane, freeze-dry the hollow fiber membrane and observe 100 or more pores in one field of view using an electron microscope (manufactured by Keyence Corporation, VE-9800). Each of the 100 observed pores is subjected to circular approximation, and the average diameter obtained from the areas of the 100 circularly approximated pores is calculated as the average pore size.

The inner diameter of the hollow portion of the hollow fiber membrane is, for example, 1000 μm or more and 2000 μm or less, 1000 μm or more and 1500 μm or less, or 1100 μm or more and 1400 μm or less. When the inner diameter of the hollow portion is 1000 μm or more, the opening of the hollow portion tends to be less likely to be clogged with 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 the filtration performance tends to be excellent.

The thickness of the hollow fiber membrane is, for example, 300 μm or more and 1000 μm or less, 350 μm or more and 800 μm or less, or 350 μm or more and 500 μm or less. When the film thickness is 300 μm or more, the removed substances can be retained inside the film, and the effect of depth filtration tends to be exhibited easily. Furthermore, it tends to be easy to maintain an appropriate filtration rate. Further, when the film thickness is 1000 μm or less, the number of hollow fiber membranes constituting the filtration module increases, the effective cross-sectional area per filtration module increases, and the filtration performance tends to be excellent.

The inner diameter (μm) and outer diameter (μm) of the hollow fiber membrane can be measured by slicing the hollow fiber membrane thinly into a cylindrical shape and observing the section with an optical microscope (manufactured by Keyence Corporation, VH6100). Also, the thickness T _h (μm) of the hollow fiber membrane can be calculated from the inner diameter D _I and the outer diameter D _O using the following formula.
T _h = (D _o −D _I )/2

Impurity permeability is measured by quantifying and comparing the concentration of impurities in the permeated liquid after the filtration process and the concentration of impurities in the cell culture medium or cell lysate before the filtration process by ELISA. can be done. Moreover, when the impurity is nucleic acid, the concentration of the impurity can also be measured with an absorptiometer (for example, an absorption wavelength of 260 nm). For example, the impurity transmittance can be calculated using the following formula.
Transmittance X = (concentration of impurities in permeate)/(concentration of impurities in cell culture medium or cell lysate before filtration) x 100

A hollow fiber membrane consists of a synthetic polymer membrane, for example. Synthetic polymers are, for example, hydrophobic. Examples of synthetic polymers include, but are not limited to, polyvinylidene fluoride (PVDF). Contaminants such as cells and debris (cell fragments) contained in a cell culture medium are generally hydrophobic. Contaminants are trapped in the hollow fiber membrane through hydrophobic interactions. This makes it possible to purify a cell culture medium containing useful substances. The hollow fiber membrane according to the embodiment can be produced, for example, by referring to the method described in JP-A-2019-048290.

The culture solution before being treated with the hollow fiber membrane or the culture solution that has passed through the hollow fiber membrane may be treated with a nuclease to decompose the DNA contained in the culture solution.

The present invention will be described in detail below with reference to Examples, Comparative Examples, and Experimental Examples, but the present invention is not limited to the following Examples, Comparative Examples, and Experimental Examples. The test methods for Examples, Comparative Examples, and Experimental Examples are as follows.
(Example 1)
A hollow fiber microfilter (Microza UMP, registered trademark, Asahi Kasei Corporation) having a blocking pore size of 0.2 μm, made of polyvinylidene fluoride (PVDF) and having a uniform structure was prepared. Mini-modules of Microza UMP were made such that the membrane area was 3 cm ² .

A hollow fiber microfilter (Microza UJP, registered trademark, Asahi Kasei Corporation) having a blocking pore size of 0.65 μm, made of polyvinylidene fluoride (PVDF) and having a uniform structure was prepared. Mini-modules of Microza UJP were made such that the membrane area was 3 cm ² .

A serum-free medium (Irvine Scientific, IS CHO-CD medium) 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 contained cells at a density of about 2.0×10 ⁶ cells/mL and had a cell viability of about 57%.

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

Water was sent to each of the mini-modules by a peristaltic pump at a shear rate of 3000/sec (UMP: 48.5 mL/min, UJP: 23.5 mL/min), and the transmembrane pressure at that time was 1 of the hollow fiber membrane. The pressure was adjusted to 20 kPa using the valve at the outlet on the secondary side. After removing the water inside the tubes and modules while maintaining the valve state and maintaining the transmembrane pressure difference, the cell culture medium and the cell culture medium in which the cells were lysed and the cell viability was reduced were placed in miniaturized cells. The liquid was sent to the module, and after 150 minutes or more, the permeability of the cell-derived protein (Host Cell Protein; HCP) was measured. The concentration of HCP was measured using a CHO HCP ELISA Kit (F550 manufactured by CYGNUS). Table 1 shows the results. Since the transmittance of HCP, which is an impurity, was low, it was shown that the impurity removability was high.

(Comparative example 1)
A hollow fiber microfilter (BioOptimal MF-SL, registered trademark, Asahi Kasei Corporation) having a blocking pore size of 0.4 μm, made of polysulfone and having a gradient structure was prepared. In a hollow fiber membrane having an inclined structure, the pore size on the primary side surface is larger than the pore size on the secondary side surface, and the pore size continuously decreases from the primary side surface to the secondary side surface. Therefore, in a hollow fiber membrane having a gradient structure, the pore size distribution is asymmetrical in the film thickness direction. A mini-module of BioOptimal MF-SL was prepared so that the membrane area was 3 cm ² .

A serum-free medium (Irvine Scientific, IS CHO-CD medium) 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 contained cells at a density of about 2.0×10 ⁶ cells/mL and had a cell viability of about 57%.

Triton X-100 (Merck) with a final concentration of 0.05% was added to the cell culture medium and incubated at 37° C. for 1 hour to lyse the cells and reduce the cell viability to 10%. prepared. The cell density in the cell culture medium to which Triton X-100 was added was 1.8×10 ⁶ cells/mL.

Water was sent to the BioOptimal MF-SL mini-module at a shear rate of 3000/sec (48.5 mL/min), and the transmembrane pressure at that time was reduced to 20 kPa using the valve at the primary side outlet of the hollow fiber membrane. It was adjusted. After removing the water inside the tube and module while maintaining the valve state and maintaining the transmembrane pressure difference, transfer the cell culture medium with reduced cell viability to the mini module, and after 100 minutes or more. was measured. Table 2 shows the results.

(Example 2)
The same Microza UMP and Microza UJP mini-modules as in Example 1 were fabricated.

A serum-free medium (Irvine Scientific, IS CHO-CD medium) 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 contained cells at a density of about 2.2×10 ⁶ cells/mL and had a cell viability of about 84%.

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

While sending 60 mL of the solution of the biopharmaceutical culture solution to the module at a shear rate of 3000 / sec (UMP: 48.5 mL / min, UJP: 23.5 mL / min), the transmembrane pressure is applied to the primary side outlet. Adjusted to 20 kPa using a valve, while maintaining the transmembrane pressure, cell lysed and cell viability reduced cell culture solution was sent to each mini module, HCP permeation after 250 minutes or more. rate was measured. Table 3 shows the results.

(Comparative example 2)
The same BioOptimal MF-SL mini-module as in Comparative Example 1 was fabricated.

Cells were lysed in the same manner as in Example 2 to prepare a cell culture solution with reduced cell viability.

Cells were lysed in the same manner as in Example 2, except that a BioOptimal MF-SL minimodule was used and a shear rate of 3000/sec (48.5 mL/min) was used. The liquid was sent to the mini-module, and the HCP permeability was measured after dead-end filtration. Table 3 shows the results.

(Example 3)
The same Microza UMP and Microza UJP mini-modules as in Example 1 were fabricated.

A serum-free medium (Irvine Scientific, IS CHO-CD medium) 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 contained cells at a density of about 2.2×10 ⁶ cells/mL and had a cell viability of about 26%. Furthermore, a cell culture medium containing cells at a density of about 2.0×10 ⁶ cells/mL and having a cell viability of about 13% was prepared.

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

While feeding the biopharmaceutical culture solution solution to the module at a shear rate of 3000 / sec (UMP: 48.5 mL / min, UJP: 23.5 mL / min), the transmembrane pressure is applied to the primary outlet valve was adjusted to 20 kPa using , and while maintaining the transmembrane pressure, each of the cell culture solutions in which the cells were lysed and the cell viability was reduced was sent to each of the mini modules, and after 100 minutes or more, HCP Transmittance was measured. Table 4 shows the results.

(Comparative Example 3)
The same BioOptimal MF-SL mini-module as in Comparative Example 1 was fabricated.

Cells were lysed in the same manner as in Example 3 to prepare a cell culture medium with reduced cell viability.

The transmembrane pressure difference was adjusted to 20 kPa using the valve on the primary side outlet while feeding the dissolved solution of the biopharmaceutical culture solution to the module at a shear rate of 3000/sec (48.5 mL/min). The HCP permeability was measured after filtration for 100 minutes or more while maintaining the pressure. Table 4 shows the results.

(Comparative Example 4)
The same BioOptimal MF-SL mini-module as in Comparative Example 1 was fabricated.

A serum-free medium (Irvine Scientific, IS CHO-CD medium) 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 contained cells at a density of about 2.3×10 ⁶ cells/mL and had a cell viability of about 69%.

TritonX-100 (Merck) with a final concentration of 0.05% and 0.1% and CHAPS (Fujifilm Wako) with a final concentration of 0.5% and 1% were added to the cell culture medium and incubated at 37°C for 1 hour. After incubation, the cells were lysed to prepare a cell culture medium in which the cell viability was reduced to 15% or less.

The transmembrane pressure difference was adjusted to 20 kPa using the valve on the primary side outlet while feeding the dissolved solution of the biopharmaceutical culture solution to the module at a shear rate of 3000/sec (48.5 mL/min). While maintaining the pressure, the HCP permeability was measured after 150 minutes or longer. Table 5 shows the results.

(Example 4)
A medium in which human embryonic kidney cells (HEK) 293 cells (293AAV, Cell Biolabs) were cultured was prepared as a cell culture medium for producing raw materials for pharmaceutical substances. The cell culture medium contained cells at a density of approximately 2.0 x ¹⁰⁶ cells/mL.

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

A KrosFlo KR2i TFF System (Repligen) was used for each module, and a shear rate of 3000/sec (MF-SL, UMP: 48.5 mL/min. UJP: 23.5 mL/min) and a transmembrane pressure difference were measured through hollow fibers. The pressure was adjusted to 20 kPa using the valve on the primary side outlet of the membrane. The opening/closing condition of the valve is automatically adjusted so as to keep the transmembrane pressure difference at 20 kPa. About 100 mL of each of the cell culture solutions with reduced cell viability is sent to a mini-module, and the HCP permeability is measured at dead end filtration or after filtration for 120 minutes. The concentration of HCP is measured using HEK 293 HCP ELISA Kit (F650S manufactured by CYGNUS).

(Experimental example 1)
The same Microza UMP and Microza UJP mini-modules as in Experimental Example 1, and the same BioOptimal MF-SL mini-modules as in Comparative Example 1 are produced.

A medium in which human embryonic kidney (HEK) 293 cells are cultured is prepared as a cell culture medium for producing a raw material for a pharmaceutical substance.

Plasmid DNA was transfected into the cell culture medium, and after culturing for several days, Triton X-100 and CHAPS were added and incubated at 37°C for 1 hour to lyse the cells and reduce cell viability. Prepare liquid.

The transmembrane pressure difference was adjusted to 20 kPa using the primary outlet valve while sending the cell culture medium with reduced cell viability to each module at a shear rate of 3000/sec. While maintaining, the HCP permeability is measured after dead end filtration or filtration for 100 minutes or more.

(Experimental example 2)
The same BioOptimal MF-SL mini-modules as in Experimental Example 1 and the same Microza UMP and Microza UJP mini-modules as in Comparative Example 1 are produced.

A medium in which human embryonic kidney (HEK) 293 cells are cultured is prepared as a cell culture medium for producing a raw material for a pharmaceutical substance.

A cell culture medium is transfected with plasmid DNA and cultured for several days to prepare a cell culture medium in which the cell viability is reduced.

The transmembrane pressure difference was adjusted to 20 kPa using the primary outlet valve while sending the cell culture medium with reduced cell viability to each module at a shear rate of 3000/sec. While maintaining, the HCP permeability is measured after dead end filtration or filtration for 100 minutes or more.

(Experimental example 3)
The same BioOptimal MF-SL mini-modules as in Experimental Example 1 and the same Microza UMP and Microza UJP mini-modules as in Comparative Example 1 are produced.

A medium in which human embryonic kidney (HEK) 293 cells are cultured is prepared as a cell culture medium for producing a raw material for a pharmaceutical substance.

Plasmid DNA is transfected into the cell culture medium, and after culturing for several days, an acidic substance is added and incubated at 37° C. for 1 hour to lyse the cells and prepare a cell culture medium with reduced cell viability. .

The transmembrane pressure difference was adjusted to 20 kPa using the primary outlet valve while sending the cell culture medium with reduced cell viability to each module at a shear rate of 3000/sec. While maintaining, the HCP permeability is measured after dead end filtration or filtration for 100 minutes or more.

Claims (10)

A method for filtering a cell culture medium, comprising filtering a cell culture medium in which cells are cultured by tangential flow filtration using a hollow fiber membrane,
adding a chemical to the cell culture medium prior to the filtration;
In the hollow fiber membrane, the average pore size is uniform in the film thickness direction,
A method for filtering a cell culture medium. 2. The method for filtering a cell culture medium according to claim 1, wherein the step of adding the chemical substance to the cell culture medium is a step of transfecting the cells. The method for filtering a cell culture solution according to claim 1, wherein the step of adding the chemical substance to the cell culture solution is a step of adding a surfactant, an acidic substance, or a basic substance to the cell culture solution. 4. The method according to any one of claims 1 to 3, wherein the viability of the cells in the cell culture medium before being filtered is 60% or less. 4. The method according to any one of claims 1 to 3, wherein the viability of the cells in the cell culture medium before being filtered is 30% or less. 4. The method for filtering a cell culture fluid according to claim 3, wherein the chemical treatment is lysis of the cells with a surfactant. 7. The method for filtering a cell culture fluid according to claim 6, wherein the surfactant is a nonionic surfactant or a zwitterionic surfactant. The method for filtering a cell culture solution according to any one of claims 1 to 7, wherein the hollow fiber membrane has a blocking pore size of 0.05 µm or more and 20 µm or less. The method for filtering a cell culture solution according to any one of claims 1 to 8, wherein the hollow fiber membrane is made of a synthetic polymer membrane. 10. The method for filtering a cell culture solution according to claim 9, wherein the synthetic polymer is polyvinylidene fluoride.