Methods for harvesting cell products

Abstract

A method for recovering cell products with a high recovery rate is provided. [Solution] A method for recovering cell products, comprising: lysing cells with a surfactant containing a polyethylene glycol chain with a repeating unit number of 6 to 22 and no benzene ring; and filtering a solution containing the lysed cells using a hollow fiber membrane.

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 5). [Prior art documents] [Patent documents]

[0003] [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 [Patent Document 5] International Publication No. 2023 / 277173 Summary of the Invention [Problem to be solved by the invention]

[0004] When producing cell products such as viruses used in biopharmaceuticals, it is necessary to culture the cells that produce the cell products, remove the cells from the cell culture medium, and purify the cell products. Conventional methods for removing cells from cell culture medium include gel filtration, centrifugation, adsorption separation, precipitation, and membrane filtration.

[0005] 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 is only applicable when the viscosity of the solution is low, making it difficult to introduce large-scale equipment. Sedimentation alone has the problem of being unable to completely remove cells.

[0006] Membrane filtration using microfiltration 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, causing an increase in filtration pressure and a decrease in filtration rate over time.

[0007] Furthermore, filtration membranes have the problem that their permeability to cell products decreases with continued use. When the permeability of filtration membranes to cell products decreases, the recovery rate of cell products can decrease. A decrease in the recovery rate of cell products can lead to an increase in the manufacturing costs of pharmaceuticals that use cell products as raw materials, and an increase in medical expenses.

[0008] Therefore, one of the objects of the present invention is to provide a method for recovering cell products with a high recovery rate. [Means for solving the problem]

[0009] [1] A method for recovering cell products, comprising: lysing cells with a surfactant containing polyethylene glycol chains with a repeating unit number of 6 to 22 and not containing a benzene ring; and filtering a solution containing the lysed cells using a hollow fiber membrane.

[0010] [2] The method of [1], wherein cellular debris is removed from the solution by filtration.

[0011] [3] The method according to [1] or [2], further comprising recovering the solution containing the cell products filtered through the hollow fiber membrane.

[0012] [4] The method according to any one of [1] to [3], wherein the concentration of the surfactant is 1 mmol / L or more.

[0013] [5] The method according to any one of [1] to [4], wherein the pore size on the primary side of the hollow fiber membrane is 1 μm or more and 100 μm or less.

[0014] [6] The method according to any one of [1] to [5], 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.

[0015] [7] The method according to any one of [1] to [6], wherein the filtration is tangential flow filtration.

[0016] [8] The method according to [7], wherein the shear rate of the tangential flow filtration is 260 / s or more.

[0017] [9] The method according to any one of [1] to [8], wherein the cellular product is a gene product of the cell.

[0018]

[10] The method according to any one of [1] to [8], wherein the cell product is a product of a gene introduced into the cell.

[0019]

[11] The method according to any one of [1] to [9], wherein the cellular product is a virus.

[0020]

[12] The method according to any one of [1] to [9], wherein the cellular product is a viral vector.

[0021]

[13] The method according to any one of [1] to [9], wherein the cell product is an adeno-associated virus.

[0022]

[14] The method according to any one of [1] to [9], wherein the cellular product is an adenovirus.

[0023]

[15] The method according to any one of [1] to

[14] , wherein the filtration of the solution containing the lysed cells using the hollow fiber membrane is carried out once.

[0024]

[16] The method according to any one of [1] to

[15] , wherein the filtration of the solution containing lysed cells using a hollow fiber membrane is not performed multiple times. 4 [Effects of the Invention]

[0025] According to the present invention, it is possible to provide a method for recovering cell products with a high recovery rate. [Brief explanation of the drawings]

[0026] [Figure 1] FIG. 1 is a schematic diagram showing a filtration method according to an embodiment. [Figure 2] This is a photograph of debris generated when cells are lysed with PEG LE1 (Laureth 9). [Figure 3] Photograph of micelles formed upon cell solubilization with laureth-5. [Figure 4] FIG. 1 is a schematic diagram showing a filtration method according to an embodiment. DETAILED DESCRIPTION OF THE INVENTION

[0027] 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.

[0028] The method for recovering cell products according to the embodiment includes lysing cells with a surfactant containing polyethylene glycol (PEG) chains with a repeating unit number of 6 to 22 and no benzene ring, and filtering the solution containing the lysed cells using a hollow fiber membrane.

[0029] 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 that can be used in pharmaceuticals, reagents, foods, etc. 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.

[0030] The cells may be, for example, transfected. Transfection introduces factors, such as nucleic acids, that produce cellular products into the cells. Introduction of factors by transfection includes, for example, introduction of factors by electroporation, introduction of factors by lipofection, and introduction of factors by viral vectors.

[0031] Cells are cultured in a solution such as a cell culture medium. The cells may be cultured by adherent culture or suspension culture. In adherent culture, the cells adhere to the inner surface of a culture vessel. In suspension culture, the cells are suspended in the cell culture medium.

[0032] 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.

[0033] 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.

[0034] A cellular product is a substance produced by a cell, such as a virus, nucleic acid, or protein. A cellular product may be a gene product of the cell. A cellular product may also be the product of a gene artificially introduced into the cell.

[0035] 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.

[0036] 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. Non-enveloped viruses tend to be stable and not killed by ethanol. Non-enveloped viruses can be inactivated by heat treatment, hypochlorite treatment, and UV irradiation. Examples of enveloped viruses include, but are not limited to, retrovirus, lentivirus, Sendai virus, rabies virus, Sindbis virus, and herpes simplex virus.

[0037] AAV has low pathogenicity and has been applied to pharmaceuticals in recent years. AAV has a weaker charge than adenovirus and tends to adsorb easily to hollow fiber membranes. AAV is smaller than adenovirus. The particle size of adenovirus is 100 nm, and the genome size that can be introduced into adenovirus is 8 kbp. In contrast, the particle size of AAV is 20 nm, and the genome size that can be introduced into AAV is 4.5 kbp.

[0038] An example of a protein is an antibody. As generally defined in biochemistry, an antibody is a glycoprotein molecule (also called gamma globulin or immunoglobulin) produced by B lymphocytes as a defense mechanism against infection in vertebrates. For example, antibodies are used as human pharmaceuticals and have a structure substantially identical to that of antibodies present in the human body to which they are administered.

[0039] The antibody may be a human antibody, or may be an antibody derived from a non-human mammal such as a bovine or a mouse. Alternatively, the antibody may be a chimeric antibody with human IgG, or a humanized antibody. A chimeric antibody with human IgG is an antibody in which the variable region is derived from a non-human organism such as a mouse, but the other constant regions are substituted with immunoglobulins derived from humans. A humanized antibody is an antibody in which the complementarity-determining region (CDR) of the variable region is derived from a non-human organism, but the other framework region (FR) is derived from humans. Humanized antibodies have even reduced immunogenicity compared to chimeric antibodies.

[0040] The class (isotype) and subclass of the antibody are not particularly limited. For example, antibodies are classified into five classes, IgG, IgA, IgM, IgD, and IgE, based on differences in the structure of their constant regions. The antibodies used in the methods of the embodiments may belong to any of the five classes. Furthermore, among human antibodies, IgG has four subclasses, IgG1 to IgG4, and IgA has two subclasses, IgA1 and IgA2. The subclass of the antibodies used in the methods of the embodiments may be any of these. Note that antibody-related proteins, such as Fc fusion proteins in which a protein is bound to the Fc region, may also be included in the antibodies used in the methods of the embodiments.

[0041] Antibodies can also be classified by origin. The antibodies used in the methods of the present invention may be any of natural antibodies, recombinant antibodies produced by genetic engineering techniques, monoclonal antibodies, and polyclonal antibodies.

[0042] The isoelectric point (pI) of the antibody is not particularly limited, but is, for example, 6.0 or higher and 8.5 or lower. The pI of the antibody may be 6.5 or higher, or 7.0 or higher. The pI of the antibody may be 8.0 or lower.

[0043] By adding a surfactant to the cell culture solution in the culture vessel, the cells are lysed by the surfactant, and the cell products inside the cells are diffused into the cell culture solution. An example of a surfactant according to an embodiment is an ether. An example of a surfactant according to an embodiment is a nonionic surfactant. Nonionic surfactants generally tend to denature proteins less than ionic surfactants.

[0044] The number of repeating units in the PEG chain contained in the surfactant according to the embodiment may be 20 or less, 18 or less, 16 or less, 14 or less, 12 or less, or 10 or less. Examples of surfactants according to the embodiment include laureth-6, laureth-7, Tergitol (registered trademark) TMN 6, laureth-8, Genapol (registered trademark) X-080, PEG LE (laureth-9), and laureth-10, and salts thereof.

[0045] Laureth-6, also known as hexaethylene glycol monolauryl ether, is a nonionic surfactant containing a PEG chain with six repeating units, with the PEG chain and alkyl chain bonded via an ether bond. Its molecular formula is C 24 H 50 O7 and the rational formula is CH3(CH2) 11 (CH2CH2O)6OH and has a molecular weight of 450.65. [ka]

[0046] Laureth-7, also known as heptaethylene glycol monododecyl ether, is a nonionic surfactant containing a PEG chain with seven repeating units, with the PEG chain and alkyl chain bonded via an ether bond. Its molecular formula is C 26 H 54 O8 and the rational formula is CH3(CH2) 11 (OCH2CH2)7OH and has a molecular weight of 494.70. [ka]

[0047] Tergitol® TMN-6 is a nonionic surfactant, also known as poly(oxyethylene) 3,5-dimethyl-1-(2-methylpropyl)hexyl ether, which contains 8 repeating units of a PEG chain, with the PEG chain and alkyl chain bonded via an ether bond. Its molecular formula is C 28 H 58 O9, the rational formula is (CH3)2CHCH2CH(CH3)CH2CH(CH2CH(CH3)2)(OCH2CH2)8OH, and the molecular weight is 538. [ka]

[0048] Laureth-8, also known as octaethylene glycol monododecyl ether, is a nonionic surfactant containing a PEG chain with eight repeating units, with the PEG chain and alkyl chain bonded via an ether bond. Its molecular formula is C 28 H 58 O9 and the rational formula is CH3(CH2) 11 (OCH2CH2)8OH and has a molecular weight of 538.75. [ka]

[0049] Genapol® X-080, also known as polyethylene glycol monoalkyl ether, is a nonionic surfactant containing a PEG chain with eight repeating units, with the PEG chain and alkyl chain bonded via an ether bond, and has the molecular formula C 29 H 60 O9, with the rational formula (CH3)2CH(CH2) 10 (OCH2CH2)8OH and has a molecular weight of 552.78. [ka]

[0050] PEG LE (Laureth-9), also known as nonaethylene glycol monododecyl ether, is a nonionic surfactant containing a PEG chain with nine repeating units, with the PEG chain and alkyl chain bonded via an ether bond. Its molecular formula is C 30 H 62 O 10 and the rational formula is CH3(CH2) 11 (OCH2CH2)9OH and has a molecular weight of 582.82. [ka]

[0051] Laureth-10, also known as decaethylene glycol monododecyl ether, is a nonionic surfactant containing 10 repeating units of a PEG chain, with the PEG chain and alkyl chain bonded via an ether bond. Its molecular formula is C 32 H 66 O 11 and the rational formula is CH3(CH2) 11 (OCH2CH2) 10 OH and has a molecular weight of 626.9. [ka]

[0052] Ionic surfactants such as CHAPS and CHAPSO are characterized by their activity determined by their acid dissociation constant (pK a ) Therefore, the activity of ionic surfactants can vary depending on the pH of the cell culture medium. Therefore, the solubility of cells by ionic surfactants can also vary depending on the pH of the cell culture medium, and the recovery rate of cell products can also vary depending on the pH of the cell culture medium. Therefore, it is not preferable to use ionic surfactants for cell lysis.

[0053] The density of cells in the cell culture medium before lysis with a surfactant is, for example, 1.0 × 10 3 cells / mL or more, 1.0×10 4 cells / mL or more, 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 9 cells / mL or less, 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 7cells / 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).

[0054] When the cell density in the cell culture medium is as described above, the concentration of the surfactant in the cell culture medium is, for example, preferably 1 mmol / L or more, and from the viewpoint of releasing intracellular products, more preferably 2 mmol / L or more, 3 mmol / L or more, 4 mmol / L or more, 5 mmol / L or more, 10 mmol / L or more, 15 mmol / L or more, 18 mmol / L or more, 20 mmol / L or more, 25 mmol / L or more, or 30 mmol / L or more. Furthermore, the concentration is preferably 100 mmol / L or less, and from the viewpoint of removing the surfactant used in the downstream purification step, more preferably 90 mmol / L or less, 85 mmol / L or less, 80 mmol / L or less, 75 mmol / L or less, 70 mmol / L or less, 65 mmol / L or less, 60 mmol / L or less, 55 mmol / L or less, or 50 mmol / L or less.

[0055] A solution containing cells lysed with a surfactant is filtered through a hollow fiber membrane. By filtering a solution containing cells lysed with a surfactant using a hollow fiber membrane, cell debris in the solution is removed, and cell products in the solution permeate the hollow fiber membrane, where they are purified and recovered. The surface of the hollow fiber membrane onto which the solution to be filtered is supplied is called the primary side of the hollow fiber membrane. The surface from which the permeate that has permeated the hollow fiber membrane flows out is called the secondary side of the hollow fiber membrane. When the solution to be filtered is supplied to the inner surface of the hollow fiber membrane, the inner surface of the hollow fiber membrane is the primary side, and the outer surface of the hollow fiber membrane is the secondary side. When the solution to be filtered is supplied to the outer surface of the hollow fiber membrane, the outer surface of the hollow fiber membrane is the primary side, and the inner surface of the hollow fiber membrane is the secondary side.

[0056] The culture tank may be provided with an outlet for feeding the solution to the primary surface of the hollow fiber membrane, and an inlet for returning the 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 tank. The outlet and the inlet may be the same or different.

[0057] The hollow fiber membrane is included in, for example, a filtration module. A plurality of hollow fiber membranes may be included in the filtration module. The filtration module may be provided with an inlet for the inflow of a solution delivered from a culture tank, the inlet communicating with the primary surface of the hollow fiber membrane, and an outlet for returning the 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 tank. The inlet and outlet may be the same or different. The filtration module may also be provided with a permeate outlet for discharging the permeate containing the cell product that has permeated the interior of the hollow fiber membrane and flowed out from the secondary surface of the hollow fiber membrane.

[0058] 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.

[0059] The filtration may be tangential flow filtration. Tangential flow filtration (TFF) is a filtration method in which a solution is made to flow 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 solution is made to flow 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 solution is made to flow back and forth in the hollow portion of a hollow fiber membrane.

[0060] The shear rate of tangential flow filtration is, for example, 260 / s or more, 500 / s or more, or 1000 / s or more, and is, for example, 6000 / s or less, 4000 / s or less, or 3000 / s or less.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] The average pore size on the surface of a hollow fiber membrane is measured using the following method. 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.

[0065] 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.

[0066] The blocking pore size is exemplified as the pore size of particles at which the permeation rejection rate of the particles is 90% when a particle dispersion containing particles of a certain pore size is filtered using a porous hollow fiber membrane. It is also sometimes called the minimum pore size. The blocking pore size is measured using the following method. 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 changing the latex particle size in approximately 0.1 μm increments from 0.1 μm, and a rejection curve for the latex particles is created. From this rejection curve, the particle size that allows 90% permeation rejection is read, and this diameter is used as the blocking pore size of the hollow fiber membrane.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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

[0071] 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.

[0072] The recovery rate of a cell product purified using a hollow fiber membrane can be measured by comparing the amount of the cell product in the permeate after the filtration step with the amount of the cell product in the solution before the filtration step. For example, the recovery rate of the cell product can be calculated using the following formula: The concentration and amount of the cell product can be measured, for example, by polymerase chain reaction (PCR). Permeability X = (amount of cell product in the permeate) / (amount of cell product in the solution before filtration) x 100

[0073] Filtration of the solution containing lysed cells using the hollow fiber membrane is carried out, for example, once.

[0074] When filtering a solution containing lysed cells using a hollow fiber membrane, a solvent may be added to the solution containing lysed cells to dilute the solution containing lysed cells. Dilution reduces the concentration of cell lysates and cell products. During dilution, the solvent may be prepared so that the surfactant concentration remains constant. The solvent is, for example, a buffer solution. The solvent contains, for example, the same surfactant as that contained in the solution containing lysed cells. The solvent contains, for example, the same surfactant concentration as that contained in the solution containing lysed cells.

[0075] When diluting the solution containing lysed cells, the solvent may be added continuously to the solution containing lysed cells. Alternatively, the solvent may be added intermittently to the solution containing lysed cells. The solution containing lysed cells may be supplied to the hollow fiber membrane while being diluted. The amount of solvent added to the solution containing lysed cells is, for example, 0.1-fold, 0.2-fold, 0.4-fold, 1-fold, 5-fold, or 10-fold the volume of the solution containing lysed cells. [Example]

[0076] EXAMPLES The present invention will be described in detail below with reference to examples and comparative examples, but the present invention is not limited to the following examples and comparative examples.

[0077] Example 1 (cells, reagents, and culture equipment) HEK293T cells (AAVpro 293T cell line), AAVpro (registered trademark) packaging plasmid (AAV2), and pAAV ZsGreen1 vector were obtained from Takara Bio. The AAVpro (registered trademark) packaging plasmid includes the pHelper vector and the pRC2-mi342 vector. The transfection reagent PEI-MAX (registered trademark) was obtained from Cosmo Bio. The culture area was 636 cm. 2 A CellSTACK® 1 chamber was obtained from Corning. Dulbecco's modified Eagle's medium (D-MEM) and 100 mmol / L sodium pyruvate solution were obtained from Sigma-Aldrich. Penicillin-streptomycin mixed solution was obtained from Nacalai Tesque. Fetal bovine serum (FBS) was obtained from BioWest.

[0078] (Surfactants according to the examples) Hexaethylene glycol dodecyl ether (Laureth 6) and octaethylene glycol monododecyl ether (Laureth 8) were obtained from Fujifilm Wako Pure Chemical Industries, Ltd. Polyethylene glycol lauryl ether (Laureth 9, PEG LE) was obtained from Nacalai Tesque, Inc. Decaethylene glycol monododecyl ether (Laureth 10) was obtained from Sigma-Aldrich. Genapol X-80 was obtained from Merck. Tergitol® TMN-6 was obtained from Sigma-Aldrich.

[0079] (Surfactant according to comparative examples) Pentaethylene glycol dodecyl ether (Laureth 5) was obtained from Fujifilm Wako Pure Chemical Industries. Laureth 5 contains a PEG chain with five repeating units. Triton X-100 was obtained from Tokyo Chemical Industry Co., Ltd. Triton X-100 contains a benzene ring. Brij® 35 30% solution was obtained from Sigma-Aldrich. Brij® 35 contains a PEG chain with 23 repeating units.

[0080] (Buffers and reagents used for filtration) HEPES buffer (pH 8.2) was obtained from Fujifilm Wako Pure Chemical Industries, Ltd. Magnesium chloride was obtained from Nacalai Tesque, Inc. Benzonase (nucleolytic enzyme) was obtained from Merck, Inc. NaCl was obtained from Sigma-Aldrich.

[0081] (filtration module) 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 so that the following was achieved: As in Patent Document 1 (JP 2018-076291 A), the pore size, inner diameter, outer diameter, and membrane thickness of the BioOptimal MF-SL hollow fiber membrane were measured, and the average pore size on the primary 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.

[0082] The PharMed BPT pump tubing L / S16, peroxide-treated silicone pump tubing L / S16 for the liquid feed pump, and the liquid feed pump (Masterflex) were purchased from Yamato Scientific. The Luer lock fittings used as tubing connectors were purchased from Isis. These tubing and pumps were used to connect the culture chamber and the filtration module.

[0083] (Adhesive culture) 2.52×10 7 HEK293T cells were suspended in 80 mL of medium (D-MEM, 1 mmol / L sodium pyruvate, 1% penicillin-streptomycin mixed solution, and 10% FBS) at a density of 4 × 10 4 cells / cm 2 The cells were seeded in a CellSTACK (registered trademark) 1 chamber so that the cells were adherently cultured at 37°C in a 5% CO2 atmosphere for 4 days.

[0084] (AAV production) After 4 days, 99.2 μg of pHelper Vector, 48 μg of pRC2-mi342 Vector, 48 μg of pAAV ZsGreen1 Vector, and 2 mg / mL of PEI-MAX were added to 5 mL of serum-free medium (D-MEM, 1 mmol / L sodium pyruvate, and 1% penicillin-streptomycin mixture) to prepare a plasmid mixture. The plasmid mixture was then incubated at room temperature for 15 minutes. The medium from the cells seeded in the CellSTACK®1 chamber was removed, and 80 mL of fresh serum-free medium was added. The entire plasmid mixture, which had been incubated for 15 minutes, was then added to the cells. The cells were then cultured at 37°C in a 5% CO2 atmosphere for 4 days to allow the cells to produce AAV2.

[0085] (cell solubilization) After allowing the cells to produce AAV2, 100 mmol / L HEPES buffer, 2 mmol / L MgCl2, 1 U / mL Benzonase, and 30 mmol / L surfactant (final concentrations) were added to the cell culture medium, and the solution was mixed at 37°C for 1 hour at 120 rpm to solubilize the cells and obtain a cell lysate.

[0086] (filtration) As shown in Figure 1, a pump pumps 60 mL of cell lysate into the MF-SL mini module at a shear rate of 2900 / s (46.8 mL / min) (200 L / m relative to the membrane area). 2 The cell lysate was circulated between the culture chamber and the MF-SL mini module. The transmembrane pressure was then adjusted to 20 kPa using the valve at the primary outlet of the hollow fiber membrane. The filtrate was collected from the secondary side while maintaining the valve in place. When no more cell lysate was left to filter, the remaining solution in the flow channel was pushed toward the filtrate side with a wash buffer (a mixture of 50 mmol / L HEPES, 100 mmol / L NaCl, and 1 mmol / L MgCl2) in a volume equal to one-quarter of the filtered cell lysate.

[0087] (Calculation of AAV recovery rate) Using a Droplet Digital (registered trademark) PCR device and ddPCR EvaGreen Supermix (BioRad), the AAV concentrations of the original solution before filtration and the filtrate were measured, and the total AAV amounts of the original solution before filtration and the filtrate were calculated. Furthermore, the AAV recovery rate was calculated using the following formula. The original solution to be filtered was not subjected to heating or alkali treatment, but when measuring the AAV concentration of the original solution, the original solution was heated and alkali treated to extract the genome contained within the capsid of the AAV in the original solution. When measuring the AAV concentration of the filtrate, the filtrate was also heated and alkali treated to extract the AAV genome. AAV recovery rate = (total AAV amount in the filtrate) / (total AAV amount in the original solution) × 100 The AAV recovery rates for each surfactant are shown in Tables 1 and 2. The AAV recovery rates when the surfactants of the Examples were used were higher than the AAV recovery rates when the surfactants of the Comparative Examples were used. [Table 1] [Table 2]

[0088] As shown in Figure 2, when PEG LE1 (Laureth 9) was used, the average size of debris formed by cell lysis was approximately 100 µm. This was also the case with the surfactants of the other Examples. The average pore size on the primary surface of the hollow fiber membrane was 30 µm or more and 80 µm or less. Therefore, it is believed that debris did not enter the pores of the hollow fiber membrane, and as a result, clogging of the hollow fiber membrane did not occur, resulting in a high AAV recovery rate.

[0089] As shown in Figure 3, when cells were solubilized with laureth-5, a large amount of micelles was observed to be formed. In laureth-5, the alkyl chain has 12 carbon atoms and the PEG chain has 5 repeating units. This is thought to have caused micellization of the surfactant, leading to self-aggregation and insufficient cell solubilization, resulting in a decrease in AAV recovery rate.

[0090] Example 2 The cells were solubilized using 30 mmol / L, 10 mmol / L, 3 mmol / L, and 1 mmol / L laureth-9 as a surfactant in the same manner as in Example 1, and the cell lysate was filtered to measure the AAV recovery rate. The results are shown in Table 3. [Table 3]

[0091] Example 3 Cells were solubilized using 30 mmol / L laureth-9 as a surfactant in the same manner as in Example 1. The cell lysate was filtered at a shear rate of 290 / s (4.7 mL / min) or 2900 / s (46.8 mL / min) to measure the recovery rate of AAV. The results are shown in Table 4. [Table 4]

[0092] Example 4 (cells, reagents, and culture equipment) Viral Production Cells 2.0, a clone of HEK293F cell line, Viral Production Medium, AAV-MAX transfection kit, Viral-Plex Complexation buffer, and 200 mmol / L GlutaMAX Supplement were purchased from Thermo Fisher Scientific. AAVpro® packaging plasmid (AAV2) and pAAV ZsGreen1 vector were purchased from Takara Bio. A 250 mL vented filter-capped sterile Erlenmeyer flask was purchased from Thermo Fisher Scientific.

[0093] (suspension culture) Viral Production Cells 2.0 cells were cultured in Viral Production Medium (Thermo Fisher) supplemented with 4 mmol / L GultaMAX (Thermo Fisher) at 37°C, 8% CO2, and 135 rpm with shaking. The cell concentration was 5 × 106 After reaching a cell / mL, the cell concentration was increased to 3 × 10 cells / mL with fresh medium. 6 The cells were diluted to 100 cells / ml.

[0094] (AAV production) 60 mL of the diluted suspension cell culture was transferred to a 125 mL sterile Erlenmeyer flask (Thermo Fisher), and 600 μL of AAV-MAX Enhancer from the AAV-MAX transfection kit (Thermo Fisher) was added to the suspension cell culture. The suspension cell culture was then cultured with shaking at 37°C, 8% CO2, and 135 rpm.

[0095] Next, pHelper vector (90 μg, Takara Bio), pRC2-mi342 vector (45 μg, Takara Bio), and pAAV ZsGreen1 vector (45 μg, Takara Bio) were added to 6 mL of Viral-Plex Complexation buffer (Thermo Fisher). A mixture of 360 μL of AAV-MAX Transfection Reagent and 180 μL of AAV-MAX Transfection Booster from the AAV-MAX Transfection Kit (Thermo Fisher) was then added to prepare a plasmid mixture. The plasmid mixture was mixed by inversion and then allowed to stand at room temperature for 30 minutes. The entire plasmid mixture was then added to the suspension cell culture, which had been cultured with shaking at 37°C, 8% CO2, and 135 rpm for 4 days to allow AAV2 production in the suspension cells. (cell solubilization) After allowing the cells to produce AAV2, 100 mmol / L HEPES buffer, 2 mmol / L MgCl2, 10 U / mL Benzonase, and 30 mmol / L Laureth 9 (PEG LE, Nacalai Tesque) (final concentrations) were added to the cell culture medium, and the solution was mixed at 37°C, 1 hour, and 135 rpm to solubilize the cells and obtain a cell lysate.

[0096] (filtration with dilution) The cell lysate was delivered to the MF-SL mini module by a pump at a shear rate of 2900 / s (46.8 mL / min) at a rate of 60 mL (200 L / m relative to the membrane area). 2 A total of 100 ml of lysate was sent to the culture chamber, and the cell lysate was circulated between the culture chamber and the MF-SL mini module. The transmembrane pressure was then adjusted to 20 kPa using the valve at the primary outlet of the hollow fiber membrane. The filtrate coming out of the secondary side was collected while the valve was kept in place.

[0097] As shown in Figure 4, every time 10 mL of filtrate was collected, 10 mL of additional solvent (a mixture of 100 mmol / L HEPES buffer (pH 8.2, Fujifilm Wako Pure Chemical Industries, Ltd.), 2 mmol / L MgCl2 (Nacalai Tesque), and 30 mmol / L PEG-LE (Nacalai Tesque)) was added to the filtered cell lysate to dilute it.

[0098] After adding the same amount of solvent (60 mL) as the original volume of the cell lysate, the cell lysate was filtered without adding any more solvent until the cell lysate was gone. When the cell lysate was gone, the remaining solution in the channel was pushed out to the filtrate side with a wash buffer (a mixture of 50 mmol / L HEPES, 100 mmol / L NaCl, and 1 mmol / L MgCl2) in a volume equal to 1 / 4 of the filtered cell lysate.

[0099] (filtration without dilution) The cell lysate was filtered in the same manner as the filtration with dilution, except that no dilution with additional solvent was performed.

[0100] (Calculation of AAV recovery rate and DNA removal rate) The AAV recovery rate was calculated in the same manner as in Example 1. The DNA removal rate was calculated using the following formula. DNA removal rate = {1 - (total DNA amount in filtrate) / (total DNA amount in original solution)} x 100 As a result, as shown in Table 5, it was shown that filtering the cell lysate while diluting it resulted in a high AAV recovery rate and a high DNA removal rate. [Table 5]

Claims

1. 1. A method for recovering a cellular product, comprising: lysing the cells with a surfactant containing a polyethylene glycol chain having a repeating unit number of 6 to 22 and not containing a benzene ring; filtering the solution containing the lysed cells using a hollow fiber membrane; A method comprising:

2. 10. The method of claim 1, wherein the filtering removes the cellular debris from the solution.

3. 10. The method of claim 1, further comprising recovering the solution containing the cell products filtered through the hollow fiber membrane.

4. The method of claim 1 , wherein the concentration of the surfactant is 1 mmol / L or more.

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

6. The method according to claim 1, 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.

7. The method of claim 1 , wherein the filtration is tangential flow filtration.

8. 8. The method of claim 7, wherein the shear rate of the tangential flow filtration is 260 / s or greater.

9. The method of claim 1 , wherein the cellular product is a gene product of the cell.

10. The method of claim 1 , wherein the cellular product is the product of a gene introduced into the cell.

11. The method of claim 1 , wherein the cellular product is a virus.

12. The method of claim 1 , wherein the cellular product is a viral vector.

13. The method of claim 1 , wherein the cellular product is an adeno-associated virus.

14. The method of claim 1 , wherein the cellular product is an adenovirus.

15. The method of claim 1 , wherein the solution containing the lysed cells is diluted and the diluted solution is filtered through the hollow fiber membrane.

16. 16. The method of claim 15, wherein a buffer solution is added to the solution containing the lysed cells to dilute the solution.

17. 1. A method for recovering a cellular product, comprising: Lysing cells with detergent, diluting the solution containing the lysed cells and filtering the diluted solution through a hollow fiber membrane; A method comprising:

18. 18. The method of claim 17, wherein a buffer solution is added to the solution containing the lysed cells to dilute the solution.

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