Method for determining virus clearance performance

Abstract

To provide a method for designing and performing a virus clearance test so as to obtain an LRV as high as possible.SOLUTION: The method comprises: a first filtration step in which a virus solution containing viruses is filtered through a virus removal medium at a first filtration rate so as to obtain the maximum virus retention capacity per unit membrane area of the virus removal medium; a second filtration step in which, using the virus removal medium at a second filtration rate, a virus addition solution is filtered to obtain a filtrate, the virus addition solution containing viruses in an amount that does not exceed the maximum virus retention capacity over the entire membrane area of the virus removal medium and is at least one tenth of the maximum virus retention capacity over the entire membrane area of the virus removal medium; a step of measuring respective virus concentrations in the solution before and after the filtration in the second filtration step; and a step of determining the virus clearance performance of the virus removal medium based on a difference between the measured virus concentrations in the solution before and after the filtration in the second filtration step.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for determining the viral clearance performance of a viral removal medium. [Background technology]

[0002] When manufacturing biological preparations such as biopharmaceuticals and plasma fractionated preparations, there is a risk of viruses being mixed into the preparation. Generally, biological preparations must be guaranteed to be safe against viruses, so it is important to fully inactivate or remove viruses in the preparation when manufacturing biological preparations. Virus inactivation or removal in biological preparations can be achieved by incorporating a virus clearance process (virus inactivation or removal process) into the manufacturing process of the biological preparation.

[0003] The virus inactivation or removal step can be carried out, for example, by filtering the biological substance-containing preparation using a virus removal medium to separate and remove viruses from the biological substance-containing preparation. Separation and removal of viruses by filtration using a virus removal medium is effective as a physical virus removal method that does not involve chemical denaturation of the biological substance-containing preparation (see, for example, Patent Document 1).

[0004] The virus clearance capability of the virus clearance step can be evaluated by conducting a virus clearance study (VCS) using a scaled-down system of the production line for the actual production of a biological substance-containing preparation before the actual production of the biological substance-containing preparation. For example, the virus clearance capability of a virus removal step using a virus removal medium can be evaluated by subjecting the virus removal medium to a virus clearance test. Typically, a virus clearance test is performed by adding a virus-containing liquid to a biological substance-containing solution before the virus removal step (virus spike), measuring the virus concentration before and after contacting the virus-spiked biological substance-containing solution with the virus removal medium, and calculating the logarithmic reduction value (LRV) of the virus concentration (see, for example, Patent Document 2). The LRV can be expressed as the value obtained by subtracting the logarithmic value of the virus concentration of the solution after contacting the virus removal medium from the logarithmic value of the virus concentration of the solution before contacting the virus removal medium. For example, if the virus concentration of the virus-spiked biological substance-containing solution is 10 n If the virus concentration in the solution after contacting it with the virus removal medium is 0, the virus clearance ability of this virus removal medium is LRV=log 10 (10 n ) - 0 = n. In a virus clearance process, it is necessary to combine multiple virus inactivation and / or removal processes that operate on different principles. In a virus removal process, a virus clearance capacity of LRV4 or greater is considered to be an effective process (see, for example, Non-Patent Document 1). Therefore, in a virus clearance test, when a virus removal medium used in the virus removal process is contacted with a virus-spiked biological substance-containing solution, the tendency is to seek a result in which the log reduction in virus concentration is 4 or greater.

[0005] Since LRV is expressed by subtracting the logarithm of the virus concentration in a solution after contact with a virus removal medium from the logarithm of the virus concentration in the solution before contact with the virus removal medium, a high LRV requires a high virus concentration in the solution before contact with the virus removal medium. However, when a high-concentration virus-containing solution is used in a virus clearance test by spiking a large amount of virus into a biological substance-containing solution to obtain a high LRV, virus breakthrough often occurs, in which the virus removal medium is unable to completely remove the virus and the virus is contained in the solution after contact with the virus removal medium (see, for example, Non-Patent Document 2). On the other hand, if an insufficient amount of virus is spiked into a biological substance-containing solution in a virus clearance test, the virus removal medium's virus clearance performance cannot be fully utilized, even though the virus removal medium can process a higher-concentration virus-containing solution without virus breakthrough. As a result, an LRV value obtained in the virus clearance test is lower than the maximum LRV that can be obtained in the virus removal process, resulting in an underestimation of the virus clearance performance of the virus removal medium.

[0006] Traditionally, virus clearance tests have been designed with reference to past data and literature values. For example, when the virus removal medium is a virus removal membrane that separates and removes viruses by filtration, the virus clearance test has been designed with reference to the type of biological substance-containing preparation to be filtered and the results of past virus clearance tests with different filtration conditions (see, for example, Non-Patent Document 3). However, these methods do not allow for an accurate understanding of the virus clearance performance of the virus removal medium in the virus clearance test, and it has not been possible to design a virus clearance test that maximizes the virus clearance performance of the virus removal medium to obtain the highest possible LRV. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. 2012 / 176876A1 [Patent Document 2] International Publication No. 2021 / 172573A1 [Non-patent literature]

[0008] [Non-Patent Document 1] ICH Q5A(R2) Guideline on viral safety evaluation of biotechnology products derived from cell lines of human or animal origin-Scientific guideline [Non-patent document 2] Kayukawa et al.,Biotechnol Prog. 2022 Mar;38(2):e3237 [Non-patent document 3] Burnham et al., Advanced Viral Clearance Study Design: A Total Viral Challenge Approach to Virus Filtration, March 24, 2018(https: / / www.bioprocessintl.com / viral-clearance / advanced-viral-clearance-study-design-a-total-viral-challenge-approach-to-virus-filtration). Summary of the Invention [Problem to be solved by the invention]

[0009] An object of the present invention is to provide a method for designing and executing a virus clearance test so as to obtain the highest possible LRV by making the most of the virus clearance performance of a virus removal medium.

[0010] Another object of the present invention is to provide a method for designing and executing a viral clearance test without causing a viral breakthrough.

[0011] The present invention also aims to achieve the highest possible filtration rate [L / m 2 One of the objectives of the present invention is to provide a method for designing and executing a viral clearance test so as to obtain the following. [Means for solving the problem]

[0012] As a result of extensive research, the inventors have found that virus clearance performance can be evaluated as highly as possible in a virus clearance test by determining in advance the maximum amount of virus that a virus removal medium can hold (maximum virus retention amount), and then designing and executing a virus clearance test so that the amount of virus loaded into the virus removal medium is within a certain range of the maximum virus retention amount.

[0013] The inventors also found that the virus clearance performance of a virus removal medium is related to the filtration rate, and that if the filtration rate is below a certain rate, the virus clearance performance of the virus removal medium decreases, which may cause virus breakthrough.

[0014] Therefore, one aspect of the present invention provides a method for determining the viral clearance performance of a viral removal medium, comprising the steps of: a first filtration step of filtering a virus solution containing viruses through a virus removal medium at a first filtration speed to determine the maximum virus retention amount per unit membrane area of ​​the virus removal medium; a second filtration step of filtering the virus-added solution containing viruses in an amount that does not exceed the maximum virus retention capacity of the entire membrane area of ​​the virus removal medium and is at least one-tenth of the maximum virus retention capacity of the entire membrane area of ​​the virus removal medium at a second filtration speed, to obtain a filtrate; a step of measuring the virus concentration of the virus-added solution before filtration in the second filtration step and the virus concentration of the filtrate after filtration in the second filtration step; and determining the virus clearance performance of the virus removal medium based on the difference between the measured virus concentration of the virus-added solution before filtration in the second filtration step and the virus concentration of the filtered solution after filtration in the second filtration step; Includes:

[0015] In one embodiment, the maximum virus retention per unit membrane area of ​​the virus removal medium determined in the first filtration step is 10 C and in the second filtration step, the amount D of virus contained in the virus-added solution may satisfy formula (1). A x 10 C-1 ≦ D ≦ A×10 C (1) Here, A is the total membrane area of ​​the virus removal medium used in the second filtration step.

[0016] In one embodiment, the maximum virus retention amount per unit membrane area of ​​the virus removal medium determined in the first filtration step may be P, and the amount of virus contained in the virus-added solution in the second filtration step, D, may satisfy formula (2). A×P×1 / 10 ≦ D ≦ A×P (2) Here, A is the total membrane area of ​​the virus removal medium used in the second filtration step.

[0017] In one embodiment, in formula (1) or (2), D and 10 C Or the unit of P is TCID 50 The number of virus particles may be any of pfu, genome copies, protein amount (mg or mol), and number of virus particles.

[0018] In one embodiment, the second filtration rate may be 74% or more of the first filtration rate, and the second filtration rate may be in the range of 74% or more and 126% or less of the first filtration rate.

[0019] In one embodiment, the virus removal medium may comprise regenerated cellulose.

[0020] In one embodiment, the virus solution and / or the virus-added solution may be purified using an ultracentrifuge or by density gradient ultracentrifugation, which may be iodixanol density gradient ultracentrifugation.

[0021] In one embodiment, the virus may be free of empty viruses.

[0022] In one embodiment, the virus may be a parvovirus.

[0023] In one embodiment, the virus may be porcine parvovirus (PPV), minute virus of mice (MVM), or bovine viral diarrhea virus (BVDV).

[0024] In one embodiment, the average particle size of the virus may be 18 nm or more and 120 nm or less.

[0025] In one embodiment, the virus removal medium may be a flat membrane or a hollow fiber membrane.

[0026] In one embodiment, the virus removal medium may have an average pore size of 15 nm or more and 75 nm or less.

[0027] In one embodiment, the virus removal medium may be a flat membrane or hollow fiber membrane having a structure in which the porosity decreases from the first surface toward the interior, and after passing through at least one minimum porosity portion, the porosity increases again at the second surface.

[0028] In one embodiment, the virus concentration may be measured by any of the infectivity titer method, quantitative PCR (qPCR), ELISA (Enzyme-Linked Immunosorbent Assay), HA assay, and particle concentration measurement by transmission electron microscope.

[0029] In one embodiment, the viral clearance performance may be a Log Reduction Value (LRV).

[0030] In one aspect, the first filtration step may include a step of filtering the virus solution, collecting aliquots of the filtered solution obtained at a fixed volume, and measuring the virus concentration of each of the collected aliquots of the filtered solution.

[0031] In one embodiment, the first filtration step may be carried out multiple times. [Effects of the Invention]

[0032] According to one aspect of the present invention, a viral clearance test can be designed and performed to maximize the viral clearance performance of the viral removal medium and obtain the highest possible LRV.

[0033] Furthermore, according to one aspect of the present invention, it is possible to design and execute a viral clearance test so as to prevent viral breakthrough.

[0034] According to one aspect of the present invention, the filtration rate [L / m 2 It is possible to design and perform viral clearance studies to obtain the following: DETAILED DESCRIPTION OF THE INVENTION

[0035] The following describes modes for carrying out the present invention (hereinafter sometimes referred to as "embodiments"). The present invention is not limited to the following embodiments, and can be practiced in various modifications within the scope of the gist of the invention. Furthermore, the embodiments shown below are merely examples of methods for embodying the technical ideas of the present invention, and the present invention is not limited to these examples.

[0036] A method for determining the virus clearance performance of a virus removal medium according to an embodiment includes: a first filtration step of filtering a virus-containing virus solution through the virus removal medium at a first filtration speed to determine the maximum virus retention per unit membrane area of ​​the virus removal medium; a second filtration step of filtering a virus-added solution through the virus removal medium at a second filtration speed, the virus content of the solution not exceeding the maximum virus retention for the entire membrane area of ​​the virus removal medium and containing virus in an amount that is at least one-tenth of the maximum virus retention for the entire membrane area of ​​the virus removal medium, to obtain a filtered solution; a virus concentration measurement step of measuring the virus concentration of the virus-added solution before filtration in the second filtration step and the virus concentration of the filtered solution after filtration in the second filtration step; and a virus clearance performance determination step of determining the virus clearance performance of the virus removal medium based on the difference between the measured virus concentration of the virus-added solution before filtration in the second filtration step and the virus concentration of the filtered solution after filtration in the second filtration step.

[0037] (First filtration step) In the first filtration step, the virus solution is filtered through the virus removal medium at a first filtration speed, and the maximum virus retention amount per unit membrane area of ​​the virus removal medium is determined.

[0038] In one embodiment, the virus contained in the virus solution is exemplified by a virus that encapsulates nucleic acid (viral nucleic acid) in a viral capsid (shell). Examples of viruses that contain nucleic acid (viral nucleic acid) include infectious and non-infectious viruses. An "infectious virus" refers to a virus that has the ability to invade cells and amplify within cells, and that causes a cytopathic effect on host cells or viral release accompanied by cell death after invasion. A "non-infectious virus" refers to a virus that may have the ability to invade cells and amplify within cells, but does not cause a cytopathic effect on host cells or viral release accompanied by cell death after invasion. The virus contained in the virus solution may be an infectious or non-infectious virus, or may be an empty virus particle that has neither infectious properties nor nucleic acid (viral nucleic acid). An "empty virus particle" is a particle that does not contain nucleic acid (viral nucleic acid) within the viral capsid, and is also called an empty capsid or virus-like particle. The infectious virus, non-infectious virus, or virus-like particle may be naturally occurring or may be synthetically produced, such as by recombinant expression or chemical synthesis.

[0039] In one embodiment, the virus contained in the virus solution is a virus that is or may be contained in a biological material, and examples thereof include minute virus of mice (MVM), porcine parvovirus (PPV), reovirus type 3, poliovirus, pseudorabies virus, human herpes virus 1, murine leukemia virus, and bovine viral diarrhea virus (BVDV). In one embodiment, minute virus of mice (MVM) and porcine parvovirus (PPV) are exemplified, in another embodiment, minute virus of mice (MVM), and in another embodiment, bovine viral diarrhea virus (BVDV).

[0040] In one embodiment, when the virus is a parvovirus, the virus has an average particle size of 20 nm or more and 40 nm or less. Specific examples include porcine parvovirus (PPV) and minute virus of mice (MVM). Porcine parvovirus (PPV) and minute virus of mice (MVM) are similar in size and structure. In one embodiment, the average particle size of the virus may be 18 nm or more, 20 nm or more, 80 nm or more, 26 nm or less, 100 nm or less, or 120 nm or less. The average particle size of the virus can be determined by any combination of these upper and lower limits.

[0041] In one embodiment, the virus solution is not particularly limited as long as it contains infectious viruses, non-infectious viruses, and / or hollow virus particles. Examples of virus solutions include solutions containing at least one of infectious viruses, non-infectious viruses, and hollow virus particles, solutions containing at least one of infectious viruses and non-infectious viruses, and solutions containing no hollow virus particles. A "virus solution containing no hollow virus particles" is a virus solution that is substantially free of hollow virus particles. For example, the content of hollow virus particles in the total virus capsids contained in the virus solution is 10% or less, 5% or less, 3% or less, 1% or less, 0.5% or less, 0.1% or less, or 0.01% or less. Examples of "virus solutions containing no hollow virus particles" include a virus solution that contains no hollow virus particles (0%) and a virus solution in which the number of hollow virus particles is below the detection limit in a transmission electron microscope observation and measurement method.

[0042] A virus culture medium obtained by culturing host cells infected with a virus in a medium is also an example of a virus solution. The virus may be present in a state in which it has infected the host cells, or may be present in a free state in the virus solution (e.g., virus culture medium). The virus solution may be a culture supernatant of a culture obtained by infecting host cells with a virus and culturing them, and / or a lysate of the infected cells (hereinafter simply referred to as "infected cell lysate"). An example of the "culture supernatant" is the medium after infection and culture. An example of the "infected cell lysate" is a so-called lysate obtained by disrupting cells after infection and culture by freezing and thawing or homogenization.

[0043] In one embodiment, the virus solution may be a solution, a suspension, or a gel, with a solution being a preferred embodiment. An example of the solution is a virus culture medium.

[0044] In one embodiment, the virus solution may contain substances other than viruses. For example, the virus solution may contain biological substances that may be contained in a biological substance-containing preparation. Examples of biological substances are not particularly limited, but include proteins, viruses, and nucleic acids. Examples of proteins include albumin, globulin, and fibrinogen. Examples of proteins include antibodies, and the virus solution may contain, for example, human IgG antibodies. Examples of biological substances include biologics and plasma fraction preparations. Biological substances can be commercially available or can be produced by conventional methods. Examples of viruses include adeno-associated viruses (AAV).

[0045] In one embodiment, the biological substance concentration (mg / mL) of the virus solution is, for example, 200 mg / mL or less, 100 mg / mL or less, or 10 mg / mL or less, and is, for example, more than 0 mg / mL, 5 mg / mL or more, 10 mg / mL or more, or 20 mg / mL or more. The range of the biological substance concentration of the virus solution can be determined by any combination of these upper and lower limits.

[0046] In one embodiment, examples of the solvent for the virus solution include an aqueous solution, a phosphate buffer, a sodium acetate buffer, or a Tris buffer, and in one aspect, an aqueous solution.

[0047] In one embodiment, the pH of the virus solution is, for example, not more than 12, not more than 10, not more than 9, not more than 8, or not more than 5, and is, for example, not less than 2, not less than 3, or not less than 4. The pH range of the virus solution can be determined by any combination of these upper and lower limits.

[0048] In one embodiment, the virus solution can be produced, for example, by the following method. To produce the virus solution, first, host cells for the virus are cultured. Any host cells can be used as long as they are capable of infecting and amplifying the virus. When the virus is a parvovirus, any cells can be used as long as they are capable of infecting and amplifying the parvovirus. When the virus is porcine parvovirus (PPV), PK-13 cells (ATCC) and PK-15 cells (ATCC) can be used. When the virus is minute virus of mice (MVM), mouse fibroblast A9, simian virus 40-transformed human fibroblasts (324K cells, NB324K cells, etc.), Chinese hamster ovary cells (CHO), etc. can be used. When the virus is bovine viral diarrhea virus (BVDV), bovine kidney-derived cells (MDBK) can be used.

[0049] In one embodiment, the method for culturing the host cells is not limited. Any method can be used, such as a method using a culture flask, a method using agitation culture with cells immobilized on microcarriers, a method using roller bottles, or a method using a bioreactor. In addition, both adherent culture and suspension culture can be used.

[0050] In one embodiment, a commercially available culture flask can be used, for example, a tissue culture flask manufactured by Falcon or a multi-well plate.

[0051] In one embodiment, commercially available microcarriers can be used, for example, Cytodex and Cytopore manufactured by GE Healthcare.

[0052] In one embodiment, a commercially available roller bottle can be used, for example, CELLMASTER cell culture roller bottle manufactured by Greiner Bio-One.

[0053] In either culture method, the culture temperature is typically 33°C to 39°C, for example 37°C, which is commonly used for culturing mammalian cells that are host cells for viruses. The pH of the medium is typically 7 to 8, which is commonly used for culturing mammalian cells. The concentration of the medium is preferably a concentration that is isotonic with the host cells (equal to the osmotic pressure).

[0054] In one embodiment, examples of the medium include DMEM medium, MEM medium, CD medium, SFM medium, OptiPro SMF medium, Viral Vaccine Platform medium (SFM4Mega Vir medium, Vaccine Xpress medium, and CDM4Avian medium), etc. These media are available from Gibco, Thermo Fisher, GE Healthcare, etc.

[0055] Cultured uninfected host cells are infected with virus seeds. Seed viruses can be purchased from ATCC or other retailers, multiplied, aliquoted into cryotubes, and stored frozen at -80°C. Examples of commercially available seed viruses include VR-742 (ATCC) for porcine parvovirus (PPV) and VR-1346 (ATCC) for minute virus of mice (MVM).

[0056] To infect host cells with a seed virus, a predetermined amount of virus (e.g., suspended in a medium) is added to a culture vessel containing cultured (uninfected) host cells.

[0057] To minimize the amount of nonviral impurities in the culture medium, the medium may be serum-free. However, efficient virus amplification can be achieved by culturing in a medium containing serum, such as fetal bovine serum (FBS) or horse serum. Therefore, by replacing the culture medium from serum-containing medium to serum-free medium at least 24 hours before harvesting the virus culture medium, the virus can be produced from cells in a medium free of serum-derived impurities and then harvested. Prior to the subsequent ultracentrifugation step, low-speed centrifugation and bacterial membrane filtration can be performed to remove large particle-sized contaminants, such as cell debris, present in the virus culture medium. Low-speed centrifugation can be performed at a gravitational acceleration that does not precipitate the virus but does precipitate the cell debris. For example, centrifugation is performed at 3,000 rpm for 20 minutes, but conditions are not limited to these. Furthermore, bacterial membrane filtration can be performed using membranes with pore sizes of 0.45 μm, 0.22 μm, or 0.1 μm. Examples of membranes having these pore sizes include a 0.45 μm bottle top filter manufactured by Thermo Fisher Scientific, and a 0.22 μm bottle top filter manufactured by Thermo Fisher Scientific.

[0058] To obtain a higher concentration of the virus-containing solution for testing, the recovered virus culture solution may be concentrated. Many known methods can be used for concentration. For example, salting-out precipitation using CsCl2, polyethylene glycol (PEG) precipitation, ultrafiltration membrane methods, and ultracentrifugation are available. Among these, ultracentrifugation and PEG precipitation have the advantages of easy solvent exchange and high virus recovery.

[0059] For example, when ultracentrifugation is used, ultracentrifugation is performed at a relative centrifugal force (g) and for a time period that allows the virus to precipitate. After the virus is precipitated, the supernatant is removed and the precipitated fraction is resuspended in an appropriate solvent. When the virus is a parvovirus, known ultracentrifugation conditions for precipitating the virus can be used. For example, when using a Beckman Optima L-90K ultracentrifuge with a Type 45 Ti rotor, parvovirus is precipitated by ultracentrifugation at 29,400 rpm for 2 hours. Solvents that can be used for resuspension include commonly used TNE (Tris buffer + NaCl + EDTA) buffer and PBS buffer. In one embodiment, PBS buffer is used. A virus solution obtained by concentrating a virus culture medium or the like using an ultracentrifuge can be called a "virus solution purified using an ultracentrifuge."

[0060] When using the PEG precipitation method, polyethylene glycol and salt are added in an amount sufficient to cause virus coagulation, followed by centrifugation to precipitate the virus. The supernatant is then removed and the precipitated fraction is resuspended in an appropriate solvent. Experimental conditions commonly used for virus precipitation can be utilized; for example, PEG6000 can be used as the polyethylene glycol. Furthermore, when the virus is a parvovirus, the parvovirus is precipitated by adding PEG6000 to a final concentration of 10.0 w / v% and sodium chloride to a final concentration of 0.1 mol / L, stirring at 4°C for 2 hours, and then centrifugation at 10,000 x g for 30 minutes. Commonly used solvents such as TNE (Tris buffer + NaCl + EDTA) buffer and PBS buffer can be used for resuspension. In one embodiment, PBS buffer is used.

[0061] In one embodiment, a virus solution free of hollow virus particles can be produced by, for example, separating and removing hollow virus particles from a virus culture solution, using methods such as density gradient ultracentrifugation and ion exchange chromatography.

[0062] In density gradient ultracentrifugation, hollow virus particles are separated by utilizing the difference in buoyancy density between viruses containing nucleic acid and hollow virus particles not containing nucleic acid. The ultracentrifugation conditions are not limited to specific conditions, and any ultracentrifugation conditions may be used as long as they allow separation of viruses containing nucleic acid and hollow virus particles.

[0063] Density gradient ultracentrifugation may be iodixanol density gradient ultracentrifugation using iodixanol. For example, 1 mL of the virus culture solution is loaded onto the top of a discontinuous density gradient (55% - 2 mL, 45% - 2 mL, 40% - 2 mL, 35% - 2 mL, 25% - 2 mL, 15% - 1 mL) of an aqueous iodixanol solution (Optiprep, Axis-Shield) in PBS-1 mmol / L MgCl-2.5 mmol / L KCl buffer (pH 7.2) formed in a UC tube (13.2 mL, #344059, Beckman Coulter), and ultracentrifugation is performed at 35,000 rpm, 4°C, and 18 hours in an SW41 rotor in an ultracentrifuge (Optima L-90K, Beckman Coulter) to separate nucleic acid-containing viruses from empty virus particles. By collecting only the fraction corresponding to the buoyant density of the nucleic acid-containing virus from the ultracentrifuged sample, the hollow virus particles are removed from the virus culture medium. When the virus is a parvovirus, the buoyant density of the hollow virus particles is 1.1 g / mL or more and 1.2 g / mL or less, and the buoyant density of the nucleic acid-containing virus is 1.2 g / mL or more and 1.3 g / mL or less. This ultracentrifugation may be repeated a total of two times to increase the degree of separation of the hollow virus particles.

[0064] The fraction containing nucleic acid-containing viruses obtained by ultracentrifugation becomes a highly concentrated aqueous solution of iodixanol, so the iodixanol concentration can be reduced by solvent exchange. The solvent exchange method can be selected from known solvent exchange methods. Examples include desalting, UF membrane filtration, and dialysis, with desalting being preferred. Desalting using Zeba Spin Desalting Columns 40K MWCO (ThermoFisher) is more preferred.

[0065] A virus solution obtained by separating and removing hollow virus particles from a virus culture medium or the like using density gradient ultracentrifugation such as iodixanol density gradient ultracentrifugation can be called a "virus solution purified by (iodixanol) density gradient ultracentrifugation." In one embodiment, the "virus solution purified by (iodixanol) density gradient ultracentrifugation" is a virus solution that does not contain hollow virus particles, but is not limited to this.

[0066] The removal of hollow virus particles from the virus solution can be confirmed by observation using a transmission electron microscope (TEM). Examples of observation methods using a TEM include negative staining observation using 1.0% uranyl acetate or cryo-electron microscope (CryoEM) observation. In negative staining observation, viruses containing nucleic acid are observed as white particles, and hollow virus particles without nucleic acid are observed as black particles, making it possible to visually distinguish these particles. In one embodiment, if hollow virus particles are not visible using TEM observation, it can be determined that the number of hollow virus particles contained in the virus solution is below the detection limit.

[0067] In one embodiment, the form of the virus removal medium is not particularly limited as long as it is a medium that can remove viruses. Examples of the form of the virus removal medium include beads, gel, sponge, powder, fiber, and membrane. The virus removal medium may be a porous shaped body, and the porous shaped body may be a microporous membrane, nonwoven fabric, woven fabric, monolith, gel, or particle bed.

[0068] In one embodiment, the shape of the virus removal medium is exemplified as a membrane (a so-called virus removal membrane). Examples of the virus removal membrane include a flat membrane and a hollow fiber membrane. In one aspect, a flat membrane is used as the virus removal membrane, and in another aspect, a hollow fiber membrane is used as the virus removal membrane.

[0069] The material of the virus removal medium is not particularly limited as long as it is a material that can remove viruses, and examples thereof include polyethersulfone, polysulfone, polyvinylidene fluoride, cellulose, regenerated cellulose, cellulose derivatives, and mixtures thereof. "Cellulose derivatives" are compounds obtained by introducing different substituents into hydroxy groups contained in cellulose molecules. Virus removal media made from these materials and virus removal media containing these materials can each be produced by known methods.

[0070] In one embodiment, when the virus removal medium is a virus removal membrane, the virus removal membrane can be appropriately selected depending on the size of the virus to be used. The virus removal membrane is not particularly limited as long as it can separate and remove viruses, and an example is a membrane having an LRV of 4 or more against parvovirus. A filtration membrane having an LRV of 4 or more against parvovirus is a membrane having an LRV of 4 or more that can separate and remove 50 L / m of a solution containing parvovirus PPV or MVM from the filtration membrane to be used. 2 The membrane has an LRV of 4 or more when loaded. For example, a solution containing parvovirus is prepared by loading a 10 mg / mL human IgG solution (PBS buffer, pH 7.4) with a log 10 [TCID 50 A virus removal membrane having a parvovirus PPV or MVM concentration of 6.5 (given in [mg / mL]). There are no particular limitations on the average pore size of the virus removal membrane, as long as it is approximately equal to or smaller than the size of the virus being used. For example, when parvovirus is used, examples of the virus removal membrane to be used include small virus removal filters. Specific examples include Planova (manufactured by Asahi Kasei Medical Co., Ltd.), Virasolve Pro (manufactured by Merck), Pegasus Prime (manufactured by PALL), Virosart CPV (manufactured by Sartrius), Viruclear VF plus (manufactured by Cobetter), Viruclear RC (manufactured by Cobetter), Virosart HF (manufactured by Sartrius), and Pegasus SV4 (manufactured by PALL).

[0071] In one embodiment, when the virus removal medium is a virus removal membrane, an example is a virus removal membrane with a nominal pore size of about 15 nm to about 75 nm. For example, a small virus removal filter can remove, for example, parvovirus (diameter 18 nm to 26 nm), but may allow proteins as large as 160 kD (about 8 nm), such as monoclonal antibodies, to pass through. For example, a large virus removal filter can be used to remove retroviruses (diameter 80 nm to 100 nm). Examples of large virus removal filters include Planova (manufactured by Asahi Kasei Medical Co., Ltd.), Ultipor VF Grade DV50 (manufactured by PALL), and Viresolve NFR (manufactured by Merck).

[0072] In one embodiment, when the virus removal medium is a virus removal membrane, an example is a virus removal membrane having a molecular weight cutoff of 100 kD or more and 1000 kD or less. In this regard, the molecular weight cutoff (MWCO) of a membrane refers to the molecular weight (nominal molecular weight) of a substance that allows 90% of the substance to pass through the membrane when the substance is passed through the membrane. The nominal molecular weight cutoff can also be used as the molecular weight cutoff.

[0073] In one embodiment, when the virus removal medium is a virus removal membrane, an example is a virus removal membrane having a porosity of about 10% to about 90%. In one aspect, the virus removal membrane has a first surface and a second surface, and has a structure in which the porosity decreases as one moves inward from the first surface toward the second surface, and after passing through at least one minimum porosity portion, the porosity increases again at the second surface. A virus removal membrane having such a structure can be produced, for example, by the methods described in Japanese Patent No. 4024041 and Japanese Patent Laid-Open No. 1-148305.

[0074] In one embodiment, filtration of a virus solution with a virus removal medium can be carried out by applying any filtration pressure to the virus solution. The filtration pressure is not particularly limited as long as it is within a range that allows viruses to be separated and removed from the virus solution by the virus removal medium, and examples include 0.01 bar or more, 0.1 bar or more, 0.4 bar or more, or 0.5 bar or more, and 3.43 bar or less, 1.96 bar or less, 0.98 bar or less, 0.9 bar or less, or 0.8 bar or less. Specific examples include 0.4 bar to 0.9 bar, 0.4 bar to 0.8 bar, 0.5 bar to 0.9 bar, or 0.5 bar to 0.8 bar. In one embodiment, the filtration pressure is approximately 0.8 bar. In one embodiment, the term "approximately" refers to a range of ±10%, for example, "approximately 0.8 bar" refers to a range of 0.72 bar to 0.88 bar. In another embodiment, "about 0.8 bar" refers to a range of numbers that, when rounded to one decimal place, results in 0.8 bar, that is, a range of 0.75 bar to 0.84 bar.

[0075] In one embodiment, the filtration rate when filtering the virus solution through the virus removal medium is not particularly limited, and may be, for example, 0 L / m 2 / hour(LMH), 10L / m 2 / hour (LMH) or more, or 30L / m 2 / hour (LMH) or more is an example, and 750L / m 2 / hour(LMH) or less, 700L / m 2 / hour(LMH) or less, 600L / m 2 / hour(LMH) or less, 500L / m 2 / hour(LMH) or less, 400L / m 2 / hour(LMH) or less, 300L / m 2 / hour(LMH) or less, 200L / m 2 / hour(LMH) or less, 100L / m 2 / hour(LMH) or less, 90L / m 2 / hour(LMH) or less, 80L / m 2 / hour(LMH) or less, 70L / m 2 / hour (LMH) or less, or 60L / m2 The filtration rate range can be determined by any combination of these upper and lower limits. The unit of filtration rate is L / m 2 The term "filtration amount" is not limited to "per hour (LMH)" and may be any value that can express the filtration amount per unit time. Here, the filtration amount may be the filtration amount per unit membrane area.

[0076] In one embodiment, the maximum virus retention per unit membrane area of ​​a virus removal medium can be determined, for example, by the following method. First, a virus solution is filtered through a virus removal medium, and the filtrate that passes through the virus removal medium is collected into multiple fractions. The virus contained in each fraction is quantified, and a fraction in which virus is detected for the first time and continuously after passing through the virus removal medium is selected. The total amount of virus per unit membrane area loaded on the virus removal medium up until immediately before collecting the fraction is quantified, and this is defined as the maximum virus retention per unit membrane area of ​​the virus removal medium.

[0077] The method for quantifying the virus is not limited, and any known method may be used. For example, a method for quantifying the viral nucleic acid concentration or a method for quantifying the viral infectivity titer is exemplified. The infectivity titer is a unit that expresses the concentration of an infectious virus.

[0078] In one embodiment, methods for quantifying viral nucleic acid concentration include quantitative PCR (qPCR) and fluorescent flow cytometry. In one aspect, quantitative PCR is used, for example, quantitative real-time PCR. For quantitative PCR, for example, the LightCycler96 (manufactured by Roche) can be used. For fluorescent flow cytometry, for example, the Gallios (manufactured by Beckman Coulter) can be used.

[0079] For example, when quantitative PCR is employed, the virus solution subjected to filtration with a virus removal medium may or may not contain hollow virus particles that do not contain nucleic acids, but it is preferable that the solution does not contain hollow viruses. Here, the virus that contains nucleic acids may be infectious or non-infectious. Since viruses usually encapsulate one nucleic acid molecule per particle in the viral capsid, the number of nucleic acid molecules encapsulated in the virus that contains nucleic acids is equal to the number of virus particles. Therefore, the amount of virus can be expressed as the number of nucleic acid molecules encapsulated in the viral capsid, i.e., the genome copy number (or copies).

[0080] When quantitative PCR is used, nucleic acids can be extracted from viruses contained in each fraction by known methods, such as using various nucleic acid extraction kits, phenol-chloroform extraction, etc. In one embodiment, a nucleic acid extraction kit is used, such as the Hi-Pure Virus Nucleic Acid Extraction Kit (manufactured by Roche).

[0081] In one embodiment, the method for quantifying the virus infectivity (infectivity titer method) is to measure the 50% infectious end point (TCID 50 Examples include the tissue culture infectious dose (TCID) method and the plaque method. The 50% infectious end point method is an endpoint method for determining the minimum infectious unit, in which the virus is serially diluted and inoculated into a certain number of cultured cells, cultured for a certain period of time, and the positive / negative results of the infection are determined to determine the dilution ratio at which 50% infection is positive. The plaque method is a method for counting local lesions formed by the virus, inoculating the virus onto a monolayer culture of host cells, allowing the virus to adsorb, then overlaying it with a medium containing agar to solidify, and measuring the number of plaques formed equal to the number of viruses inoculated. The unit of infectious titer is TCID 50 If you use the TCID 50 When the plaque method is used, pfu (plaque forming unit) can be used.

[0082] In one embodiment, other methods for quantifying viruses include, for example, ELISA (Enzyme-Linked Immunosorbent Assay), hemagglutination (HA) assay, or particle concentration measurement using a transmission electron microscope. In the ELISA method, for example, an antibody against the virus is bound to an immobilized antigen, and after washing away the unbound sample, the bound antibody is subjected to measurement of the enzyme activity using an enzyme-labeled antibody, thereby measuring the amount of virus. In the HA assay, the amount of virus can be measured based on the presence or absence and degree of hemagglutination in the presence of hemagglutinin possessed by the virus. In the ELISA method and HA assay, the virus infectivity titer and the amount of virus protein can be determined, and the determined infectivity titer can be measured in units of, for example, TCID 50 Examples of units of the determined protein amount include mass (mg, g) or amount of substance (mol). It is also possible to determine the virus particle concentration by observing a solution containing the virus under a transmission electron microscope and counting the number of particles observed.

[0083] The amount of virus contained in a sample is also called the virus titer. For example, the number of virus genome copies quantified by quantitative PCR, or the TCID quantified by the infectivity titer method. 50 and pfu, the number of virus particles (units) determined by particle concentration measurement using a transmission electron microscope, or the amount of virus protein (mg, mol), etc. In this specification, unless otherwise specified, the virus concentration refers to the amount of virus contained in 1 mL of sample (titer / mL), and units of virus concentration are, for example, Copies / mL and TCID 50 / mL, pfu / mL, mg / mL, mol / mL, and cells / mL can be used.

[0084] In one embodiment, the unit membrane area used to determine the maximum virus retention amount per unit membrane area of ​​the virus removal medium is not limited, and may be up to 1 m 2 , 1cm2 , and 1 mm 2 In one embodiment, the unit membrane area is 1 m 2 The maximum virus retention per unit membrane area of ​​a virus removal medium may be determined by filtering a virus solution through a virus removal medium equivalent to the unit membrane area, or by filtering a virus solution through a virus removal medium with a membrane area different from the unit membrane area, determining the maximum virus retention for that area, and then calculating the maximum virus retention per unit membrane area. The membrane area is the surface area of ​​the portion of the virus removal part of the virus removal medium that comes into contact with the filtrate during filtration, regardless of the form of the virus removal medium. In an embodiment where the virus removal medium is a virus removal membrane, the membrane area is the surface area of ​​the portion of the membrane part of the virus removal membrane that comes into contact with the filtrate during filtration. In an embodiment where the virus removal medium is a hollow fiber membrane, when filtration is performed from the inside of the hollow fiber, the membrane area is the surface area of ​​the inside of the hollow fiber, and when filtration is performed from the outside of the hollow fiber, the membrane area is the surface area of ​​the outside of the hollow fiber.

[0085] In one embodiment, the unit membrane area is 1 m 2 If the maximum virus retention amount is quantified by quantitative PCR, it is expressed as Copies / m2 as the unit of maximum virus retention amount per unit membrane area. 2 is exemplified, and TCID 50 When the maximum viral load is quantified by the method, it is calculated as TCID 50 / m 2 is an example, and when the maximum amount of virus held is quantified by the plaque method, it is pfu / m 2 The maximum amount of virus retained per unit membrane area is expressed as mg / m 2 , mol / m 2 , pieces / m 2 The maximum virus retention amount per unit membrane area of ​​the virus removal medium may be expressed as a logarithmic value, for example, log 10 [Copies / m 2 ], log 10 [TCID 50 / m 2 ], log 10 [pfu / m 2 ], log 10[mg / m 2 ]log 10 [mol / m 2 ] or log 10 [pcs / m 2 ] is written as follows.

[0086] The maximum amount of virus that can be retained per unit membrane area of ​​a virus removal medium can also be expressed as the "virus capture capacity."

[0087] In one embodiment, the first filtration step may be performed multiple times under the same conditions, and the maximum virus retention per unit membrane area may be determined for each filtration step. By determining the maximum virus retention per unit membrane area through repeated experiments, the reproducibility of the experimental system can be confirmed and the range of error in the data can be determined.

[0088] (Second filtration step) In the second filtration step, the virus-added solution containing viruses in an amount that does not exceed the maximum virus retention capacity of the entire membrane area of ​​the virus removal medium and is at least one-tenth of the maximum virus retention capacity of the entire membrane area of ​​the virus removal medium is filtered through the virus removal medium at a second filtration speed to obtain a filtered solution.

[0089] For example, if the maximum virus retention per unit membrane area of ​​the virus removal medium determined in the first filtration step is 10 C In the case where the amount of virus contained in the virus-spiked solution filtered in the second filtration step is represented by D, the amount of virus may be D that satisfies formula (3). A x 10 C-1 ≦ D ≦ A×10 C (3) In equation (3), A is the total membrane area of ​​the virus removal medium used in the second filtration step.

[0090] Furthermore, for example, when the maximum virus retention amount per unit membrane area of ​​the virus removal medium determined in the first filtration step is represented by P, the amount of virus contained in the virus-added solution filtered in the second filtration step may be D, which satisfies formula (4). A×P×1 / 10 ≦ D ≦ A×P (4) In equation (4), A is the total membrane area of ​​the virus removal medium used in the second filtration step.

[0091] The term "total membrane area of ​​the virus removal medium" refers to the surface area of ​​the portion of the virus removal medium that comes into contact with the virus-added solution during filtration in the second filtration step, regardless of the form of the virus removal medium.

[0092] In one embodiment, the second filtration step includes a step of preparing a virus-added solution (step (a)) and a step of filtering the prepared virus-added solution through a virus removal medium to obtain a filtered solution (step (b)). In another embodiment, the second filtration step does not include steps (a) and (b), but includes a step of filtering the virus-added solution through a virus removal medium to obtain a filtered solution (step (c)).

[0093] (Step (a)) In one embodiment, in step (a) of preparing a virus addition solution, the virus addition solution is prepared based on the maximum virus retention capacity per unit membrane area of ​​the virus removal medium determined in the first filtration step. Specifically, the virus addition solution is prepared so that the virus addition solution contains viruses in an amount that does not exceed the maximum virus retention capacity per total membrane area of ​​the virus removal medium, and is at least one-tenth of the maximum virus retention capacity per total membrane area of ​​the virus removal medium. The maximum virus retention capacity per total membrane area of ​​the virus removal medium can be calculated, for example, by multiplying the maximum virus retention capacity per unit membrane area of ​​the virus removal medium determined in the first filtration step by the total membrane area of ​​the virus removal medium used in the second filtration step. If the total membrane area of ​​the virus removal medium used in the second filtration step is equal to the unit membrane area of ​​the virus removal medium used in the first filtration step, the maximum virus retention capacity per total membrane area of ​​the virus removal medium will be equal to the maximum virus retention capacity per unit membrane area determined in the first filtration step.

[0094] For example, in one embodiment, the maximum virus retention per unit membrane area of ​​the virus removal medium is 10 CIn the case where the amount of virus added is expressed as above, a virus addition solution containing an amount D of virus that satisfies the above formula (3) is prepared in step (a). In formula (3), A is the total membrane area of ​​the virus removal medium used in step (b). The unit of A is not limited, and it can be expressed as m 2 ,cm 2 , and mm 2 However, it must be in the same unit as the unit membrane area used in the first filtration step. C The units are Copies and TCID. 50 Examples of units of D include the amount of protein (mg or mol), the number of virus particles (units), and pfu. 50 , protein amount (mg or mol), number of virus particles (particles) or pfu are examples, and the unit of D is 10 C It is the same as the unit of

[0095] Equation (3) expresses the relationship between the amount of virus D contained in the virus-spiked solution and the maximum virus retention amount (A × 10) of the total membrane area of ​​the virus removal medium used in step (b). C ) and does not exceed one-tenth (A × 10) of the maximum virus retention amount in the total membrane area of ​​the virus removal medium used in step (b). C-1 ) or more.

[0096] Furthermore, for example, when the maximum virus retention amount per unit membrane area of ​​a virus removal medium is represented by P, a virus-added solution containing an amount D of virus that satisfies the above formula (4) is prepared in step (a). In formula (4), A is the total membrane area of ​​the virus removal medium used in step (b). The unit of A is not limited and can be m 2 ,cm 2 , and mm 2 However, it must be in the same unit as the unit membrane area used in the first filtration step. The units of P include Copies and TCID 50 Examples of units of D include the amount of protein (mg or mol), the number of virus particles (units), and pfu. 50 , protein amount (mg or mol), number of virus particles (particles) or pfu are examples, and the unit of D is the same as the unit of P.

[0097] Equation (4) means that the amount of virus D contained in the virus added solution does not exceed the maximum virus retention amount (A × P) for the total membrane area of ​​the virus removal medium used in step (b), and is at least one-tenth (A × P × 1 / 10) of the maximum virus retention amount for the total membrane area of ​​the virus removal medium used in step (b).

[0098] The present inventors have found that when performing the second filtration step using a virus removal medium with area A, the second filtration step can be performed while making maximum use of the virus clearance performance of the virus removal medium by designing the virus removal medium so that the amount of virus loaded onto the virus removal medium does not exceed the maximum virus retention capacity of the virus removal medium with area A. Furthermore, by setting the amount of virus loaded onto the virus removal medium to at least one-tenth of the maximum virus retention capacity of the virus removal medium with area A, it is possible to avoid underestimating the virus clearance performance of the virus removal medium.

[0099] The virus additive solution prepared in step (a) need only contain an amount of virus that does not exceed the maximum virus retention capacity of the entire membrane area of ​​the virus removal medium and is at least one-tenth of the maximum virus retention capacity of the entire membrane area of ​​the virus removal medium (e.g., amount D that satisfies formula (3) or (4)), and the volume and virus concentration of the virus additive solution are not limited. For example, in an embodiment in which the volume of the virus additive solution to be filtered in the subsequent step (b) is relatively large, a virus additive solution with a relatively low concentration and a large volume can be prepared in step (a). In an embodiment in which the virus concentration of the virus additive solution to be filtered in step (b) is relatively high, a virus additive solution with a relatively high concentration and a small volume can be prepared in step (a). The volume and virus concentration of the virus additive solution can be set as desired based on the value of amount D that satisfies formula (3) or (4). For example, if the volume of the virus additive solution to be filtered in step (b) is predetermined, the virus concentration of the virus additive solution to be prepared can be determined from that volume and the value of D. Furthermore, if the virus concentration of the virus-spiked solution to be filtered in step (b) is predetermined, the volume of the virus-spiked solution to be prepared can be determined from that concentration and the value of D.

[0100] In one embodiment, the viruses contained in the virus addition solution are exemplified as the same viruses as those exemplified above as the viruses contained in the virus solution. Of the viruses exemplified above as the viruses contained in the virus solution, the viruses contained in the virus addition solution used in the second filtration step may be the same as or different from the viruses contained in the virus solution used in the first filtration step. In one aspect, the viruses contained in the virus addition solution include the same viruses as the viruses contained in the virus solution used in the first filtration step.

[0101] In one embodiment, the method for preparing the virus-added solution is not limited, and for example, the virus-added solution may be prepared by adding a virus to the virus solution used in the first filtration step, or the virus-added solution may be prepared using the method exemplified above as a method for producing a virus solution. The virus-added solution may contain the same virus as the virus contained in the virus solution used in the first filtration step, or may contain a different virus. When the virus solution used in the first filtration step contains multiple viruses, the virus-added solution may contain the same combination of viruses as the multiple viruses contained in the virus solution. In one aspect, the virus-added solution contains the same virus or the same combination of viruses as the virus contained in the virus solution used in the first filtration step.

[0102] In one embodiment, the substances contained in the virus-added solution and the composition of the virus-added solution may be the same as those exemplified above for the substances contained in the virus solution and the composition of the virus solution. For example, a virus culture solution obtained by culturing virus-infected host cells in a medium may be used as the virus-added solution. The virus-added solution may contain substances other than viruses, such as biological substances that may be contained in a biological substance-containing formulation. The biological substance is not particularly limited, and examples include proteins, viruses, and nucleic acids. Examples of proteins include albumin, globulin, and fibrinogen. The protein as the biological substance contained in the virus-added solution may be an antibody, such as a human IgG antibody. Examples of viruses include adeno-associated virus (AAV). The biological substance concentration in the virus-added solution, the solvent contained in the virus-added solution, and the pH of the virus-added solution may be the same as those exemplified above for the virus solution.

[0103] In one embodiment, the virus-added solution may be prepared by adding a virus (virus spike) to a biological substance-containing solution produced by appropriately scaling down the commercial production conditions of a biological substance-containing preparation intended for commercial manufacture and sale, so that the amount of virus D satisfies formula (3) or formula (4). In this case, the virus added to the biological substance-containing solution may be the same virus or the same combination of viruses as those used in the first filtration step.

[0104] The temperature of the biological substance-containing solution when a virus is added to the biological substance-containing solution is not particularly limited as long as it is a temperature at which the biological substance contained in the biological substance-containing solution remains stable, and examples include 50°C or lower, 37°C or lower, and 25°C or lower, as well as above 0°C, 4°C or higher, and 8°C or higher. These upper and lower temperature limits can be combined arbitrarily to determine the temperature range of the biological substance-containing solution when a virus is added to the biological substance-containing solution. If the biological substance-containing solution is intended for commercial production, the manufacturing temperature for commercial production can be used.

[0105] (Step (b)) In step (b), the virus-added solution prepared in step (a) is filtered through a virus removal medium to obtain a filtrate. The membrane area of ​​the virus removal medium used for filtration in step (b) is the "total membrane area of ​​the virus removal medium" defined above, i.e., A defined in formula (3) or formula (4). The unit of membrane area A is not limited, and may be m 2 ,cm 2 , and mm 2 However, it is necessary that the unit is the same as the unit membrane area used in the first filtration step. The membrane area A may be the same as the unit membrane area, or may be larger or smaller than the unit membrane area. For example, when the unit membrane area used in the first filtration step is 1 m 2 If A is 1m 2 It may be smaller.

[0106] In one embodiment, the virus removal medium used in step (b) is exemplified by the same virus removal medium as the virus removal medium exemplified above as the virus removal medium that can be used in the first filtration step. In one aspect, the virus removal medium used in step (b) is identical in structure and composition to the virus removal medium used in the first filtration step, but the membrane area may be different. Furthermore, the virus removal medium used in step (b) is identical in structure and composition to the virus removal medium used in the first filtration step, and is prior to being subjected to filtration in the first filtration step.

[0107] In one embodiment, the filtration of the virus-added solution with the virus removal medium may be carried out by applying any filtration pressure to the virus-added solution, or may be carried out at any flow rate using a pump or the like. The filtration pressure and filtration flow rate are not particularly limited as long as they are within ranges that allow the virus to be separated and removed from the virus solution by the virus removal medium. In one aspect, the filtration pressure can be adjusted so that the filtration rate of the virus-added solution falls within the ranges described below.

[0108] In one embodiment, the filtration rate when the virus-added solution is filtered is 74% or more of the filtration rate when the virus solution is filtered in the first filtration step. In one aspect, the filtration rate when the virus-added solution is filtered is 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more of the filtration rate when the virus solution is filtered in the first filtration step. The upper limit of the filtration rate when the virus-added solution is filtered is not limited, but in one aspect, it is 126% or less, 125% or less, 120% or less, 115% or less, 110% or less, or 105% or less of the filtration rate when the virus solution is filtered in the first filtration step. Examples of the filtration rate when the virus-added solution is filtered include, but are not limited to, 74% to 126%, 75% to 125%, or 80% to 120% of the filtration rate when the virus solution is filtered in the first filtration step. The filtration rate of the virus-added solution in step (b) can be determined by any combination of the above lower and upper limits.

[0109] The present inventors have found that the virus-added solution can be filtered without virus breakthrough when the filtration rate when the virus solution is filtered through the virus removal medium in the first filtration step is 74% or more of the filtration rate when the virus solution is filtered through the virus removal medium.If the filtration rate when the virus solution is filtered through the virus removal medium in the first filtration step is less than 74% of the filtration rate when the virus solution is filtered through the virus removal medium, virus breakthrough may occur, in which the virus is contained in the solution that has passed through the virus removal medium.

[0110] Furthermore, although there is no upper limit to the filtration rate when the virus-added solution is filtered through the virus removal medium, if the filtration rate when the virus-added solution is filtered through the virus removal medium exceeds 126% of the filtration rate when the virus solution is filtered through the virus removal medium in the first filtration step, the filtration in step (b) may be performed using a virus-added solution with a relatively low virus concentration, even though the virus removal medium can process an amount of virus greater than that contained in the virus-added solution without virus breakthrough, which could result in an underestimation of the virus clearance performance of the virus removal medium. By setting the filtration rate when the virus-added solution is filtered through the virus removal medium to be between 74% and 126% of the filtration rate when the virus solution is filtered through the virus removal medium in the first filtration step, the virus clearance performance of the virus removal medium can be evaluated to the maximum extent possible without virus breakthrough.

[0111] In one embodiment, in step (b), the entire amount of the virus added solution prepared in step (a) is filtered. By filtering the entire amount of the virus added solution prepared in step (a) in step (b), an amount D of virus that satisfies formula (2) is loaded into the virus removal medium. If only a portion of the virus added solution prepared in step (a) is filtered in step (b), the virus clearance performance of the virus removal medium may not be fully utilized, and the virus clearance performance may be underestimated.

[0112] (Process (c)) In one embodiment, in the second filtration step, steps (a) and (b) are not performed, and step (c) can be performed by filtering the virus-added solution so that the virus loading amount in the virus removal medium does not exceed the maximum virus retention amount in the total membrane area of ​​the virus removal medium, and is at least one-tenth of the maximum virus retention amount in the total membrane area of ​​the virus removal medium (for example, amount D that satisfies formula (3) or (4)), to obtain a filtered solution. In formula (3) or (4), A is the membrane area of ​​the virus removal medium used in filtration. The unit of A is not limited, and m 2 ,cm 2 , and mm 2 However, it must be in the same unit as the unit membrane area used in the first filtration step. C In equation (4), P is the maximum virus retention capacity per unit membrane area of ​​the virus removal medium determined in the first filtration step. C The units of P are Copies and TCID 50 Examples of units of D include the amount of protein (mg or mol), the number of virus particles, or pfu. 50 Examples of the unit of D are the amount of protein (mg or mol), the number of virus particles (units), or pfu. C The units of D are the same as the units of P in formula (4).

[0113] The virus spiked solution used in step (c) differs from the virus spiked solution prepared in step (a) in that the amount of virus contained in the virus spiked solution prepared for filtration is not limited to the value D that satisfies formula (3) or (4) above; however, in other respects, the virus spiked solution may be the same as the virus spiked solution prepared in step (a). In step (c), the virus spiked solution is filtered using a virus removal medium so that an amount D of virus that satisfies formula (3) or formula (4) is loaded onto the virus removal medium. For example, if the prepared virus spiked solution contains an amount of virus that exceeds the value of D that satisfies formula (3) or formula (4), a portion of the prepared virus spiked solution can be subjected to filtration so that an amount D of virus that satisfies formula (3) or formula (4) is loaded onto the virus removal medium. Specifically, for example, the second filtration step can be terminated when an amount D of virus that satisfies formula (3) or formula (4) has been loaded onto the virus removal medium.

[0114] In one embodiment, the second filtration step including steps (a) and (b), or the second filtration step including step (c), uses the same virus and virus removal medium as those used in the first filtration step.

[0115] (Virus concentration measurement process) In the virus concentration measurement step, the virus concentration of the virus-added solution prepared in step (a) of the second filtration step and the virus concentration of the filtered solution after filtration in step (b) of the second filtration step are each measured. The "virus concentration of the virus-added solution" refers to the virus concentration of the virus-added solution before being subjected to filtration in step (b). Furthermore, when step (c) is performed without performing steps (a) and (b) in the second filtration step, the virus concentration of the virus-added solution before filtration and the virus concentration of the filtered solution after filtration are each measured in the virus concentration measurement step.

[0116] In one embodiment, the method for measuring the virus concentration in the virus-added solution and the virus concentration in the filtered solution is appropriately selected depending on the type of virus contained in the virus-added solution. For example, if the virus-added solution does not contain hollow virus particles, a method for quantifying the virus by detecting viral nucleic acid, such as virus quantitative PCR or fluorescent flow cytometry, can be used. Furthermore, if the virus-added solution does not contain hollow virus particles or non-infectious viruses but contains infectious viruses, the TCID 50 Infectivity titer methods that quantify viruses by measuring the infectivity titer of the virus, such as the plaque method or the smear method, can be employed. By using these methods, the virus contained in the virus-spiked solution and the filtered solution are quantified and converted into the amount of virus per unit volume, so that the virus concentration in the virus-spiked solution before filtration and the virus concentration in the filtered solution can be measured.

[0117] In one embodiment, the virus concentration of the virus-added solution and the virus concentration of the filtrate are measured by quantifying the virus using the same method. Specifically, the virus concentration of the virus-added solution and the virus concentration of the filtrate are measured by quantifying the virus using a quantitative PCR method, the virus concentration of the virus-added solution and the virus concentration of the filtrate are measured by quantifying the virus using a fluorescent flow cytometry method, and the virus concentration of the virus in the virus-added solution and the virus concentration of the filtrate are measured by TCID 50 and an embodiment in which the virus concentration in the virus-spiked solution and the virus concentration in the filtered solution are measured by quantifying the virus by the plaque method.

[0118] In one embodiment, the unit volumes of the virus concentration of the virus-added solution and the virus concentration of the filtered solution are not limited, but are usually expressed in mL. Therefore, the units of virus concentration are Copies / mL and TCID 50Examples of units of virus concentration when the virus is quantified by quantitative PCR are Copies / mL, and TCID 50 When the virus is quantified by the method, the unit of virus concentration is TCID 50 When the virus is quantified by the plaque method, the unit of the virus concentration is exemplified by pfu / mL, and when the virus is quantified by particle concentration measurement using a transmission electron microscope, the unit of the virus concentration is exemplified by particles / mL. The virus concentration may be expressed as a logarithmic value, in which case, for example, log 10 [Copies / mL], log 10 [TCID 50 / mL], log 10 [pfu / mL], log 10 [mg / mL]log 10 [mol / mL] or log 10 It is expressed in [pieces / mL].

[0119] (Virus clearance performance determination process) In the virus clearance performance determining step, the virus clearance performance of the virus removal medium is evaluated and determined based on the virus concentration of the virus-added solution and the virus concentration of the filtered solution measured in the virus concentration measuring step.

[0120] The method for evaluating the virus clearance performance of a virus removal medium is not limited to a specific method, but for example, the log reduction value (LRV) calculation method can be used. In the LRV calculation method, the virus concentration before and after filtering a virus-added solution through the virus removal medium is compared, and the log reduction value (LRV) is calculated. LRV can be calculated using the following formula (5): LRV = logarithm of virus concentration before filtration - logarithm of virus concentration after filtration (5)

[0121] In equation (5), the logarithm of the virus concentration before filtration is, for example, log 10The logarithm of the virus concentration after filtration is given as [virus amount before filtration / mL], for example, log 10 It is given as [virus amount after filtration / mL]. When the virus is quantified using the genome copy number, the virus concentration can be expressed in units of, for example, Copies / mL. Therefore, the logarithm of the virus concentration before filtration can be expressed as, for example, log 10 The virus concentration before filtration is given as [virus concentration (copies / mL)], and the logarithm of the virus concentration after filtration is given as, for example, log 10 Given as [virus concentration after filtration (Copies / mL)].

[0122] In one embodiment, the second filtration step, the virus concentration measurement step, and the virus clearance performance determination step can be performed in accordance with ICH Q5A(R2) (Non-Patent Document 1), which is a guideline for virus clearance testing. [Example]

[0123] The present invention will be described in more detail below with reference to examples and comparative examples. Note that the examples shown below are representative examples, and the present invention is not limited to the examples shown below.

[0124] Example 1: Preparation of Minute Virus of Mice (MVM) Solution Human fibroblasts transformed with siamin virus 40 were used as host cells for minute virus of mice (MVM) (VR-1346 (ATCC)). The cells were cultured in DMEM supplemented with 5% fetal bovine serum (FBS) at 37°C in a 5% CO2 atmosphere at a temperature of 75 cm. 2 The cells were subcultured in a tissue culture flask with a bottom area of ​​15 mL (hereinafter simply referred to as "flask"). The siamin virus 40-transformed human fibroblasts were detached from the flask and transferred to a new flask at a density of 1.5 × 10 6The cells were seeded at a density of 15 mL / flask in DMEM medium containing 2.5% FBS and infected with MVM at an MOI of 0.01. After four days, the medium was replaced with serum-free medium, and the cells were cultured for an additional three days. Three days after infection, the culture supernatant containing MVM was collected. After 20 minutes of centrifugation at 3,000 rpm, the supernatant was collected and filtered through a 0.45 μm filter (Nalgene) to obtain a virus culture solution. This procedure was repeated for 72 flasks, yielding a total of 720 mL of virus culture solution.

[0125] 360 mL of this virus culture was ultracentrifuged at 29,400 rpm for 2 hours in an ultracentrifuge (Beckman Coulter Optima L-90K) equipped with a Type 45 Ti rotor to precipitate MVM. After removing the supernatant, the same procedure was repeated on another 360 mL of virus culture. The pellet containing MVM was then resuspended in 36 mL of TE buffer (50 mmol / L Tris-HCl pH 8.0 / 1 mmol / L EDTA) to obtain 36 mL of concentrated MVM in TE buffer. Next, PEG6000 and NaCl were added to the concentrated MVM in TE buffer to final concentrations of 10 w / v% and 500 mmol / L, respectively, and the suspension was left overnight at 4°C. This solution was then centrifuged at 7,800 rpm for 30 minutes in an ultracentrifuge (Beckman Coulter Optima L-90K) equipped with an SW28 rotor to precipitate MVM. After removing the supernatant, the MVM-containing pellet was resuspended in 2 mL of PBS+KM (1x PBS, 2.5 mmol / L KCl, 1 mmol / L MgCl2) to obtain a concentrated MVM-containing solution (hereafter referred to as "MVM-PEG"). The MVM-PEG was purified by iodixanol density gradient ultracentrifugation. Iodixanol / PBS+KM layers were layered in Beckman Coulter UC tubes (13.2 mL, product number 344059) in the following order from the bottom: 55% - 2 mL, 45% - 2 mL, 40% - 2 mL, 35% - 2 mL, 25% - 2 mL, and 15% - 1 mL, with 1 mL of MVM-PEG layered on top. The sample was ultracentrifuged at 35,000 rpm for 18 hours at 18°C ​​in an ultracentrifuge (Beckman Coulter Optima L-90K) equipped with an SW41 rotor.

[0126] Twelve 1 mL fractions were then collected from the top of the tube. The density and MVM nucleic acid copy number of all 12 collected fractions were measured. To measure MVM nucleic acid copy number, free nucleic acids in the sample were removed by nuclease treatment, and the resulting MVM nucleic acid molecules were measured by quantitative real-time PCR. The number of viral nucleic acid molecules was quantified using a nucleic acid of known concentration as a standard. The MVM standard was prepared by cloning 4775 bases of the ATCC VR1346 strain (excluding the repeat sequences) into the pT7Blue-2 T-vector (Novagen, product code 69080), followed by digestion of the plasmid with restriction enzymes SalI-HF and EcoNI (New England BioLabs, product codes R3138S and R0521S). Fraction 10, which represented the peak in MVM nucleic acid copy number, was collected as the viral capsid-containing solution.

[0127] Subsequently, the solvent in fraction 10 was exchanged from iodixanol / PBS+KM to PBS+KM using a Zeba Spin Desalting Column 40K MWCO 10 mL (Thermo Fisher Scientific), and 2.1 mL of MVM viral capsid-containing solution (MVM FC) was obtained.

[0128] Example 2: Determining the variability in virus capture capacity that can be considered within an acceptable margin of error Human immunoglobulin G (IgG) was diluted with water for injection (Otsuka Pharmaceutical) to an antibody concentration of 10 mg / mL, and NaCl was added to a final concentration of 100 mM to prepare a biological substance-containing solution. The MVM FC produced in Example 1 was added to the prepared biological substance-containing solution to prepare a virus-added biological substance-containing solution, and the virus-added biological substance-containing solution was then passed through a virus removal membrane (membrane area 0.001 m) prepared by the method of Japanese Patent No. 4024041. 2 The filtration was performed under dead-end conditions of 78.4 kPa (0.784 bar) and 160 L / m 2The filtrate after passing through the virus removal membrane was collected as multiple fractions, and the fraction in which the virus was first and subsequently continuously detected was collected. 2 The total amount of virus per unit membrane area was taken as the maximum amount of virus retained (virus capture capacity) per unit membrane area of ​​the virus removal membrane. Since the virus was quantified using the genome copy number and the logarithm was used, the unit of virus capture capacity is log 10 [Copies / m 2 The experimental system was repeated eight times under the same conditions, and the virus capture capacity of the virus removal membrane was determined for each run. In addition, three times the standard deviation (3 s.d.) of the virus capture capacity of the virus removal membrane determined for each run was calculated. The results are shown in Table 1. [Table 1]

[0129] The variation in the virus capture capacity of the virus removal membrane is 1.12 log, which is three times the standard deviation (3s.d.). 10 [copies / m 2 ] or less, the variation in virus capture capacity was considered to be within the acceptable error range.

[0130] Example 3: Determination of filtration rates that produce variations in virus capture capacity that are considered within an acceptable error range The virus-added biological substance-containing solution used in Example 2 was passed through the same virus removal membrane (membrane area 0.001 m) as the virus removal membrane used in Example 2. 2 ) and filtration rate (Flux) (L / m 2 / hour (LMH) was changed, and the virus capture capacity (log 10 [Copies / m 2 The filtration rate (Flux) was varied by changing the filtration pressure applied to the virus-added biological material-containing solution. The results are shown in Table 2. [Table 2]

[0131] In Table 2, the filtration rate (Flux) is represented by x(LMH) and the virus capture capacity is represented by y(log 10 [Copies / m 2 ]), it can be seen that the virus capture capacity y of the virus removal membrane increases in proportion to the filtration rate (Flux) x, and the following relational equation (6) is obtained: y=0.078x+6.4 (6)

[0132] In Example 2, the variation in virus capture capacity was considered to be within the allowable error range of 1.12 log 10 [copies / m 2 Substituting [x] for y in equation (6), we obtain the value x = 14.4 (LMH). This value can be regarded as the range of filtration rate (flux) variation that results in a variation in virus capture capacity within the acceptable error range. In other words, if the variation in filtration rate (flux) is 14.4 (LMH) or less, the variation in virus capture capacity will be within the acceptable error range.

[0133] Next, using the filtration rate (flux) at a filtration pressure of 0.8 bar, the standard operating pressure, as a reference, we calculated the filtration rate at which the variation in the virus capture capacity of the virus removal membrane falls within the allowable error range. As shown in Table 2, the filtration rate (flux) at a filtration pressure of 0.8 bar is 55.4 LMH, so the variation in the filtration rate (flux) at which the variation in virus capture capacity falls within the allowable error range is within (14.4 (LMH) / 55.4 (LMH)) × 100 = 26% of the filtration rate (flux) at a filtration pressure of 0.8 bar. Therefore, it was found that if filtration is performed at a filtration rate that is at least -26% of the filtration rate in the first filtration step, i.e., at a rate that is at least 74% of the filtration rate in the first filtration step, the virus capture capacity will fall within the allowable error range of the virus capture capacity calculated in the first filtration step.

[0134] Example 4: First Filtration Step Human immunoglobulin G (IgG) was diluted with water for injection (Otsuka Pharmaceutical) to an antibody concentration of 10 mg / mL, and NaCl was added to a final concentration of 100 mM to prepare a biological substance-containing solution. The MVM FC produced in Example 1 was added to the prepared biological substance-containing solution to prepare a virus-added biological substance-containing solution, and the virus-added biological substance-containing solution was passed through the same virus removal membrane (membrane area 0.001 m) as the virus removal membrane used in Example 2. 2 The filtration conditions were 78.4 kPa (0.784 bar), 160 L / m 2 The filtration rate was 39.8 LMH (hereinafter referred to as "first filtration rate of Example 4"). As in Example 2, an experiment was performed once in which the filtrate was separated into multiple fractions to determine the virus capture capacity, and the virus was quantified using the genome copy number to determine the virus capture capacity (unit membrane area (1.0 m)). 2 The maximum amount of virus retained per 10 12.8 copies / m 2 The logarithm of the virus capture capacity and the filtration rate are shown in Table 3. [Table 3]

[0135] Example 5: Second Filtration Step The same virus removal membrane (membrane area 0.001 m) as that used in Example 2 was used. 2 The volume of the virus-spiked biological material-containing solution to be filtered was 160 mL (160 L / m 2 ) was set.

[0136] To determine the virus concentration of the virus-added biological substance-containing solution to be filtered, add 0.001 m, which is the membrane area of ​​the virus removal membrane, to A in the above formula (3). 2 and C is the virus removal membrane 1 m obtained in Example 4. 2 Substituting 12.8, the logarithm of the virus capture capacity per unit volume, to calculate the range of D (copies), we obtain (0.001 x 10 11.8 )≦ D ≦(0.001×10 12.8 )

[0137] The maximum value of the viral load D calculated by formula (2) (0.001 × 10 12.8 To convert the amount of virus per mL (Feed titer), use {0.001 × 10 12.8 (copies)} / {160(L / m 2 )×0.001(m 2 The logarithm of the calculated value was 7.6 (log 10 The minimum value of the viral load D calculated by formula (3) was 0.001 × 10 11.8 Similarly, the logarithm of the viral load per mL for each virus (copies) was calculated to be 6.6 (log 10 [copies / mL]. Therefore, the volume of the virus-spiked biological material-containing solution to be filtered was 160 mL (160 L / m 2 ), the concentration range of minute virus of mice (MVM) that should be contained in the virus-added biological substance-containing solution calculated from the range of D that satisfies the above formula (3) is 6.6 to 7.6 (log 10 [copies / mL]).

[0138] Human immunoglobulin G (IgG) was diluted with water for injection (Otsuka Pharmaceutical) to an antibody concentration of 10 mg / mL, and NaCl was added to a final concentration of 100 mM to prepare a biological substance-containing solution. MVM FC produced in Example 1 was added to the prepared biological substance-containing solution to prepare a virus-added biological substance-containing solution. The virus concentration of the virus-added biological substance-containing solution was within the concentration range calculated above (6.6 to 7.6 (log 10 [copies / mL])) is within 7.2(log 10 [copies / mL]).

[0139] The virus concentration was 7.2 (log 10 The virus-spiked biological material-containing solution ([copies / mL]) was applied to the membrane with an area of ​​0.001 m prepared above. 2 The filtration volume was 160 L / m as determined above. 2The filtration rate was 32.4 LMH, which is 81% of the first filtration rate in Example 4. As a result of the filtration, the amount of virus contained in the filtrate was 2.6 (log 10 [copies / mL] or less.

[0140] Furthermore, the minute virus of mice (MVM) contained in the virus-spiked biological material-containing solution before and after filtration was quantified using the genome copy number, and the log reduction value (LRV) was calculated using the following formula. The LRV was 4.6 or more: LRV=log 10 [Number of virus particles before filtration (copies / mL)]-log 10 [Number of virus particles (copies) / mL after filtration] = 7.2 - ≦2.6 = ≧4.6

[0141] The filtration conditions and results in Example 5 are shown in Table 4. [Table 4]

[0142] (Comparative Example 1: Second Filtration Step) The same virus removal membrane (membrane area 0.001 m) as that used in Example 2 was used. 2 The volume of the virus-spiked biological material-containing solution to be filtered was 122 mL (122 L / m 2 ) was set.

[0143] The maximum value of D in formula (3) calculated in Example 5 (0.001 × 10 12.8 To convert the amount of virus per mL, we use {0.001 × 10 12.8 (copies)} / {122(L / m 2 )×0.001(m 2 ) × 1000} was calculated. The logarithm of the calculated value was 7.7(log 10 The minimum value of the viral load D calculated by formula (3) was 0.001 × 10 11.8Similarly, the logarithm of the viral load per mL for each virus was calculated as 6.7(log 10 [copies / mL]. Therefore, the volume of the virus-spiked biological material-containing solution to be filtered was 122 mL (122 L / m 2 ), the concentration range of minute virus of mice (MVM) that should be contained in the virus-added biological substance-containing solution calculated from the range of D that satisfies the above formula (3) is 6.7 to 7.7 (log 10 [copies / mL]).

[0144] Human immunoglobulin G (IgG) was diluted with water for injection (Otsuka Pharmaceutical) to an antibody concentration of 10 mg / mL, and NaCl was added to a final concentration of 100 mM to prepare a biological substance-containing solution. MVM FC produced in Example 1 was added to the prepared biological substance-containing solution to prepare a virus-added biological substance-containing solution. The virus concentration of the virus-added biological substance-containing solution was within the concentration range calculated above (6.7 to 7.7 (log 10 [copies / mL])) is within 7.7(log 10 [copies / mL]).

[0145] The virus concentration produced was 7.7 (log 10 The virus-spiked biological material-containing solution ([copies / mL]) was applied to the membrane with an area of ​​0.001 m prepared above. 2 The filtration volume was 122 L / m as determined above. 2 The filtration rate was 23.1 LMH, which is 58% of the first filtration rate in Example 4. As a result of the filtration, a virus breakthrough occurred, and 3.3 (log 10 [copies / mL]) of virus was detected.

[0146] Furthermore, minute virus of mice (MVM) contained in the virus-spiked biological material-containing solution before and after filtration was quantified using the genome copy number, and the log reduction value (LRV) was calculated using the following formula. The LRV was 4.4: LRV=log 10[Number of virus particles before filtration (copies / mL)]-log 10 [Number of virus particles (copies) / mL after filtration] = 7.7 - 3.3 = 4.4

[0147] The filtration conditions and results in Comparative Example 1 are shown in Table 4.

[0148] (Reference: Comparative Example 2: Second Filtration Step) The same virus removal membrane (membrane area 0.001 m) as that used in Example 2 was used. 2 ), the concentration of minute virus of mice (MVM) was 6.0 (log 10 160L / m of virus-spiked biological material containing solution ([copies / mL]) 2 When filtering under these filtration conditions, the amount of virus loaded onto the virus removal membrane is within the range of the value of D in formula (3) calculated in Example 5 (6.6 to 7.6 (log 10 In this case, if the filtration rate is set to 74% or more of the first filtration rate in Example 4, no virus will be detected in the filtrate (viral breakthrough will not occur), but the LRV will be 3.4, which is expected to be less than 4.

[0149] Example 6: Preparation of bovine viral diarrhea virus (BVDV) solution Bovine kidney-derived cells (MDBK) were used as host cells for bovine viral diarrhea virus (BVDV). The cells were cultured in DMEM supplemented with 10% horse serum at 37°C in a 5% CO2 environment for 75 cm 2 Subculture was performed using a tissue culture flask with a bottom area of ​​15 mL (hereinafter simply referred to as "flask"). The MDBK was removed from the flask, and 6 × 10 cells were transferred to a new flask. 6 The cells were seeded at a density of 15 ml / flask in DMEM medium containing 10% FBS and infected with BVDV at an MOI of 0.03. Two days after infection, the culture supernatant containing BVDV was collected. After centrifugation at 3,000 rpm for 20 minutes, the supernatant was collected and filtered through a 0.45 μm filter (Nalgene) to obtain a viral culture solution.

[0150] Example 7: First Filtration Step The BVDV virus culture solution prepared in Example 6 was added to DMEM to prepare a virus-added solution, and the virus-added solution was passed through a virus removal membrane (0.001 mm) prepared by the method described in Japanese Patent Application Laid-Open No. 1-148305. 2 The filtration conditions were 40 kPa (0.4 bar), 160 L / m 2 The filtration rate was 118 LMH (hereinafter referred to as "first filtration rate of Example 7"). As in Example 2, an experiment was conducted to measure the virus capture capacity by collecting the filtrate into multiple fractions, and TCID 50 The virus capture capacity (unit membrane area (1.0 m)) was calculated using the method 2 The maximum amount of virus retained per 10 11.2 TCID 50 / m 2 The logarithm of the virus capture capacity and the filtration rate are shown in Table 5. [Table 5]

[0151] Example 8: Second Filtration Step The same virus removal membrane (membrane area 0.001 m) as that used in Example 7 was used. 2 The volume of the virus-spiked biological material-containing solution to be filtered was 160 mL (160 L / m 2 ) was set.

[0152] To determine the virus concentration of the virus-spiked solution to be filtered, add 0.001 m, the membrane area of ​​the virus removal membrane, to A in the above formula (3). 2 and C is the virus removal membrane 1 m obtained in Example 7. 2 Substituting 11.2, the logarithm of the virus capture capacity per 1000 μg / mL, gives the value of D (TCID 50 ) range is calculated as (0.001 × 10 10.2 )≦ D ≦(0.001×10 11.2 )

[0153] The maximum value of the viral load D calculated by formula (3) (0.001 × 10 11.2 (TCID 50 )) to the amount of virus per mL, {0.001 × 10 11.2 (TCID 50 )} / {160(L / m 2 )×0.001(m 2 ) × 1000} was calculated. The logarithm of the calculated value was 6.0 (log 10 [TCID 50 The minimum value of the viral load D calculated by formula (3) was 0.001 × 10 10.2 (TCID 50 Similarly, the logarithm of the viral load per mL for 10 [TCID 50 Therefore, the volume of virus-spiked solution to be filtered was 160 mL (160 L / m 2 ), the range of the concentration of bovine viral diarrhea virus (BVDV) that should be contained in the virus-spiked solution calculated from the range of D that satisfies the above formula (2) is 5.0 to 6.0 (log 10 [TCID 50 / mL).

[0154] The BVDV virus culture solution prepared in Example 6 was added to DMEM to prepare a virus-added solution. The virus concentration in the virus-added solution was within the concentration range calculated above (5.0 to 6.0 (log 10 [TCID 50 / mL])) 10 [TCID 50 / mL).

[0155] The virus concentration produced was 5.9 (log 10 [TCID 50 The virus-spiked solution ([[ / mL])) was applied to the membrane with an area of ​​0.001 m prepared above. 2 The filtration volume was 160 L / m as determined above. 2The filtration rate was 89.5 LMH, which is 76.1% of the first filtration rate in Example 7. As a result of the filtration, the amount of virus contained in the filtrate was found to be 0.43 (log 10 [TCID 50 / mL] or less.

[0156] In addition, the TCID of bovine viral diarrhea virus (BVDV) contained in the virus-spiked solution before and after filtration was 50 The logarithmic reduction value (LRV) was calculated using the following formula, and the LRV was found to be 5.5 or more: LRV=log 10 [Virus infectivity before filtration (TCID 50 ) / mL)]-log 10 [Virus infectivity after filtration (TCID 50 ) / mL]=5.9 - ≦0.43 = ≧5.5

[0157] The filtration conditions and results in Example 8 are shown in Table 6. [Table 6]

[0158] (Reference: Comparative Example 3: Second Filtration Step) The same virus removal membrane (membrane area 0.001 m) as that used in Example 7 was used. 2 ), the concentration of bovine viral diarrhea virus (BVDV) was 4.0 (log 10 [TCID 50 The virus spike solution was 160 L / m 2 When filtering under these filtration conditions, the amount of virus loaded onto the virus removal membrane is within the range of the value of D in formula (3) calculated in Example 8 (5.0 to 6.0 (log 10 [TCID 50 In this case, if the filtration rate is set to 74% or more of the first filtration rate in Example 7, no virus will be detected in the filtrate (viral breakthrough will not occur), but the LRV will be 3.6, which is expected to be less than 4.

[0159] Thus, it was found that a high LRV can be obtained without virus breakthrough when the amount of virus loaded into the virus removal medium is within the range of D in equation (3) and the filtration rate through the virus removal medium is 74% or more of the first filtration rate.

Claims

1. 1. A method for determining the viral clearance performance of a viral removal medium, comprising: a first filtration step of filtering a virus solution containing viruses through the virus removal medium at a first filtration speed to determine the maximum virus retention amount per unit membrane area of ​​the virus removal medium; a second filtration step of filtering a virus-added solution containing the virus in an amount that does not exceed the maximum virus retention capacity of the entire membrane area of ​​the virus removal medium and is at least one-tenth of the maximum virus retention capacity of the entire membrane area of ​​the virus removal medium at a second filtration speed, to obtain a filtered solution; measuring the virus concentration of the virus-added solution before filtration in the second filtration step and the virus concentration of the filtered solution after filtration in the second filtration step; and determining the virus clearance performance of the virus removal medium based on the difference between the measured virus concentration of the virus-added solution before filtration in the second filtration step and the virus concentration of the filtered solution after filtration in the second filtration step; A method comprising:

2. The maximum virus retention amount per unit membrane area of ​​the virus removal medium determined in the first filtration step is 10 C and In the second filtration step, the amount D of the virus contained in the virus added solution satisfies formula (1). A×10 C-1 ≦ D ≦ A×10 C (1) Here, A is the total membrane area of ​​the virus removal medium used in the second filtration step. The method of claim 1.

3. the maximum virus retention amount per unit membrane area of ​​the virus removal medium determined in the first filtration step is P; In the second filtration step, the amount D of the virus contained in the virus added solution satisfies formula (2). A×P×1 / 10 ≦ D ≦ A×P (2) Here, A is the total membrane area of ​​the virus removal medium used in the second filtration step. The method of claim 1.

4. 2. The method of claim 1, wherein the second filtration rate is at least 74% of the first filtration rate.

5. 2. The method of claim 1, wherein the second filtration rate is in the range of 74% to 126% of the first filtration rate.

6. 10. The method of claim 1, wherein the virus removal medium comprises regenerated cellulose.

7. 2. The method of claim 1, wherein the virus solution and the virus spiked solution are purified using an ultracentrifuge.

8. 2. The method of claim 1, wherein the virus solution and the virus spiked solution are purified by density gradient ultracentrifugation.

9. 2. The method of claim 1, wherein the virus solution and the virus spiked solution are purified by iodixanol density gradient ultracentrifugation.

10. The method of claim 1 , wherein the virus does not include hollow viruses.

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

12. 2. The method of claim 1, wherein the virus is porcine parvovirus (PPV), minute virus of mice (MVM), or bovine viral diarrhea virus (BVDV).

13. The method of claim 1, wherein the average particle size of the virus is 18 nm or more and 120 nm or less.

14. The method according to claim 1, wherein the virus removal medium is a flat membrane or a hollow fiber membrane.

15. The method of claim 1 , wherein the virus removal medium is a hollow fiber membrane.

16. The method of claim 1, wherein the virus removal medium has an average pore size of 15 nm or more and 75 nm or less.

17. The method according to claim 1, wherein the virus removal medium is a flat membrane or hollow fiber membrane having a structure in which the porosity decreases from the first surface toward the interior, and after passing through at least one minimum porosity portion, the porosity increases again at the second surface.

18. In the formula (1), D and 10 C The unit is TCID 50 , pfu, genome copy number (copies), protein amount (mg or mol), or virus particle number (pieces).

19. The method according to claim 1, wherein the virus concentration is measured by an infectivity titer method, a quantitative PCR (qPCR) method, an enzyme-linked immunosorbent assay (ELISA), an HA assay, or particle concentration measurement using a transmission electron microscope.

20. 2. The method of claim 1, wherein the viral clearance performance is a Log Reduction Value (LRV).

21. 2. The method according to claim 1, wherein the first filtration step comprises a step of collecting a fixed volume of the filtered solution obtained by filtering the virus solution, and measuring the virus concentration of each of the collected aliquots of the filtered solution.

22. The method of claim 1 , wherein the first filtration is performed multiple times.

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