Viral Clearance Testing Methodology

Abstract

A viral clearance test method that comprises: supplying a protein solution into a first channel 10, in which a protein purification part 11 is provided upstream and a virus removal filter 12 is provided downstream, and flowing the protein solution at a first constant speed to the protein purification part 11; supplying a virus solution at a second constant speed to a second channel 20, which is connected between the protein purification part 11 and the virus removal filter 12 of the first channel, and mixing the purified protein solution with the virus solution in the first channel 10; flowing the solution mixture of the protein solution with the virus solution at a third constant speed through the virus removal filter 12; and measuring the virus contained in the permeate of the solution mixture having passed through the virus removal filter 12.

Description

[Technical field]

[0001] The present invention relates to a method for viral clearance testing. [Background technology]

[0002] Biological substance-containing preparations include plasma fraction preparations purified from blood and biological preparations produced by biotechnology. These biological substance-containing preparations carry the risk of viral contamination. In general, biological substance-containing preparations must be guaranteed safe against viruses. For this reason, in the manufacture of biological substance-containing preparations, a process must be included to sufficiently remove or inactivate viruses that are contained or may be contained in the biological substance-containing preparation, i.e., a virus removal process (see, for example, Non-Patent Document 1). Testing the virus removal or inactivation ability in the virus removal process is called a virus clearance test.

[0003] Among the manufacturing processes for biological substance-containing preparations, the processes that are said to be robust virus removal processes are low pH treatment and treatment with a virus removal medium (see, for example, Non-Patent Document 2). Treatment with a virus removal medium such as a virus removal membrane is advantageous in that it can be carried out on all viruses, regardless of whether they have an envelope or not.

[0004] Typically, in a virus clearance test in the manufacturing process of a biological substance-containing preparation, the virus concentration is measured before and after any manufacturing step, and the virus clearance capability is evaluated, for example, by calculating the logarithmic reduction ratio (LRV) (see, for example, Non-Patent Document 1). For example, the virus clearance capability of the virus removal step is evaluated by mixing a biological substance-containing solution with a virus solution and measuring the virus concentration in the mixed solution before and after contacting the mixed solution with a virus removal medium.

[0005] Regarding the type of virus to be used in evaluating the virus clearance ability, the ICH (International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use) guidelines are set out in q5a, and "animal parvovirus" is listed as an example of a useful non-specific model virus (see, for example, Non-Patent Document 1). Among them, minute virus of mice (MVM) and porcine parvovirus (PPV) are frequently used in many virus clearance studies that cite ICH-q5a.

[0006] Methods for measuring virus concentration mainly include infectivity titer measurement methods, including end-point assays and local lesion counting assays, and quantitative PCR (Quantitative Polymerase Chain Reaction: qPCR) (see, for example, Non-Patent Documents 2 and 3). [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. 2014 / 080676A1 [Patent Document 2] US Patent Application Publication No. 2012 / 0088228 [Patent Document 3] Patent No. 4024041 [Non-patent literature]

[0008] [Non-Patent Document 1] Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Human or Animal Origin Q5A(R1) [Non-Patent Document 2] Note For Guidance On Virus Validation Studies:The Design, Contribution And Interpretation Of Studies Validating The Inactivation and Removal Of Viruses [Non-Patent Document 3] Guidelines for ensuring the safety of plasma derivatives against viruses (Notification No. 1047 from the Director-General of the Pharmaceutical and Medical Safety Bureau, issued on August 30, 1999) [Non-Patent Document 4] Raphael Wolfisberg et al., Journal of Virology (2016) [Non-Patent Document 5] Beatriz Maroto et al., JOURNAL OF VIROLOGY (2004) [Non-Patent Document 6] Special Topics in Experimental Virus Research, Maruzen Publishing, National Institute of Health Alumni Association, pp22-23 [Non-Patent Document 7] V. Hutornojs at. al.,(2012) Env, Exp. Biol. 10: pp.117-123 [Non-Patent Document 8] Anthony M. D'Abramo Jr. et al., 2005 Virology [Non-Patent Document 9] Joshua C Grieger et al., Molecular Therapy 2016 [Non-Patent Document 10] Pavel Plevka et al., JOURNAL OF VIROLOGY (2011) [Non-Patent Document 11] PETER TATTERSALL et al., JOURNAL OF VIROLOGY (1976) Summary of the Invention [Problem to be solved by the invention]

[0009] The present inventors have considered that a method for virus clearance testing applicable to a continuous process would be beneficial. Thus, an object of the present invention is to provide a method for virus clearance testing applicable to a continuous process. [Means for solving the problem]

[0010] [1] A method for virus clearance testing comprising: (a) supplying a protein solution to a first flow path having a protein purification section provided upstream and a virus removal filter provided downstream, and flowing the protein solution through the protein purification section at a first constant speed; (b) supplying a virus solution at a second constant speed to a second flow path connected between the protein purification section of the first flow path and the virus removal filter, and mixing the purified protein solution with the virus solution in the first flow path; (c) flowing a mixture of the protein solution and the virus solution through the virus removal filter at a third constant speed; and (d) measuring viruses contained in the permeate of the mixture that has passed through the virus removal filter.

[0011] [2] The method according to [1], wherein the first flow path is provided with a first pump for flowing the protein solution through the protein purification section at a first constant velocity.

[0012] [3] The method according to [1] or [2], wherein the second flow path is provided with a second pump for flowing the virus solution at a second constant velocity.

[0013] [4] The method according to any one of [1] to [3], wherein the protein solution is continuously passed through a protein purification section.

[0014] [5] The method according to any one of [1] to [4], wherein the virus solution is continuously flowed into the second flow path.

[0015] [6] The method according to any of [1] to [5], wherein the mixture is continuously passed through a virus removal filter.

[0016] [7] The method according to any of [1] to [6], wherein a is the first constant velocity, b is the second constant velocity, x is the virus concentration in the mixed liquid, and y is the virus concentration in the virus solution, and a, b, x, and y satisfy the following formula (1). x / y=b / (a+b) (1)

[0017] [8] The method according to any of [1] to [7], wherein the ratio of the second constant velocity to the sum of the first constant velocity and the second constant velocity is 0.1% or more and 20% or less.

[0018] [9] The infectious titer of the virus in the mixture (Log 10 TCID 50 The method according to any one of [1] to [8], wherein (unit / mL) is 2 or more and 10 or less.

[0019]

[10] The membrane area of ​​the virus removal filter is 0.0001 m 2 More than 4m 2 A method according to any one of [1] to [9],

[0020]

[11] The method according to any of [1] to

[10] , wherein the flux of the permeate in the virus removal filter is 0.1 LMH or more and 500 LMH or less.

[0021]

[12] The membrane area of ​​the virus removal filter is C (m 2 ), the minimum value of b / (a+b) is D min , the maximum value of b / (a+b) is D max , the minimum flux of the permeate through the virus removal filter is F min (LMH), the maximum flux of the permeate through the virus removal filter is F max As (LMH), Minimum value of the first uniform speed a min (mL / min) is given by the following formula (2), a min =(1-D max )(1000 / 60)×F min ×C (2) Maximum value of first uniform velocity a max(mL / min) is given by the following formula (3), a max =(1-D min )(1000 / 60)×F max ×C (3) Minimum value of the second uniform velocity b min (mL / min) is given by the following formula (4), b min =D min (1000 / 60) x F min ×C (4) Maximum value of second uniform velocity b max (mL / min) is given by the following formula (5): b max =D max (1000 / 60) x F max ×C (5) The method described in [7].

[0022]

[13] The method according to any of [1] to

[12] , further comprising: after stopping the supply of the protein solution to the first flow path, supplying a cleaning solution to the first flow path and flowing the cleaning solution through the protein purification section and the virus removal filter; and measuring viruses contained in the permeate of the cleaning solution that has passed through the virus removal filter.

[0023]

[14] The method according to

[13] , wherein the time from stopping the supply of the protein solution to the first flow path to supplying the cleaning solution to the first flow path is 0 minutes or more and 24 hours or less.

[0024]

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

[14] , wherein the virus solution contains protein.

[0025]

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

[15] , wherein the virus solution contains the same proteins as the protein solution.

[0026]

[17] The method according to

[15] or

[16] , wherein the concentration of the protein in the virus solution is the same as the concentration of the protein in the protein solution.

[0027]

[18] The method according to any of [1] to

[17] , further comprising comparing the amount of virus contained in the mixed liquid before passing through the virus removal filter with the amount of virus contained in the permeate of the mixed liquid that has passed through the virus removal filter.

[0028]

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

[18] , wherein the protein purification unit has the ability to remove viruses.

[0029]

[20] The method according to

[19] , wherein the log reduction value (LRV) in the protein purification section 11 is 0 or more and 7 or less. Effect of the Invention

[0030] According to the present invention, it is possible to provide a virus clearance test method that is applicable to a continuous process. [Brief description of the drawings]

[0031] [Figure 1] FIG. 1 is a schematic diagram of a protein purification system according to an embodiment. [Diagram 2] 1 is a table showing purification conditions for Examples 1 to 6. [Diagram 3] 1 is a table showing the purification results for Examples 1 to 6. [Figure 4] FIG. 1 is a schematic diagram of a protein purification system according to Comparative Example 1. [Diagram 5] 1 is a table showing purification conditions according to Comparative Examples 1 to 3. [Figure 6] 1 is a table showing the purification results according to Comparative Examples 1 to 3. [Figure 7] FIG. 13 is a schematic diagram of a protein purification system according to Comparative Example 4. [Figure 8] 1 is a table showing purification conditions according to Comparative Examples 4 to 9. [Figure 9] 1 is a table showing the purification results according to Comparative Examples 4 to 9. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] The following describes the form for carrying out the present invention (hereinafter, sometimes referred to as "embodiment"). The present invention is not limited to the following embodiment, and can be practiced in various modifications within the scope of the gist of the invention. Furthermore, the following embodiment is merely an example of a method for embodying the technical idea of ​​the present invention, and the present invention is not limited to these examples.

[0033] 1, a protein purification system according to an embodiment includes a first flow path 10 through which a protein solution flows, a protein purification section 11 provided upstream of the first flow path 10, a virus removal filter 12 provided downstream of the first flow path 10, and a second flow path 20 connected to the first flow path 10 between the protein purification section 11 and the virus removal filter 12. A first pump 13 for flowing the protein solution through the protein purification section 11 at a first constant speed may be provided in the first flow path 10. A second pump 21 for flowing the virus solution at a second constant speed may be provided in the second flow path 20.

[0034] The method of the virus clearance test according to the embodiment is carried out, for example, using the protein purification system shown in Fig. 1. The method of the virus clearance test according to the embodiment includes: (a) supplying a protein solution to a first flow path 10 having a protein purification section 11 provided upstream and a virus removal filter 12 provided downstream, and flowing the protein solution through the protein purification section 11 at a first constant speed; (b) supplying a virus solution at a second constant speed to a second flow path 20 connected between the protein purification section 11 and the virus removal filter 12 of the first flow path 10, and mixing the purified protein solution with the virus solution in the first flow path 10; (c) flowing a mixture of the protein solution and the virus solution through the virus removal filter 12 at a third constant speed; and (d) measuring viruses contained in the permeate of the mixture that has permeated the virus removal filter 12.

[0035] The protein solution flowed into the first flow path 10 contains a protein. The protein solution preferably does not contain a virus. The protein solution is flowed into the first flow path 10 from, for example, a protein solution tank 41.

[0036] The first pump 13 is provided, for example, but not limited to, upstream of the protein purification section 11 of the first flow path 10. A volumetric pump can be used as the first pump 13, but is not limited to this. An example of the volumetric pump includes, but is not limited to, a peristaltic pump. The first pump 13 continuously flows the protein solution into the protein purification section 11 at a first constant speed.

[0037] An example of a protein is an antibody. An antibody, as generally defined in biochemistry, is a glycoprotein molecule (also called a gamma globulin or immunoglobulin) produced by B lymphocytes as a defense mechanism against infection in vertebrates. For example, an antibody may be used as a human pharmaceutical and have a structure substantially identical to that of an antibody present in the human body to which it is administered.

[0038] The antibody may be a human antibody, or may be an antibody derived from a non-human mammal such as a cow or a mouse. Alternatively, the antibody may be a chimeric antibody with human IgG, or a humanized antibody. A chimeric antibody with human IgG is an antibody in which the variable region is derived from a non-human organism such as a mouse, but the other constant regions are replaced with immunoglobulins derived from humans. A humanized antibody is an antibody in which the complementarity-determining region (CDR) of the variable region is derived from a non-human organism, but the other framework regions (FR) are derived from humans. The immunogenicity of a humanized antibody is further reduced compared to a chimeric antibody.

[0039] The class (isotype) and subclass of an antibody are not limited. For example, antibodies are classified into five classes, IgG, IgA, IgM, IgD, and IgE, depending on the structure of the constant region. However, an antibody may be any of the five classes. In addition, in human antibodies, IgG has four subclasses, IgG1 to IgG4, and IgA has two subclasses, IgA1 and IgA2. However, an antibody may be any of the subclasses. In addition, antibody-related proteins such as Fc fusion proteins in which a protein is bound to the Fc region may also be included in antibodies.

[0040] Antibodies can be classified according to their origin. However, the antibody may be any of natural human antibodies, recombinant human antibodies produced by gene recombination technology, monoclonal antibodies, and polyclonal antibodies. From the viewpoint of the demand and importance of antibody drugs, human IgG is preferred as the antibody, but is not limited thereto.

[0041] The protein purification unit 11 removes impurities contained in the protein and purifies the protein contained in the protein solution. The protein purification unit 11 includes, for example, a chromatography column.

[0042] The chromatography column may be, for example, a cation exchange chromatography column, in which protein aggregates such as antibody multimers are adsorbed onto a cation exchange carrier as impurities, and proteins such as antibody monomers permeate the cation exchange chromatography column.

[0043] The cation exchange carrier has a cation exchange group. The cation exchange group may be a strong cation exchange group, a weak cation exchange group, or both.

[0044] Generally, a strong cation exchange group is charged in the pH range of an antibody solution, and therefore the charge amount is constant. Therefore, if a cation exchange carrier has a strong cation exchange group, a charge amount of at least a certain amount is always guaranteed. Therefore, if a cation exchange carrier has a strong cation exchange group, the change in charge amount with respect to pH is suppressed, and the reproducibility of purification characteristics can be improved. An example of a strong cation exchange group is a sulfonic acid group.

[0045] The charge amount of the weak cation exchange group can be changed by changing the pH of the mobile phase. Therefore, the charge density of the cation exchange carrier can be adjusted by changing the pH of the mobile phase. Therefore, any impurity can be removed by adjusting the pH according to the characteristics of the impurity to be removed. Examples of weak cation exchange groups include carboxyl groups, phosphonic acid groups, and phosphate groups.

[0046] Examples of the shape of the cation exchange support include, but are not limited to, membranes, beads, and monoliths.

[0047] Examples of membrane-type cation exchange carriers include, but are not limited to, Mustang™ S (Pall Corporation), Sartobind® S (Sartorius Stedim Biotech), and Natrix HD-Sb and Natrix HD-C (Natrix Separations).

[0048] Examples of beaded cation exchange carriers include SP Sepharose™ Fast Flow, High Performance, XL, Capto™ S (GE Healthcare), Fractogel™ COO - , SO3 -, SE Highcap, Eshumuno® S, CPX (Merck Millipore Corporation), POROS® XS, HS (ThermoFisher), Nuvia™ S, HR-S, UNOsphere™ S, Rapid S, Macro-Prep® High S, CM, 25 S (Bio-Rad), and Cellufine® Max CM, Max S (JNC), Cellufine® DexS-HbP (JNC).

[0049] Examples of monolithic cation exchange carriers include, but are not limited to, CIM (registered trademark) SO3 (BIA Separations).

[0050] The chromatography column may be, for example, an anion exchange chromatography column, in which impurities with low isoelectric points, such as host cell-derived proteins (HCPs), nucleic acids, and viruses, are adsorbed to an anion exchange carrier, and proteins, such as antibody monomers, permeate the anion exchange chromatography column.

[0051] The anion exchange carrier has an anion exchange group. The anion exchange group may be a strong anion exchange group, a weak anion exchange group, or both.

[0052] Examples of strong anion exchange groups include quaternary ammonium having a trimethylamino group, a triethylamino group, and the like.

[0053] Examples of weak anion exchange groups include, but are not limited to, tertiary amines.Tertiary amines having two or more alkyl groups with a carbon number of two or more can have moderate hydrophobicity.Examples of tertiary amines include diethylamino, dipropylamino, diisopropylamino, and dibutylamino.

[0054] Examples of the shape of the anion exchange support include, but are not limited to, membranes, beads, and monoliths.

[0055] Examples of membrane-type anion exchange carriers include, but are not limited to, Chromasorb™ (Merck Millipore Corporation), Mustang™ Q (Pall Corporation), Sarotibind™ Q, STIC™ PA (Sartorius Stedim Biotech), NatriFlo™ HD-Q (Natrix Separations), and QyuSpeed™ D (Asahi Kasei Medical Co., Ltd.).

[0056] Examples of beaded anion exchange supports include, but are not limited to, Q Sepharose™ Fast Flow, High Performance, XL, QAE Sephadex™ (GE Healthcare), Fractogel™ TMAE, TMAE Highcap, DMAE, DEAE, Eshmuno™ Q (Merck Millipore Corporation), POROS™ XQ, HQ, D, PI (ThermoFisher), DEAE-Cellulose (Sigma-Aldrich), Nuvia™ Q, UNOsphere™ Q, Macro-Prep™ High Q, DEAE, 25 Q (Bio-Rad), CaptoQ (GE Healthcare Japan, Ltd.), Cellufine™ Max DEAE, and Max Q (JNC).

[0057] Examples of monolithic anion exchange supports include, but are not limited to, CIM® QA, DEAE, and EDA (BIA Separations).

[0058] The chromatography column may be, for example, a mixed-mode chromatography column. In the mixed-mode chromatography column, reverse phase chromatography and ion exchange chromatography are combined to purify a protein solution. An example of a carrier used in mixed-mode chromatography is Cellufine MAX IB (JNC).

[0059] Protein purification unit 11 may or may not have the ability to remove viruses. The log reduction value (LRV) in protein purification unit 11 may be, for example, 0 or more and 7 or less. The lower limit of the LRV in protein purification unit 11 may be 0 or more, 1 or more, 2 or more, 3 or more, 4 or more, or 5 or more. The upper limit of the LRV in protein purification unit 11 may be 7 or less, or 6 or less.

[0060] A protein concentration meter 31 that measures the protein concentration of the protein solution that has passed through protein purification unit 11 may be provided downstream of protein purification unit 11 in first flow path 10. Protein concentration meter 31 measures the protein concentration of the protein solution that has passed through protein purification unit 11 by, for example, ultraviolet absorption method.

[0061] A conductivity meter 32 that measures the conductivity of the protein solution that has permeated through protein purification unit 11 may be provided downstream of protein purification unit 11 in first flow path 10. Conductivity meter 32 measures the conductivity of the protein solution that has permeated through protein purification unit 11 by, for example, an AC two-electrode method or an electromagnetic induction method.

[0062] Downstream of protein purification section 11 in first flow path 10 may be provided a pH meter for measuring the pH of the protein solution that has permeated through protein purification section 11, a thermometer for measuring the temperature, and a pressure gauge for measuring the pressure.

[0063] The virus solution that is caused to flow into the second flow path 20 contains viruses. The virus solution is caused to flow into the second flow path 20 from a virus solution tank 42, for example.

[0064] The second pump 21 may be, but is not limited to, a volumetric pump. An example of a volumetric pump is, but is not limited to, a peristaltic pump. The second pump 21 continuously flows the virus solution into the second flow path 20 at a second constant speed. Since the second flow path 20 is connected to the first flow path 10, the protein solution and the virus solution are mixed downstream of the first flow path 10 from the connection point of the second flow path 20 and the first flow path 10 to form a mixed solution. An in-line mixer 33 may be provided downstream of the first flow path 10 from the connection point of the second flow path 20 and the first flow path 10. The in-line mixer 33 promotes mixing of the protein solution and the virus solution.

[0065] The virus may be an infectious virus. The virus may be a naturally occurring virus. A naturally occurring virus includes a virus obtained by culturing a host cell infected with the virus in a medium, and a virus obtained by transfecting a cell with a viral nucleic acid and culturing the cell.

[0066] Examples of viruses include, but are not limited to, Minute virus of mice (MVM), Porcine parvovirus (PPV), ReoVirus Type 3, PolioVirus, PseudorabiesVirus, Human HerpesVirus 1, Xenotropic murine leukemia virus (X-MuLV), and Bovine Viral Diarrhea Virus.

[0067] For example, the virus solution supplied to the second flow path 20 contains the same protein as the protein solution supplied to the first flow path 10. Also, for example, the protein concentration in the virus solution supplied to the second flow path 20 is the same as the protein concentration in the protein solution supplied to the first flow path 10. As a result, the protein concentration in the mixture of the protein solution and the virus solution becomes the same as the protein concentration in the protein solution supplied to the first flow path 10.

[0068] The virus infectivity value (Log 10 TCID 50 (unit / mL)) is, for example, 2 or more, 3 or more, or 4 or more. 10 TCID 50 (unit / mL) is, for example, 10 or less, 9 or less, 8 or less, or 7 or less.

[0069] The first constant velocity, the second constant velocity, the supply amount of the protein solution, the supply amount of the virus solution, and the virus concentration in the virus solution may be adjusted so that a, b, x, and y satisfy the following formula (6), where a is the first constant velocity, b is the second constant velocity, x is the virus concentration in the mixed liquid, and y is the virus concentration in the virus solution. x / y=b / (a+b) (6)

[0070] The ratio of the second constant velocity to the sum of the first constant velocity and the second constant velocity is, for example, 0.1% or more, 0.5% or more, 1.0% or more, 1.5% or more, 2.0% or more, or 3.0% or more. The ratio of the second constant velocity to the sum of the first constant velocity and the second constant velocity is, for example, 20% or less, 15% or less, 10% or less, 9% or less, 8% or less, or 7% or less. By setting the ratio of the second constant velocity to the sum of the first constant velocity and the second constant velocity within the above range, the flow of the protein solution tends to be less significantly affected by the flow of the virus solution.

[0071] The mixture of the protein solution and the virus solution flows continuously through the virus removal filter 12 at a third constant velocity.

[0072] The membrane area of ​​the virus removal filter 12 is, for example, 0.0001 m 2 Above 0.0002m 2 More than 0.0003m 2 More than 0.0006m 2 Above 0.0009m 2 or more than 0.0015m 2 The membrane area of ​​the virus removal filter 12 is, for example, 4 m 2 Below, 3m 2 Below, 2m 2 Less than or equal to 1m 2 The following is the result.

[0073] The virus removal filter 12 may have a hollow fiber shape or a flat membrane shape.

[0074] Examples of hollow fiber virus removal filters include Planova 15N, 20N, 35N, and BioEX (Asahi Kasei Medical).

[0075] Examples of flat membrane virus removal filters include Viresolve Pro (EMD Millipore Corporation), Ultipor VF Grade DV20, DV50, Pegasus (trademark) SV4, Grade LV6 (Pall Corporation), Virosart CPV, HC, HF (Sartorius Stedim Biotech), and NFP (Merck Millipore Corporation).

[0076] The flux of the permeate through the virus removal filter 12 is, for example, 0.1 LMH or more, 1.0 LMH or more, 2.0 LMH or more, 4.0 LMH or more, or 10.0 LMH or more. The flux of the permeate through the virus removal filter 12 is, for example, 500 LMH or less, 400 LMH or less, 300 LMH or less, 200 LMH or less, or 100 LMH or less. The flux of the permeate through the virus removal filter 12 is adjusted by the first pump 13 and the second pump 21.

[0077] The membrane area of ​​the virus removal filter 12 is C (m 2 ), the first uniform speed is a, the second uniform speed is b, and the minimum value of b / (a+b) is D. min , the maximum value of b / (a+b) is D max , the minimum flux of the permeate through the virus removal filter is F min (LMH), the maximum flux of the permeate through the virus removal filter is F max (LMH), the minimum value of the first uniform velocity a min (mL / min) is given, for example, by the following formula (7): a min =(1-D max )(1000 / 60)×F min ×C (7)

[0078] Maximum value of first uniform speed a max (mL / min) is given, for example, by the following formula (8): a max =(1-D min )(1000 / 60)×F max ×C (8)

[0079] Minimum value of the second uniform velocity b min (mL / min) is given, for example, by the following formula (9): b min =D min (1000 / 60) x F min ×C (9)

[0080] Maximum value of second uniform velocity b max(mL / min) is given, for example, by the following formula (10): b max =D max (1000 / 60) x F max ×C (10)

[0081] The protein solution supplied to the first flow path 10 is continuously flowed through the first flow path 10 provided with a protein purification section 11 and a virus removal filter 12. Moreover, the virus solution supplied to the second flow path 20 is continuously flowed through the second flow path 20 and the first flow path 10 provided with a virus removal filter 12. Here, "continuously flowed" means that the solution is flowed without being pooled midway through the flow path.

[0082] The permeate of the mixed liquid that has permeated through the virus removal filter 12 is collected, for example, in a permeate collection container 43. Examples of methods for measuring viruses contained in the permeate of the mixed liquid that has permeated through the virus removal filter 12 include, but are not limited to, an infectivity titer measurement method and a quantitative PCR method.

[0083] In infectivity titer measurement, the infectivity titer is a unit that expresses the concentration of an infectious virus. There are two types of infectivity titer measurement: the endpoint method, which determines the minimum infectious unit, and the method that calculates local lesions formed by the virus. In the endpoint method, the virus is serially diluted and inoculated into a certain number of cultured cells, which are then cultured for a certain period of time, and the infection is judged to be positive or negative, to determine the dilution ratio at which 50% infection is positive, the 50% infection endpoint (TCID 50 The most common method for calculating local lesions is the plaque method, in which the virus is inoculated onto cells cultured in a sheet form, the cells are covered with a layer of agar-containing medium, and the number of plaques formed is counted according to the number of viruses inoculated. The unit of infectious dose is TCID 50 If you use the TCID 50 When the plaque method is used, it is called pfu. pfu is an abbreviation of plaque forming unit. Also, TCID 50The infectious titer per mL is expressed in units of / mL and pfu / mL.

[0084] Quantitative PCR quantifies the amount of nucleic acid contained in the virus. Normally, a virus contains one nucleic acid molecule in its capsid per particle, so the number of nucleic acid molecules is equal to the number of virus particles.

[0085] The virus removal filter 12 is evaluated for its virus clearance capability based on the amount of virus contained in the mixed liquid before passing through the virus removal filter 12 and the amount of virus contained in the mixed liquid after passing through the virus removal filter 12. The amount of virus may be expressed as an infectivity titer or as the number of particles. The virus clearance capability of the virus removal filter 12 is evaluated, for example, by the logarithmic removal value (LRV) given by the following formula (11). LRV=Log 10 T1-Log 10 T2(11) Here, T1 represents the amount of viruses contained in the mixed liquid before it passes through the virus removal filter 12, and T2 represents the amount of viruses contained in the mixed liquid after it passes through the virus removal filter 12.

[0086] It can be evaluated that the larger the LRV, the higher the virus clearance capability of the virus removal filter 12.

[0087] After stopping the supply of the protein solution to the first flow path 10, a cleaning liquid may be supplied to the first flow path 10. The cleaning liquid is, for example, flowed from a cleaning liquid tank 44 to the first flow path 10. The cleaning liquid is a solvent that does not contain proteins and viruses. The cleaning liquid may be flowed through the protein purification unit 11 and the virus removal filter 12, and the viruses contained in the permeate of the cleaning liquid that has passed through the virus removal filter 12 may be measured. If the amount of viruses contained in the permeate of the cleaning liquid is small, it is possible to evaluate that the virus retention ability of the virus removal filter 12 is high.

[0088] The time from when the supply of the protein solution to the first flow path 10 is stopped until the cleaning solution is supplied to the first flow path 10 is, for example, 0 minutes or more, 5 minutes or more, 10 minutes or more, or 30 minutes or more. Also, the time from when the supply of the protein solution to the first flow path 10 is stopped until the cleaning solution is supplied to the first flow path 10 (process pause) is, for example, 24 hours or less, 20 hours or less, 10 hours or less, 5 hours or less, or 1 hour or less.

[0089] According to the method of the virus clearance test of the embodiment, the virus solution is supplied to the second flow path 20 at a constant speed, so that the virus load condition on the virus removal filter 12 becomes constant, and it is possible to carry out a virus clearance test with high reproducibility. If the virus solution is not supplied to the second flow path 20 at a constant speed, the reproducibility of the virus clearance test may decrease. Furthermore, if the virus solution is not supplied to the second flow path 20 at a constant speed, the amount of virus spikes may vary, making it impossible to calculate the logarithmic removal rate (LRV).

[0090] Example 1 A protein purification system similar to that shown in FIG. 1 was prepared. A 0.5 mL column packed with a mixed mode chromatography carrier (Cellufine MAX IB, JNC) was used as the protein purification section 11. Cellufine MAX IB has a ligand in which a portion of a polyamine is modified with a butyl group. A 0.0003 m 2 Planova BioEX (Asahi Kasei Medical) was used.

[0091] A protein solution containing 5 mg / mL IgG was prepared using a solvent containing 20 mmol / L Tris-acetate and 100 mmol / L NaCl at pH 6.5. MVM was added to the protein solution to prepare a virus solution containing 10% MVM.

[0092] Using the first pump 13, 27 mL of the protein solution was flowed into the first flow path 10 at a first constant rate of 0.225 mL / min. Also, using the second pump 21, 3 mL of the virus solution was flowed into the second flow path 20 at a second constant rate of 0.025 mL / min. The ratio of the second constant rate to the sum of the first and second constant rates was 10%. This resulted in the virus infectivity titer (Log 10 TCID 50 The mixture having a pH (unit / mL) of 6.884 flowed through the virus removal filter 12, and the flux of the permeate was 50 LHM. The permeate of the mixture was collected.

[0093] After 30 mL of the solution had flowed through the virus removal filter 12, the first pump 13 and the second pump 21 were stopped and the filter was left to stand for 35 minutes. Then, a solvent containing 20 mmol / L tris-acetate and 100 mmol / L NaCl with a pH of 6.5 was used as a cleaning solution and was passed through the first flow path 10 at a constant rate of 0.25 mL / min using the first pump 13. The permeate of the cleaning solution that had permeated the protein purification section 11 and the virus removal filter 12 was collected.

[0094] The virus infectivity titer in the permeate of the recovered mixed liquid was measured, and the logarithmic removal value (LRV) of the virus removal filter 12 calculated from the virus infectivity titer in the permeate of the mixed liquid and the virus infectivity titer in the mixed liquid before passing through the virus removal filter 12 was 5.56 or more.

[0095] The virus infectivity titer in the permeated liquid of the recovered cleaning liquid was measured, and the logarithmic removal value (LRV) of the virus removal filter 12 was calculated from the virus infectivity titer in the permeated liquid of the mixed liquid, the virus infectivity titer in the permeated liquid of the cleaning liquid, and the virus infectivity titer in the mixed liquid before passing through the virus removal filter 12, and was 5.19 or more. The purification conditions and purification results of Example 1 are shown in FIGS. 2 and 3.

[0096] Example 2 The protein solution and the virus solution were run through the protein purification system in the same manner as in Example 1, except that a 0.5 mL column packed with a strong cation exchange chromatography carrier (Cellufine MAX GS, JNC) was used as the protein purification section 11. In this case, the virus infectivity (Log 10 TCID 50 (unit / mL) was 6.813.

[0097] The logarithmic removal value (LRV) of the virus removal filter 12, calculated from the virus infectivity titer in the permeated liquid of the mixed liquid and the virus infectivity titer in the mixed liquid before passing through the virus removal filter 12, was 5.50 or more.

[0098] The logarithmic removal value (LRV) of the virus removal filter 12 was 5.13 or more, calculated from the virus infectivity titer in the permeated liquid of the mixed liquid, the virus infectivity titer in the permeated liquid of the cleaning liquid, and the virus infectivity titer in the mixed liquid before passing through the virus removal filter 12. The purification conditions and purification results of Example 2 are shown in Figures 2 and 3.

[0099] Example 3 The protein solution and the virus solution were run through the protein purification system in the same manner as in Example 1, except that a 0.5 mL column packed with a strong cation exchange chromatography carrier (Cellufine DexS-HbP, JNC) was used as the protein purification section 11. In this case, the virus infectivity (Log 10 TCID 50 (unit / mL) was 6.875.

[0100] The logarithmic removal value (LRV) of the virus removal filter 12, calculated from the virus infectivity titer in the permeated liquid of the mixed liquid and the virus infectivity titer in the mixed liquid before passing through the virus removal filter 12, was 5.56 or more.

[0101] The logarithmic removal value (LRV) of the virus removal filter 12 was 5.19 or more, calculated from the virus infectivity titer in the permeated liquid of the mixed liquid, the virus infectivity titer in the permeated liquid of the cleaning liquid, and the virus infectivity titer in the mixed liquid before passing through the virus removal filter 12. The purification conditions and purification results of Example 3 are shown in Figures 2 and 3.

[0102] Example 4 The protein solution and the virus solution were run through the protein purification system in the same manner as in Example 1, except that a virus solution containing 10% x-MuLV was used. In this case, the virus infectivity titer (Log 10 TCID 50 (unit / mL) was 5.075.

[0103] The logarithmic removal value (LRV) of the virus removal filter 12, calculated from the virus infectivity titer in the permeated liquid of the mixed liquid and the virus infectivity titer in the mixed liquid before passing through the virus removal filter 12, was 3.75 or more.

[0104] The logarithmic removal value (LRV) of the virus removal filter 12 was 3.39 or more, calculated from the virus infectivity titer in the permeated liquid of the mixed liquid, the virus infectivity titer in the permeated liquid of the cleaning liquid, and the virus infectivity titer in the mixed liquid before passing through the virus removal filter 12. The purification conditions and purification results of Example 4 are shown in Figures 2 and 3.

[0105] Example 5 The protein solution and the virus solution were run through the protein purification system in the same manner as in Example 2, except that a virus solution containing 10% x-MuLV was used. In this case, the virus infectivity titer (Log 10 TCID 50 (unit / mL) was 4.939.

[0106] The logarithmic removal value (LRV) of the virus removal filter 12, calculated from the virus infectivity titer in the permeated liquid of the mixed liquid and the virus infectivity titer in the mixed liquid before passing through the virus removal filter 12, was 3.62 or more.

[0107] The logarithmic removal value (LRV) of the virus removal filter 12 was 3.26 or more, calculated from the virus infectivity titer in the permeated liquid of the mixed liquid, the virus infectivity titer in the permeated liquid of the cleaning liquid, and the virus infectivity titer in the mixed liquid before passing through the virus removal filter 12. The purification conditions and purification results of Example 5 are shown in Figures 2 and 3.

[0108] Example 6 The protein solution and the virus solution were run through the protein purification system in the same manner as in Example 3, except that a virus solution containing 10% x-MuLV was used. In this case, the infectivity of the virus in the mixed solution (Log 10 TCID 50 (unit / mL) was 4.809.

[0109] The logarithmic removal value (LRV) of the virus removal filter 12, calculated from the virus infectivity titer in the permeated liquid of the mixed liquid and the virus infectivity titer in the mixed liquid before passing through the virus removal filter 12, was 3.50 or more.

[0110] The logarithmic removal value (LRV) of the virus removal filter 12 was 3.12 or more, calculated from the virus infectivity titer in the permeated liquid of the mixed liquid, the virus infectivity titer in the permeated liquid of the cleaning liquid, and the virus infectivity titer in the mixed liquid before passing through the virus removal filter 12. The purification conditions and purification results of Example 6 are shown in Figures 2 and 3.

[0111] Comparative Example 1 A protein purification system according to Comparative Example 1 was prepared as shown in Fig. 4. The protein purification system according to Comparative Example 1 was provided with a flow path 110, a pump 113 provided in the flow path 110, and a virus removal filter 12 provided in the flow path 110. The virus removal filter 12 was a 0.0003 m 2 Planova BioEX (Asahi Kasei Medical) was used. The protein purification system of Comparative Example 1 did not include a protein purification section. Furthermore, the protein purification system of Comparative Example 1 did not include a second flow path and a second pump.

[0112] A virus solution containing 1% MVM was prepared using the same materials as in Example 1, and 30 mL of the virus solution was flowed through the flow channel 110 at a constant rate of 0.025 mL / min using the pump 113. This resulted in a virus infectivity titer (Log 10 TCID 50 A virus solution having a molecular weight (Mw / mL) of 6.13 was passed through the virus removal filter 12, and the flux of the permeate was 5 LHM. The permeate of the virus solution was collected.

[0113] After 30 mL of the solution had flowed through the virus removal filter 12, the pump 113 was stopped and the filter was left to stand for 35 minutes. Then, the same cleaning solution as in Example 1 was made to flow through the flow path 110 at a constant rate of 0.25 mL / min using the pump 113. The permeate of the cleaning solution that had permeated the virus removal filter 12 was collected.

[0114] The virus infectivity titer in the permeate of the recovered virus solution was measured, and the logarithmic removal value (LRV) of the virus removal filter 12, calculated from the virus infectivity titer in the permeate of the virus solution and the virus infectivity titer in the virus solution before passing through the virus removal filter 12, was 5.27 or more.

[0115] The virus infectivity titer in the permeate of the recovered cleaning liquid was measured, and the logarithmic removal value (LRV) of the virus removal filter 12 was calculated to be 5.13 or more from the virus infectivity titer in the permeate of the virus solution, the virus infectivity titer in the permeate of the cleaning liquid, and the virus infectivity titer in the virus solution before passing through the virus removal filter 12. The purification conditions and purification results of Comparative Example 1 are shown in Figs.

[0116] The LRV obtained in Comparative Example 1 is similar to the LRV obtained in Examples 1 to 3. This indicates that in Examples 1 to 3, even if the protein purification section 11 was located upstream of the virus removal filter 12, the virus removal ability of the virus removal filter 12 alone could be tested.

[0117] Comparative Example 2 The virus solution was passed through the protein purification system according to Comparative Example 1 in the same manner as in Comparative Example 1, except that the virus solution was passed through the flow channel 110 at a constant rate of 0.05 mL / min. In this case, the virus infectivity titer (Log 10 TCID 50 (unit / mL) was 6.25, and the permeate flux was 10 LHM.

[0118] The virus infectivity titer in the permeate of the recovered virus solution was measured, and the logarithmic removal value (LRV) of the virus removal filter 12, calculated from the virus infectivity titer in the permeate of the virus solution and the virus infectivity titer in the virus solution before passing through the virus removal filter 12, was 5.40 or more.

[0119] The virus infectivity titer in the permeate of the recovered cleaning liquid was measured, and the logarithmic removal value (LRV) of the virus removal filter 12 was calculated to be 5.24 or more from the virus infectivity titer in the permeate of the virus solution, the virus infectivity titer in the permeate of the cleaning liquid, and the virus infectivity titer in the virus solution before passing through the virus removal filter 12. The purification conditions and purification results of Comparative Example 2 are shown in Figs.

[0120] Comparative Example 3 The virus solution was passed through the protein purification system according to Comparative Example 1 in the same manner as in Comparative Example 1, except that the virus solution was passed through the flow channel 110 at a constant rate of 0.1 mL / min. In this case, the virus infectivity (Log 10 TCID 50 (unit / mL) was 6.44, and the permeate flux was 20 LHM.

[0121] The virus infectivity titer in the permeate of the recovered virus solution was measured, and the logarithmic removal value (LRV) of the virus removal filter 12, calculated from the virus infectivity titer in the permeate of the virus solution and the virus infectivity titer in the virus solution before passing through the virus removal filter 12, was 5.59 or more.

[0122] The virus infectivity titer in the permeate of the recovered cleaning liquid was measured, and the logarithmic removal value (LRV) of the virus removal filter 12 was calculated to be 5.43 or more from the virus infectivity titer in the permeate of the virus solution, the virus infectivity titer in the permeate of the cleaning liquid, and the virus infectivity titer in the virus solution before passing through the virus removal filter 12. The purification conditions and purification results of Comparative Example 3 are shown in Figs.

[0123] Comparative Example 4 A protein purification system according to Comparative Example 4 was prepared as shown in FIG. 7. The protein purification system according to Comparative Example 4 included a flow path 210, a pump 213 provided in the flow path 210, and a protein purification unit 11 provided in the flow path 210. A 0.5 mL column packed with a mixed mode chromatography carrier (Cellufine MAX IB, JNC) was used as the protein purification unit 11. The protein purification system according to Comparative Example 4 did not include a virus removal filter. In addition, the protein purification system according to Comparative Example 4 did not include a second flow path and a second pump.

[0124] A virus solution containing 5% MVM was prepared using the same materials as in Example 1, and 30 mL of the virus solution was flowed through the flow channel 110 at a constant rate of 0.25 mL / min using the pump 213. This resulted in a virus infectivity titer (Log 10 TCID 50 A virus solution with a pH of 7.741 (unit / mL) was passed through the protein purification section 11. The permeate of the virus solution was collected.

[0125] After 30 mL of the solution had flowed through protein purification section 11, pump 213 was stopped and the solution was allowed to stand for 0.05 minutes. Then, the same washing solution as in Example 1 was made to flow through flow channel 210 at a constant rate of 0.25 mL / min using pump 213. The permeate of the washing solution that had permeated protein purification section 11 was collected.

[0126] The virus infectivity in the permeate of the recovered virus solution was measured, and the logarithmic removal value (LRV) in protein purification section 11 calculated from the virus infectivity in the permeate of the virus solution and the virus infectivity in the virus solution before passing through protein purification section 11 was 3.94.

[0127] The virus infectivity in the permeate of the recovered washing liquid was measured, and the logarithmic removal value (LRV) in protein purification unit 11 was calculated to be 1.96 from the virus infectivity in the permeate of the virus solution, the virus infectivity in the permeate of the washing liquid, and the virus infectivity in the virus solution before passing through protein purification unit 11. The purification conditions and purification results for Comparative Example 4 are shown in Figures 8 and 9.

[0128] This result indicates that viruses are removed in the protein purification unit 11. Therefore, this result indicates that in the system shown in Figure 1, if a virus solution is passed upstream of the protein purification unit 11, the LRV of the virus removal filter 12 alone cannot be accurately measured.

[0129] Comparative Example 5 The virus solution was passed through the protein purification system in the same manner as in Comparative Example 4, except that a 0.5 mL column packed with a strong cation exchange chromatography carrier (Cellufine MAX GS, JNC) was used as the protein purification section 11. As a result, the virus infectivity (Log 10 TCID 50 A virus solution with a titer (unit / mL) of 7.741 was passed through the protein purification section 11.

[0130] The virus infectivity in the permeate of the recovered virus solution was measured, and the logarithmic removal value (LRV) in protein purification section 11, calculated from the virus infectivity in the permeate of the virus solution and the virus infectivity in the virus solution before passing through protein purification section 11, was 0.25.

[0131] The virus infectivity in the permeate of the recovered washing liquid was measured, and the logarithmic removal value (LRV) in protein purification unit 11 was calculated to be 0.21 from the virus infectivity in the permeate of the virus solution, the virus infectivity in the permeate of the washing liquid, and the virus infectivity in the virus solution before passing through protein purification unit 11. The purification conditions and purification results for Comparative Example 5 are shown in Figures 8 and 9.

[0132] Comparative Example 6 The virus solution was passed through the protein purification system in the same manner as in Comparative Example 4, except that a 0.5 mL column packed with a strong cation exchange chromatography carrier (Cellufine DexS-HbP, JNC) was used as the protein purification section 11. As a result, the virus infectivity (Log 10 TCID 50 A virus solution with a titer (unit / mL) of 7.738 was passed through the protein purification section 11.

[0133] The virus infectivity in the permeate of the recovered virus solution was measured, and the logarithmic removal value (LRV) in protein purification section 11, calculated from the virus infectivity in the permeate of the virus solution and the virus infectivity in the virus solution before passing through protein purification section 11, was -0.39.

[0134] The virus infectivity in the permeate of the recovered washing liquid was measured, and the logarithmic removal value (LRV) in protein purification unit 11 was calculated to be 0.38 from the virus infectivity in the permeate of the virus solution, the virus infectivity in the permeate of the washing liquid, and the virus infectivity in the virus solution before passing through protein purification unit 11. The purification conditions and purification results for Comparative Example 6 are shown in Figures 8 and 9.

[0135] Comparative Example 7 Except for using a virus solution containing 10% x-MuLV, the virus solution was run through the protein purification system according to Comparative Example 4 in the same manner as in Comparative Example 4. In this case, the virus infectivity (Log 10 TCID 50 (unit / mL) was 6.614.

[0136] The virus infectivity in the permeate of the recovered virus solution was measured, and the logarithmic reduction value (LRV) in protein purification section 11, calculated from the virus infectivity in the permeate of the virus solution and the virus infectivity in the virus solution before passing through protein purification section 11, was 2.25.

[0137] The virus infectivity in the permeate of the recovered washing liquid was measured, and the logarithmic removal value (LRV) in protein purification unit 11 was calculated to be 1.96 from the virus infectivity in the permeate of the virus solution, the virus infectivity in the permeate of the washing liquid, and the virus infectivity in the virus solution before passing through protein purification unit 11. The purification conditions and purification results for Comparative Example 7 are shown in Figures 8 and 9.

[0138] Comparative Example 8 Except for using a virus solution containing 10% x-MuLV, the virus solution was run through the protein purification system of Comparative Example 4 in the same manner as in Comparative Example 5. In this case, the virus infectivity (Log 10 TCID 50 (unit / mL) was 6.239.

[0139] The virus infectivity in the permeate of the recovered virus solution was measured, and the logarithmic removal value (LRV) in protein purification section 11 calculated from the virus infectivity in the permeate of the virus solution and the virus infectivity in the virus solution before passing through protein purification section 11 was 0.13.

[0140] The virus infectivity in the permeate of the recovered washing liquid was measured, and the logarithmic removal value (LRV) in protein purification unit 11 was calculated to be 0.21 from the virus infectivity in the permeate of the virus solution, the virus infectivity in the permeate of the washing liquid, and the virus infectivity in the virus solution before passing through protein purification unit 11. The purification conditions and purification results for Comparative Example 8 are shown in Figures 8 and 9.

[0141] Comparative Example 9 Except for using a virus solution containing 10% x-MuLV, the virus solution was run through the protein purification system of Comparative Example 4 in the same manner as in Comparative Example 6. In this case, the virus infectivity (Log 10 TCID 50 (unit / mL) was 6.368.

[0142] The virus infectivity in the permeate of the recovered virus solution was measured, and the logarithmic removal value (LRV) in protein purification section 11 calculated from the virus infectivity in the permeate of the virus solution and the virus infectivity in the virus solution before passing through protein purification section 11 was 0.44.

[0143] The virus infectivity in the permeate of the recovered washing liquid was measured, and the logarithmic removal value (LRV) in protein purification unit 11 was calculated to be 0.38 from the virus infectivity in the permeate of the virus solution, the virus infectivity in the permeate of the washing liquid, and the virus infectivity in the virus solution before passing through protein purification unit 11. The purification conditions and purification results for Comparative Example 9 are shown in Figures 8 and 9. [Explanation of symbols]

[0144] 10...First flow path, 11...Protein purification section, 12...Virus removal filter, 13...First pump, 20...Second flow path, 21...Second pump, 31...Protein concentration measuring device, 32...Conductivity measuring device, 33...In-line mixer

Claims

1. supplying a protein solution to a first flow path having a protein purification section provided upstream and a virus removal filter provided downstream, and flowing the protein solution through the protein purification section at a first constant velocity; supplying a virus solution at a second constant speed to a second flow path connected between the protein purification unit and the virus removal filter of the first flow path, and mixing the virus solution with the protein solution purified in the protein purification unit in the first flow path; flowing the mixture of the protein solution and the virus solution through the virus removal filter at a third constant speed; measuring viruses contained in the permeate of the mixed liquid that has passed through the virus removal filter; supplying a cleaning solution to the first flow path after stopping the supply of the protein solution to the first flow path, and causing the cleaning solution to flow through the protein purification unit and the virus removal filter; measuring viruses contained in the permeate of the cleaning solution that has passed through the protein purification unit and the virus removal filter; calculating a virus clearance capability of the virus removal filter from the amount of the virus in the mixed liquid before passing through the virus removal filter, the amount of the virus contained in the permeate of the mixed liquid that has passed through the virus removal filter, and the amount of the virus contained in the permeate of the cleaning liquid that has passed through the virus removal filter; A method for viral clearance testing, comprising:

2. The method according to claim 1 , wherein the first flow path is provided with a first pump for flowing the protein solution through the protein purification section at the first constant velocity.

3. The method according to claim 1 or 2, further comprising providing a second pump in the second flow path for flowing the virus solution at the second constant velocity.

4. The method according to claim 1 , wherein the protein solution is passed through the protein purification section continuously.

5. The method of claim 1 , wherein the virus solution is continuously passed through the second flow path.

6. The method according to any one of claims 1 to 5, wherein the mixed liquid is continuously passed through the virus removal filter.

7. 7. The method according to claim 1, wherein a, b, x, and y satisfy the following formula (1), where a is the first constant velocity, b is the second constant velocity, x is a virus concentration in the mixed liquid, and y is a virus concentration in the virus solution. x / y=b / (a+b) (1)

8. The method according to claim 1 , wherein a ratio of the second constant velocity to a sum of the first constant velocity and the second constant velocity is 0.1% or more and 20% or less.

9. The infectivity of the virus in the mixture (Log 10 T.C.I.D. 50 The method according to any one of claims 1 to 8, wherein the concentration of the serotonin in the blood is 2 or more and 10 or less.

10. The membrane area of ​​the virus removal filter is 0.0001 m 2 4m or more 2 10. The method of any one of claims 1 to 9, wherein:

11. The method according to any one of claims 1 to 10, wherein the flux of the permeate through the virus removal filter is 0.1 LMH or more and 500 LMH or less.

12. The membrane area of ​​the virus removal filter is C (m 2 ), b / (a+b) is the minimum value D min , b / (a+b) is the maximum value D max , the minimum flux of the permeate in the virus removal filter is F min (LMH), the maximum flux of the permeate in the virus removal filter is F max As (LMH), The minimum value a of the first uniform velocity a min (mL / min) is given by the following formula (2): a min =(1-D max )(1000 / 60)×F min ×C (2) The maximum value a of the first uniform velocity a max (mL / min) is given by the following formula (3): a max =(1-D min )(1000 / 60)×F max ×C (3) The minimum value b of the second constant speed b min (mL / min) is given by the following formula (4): b min =D min (1000 / 60)×F min ×C (4) The maximum value b of the second constant speed b max (mL / min) is given by the following formula (5): b max =D max (1000 / 60)×F max ×C (5) The method of claim 7.

13. The method according to claim 1 , wherein a time period from when the supply of the protein solution to the first flow path is stopped until the supply of the cleaning solution to the first flow path is 0 minutes or more and 24 hours or less.

14. 14. The method of any one of claims 1 to 13, wherein the virus solution comprises a protein.

15. 15. The method of any one of claims 1 to 14, wherein the virus solution contains the same proteins as the protein solution.

16. 16. The method of claim 14 or 15, wherein the concentration of the protein in the virus solution is the same as the concentration of the protein in the protein solution.

17. 17. The method according to any one of claims 1 to 16, wherein a logarithmic removal value (LRV) of the virus in the virus removal filter is calculated from the infectivity titer of the virus contained in the mixed liquid before passing through the virus removal filter, the infectivity titer of the virus contained in a permeate of the mixed liquid that has passed through the virus removal filter, and the infectivity titer of the virus contained in a permeate of the cleaning liquid that has passed through the virus removal filter.

18. The method according to any one of claims 1 to 17, wherein the protein purification unit has the ability to remove viruses.

19. 19. The method of claim 18, wherein the log reduction value (LRV) in the protein purification section is 0 to 7.

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