Method for evaluating virus removal membrane

Abstract

To provide a method for evaluating the performance of a virus-removing membrane even by using an antibody filtered through the virus-removing membrane at least once.SOLUTION: A method for evaluating a virus removing membrane, comprising a step of filtering a liquid containing antibodies through a virus removing membrane, wherein the antibodies have been filtered through the virus removing membrane one or more times, and the liquid contains arginine in an amount of 350mmol / L or more and 650mmol / L or less.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for evaluating a virus removal membrane. [Background technology]

[0002] When producing protein preparations such as biopharmaceuticals and plasma fraction preparations, it is important to inactivate or remove viruses from the preparations. As described in Patent Document 1, viruses are separated and removed by filtration using a virus removal membrane as a physical virus removal method that does not involve chemical denaturation.

[0003] Virus removal membranes are evaluated for their performance, including virus removal, by filtering a virus-containing protein solution. Antibodies are sometimes used as proteins. When antibodies are used, they often aggregate during performance evaluation tests, making it difficult to accurately evaluate the performance of the virus removal membrane. Furthermore, while evaluation of antibody-based virus removal membranes requires multiple experiments, monoclonal antibodies are very expensive, so it is desirable for them to be reusable.

[0004] To address the above issues, it is possible to remove impurities such as aggregates using column chromatography or a prefilter before using the membrane to evaluate the performance of the virus removal membrane. However, column chromatography is time-consuming and labor-intensive, placing a significant burden on the operator. Non-Patent Document 2 also shows that while pre-filtering improves the filtration performance of virus removal membranes for many proteins, there are also cases where pre-filtering does not improve filtration performance, and does not clearly explain why filtration performance does not improve. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2012 / 176876 [Patent Document 2] Patent No. 6385444 [Patent Document 3] Patent No. 4024041 [Patent Document 4] International Publication No. 2022 / 118943 [Patent Document 5] Patent No. 4699207 [Non-patent literature]

[0006] [Non-Patent Document 1] Biotechnology and Bioengineering, Vol.119,p.743(2022) Summary of the Invention [Problem to be solved by the invention]

[0007] The problem to be solved by the present invention is to provide a method that can evaluate the performance of a virus removal membrane even when an antibody that has been filtered through the virus removal membrane more than once is used. [Means for solving the problem]

[0008] As a result of intensive research to solve the above problems, the present inventors discovered that the above problems can be solved by adding a high concentration of arginine to a solution containing an antibody and filtering the solution using a virus removal membrane, and thus completed the present invention.

[0009] That is, the present invention is as follows.

[0010] [1] A method for evaluating a virus removal membrane, comprising a step of filtering a solution containing an antibody through the virus removal membrane, wherein the antibody has been filtered through the virus removal membrane one or more times, and the solution contains 350 mmol / L or more and 650 mmol / L or less of arginine.

[0011] [2] The method according to [1], wherein the antibody has been frozen and thawed one or more times.

[0012] [3] The method according to [1] or [2], wherein the antibody has been solution-exchanged one or more times by a procedure including a concentration step.

[0013] [4] The method according to any one of [1] to [3], wherein the antibody is an antibody that has been filtered through a virus removal membrane at least 10 times.

[0014] [5] The method according to any one of [1] to [4], wherein the antibody is a monoclonal antibody.

[0015] [6] The method according to any one of [1] to [5], wherein the antibody concentration is 0.5 g / L or more and less than 20 g / L.

[0016] [7] The method according to any one of [1] to [6], wherein the antibody has a pI of 6.0 or more and 8.5 or less.

[0017] [8] The method according to any one of [1] to [7], wherein the pH range of the solution is 4.5 or more and 8.0 or less, and the pH value of the solution is 0.5 or more lower than the pI value of the antibody.

[0018] [9] The method according to any one of [1] to [8], wherein the solution is an acetate buffer or a Tris-HCl buffer.

[0019]

[10] The method according to any one of [1] to [9], wherein the filtration pressure in the filtration step is 0.5 bar or more and 3.5 bar or less.

[0020]

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

[10] , further comprising evaluating the antibody permeability of the virus removal membrane.

[0021]

[12] The pure water permeability of the virus removal membrane is 40 L / m 2 / hr / bar or more 400L / m 2 The method according to any one of [1] to

[11] , wherein the value is 1 / hr / bar or less.

[0022]

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

[12] , wherein the virus removal membrane comprises a hydrophobic polymer and a hydrophilic polymer.

[0023]

[14] The method according to

[13] , wherein the hydrophobic polymer is polyvinylidene fluoride, polyethersulfone, polysulfone, or polyethylene.

[0024]

[15] The method according to

[13] or

[14] , wherein the hydrophilic polymer is coated on a substrate film containing a hydrophobic polymer.

[0025]

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

[12] , wherein the virus removal membrane comprises cellulose.

[0026]

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

[16] , wherein the antibody is not treated by column chromatography.

[0027]

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

[16] , wherein the antibody is not pre-filtered.

[0028]

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

[18] , wherein the solution contains arginine at 400 mmol / L or more and 600 mmol / L or less. [Effects of the Invention]

[0029] According to the present invention, when an antibody that has been filtered through a virus removal membrane one or more times is used to evaluate the performance of the virus removal membrane, a stable and cost-effective evaluation method can be provided in which antibody aggregation is suppressed and the influence of aggregates remaining in the protein solution is suppressed by adding a high concentration of arginine and filtering through the virus removal membrane. [Brief explanation of the drawings]

[0030] [Figure 1] FIG. 1 is a schematic perspective view of a virus removal membrane according to an embodiment. [Figure 2] FIG. 1 is a schematic perspective view of a virus removal membrane according to an embodiment. [Figure 3] 1 is a graph showing the results of size exclusion chromatography in Reference Example 5. [Figure 4] 4 is an enlarged view of a portion of the graph in FIG. 3. DETAILED DESCRIPTION OF THE INVENTION

[0031] The following describes embodiments of the present invention. However, it should not be understood that the following embodiments limit the present invention. From this disclosure, various alternative embodiments, examples, and operating techniques will become apparent to those skilled in the art. It should be understood that the present invention encompasses various embodiments not described herein.

[0032] A method for evaluating a virus removal membrane according to an embodiment includes filtering a solution containing an antibody through the virus removal membrane. The antibody is an antibody that has been filtered through the virus removal membrane one or more times. The solution containing the antibody contains arginine at 350 mmol / L or more and 650 mmol / L or less. By filtering the solution containing the antibody through the virus removal membrane, for example, the permeability of the antibody through the virus removal membrane can be evaluated. Furthermore, by filtering a solution containing an antibody and a virus through the virus removal membrane, for example, the virus removal performance of the virus removal membrane can be evaluated.

[0033] An antibody, an example of a physiologically active substance, is generally defined in biochemistry as a glycoprotein molecule (also called gamma globulin or immunoglobulin) produced by B lymphocytes as a defense mechanism against infection in vertebrates. For example, antibodies are used as human pharmaceuticals and have a structure substantially identical to that of antibodies present in the human body to which they are administered.

[0034] The antibody may be a human antibody, or may be an antibody derived from a non-human mammal such as a bovine or a mouse. Alternatively, the antibody may be a chimeric antibody with human IgG, or a humanized antibody. A chimeric antibody with human IgG is an antibody in which the variable region is derived from a non-human organism such as a mouse, but the other constant regions are substituted with immunoglobulins derived from humans. A humanized antibody is an antibody in which the complementarity-determining region (CDR) of the variable region is derived from a non-human organism, but the other framework region (FR) is derived from humans. Humanized antibodies have even reduced immunogenicity compared to chimeric antibodies.

[0035] The class (isotype) and subclass of the antibody are not particularly limited. For example, antibodies are classified into five classes, IgG, IgA, IgM, IgD, and IgE, based on differences in the structure of their constant regions. The antibodies used in the methods of the embodiments may belong to any of the five classes. Furthermore, among human antibodies, IgG has four subclasses, IgG1 to IgG4, and IgA has two subclasses, IgA1 and IgA2. The subclass of the antibodies used in the methods of the embodiments may be any of these. Note that antibody-related proteins, such as Fc fusion proteins in which a protein is bound to the Fc region, may also be included in the antibodies used in the methods of the embodiments.

[0036] Antibodies can also be classified by origin. The antibodies used in the methods of the present invention may be any of natural antibodies, recombinant antibodies produced by genetic engineering techniques, monoclonal antibodies, and polyclonal antibodies.

[0037] The isoelectric point (pI) of the antibody is not particularly limited, but is, for example, 6.0 or higher and 8.5 or lower. The pI of the antibody may be 6.5 or higher, or 7.0 or higher. The pI of the antibody may be 8.0 or lower.

[0038] For example, as shown in FIG. 1, a virus removal membrane 10 has a primary surface 1 to which an antibody-containing solution is supplied, and a secondary surface 2 from which the permeate that has permeated the virus removal membrane 10 is discharged.

[0039] The virus removal membrane 10 has virus capture sites where viruses are captured in its cross section. It is preferable that the amount of viruses captured in the virus capture sites be uniform across the cross section, regardless of the location on the filtration surface (primary surface 1) where the solution enters.

[0040] Furthermore, it is preferable that the thickness of the virus capture site of the virus removal membrane 10 is uniform. Furthermore, when the virus removal membrane 10 has the shape of a hollow fiber membrane, it is preferable that the thickness of the virus capture site is uniform in the circumferential direction.

[0041] It can be difficult to visually detect viruses captured by the virus removal membrane 10. In contrast, gold colloids have a diameter similar to that of viruses but do not transmit light, making them easy to detect visually. Therefore, for example, the characteristics of the virus removal membrane 10 can be evaluated by filtering a solution containing gold colloids through the virus removal membrane 10 and then measuring the relative brightness of the gold colloid capture site on a cross section of the virus removal membrane 10 where the virus removal membrane 10 has captured the gold colloid.

[0042] When a solution containing 20 nm diameter gold colloids is supplied to the virus removal membrane 10 from the primary surface 1 to capture the gold colloids in the virus removal membrane 10 and the luminance is measured at a cross section of the virus removal membrane 10, the value obtained by dividing the standard deviation of the area values ​​of the luminance shift spectrum by the average area values ​​of the luminance shift spectrum is, but is not particularly limited to, for example, 0.01 to 1.50. This value indicates the coefficient of variation of the amount of gold colloid captured in the virus removal membrane 10; a smaller value indicates greater uniformity in the amount of gold colloid captured at the gold colloid capture sites in the virus removal membrane 10.

[0043] For the virus removal membrane 10, the value indicating the coefficient of variation is, for example, 0.01 or more and 1.50 or less, 0.01 or more and 1.20 or less, or 0.01 or more and 1.00 or less, 0.01 or more and 0.90 or less, or 0.01 or more and 0.80 or less.

[0044] If the above coefficient of variation is 0.01 or more and 1.50 or less, viruses will be captured uniformly at the virus capture site of the membrane (in the circumferential direction for hollow fiber membranes), and high virus removal performance can be maintained even when the total amount of virus loaded on the virus removal membrane (spike amount in the virus protein preparation or total filtration amount) increases.

[0045] The coefficient of variation is measured, for example, by the following method. A section is cut from the virus removal membrane after filtering the gold colloid solution, and the brightness profile of multiple locations on the cross section of the section stained with gold colloid is measured using an optical microscope. Because gold colloid absorbs light, the brightness variation depends on the amount of gold colloid captured. If necessary, background noise may be removed from the brightness profile. A graph is then created with film thickness on the horizontal axis and brightness variation on the vertical axis, and the area of ​​the spectrum of brightness variation displayed on the graph is calculated. Furthermore, the standard deviation of the area of ​​the spectrum of brightness variation at multiple locations is divided by the average area of ​​the spectrum of brightness variation at multiple locations to calculate a value indicating the coefficient of variation of the amount of gold colloid captured at the gold colloid capture site on the virus removal membrane 10.

[0046] In the cross section of the virus removal membrane 10 in a wet state, the thickness of the portion (dense layer) that captures gold colloids with a diameter of 20 nm to 30 nm is not particularly limited, but may be, for example, 10 μm to 30 μm, 10 μm to 29 μm, 10 μm to 28 μm, 10 μm to 20 μm, 11 μm to 20 μm, or 12 μm to 20 μm.

[0047] The thickness of a gold colloid capture site with a diameter of 20 nm or more and 30 nm or less can be obtained, for example, by the following method. Sections are cut from a virus removal membrane through which gold colloid solutions with diameters of 20 nm and 30 nm have been filtered. The brightness profile of multiple areas stained with gold colloid on the cross section of the section is measured using an optical microscope. Here, a first distance a is measured in the film thickness direction from the primary surface 1 of the virus removal membrane 10 to the part of the gold colloid capture site closest to the primary surface. A second distance b is also measured in the film thickness direction from the primary surface 1 of the virus removal membrane 10 to the part of the gold colloid capture site closest to the secondary surface 2.

[0048] Next, for each of the multiple locations, the first distance a is divided by the membrane thickness c of the wet virus removal membrane to calculate a percentage value A (= a / c as a percentage), and the average value of values ​​A at the multiple locations is calculated as the first degree of reach.Furthermore, the second distance b is divided by the membrane thickness c of the wet virus removal membrane to calculate a percentage value B (= b / c as a percentage), and the average value of values ​​B at the multiple locations is calculated as the second degree of reach.

[0049] Furthermore, as shown in the following formula (1), the average value B of the second degree of penetration in the virus removal membrane through which gold colloids with a diameter of 20 nm were filtered 20 and the average value A of the first penetration rate in the virus removal membrane through which gold colloids with a diameter of 30 nm were filtered. 30 The difference between and is the average thickness C of the wet virus removal membrane through which 20 nm diameter gold colloids were filtered. 20 and the average thickness C of the wet virus removal membrane after filtering 30 nm diameter gold colloids. 30 Average value of C AVE When gold colloids with a diameter of 20 nm and gold colloids with a diameter of 30 nm are passed through, the resulting value is calculated as the thickness T of the section of the virus removal membrane 10 where gold colloids with a diameter of 20 nm to 30 nm are captured. The thickness T of the gold colloid capture section is also expressed as the thickness T of the dense layer of the virus removal membrane. T=(B 20 -A 30 )×CAVE (1)

[0050] In the cross section of the virus removal membrane 10 in a wet state, the thickness of the portion capturing 15 nm diameter gold colloids (the densest layer) is not particularly limited, but is preferably 2 μm to 10 μm, more preferably 3 μm to 10 μm.

[0051] The thickness of a 15 nm diameter gold colloid capture site is obtained, for example, by the following method. A section is cut from a virus removal membrane through which a 15 nm diameter gold colloid solution has been filtered. The brightness profile of multiple areas stained with gold colloid on the cross section of the section is measured using an optical microscope. Here, a first distance d is measured in the film thickness direction from the primary surface 1 of the virus removal membrane 10 to the part of the gold colloid capture site closest to the primary surface. In addition, a second distance e is measured in the film thickness direction from the primary surface 1 of the virus removal membrane 10 to the part of the gold colloid capture site closest to the secondary surface 2.

[0052] Next, for each of the multiple locations, the first distance d is divided by the membrane thickness f of the wet virus removal membrane to calculate a percentage value D (= percentage of d / f), and the average value of values ​​D at the multiple locations is calculated as the first degree of reach.Furthermore, the second distance e is divided by the membrane thickness f of the wet virus removal membrane to calculate a percentage value E (= percentage of e / f), and the average value of values ​​E at the multiple locations is calculated as the second degree of reach.

[0053] Furthermore, as shown in the following formula (2), the difference between the average second attainment level E and the average first attainment level D is multiplied by the average membrane thickness F of the filtered virus removal membrane in a wet state to calculate the thickness T of the region on the cross section of the virus removal membrane 10 where a 15 nm diameter gold colloid is captured when a 15 nm diameter gold colloid is passed through. The thickness T of the 15 nm diameter gold colloid capture region can also be expressed as the thickness T of the densest layer of the virus removal membrane. T = (ED) × F (2)

[0054] When a solution containing 30 nm diameter gold colloids is filtered through the virus removal membrane 10, the region where the 30 nm diameter gold colloids are captured on the cross section of the wet virus removal membrane 10 is measured with an optical microscope, and is not particularly limited, but is, for example, located between 15% and 60% of the film thickness, or between 20% and 55% of the film thickness, from the primary surface 1. Even if a small amount of 30 nm diameter gold colloids is captured in a region less than 15% or more than 60% of the film thickness from the primary surface 1, if the absolute value of the spectrum obtained by subtracting the measured brightness profile from the constant (255) during optical microscope observation is 10% or less of the maximum absolute value of the spectrum, the capture of gold colloids in that region can be considered to be within the error range from the perspective of the virus removal ability of the virus removal membrane, and therefore the region where the 30 nm diameter gold colloids are captured can be considered to be located between 15% and 60% of the film thickness from the primary surface 1.

[0055] When a solution containing 20 nm diameter gold colloids is filtered through virus removal membrane 10, the location where the 20 nm diameter gold colloids are captured on the cross section of the wet virus removal membrane 10 is measured with an optical microscope, and is not particularly limited, but is, for example, located between 25% and 85% of the film thickness, or between 30% and 85% of the film thickness, from the primary surface 1. As with the 30 nm diameter gold colloids, even if gold colloids are observed in a region less than 25% or more than 85% of the film thickness from the primary surface 1, the deviation in brightness obtained by subtracting the measured brightness profile from the constant (255) when observed with an optical microscope can be considered within the error range if the absolute value of the spectrum is 10% or less of the maximum absolute value of the spectrum.

[0056] When a solution containing 15 nm diameter gold colloids is filtered through virus removal membrane 10, the location where the 15 nm diameter gold colloids are captured on the cross section of the wet virus removal membrane 10 is measured with an optical microscope, but is not particularly limited thereto, and is, for example, located between 60% and 100%, or between 65% and 100%, of the film thickness from the primary surface 1. As with gold colloids with diameters of 30 nm and 20 nm, even if gold colloids are observed in an area less than 60% of the film thickness from the primary surface 1, the deviation in brightness obtained by subtracting the measured brightness profile from the constant (255) when observed with an optical microscope can be considered within the error range if it is 10% or less of the maximum absolute value of the spectrum.

[0057] When a solution containing 10 nm diameter gold colloids is filtered through the virus removal membrane 10, the 10 nm diameter gold colloids are hardly captured on the cross section of the virus removal membrane 10. This can be confirmed by the fact that no significant value can be detected in the brightness spectrum when observed using an optical microscope (Biozero, BZ8100, manufactured by Keyence Corporation). This can also be confirmed by the fact that the logarithmic removal value (LRV), described below, becomes low. The fact that 10 nm diameter gold colloids are not captured indicates that high permeability can be achieved for useful proteins with diameters of approximately 10 nm, such as IgG.

[0058] Examples of synthetic polymers that can be used to form the virus removal membrane 10 include thermoplastic resins, which are easy to process using techniques such as compression, extrusion, injection, inflation, and blow molding, and have excellent pressure resistance during filtration. From the perspectives of heat resistance and moldability, examples of thermoplastic resins include polyolefin resins and fluorine-based resins. An example of a fluorine-based resin is polyvinylidene fluoride resin. Other examples of thermoplastic resins include polyethersulfone, polysulfone, and polyethylene.

[0059] Thermoplastic crystalline resins, which are hydrophobic polymers, are prone to adsorption of proteins and the like, membrane contamination and clogging, which can cause a rapid decrease in filtration rate. Therefore, when a hydrophobic polymer is used as the material for a virus removal membrane, a base membrane containing a hydrophobic polymer membrane may be coated with a hydrophilic polymer to prevent clogging due to adsorption of proteins and the like. For example, to impart hydrophilicity, the hydrophobic polymer membrane may be covered with hydrophilic graft chains by graft polymerization.

[0060] Alternatively, the virus removal membrane 10 may be made of cellulose. Because cellulose is hydrophilic, it is less susceptible to protein adsorption, membrane contamination, and clogging. Examples of cellulose include regenerated cellulose, natural cellulose, and cellulose acetate. Methods for producing regenerated cellulose include a method in which it is produced from a cuprammonium cellulose solution (the cuprammonium method) and a method in which cellulose acetate is saponified with an alkali (the saponification method).

[0061] The virus removal membrane 10 has the shape of a hollow fiber membrane, for example. Alternatively, the virus removal membrane 10 may have the shape of a flat membrane, as shown in Fig. 2. A hollow fiber membrane is preferred because, even if the membrane area is large, the membrane can be loaded into a container to create a small filter.

[0062] 1 is not particularly limited, but is, for example, 40.0 μm or more and 60.0 μm or less, more preferably 42.0 μm or more and 55.0 μm or less, in a dry state. In the cross section of the virus removal membrane 10, the pore size of the pores decreases from the primary side to the secondary side and then becomes constant, and preferably, the virus removal membrane 10 may have the densest layer near the outermost layer on the secondary side.

[0063] The logarithmic reduction value (LRV) of viruses by a virus removal membrane is not particularly limited, but is preferably 4.00 or higher because viruses are sufficiently removed by membrane filtration, and more preferably 4.50 or higher, 5.00 or higher, or 6.00 or higher. If the logarithmic reduction value of viruses is 6.00 or higher, it is considered that viruses are removed and almost no viruses leak out.

[0064] The logarithmic removal value (LRV) of 30 nm diameter gold colloids by the virus removal membrane is not particularly limited, but is, for example, 1.00 or more, preferably 1.20 or more. The logarithmic removal value of 20 nm diameter gold colloids by the virus removal membrane 10 is, for example, 1.00 or more, preferably 1.20 or more. The logarithmic removal value of 15 nm diameter gold colloids by the virus removal membrane 10 is, for example, 0.10 or more, preferably 0.20 or more. The logarithmic removal value of 10 nm diameter gold colloids by the virus removal membrane 10 is, for example, less than 0.10.

[0065] The bubble point measured for a virus removal membrane is not particularly limited, but is, for example, 1.30 MPa or more and 1.80 MPa or less, preferably 1.40 MPa or more and 1.80 MPa or less, 1.45 MPa or more and 1.80 MPa or less, or 1.50 MPa or more and 1.80 MPa or less. Furthermore, the characteristics of a virus removal membrane can also be expressed as the ratio of the bubble point (MPa) to the surface tension (N / m) of the solvent used for measurement. When a hydrofluoroether with a surface tension of 13.6 mN / m is used as the test liquid in which the membrane is immersed, the ratio of the bubble point to the surface tension is 96 to 133. More preferably, it is 103 to 133, 106 to 133, or 110 to 133.

[0066] The amount of pure water that can pass through the virus removal membrane is not particularly limited, but is, for example, 40 L / m 2 / hr / bar or more, 45L / m 2 / hr / bar or more, or 50L / m 2 / hr / bar or more. The permeation rate of pure water through the virus removal membrane is, for example, 400 L / m2 / hr / bar or less, 380L / m 2 / hr / bar or less, 360L / m 2 / hr / bar or less, or 340L / m 2 / hr / bar or less.

[0067] By including arginine in the antibody-containing solution to be filtered through the virus removal membrane, a decrease in the permeation flux (Flux) of the antibody-containing solution through the virus removal membrane is suppressed, even when the antibody-containing solution is filtered through the virus removal membrane multiple times. Therefore, the antibody filtered through the virus removal membrane can be reused repeatedly. Because antibodies are expensive, if the antibody filtered through the virus removal membrane can be reused, the cost required to implement the virus removal membrane evaluation method can be reduced.

[0068] The arginine may be L-arginine or D-arginine. The concentration of arginine in the antibody-containing solution is 350 mmol / L or more and 650 mmol / L or less. The concentration of arginine in the antibody-containing solution may be 400 mmol / L or more. The concentration of arginine in the antibody-containing solution may be 600 mmol / L or less.

[0069] Furthermore, when a solution containing an antibody is supplemented with polysorbate or sorbitol, which are known to inhibit antibody aggregation, and the solution is then filtered multiple times through a virus removal membrane, the flux of the solution containing the antibody through the virus removal membrane decreases.

[0070] The number of times an antibody has been filtered multiple times through a virus removal membrane is not particularly limited, but may be, for example, 2 or more times, 5 or more times, or 10 or more times, and 100 or less times, 50 or less times, 30 or less times, or 20 or less times.

[0071] In another aspect, the number of times an antibody has been filtered multiple times through a virus removal membrane is not particularly limited, but may be, for example, 2 or more times, 3 or more times, or 5 or more times, and 20 or less times, 15 or less times, or 10 or less times.

[0072] The antibody that has been filtered multiple times through a virus removal membrane may be an antibody that has been frozen and thawed one or more times. When frozen, the antibody may be contained in a solution containing arginine. The concentration of arginine in the solution containing the antibody when frozen is, for example, 350 mmol / L or more and 650 mmol / L or less. The concentration of arginine in the solution containing the antibody when frozen may be 400 mmol / L or more. The concentration of arginine in the solution containing the antibody when frozen may be 600 mmol / L or less.

[0073] The number of times an antibody is frozen and thawed multiple times is not particularly limited, but may be, for example, 2 or more times, 5 or more times, or 10 or more times. Furthermore, the number of times an antibody is frozen and thawed multiple times is not particularly limited, but may be, for example, 100 or less times, 50 or less times, 30 or less times, or 20 or less times.

[0074] An antibody that has been filtered multiple times through a virus removal membrane may be an antibody that has been solution-exchanged one or more times by an operation including a concentration step. The concentration step is a step of increasing the antibody concentration in an antibody-containing solution, for example, by centrifugation. The solution exchange is a step of exchanging the antibody-containing solution using a dialysis membrane, for example.

[0075] In the concentration step, the antibody may be contained in a solution containing arginine. The concentration of arginine in the solution containing the antibody in the concentration step is, for example, 350 mmol / L or more and 650 mmol / L or less. The concentration of arginine in the solution containing the antibody in the concentration step may be 400 mmol / L or more. The concentration of arginine in the solution containing the antibody in the concentration step may be 600 mmol / L or less.

[0076] In the solution exchange step, the antibody may be contained in a solution containing arginine. The concentration of arginine in the antibody-containing solution during the solution exchange step is, for example, 350 mmol / L or more and 650 mmol / L or less. The concentration of arginine in the antibody-containing solution during the solution exchange step may be 400 mmol / L or more. The concentration of arginine in the antibody-containing solution during the solution exchange step may be 600 mmol / L or less.

[0077] The number of times that an antibody undergoes solution exchange multiple times, including a concentration step, is not particularly limited, but may be, for example, 2 or more times, 5 or more times, or 10 or more times. Furthermore, the number of times that an antibody undergoes solution exchange multiple times, including a concentration step, is not particularly limited, but may be, for example, 100 or less times, 50 or less times, 30 or less times, or 20 or less times.

[0078] The antibody concentration in the antibody-containing solution when filtered through the virus removal membrane is not particularly limited, but may be, for example, 0.5 g / L or more and less than 20 g / L. The antibody concentration is not particularly limited, but may be, for example, 0.6 g / L or more, 0.8 g / L or more, or 1 g / L or more. The antibody concentration is not particularly limited, but may be, for example, 19 g / L or less, 17 g / L or less, or 15 g / L or less.

[0079] The pH range of the antibody-containing solution is not particularly limited, but may be, for example, 4.5 or more and 8.0 or less. The pH range of the antibody-containing solution is not particularly limited, but may be, for example, 5.0 or more. The pH range of the antibody-containing solution is not particularly limited, but may be, for example, 7.5 or less, or 7.0 or less. The pH of the antibody-containing solution is not particularly limited, but may be, for example, 0.5 or more, 1.0 or more, 1.5 or more, or 2.0 or more lower than the pI value of the antibody.

[0080] The solution containing the antibody may contain a buffer solution, examples of which include, but are not limited to, acetate buffer and Tris-HCl buffer.

[0081] The filtration pressure when an antibody-containing solution is filtered through a virus removal membrane is not particularly limited, but may be, for example, 0.5 bar or more and 3.5 bar or less. The filtration pressure when an antibody-containing solution is filtered through a virus removal membrane is not particularly limited, but may be, for example, 0.8 bar or more or 1.0 bar or more. The filtration pressure when an antibody-containing solution is filtered through a virus removal membrane is not particularly limited, but may be, for example, 3.0 bar or less, 2.5 bar or less, or 2.0 bar or less. [Example]

[0082] The present invention will be described in detail below with reference to examples, but the present invention is not limited to the following examples. Test methods shown in the examples are as follows.

[0083] The monoclonal antibodies used for filtration were a Chinese hamster ovary cell-derived monoclonal antibody with a pI of 8.0 (nivolumab) or a Chinese hamster ovary cell-derived monoclonal antibody with a pI of 7.0 (pembrolizumab).

[0084] (1) Water permeability measurement Using the polyethersulfone (PES) membrane of Example 1 of Japanese Patent No. 6385444, the effective area of ​​the membrane was 3.3 cm 2 A virus removal membrane of this type was assembled. Pure water at 25°C was filtered through the virus removal membrane by constant-pressure dead-end filtration at 1.0 bar, the filtration volume was measured for 10 minutes, and the pure water permeability through the virus removal membrane was calculated from the filtration volume, effective area of ​​the membrane, filtration time, and filtration pressure. In the following examples, unless otherwise specified, the virus removal membrane used is the virus removal membrane using the PES membrane described in Example 1 of Japanese Patent No. 6385444.

[0085] (2) Preparation of antibody solution A culture medium containing nivolumab-producing CHO cells was filtered using a filtration membrane (manufactured by Asahi Kasei Medical Corporation, trade name BioOptimal (registered trademark) MF-SL) to obtain a culture supernatant containing impurities and nivolumab. The resulting culture supernatant was purified by protein A affinity chromatography and ion exchange chromatography to prepare an antibody solution containing nivolumab. Using a similar method, an antibody solution containing pembrolizumab was prepared from a culture medium containing pembrolizumab-producing CHO cells.

[0086] An antibody solution containing nivolumab or pembrolizumab was concentrated using Ultrafiltration Discs 30 kDa (Merck) to an antibody concentration of 17 g / L (hereinafter referred to as the "concentration step"). The concentrated antibody solution was dialyzed using Slide-A-Lyzer Dialysis Devices 20K (ThermoFisher) against a dialysis buffer containing 50 mmol / L acetate buffer, 200 mmol / L sodium chloride, and 400 mmol / L arginine (Fujifilm Wako Pure Chemical Industries, Ltd.) and having a pH of 5.0, to exchange the antibody solution (hereinafter referred to as the "solution exchange step"). The antibody solution was diluted with a buffer having the same composition as the dialysis buffer so that the antibody concentration of the dialyzed antibody solution was 1 g / L. In the following examples, unless otherwise specified, the composition of the antibody solution is the same as the monoclonal antibody solution described in this paragraph. One liter of the monoclonal antibody solution was dialyzed using a dialysis buffer with a membrane area of ​​34 cm. 2 The solution was filtered at a constant pressure of 0.5 bar and a temperature of 25°C using a PES-0.22 µm filter and a PVDF-0.1 µm filter.

[0087] (3) Antibody solution filtration test The permeability of monoclonal antibodies through virus removal membranes was determined by the following experiment. Using the same virus removal membrane as in (1), the antibody solution (nivolumab solution or pembrolizumab solution) prepared in (2) was filtered at a constant pressure of 2.0 bar (hereinafter referred to as the "filtration step"). The flux ratio (3-hour flux / permeation volume) was calculated by dividing the flux 3 hours after the start of filtration (3-hour flux) by the permeation volume in (1). A flux ratio of 0.8 or higher was evaluated as indicating that the antibody had permeated the virus removal membrane without clogging due to aggregates.

[0088] (4) Reuse of antibody solution After the filtration test in (3), all of the antibody solution was collected in a wide-mouth bottle (hereinafter referred to as the "collection step") and stored frozen at -78°C (hereinafter referred to as the "freezing step"). When reusing the antibody, the frozen antibody solution was thawed in a water bath at 25°C (hereinafter referred to as the "thawing step"), an antibody solution was prepared according to the procedure in (2), and a filtration test was carried out according to the procedure in (3). In the following examples, one cycle of the antibody filtration step, collection step, freezing step, thawing step, concentration step, and solution exchange step was counted as one reuse.

[0089] Example 1 Filtration studies were performed using a 1 g / L nivolumab solution that was reused 10 times. The flux ratio was 0.86.

[0090] Example 2 A filtration test was carried out in the same manner as in Example 1, except that the buffer type was changed from acetic acid to Tris-HCl and the pH was changed from 5.0 to 7.0. The flux ratio was 0.86.

[0091] Example 3 Filtration was carried out in the same manner as in Example 1, except that the arginine concentration was changed from 400 mmol / L to 600 mmol / L. The flux ratio was 0.86.

[0092] Example 4 Filtration was carried out in the same manner as in Example 2, except that the arginine concentration was changed from 400 mmol / L to 600 mmol / L. The flux ratio was 0.86.

[0093] Example 5 Filtration was carried out in the same manner as in Example 1, except that the antibody concentration in the diluted antibody solution was changed from 1 g / L to 15 g / L. The flux ratio was 0.97.

[0094] Example 6 Filtration was carried out in the same manner as in Example 5, except that the buffer type was changed from acetic acid to Tris-HCl and the pH was changed from 5.0 to 7.0. The flux ratio was 0.98.

[0095] Example 7 Filtration was carried out in the same manner as in Example 5, except that the arginine concentration was changed from 400 mmol / L to 600 mmol / L. The flux ratio was 0.92.

[0096] Example 8 Filtration was carried out in the same manner as in Example 6, except that the arginine concentration was changed from 400 mmol / L to 600 mmol / L. The flux ratio was 0.95.

[0097] Example 9 Filtration was performed in the same manner as in Example 7, except that a nivolumab solution that had been reused once was used. The flux ratio was 0.86.

[0098] Example 10 Filtration was performed in the same manner as in Example 7, except that a nivolumab solution that had been reused 15 times was used. The flux ratio was 0.87.

[0099] Example 11 Filtration was performed in the same manner as in Example 7, except that a nivolumab solution that had been reused 20 times was used. The flux ratio was 0.88.

[0100] Example 12 Filtration was carried out in the same manner as in Example 7, except that the PES membrane of Example 1 of Japanese Patent No. 6,385,444 was changed to the cellulose membrane of Example 1 of Japanese Patent No. 4,024,041, and the filtration pressure was changed from 2.0 bar to 1.0 bar. The flux ratio was 0.94.

[0101] Example 13 Filtration was carried out in the same manner as in Example 7, except that the PES membrane of Example 1 of Japanese Patent No. 6385444 was changed to the cellulose membrane of Example 1 of WO 2022 / 118943. The flux ratio was 0.93.

[0102] Example 14 Filtration was carried out in the same manner as in Example 7, except that the PES membrane of Example 1 of Japanese Patent No. 6385444 was changed to the polyvinylidene fluoride (PVDF) membrane of Example 1 of Japanese Patent No. 4699207. The flux ratio was 0.96.

[0103] Example 15 Filtration was performed in the same manner as in Example 7, except that nivolumab was changed to pemprolizumab. The flux ratio was 0.81.

[0104] (Reference example 1) Filtration was performed in the same manner as in Example 7, except that new nivolumab was used instead of recycled nivolumab. The flux ratio was 0.87.

[0105] (Reference example 2) Except for using new pemprolizumab rather than recycled pemprolizumab, filtration was carried out in the same manner as in Reference Example 1. The flux ratio was 0.85.

[0106] (Reference example 3) Filtration was carried out in the same manner as in Reference Example 1, except that the arginine concentration was changed from 400 mmol / L to 0 mmol / L. The flux ratio was 0.91.

[0107] (Reference example 4) Filtration was carried out in the same manner as in Reference Example 2, except that the arginine concentration was changed from 400 mmol / L to 0 mmol / L. The flux ratio was 0.85.

[0108] [Table 1] [Table 2] [Table 3]

[0109] (Comparative Example 1) Filtration was carried out in the same manner as in Example 5, except that the arginine concentration was changed from 400 mmol / L to 200 mmol / L. The flux ratio was 0.37.

[0110] (Comparative Example 2) Filtration was carried out in the same manner as in Example 6, except that the arginine concentration was changed from 400 mmol / L to 200 mmol / L. The flux ratio was 0.68.

[0111] (Comparative Example 3) Filtration was carried out in the same manner as in Example 5, except that the arginine concentration was changed from 400 mmol / L to 700 mmol / L. The flux ratio was 0.75.

[0112] Comparative Example 4 Filtration was carried out in the same manner as in Example 6, except that the arginine concentration was changed from 400 mmol / L to 700 mmol / L. The flux ratio was 0.60.

[0113] (Comparative Example 5) Filtration was carried out in the same manner as in Example 5, except that arginine was replaced with polysorbate (PS80, Tokyo Chemical Industry Co., Ltd.) and the concentration of polysorbate was changed to 0.02%. The flux ratio was 0.45.

[0114] (Comparative Example 6) Except for changing the polysorbate concentration to 0.2%, filtration was carried out in the same manner as in Comparative Example 5. The flux ratio was 0.27.

[0115] (Comparative Example 7) Filtration was carried out in the same manner as in Example 5, except that arginine was replaced with sorbitol (Fujifilm Wako Pure Chemical Industries, Ltd.) and the sorbitol concentration was changed to 200 mmol / L. The flux ratio was 0.14.

[0116] (Comparative Example 8) Filtration was carried out in the same manner as in Comparative Example 7, except that the sorbitol concentration was changed to 700 mmol / L. The flux ratio was 0.30.

[0117] (Comparative Example 9) Except for changing nivolumab to pemprolizumab, filtration was carried out in the same manner as in Comparative Example 1. The flux ratio was 0.1.

[0118] [Table 4]

[0119] (Reference example 5) The aggregate formation state of monoclonal antibodies due to the addition of arginine was confirmed by size exclusion chromatography and particle size measurement.

[0120] (1) Size exclusion chromatography measurement of monoclonal antibody solutions A nivolumab solution containing nivolumab was prepared so that the acetate buffer concentration was 50 mmol / L, the sodium chloride concentration was 200 mmol / L, and the pH was 5.0, and arginine was added at 600 mmol / L or 0 mmol / L and the solution was allowed to stand at room temperature for 24 hours.

[0121] A size-exclusion chromatography (SEC) system was configured using an ACQUITY UPLC H-Class Bio System (Waters) as the ultra-performance, high-resolution liquid chromatograph (UPLC) system, an ACQUITY UPLC BEH200 SEC 1.7µm 4.6 x 150mmol / L (Waters) column, an ACQUITY UPLC BEH SEC Guard Column, 200A, 1.7µm (Waters) as the guard column, and a UV-visible detector (280nm). Nivolumab solutions were analyzed using a mixed buffer (pH 6.8) of 0.3mol / L sodium phosphate, 0.23mol / L arginine, and 0.13mol / L NaCl. In SEC, larger molecules elute faster, while smaller molecules elute slower. The measurement temperature was 25°C, the flow rate was 0.4 mL / min, the retention time was 7 minutes, and the injection volume was 1 μL.

[0122] As a result, as shown in Figures 3 and 4, it was confirmed that the amount of dimers and higher aggregates was reduced by adding arginine to the nivolumab solution.

[0123] (2) Particle size measurement of monoclonal antibody solution The dynamic light scattering (DLS) system used was a Zetasizer Nano ZSP (Malvern). Measurement conditions were as follows: sample volume 80 μL, equilibration time 120 seconds, equilibration temperature 25°C, measurement count 3, detection angle 173° Backscatter, and measurement interval 0 seconds. As shown in Table 3, it was confirmed that the average particle size decreased by adding arginine to the nivolumab solution. This indicates that the amount of dimers and higher aggregates was reduced. [Table 5]

Claims

1. A method for evaluating a virus removal membrane, comprising a step of filtering a solution containing an antibody through the virus removal membrane, the antibody has been filtered through the virus removal membrane one or more times, The solution contains arginine at 350 mmol / L or more and 650 mmol / L or less. method.

2. 10. The method of claim 1, wherein the antibody is an antibody that has been frozen and thawed one or more times.

3. The method of claim 1 , wherein the antibody is an antibody that has been solution-exchanged one or more times in an operation that includes a concentration step.

4. The method of claim 1 , wherein the antibody is a monoclonal antibody.

5. The method of claim 1 , wherein the concentration of the antibody is greater than or equal to 0.5 g / L and less than 20 g / L.

6. The method of claim 1, wherein the antibody has a pI of 6.0 or greater and 8.5 or less.

7. 2. The method of claim 1, wherein the pH range of the solution is 4.5 or more and 8.0 or less, and the pH value of the solution is 0.5 or more lower than the pI value of the antibody.

8. 2. The method of claim 1, wherein the solution is an acetate buffer or a Tris-HCl buffer.

9. The method according to claim 1, wherein the filtration pressure in the filtering step is 0.5 bar or more and 3.5 bar or less.

10. The method of claim 1, further comprising evaluating the antibody permeability of the virus removal membrane.

11. The pure water permeability of the virus removal membrane is 40 L / m 2 / hr / bar or more 400L / m 2 10. The method of claim 1, wherein the pressure is 1000 bar / hr / bar or less.

12. The method of claim 1 , wherein the virus removal membrane comprises a hydrophobic polymer and a hydrophilic polymer.

13. The method of claim 12, wherein the hydrophobic polymer is polyvinylidene fluoride, polyethersulfone, polysulfone, or polyethylene.

14. The method of claim 12 , wherein the hydrophilic polymer is coated onto a substrate membrane containing the hydrophobic polymer.

15. The method of claim 1 , wherein the virus removal membrane comprises cellulose.

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