Porous hollow fiber membrane and integrity test method

Abstract

The present invention is a porous hollow-fiber membrane that includes recycled cellulose, wherein the elastic limit pressure of the porous hollow fiber membrane is 200 kPa or more.

Description

[Technical field]

[0001] The present invention relates to a porous hollow fiber membrane containing regenerated cellulose, and to a method for testing the integrity of a membrane module filled with the porous membrane. [Background technology]

[0002] In biological preparations such as plasma fraction preparations derived from human blood and biopharmaceuticals, a virus removal / inactivation process has been introduced into the manufacturing process as a measure to improve safety against viruses. Among these, a virus removal method by filtration using a porous membrane is an effective method that can reduce viruses without denaturing useful proteins. Porous membranes containing regenerated cellulose have the characteristic of low protein adsorption to the membrane due to their excellent hydrophilicity, and are widely used for virus removal applications in various biological preparations (see, for example, Patent Documents 1 and 2).

[0003] In virus removal methods that involve filtration using porous membranes, integrity testing of the membrane module is required to confirm that the virus removal membrane functions effectively in the virus removal process in order to ensure the safety of the manufactured pharmaceuticals.

[0004] When large pores other than the original pore size distribution of the porous membrane, such as pinholes with a diameter of about 100 μm, occur in the porous membrane, the decrease in virus removal performance cannot be confirmed by evaluation using virus-substitute microparticles. In contrast, a method for testing the integrity of a membrane module made of porous hollow fiber membranes made of cuprammonium regenerated cellulose with an average pore size of 35 nm is to test the membrane module at a test pressure of 1 kgf / cm. 2 It is known that a leak test at 98 kPa (98 kPa) can evaluate the deterioration of virus removal performance (see, for example, Patent Document 3).

[0005] On the other hand, methods for testing the integrity of membrane modules include leak tests and diffusion tests to determine the pinhole diameter allowable for the desired virus removal performance and confirm that no defects larger than that pinhole diameter have occurred. In virus removal membranes intended to remove parvoviruses with a diameter of about 20 nm, the pinholes that affect the decrease in virus removal performance are very small, so the test pressure in leak tests, etc. must be set high. However, integrity tests that can be performed using this method are limited to porous membranes made of polyvinylidene fluoride or polysulfone-based synthetic polymers. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2015 / 156401 [Patent Document 2] International Publication No. 2017 / 170874 [Patent Document 3] Japanese Patent Application Publication No. 7-132215 Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide a porous hollow fiber membrane containing regenerated cellulose that enables evaluation of high fine particle removal performance by leak tests, etc. Another object of the present invention is to provide an integrity test method by a leak test method for a membrane module containing the porous hollow fiber membrane. [Means for solving the problem]

[0008] As described above, in a virus removal membrane intended to remove parvoviruses having a diameter of about 20 nm, the pinholes that affect the decrease in virus removal performance are very small in a leak test, so the test pressure in the leak test must be set high to improve the detection accuracy of such very small pinholes. However, the integrity test can be performed by this method only on porous membranes made of polyvinylidene fluoride or polysulfone-based synthetic polymers. If the test pressure is set high for a regenerated cellulose membrane to improve the detection accuracy of very small pinholes, the regenerated cellulose membrane cannot withstand the test pressure.

[0009] As a result of extensive research to solve the above-mentioned problems, the inventors have discovered that the elastic limit pressure of a porous hollow fiber membrane containing regenerated cellulose can be set to a specific value or higher, and that by providing such a porous hollow fiber membrane containing regenerated cellulose, it is possible to improve the accuracy of detection of minute pinholes and the like in a porous hollow fiber membrane containing regenerated cellulose in an integrity testing method by a leak test method using a membrane module containing the porous hollow fiber membrane, thereby completing the present invention.

[0010] That is, the details of the present invention are as follows. [1] A porous hollow fiber membrane containing regenerated cellulose, the porous hollow fiber membrane having an elastic limit pressure of 200 kPa or more.

[0011] [2] The porous hollow fiber membrane according to [1], wherein the ratio (R / t) of the inner diameter (R) to the membrane thickness (t) of the porous hollow fiber membrane is 8.4 or less.

[0012] [3] The porous hollow fiber membrane according to [1] or [2], wherein the membrane thickness (t) of the porous hollow fiber membrane is in the range of 20 μm or more and 70 μm or less.

[0013] [4] The porous hollow fiber membrane according to any one of [1] to [3], wherein the regenerated cellulose is regenerated cellulose by a cuprammonium method.

[0014] [5] The porous hollow fiber membrane according to any one of [1] to [4], wherein the pore size on the inner surface of the porous hollow fiber membrane is larger than the pore size on the outer surface.

[0015] [6] The porous hollow fiber membrane according to any one of [1] to [5], having a gradient structure in which the pore size decreases from the inner surface side to the outer surface side of the porous hollow fiber membrane.

[0016] [7] The permeability at filtration pressure of 27 kPa and 37°C is 10 L / (m 2 ·hr) or more 50L / (m 2 1. The porous hollow fiber membrane according to any one of [1] to [6], wherein the internal wall thickness is less than or equal to 1.

[0017] [8] The porous hollow fiber membrane according to any one of [1] to [7], having a bubble point of 1.2 MPa or more.

[0018] [9] A porous hollow fiber membrane described in any one of [1] to [8], which is used for virus removal.

[0019]

[10] The porous hollow fiber membrane described in [9], having a parvovirus removal rate (LRV) of 4.0 or more.

[0020]

[11] A method for filtering a liquid containing a biological agent using the porous hollow fiber membrane described in any one of [1] to

[10] , wherein the transmembrane pressure difference of the porous hollow fiber membrane during filtration is 150 kPa or more.

[0021]

[12] The filtration method described in

[11] , wherein the biological product-containing liquid contains at least one of immunoglobulin (polyclonal antibody), albumin, blood coagulation factor, prothrombin complex, culture medium, monoclonal antibody, antibody-drug complex, vaccine, recombinant protein, viral vector, DNA, and RNA.

[0022]

[13] The filtration method described in

[11] or

[12] , which is a filtration method for removing viruses.

[0023]

[14] A method for complete testing of a membrane module filled with the porous hollow fiber membrane according to any one of [1] to

[10] , comprising: The membrane module has an outer surface side space in contact with the outer surface of the porous hollow fiber membrane and an inner surface side space in contact with the inner surface of the porous hollow fiber membrane, Filling the outer surface side space with a liquid; Pressurizing the inner surface side space with air so that the transmembrane pressure difference of the porous hollow fiber membrane is greater than 98 kPa and is equal to or less than the elastic limit pressure of the porous hollow fiber membrane; Integrity test methods including:

[0024]

[15] A complete test method for a membrane module filled with regenerated cellulose porous hollow fiber membranes, comprising: The membrane module has an outer surface side space in contact with the outer surface of the porous hollow fiber membrane and an inner surface side space in contact with the inner surface of the porous hollow fiber membrane, Pressurizing the inner surface side space so that the transmembrane pressure difference of the porous hollow fiber membrane is 98 kPa or more and is within a range not exceeding the elastic limit pressure of the porous hollow fiber membrane; Integrity test methods including:

[0025]

[16] An integrity test method according to

[14] or

[15] , wherein the regenerated cellulose porous hollow fiber membrane is a regenerated cellulose porous hollow fiber membrane having an elastic limit pressure of 200 kPa or more.

[0026]

[17] The integrity test method according to any one of

[14] to

[16] , wherein the ratio (R / t) of the inner diameter (R) to the membrane thickness (t) of the porous hollow fiber membrane is 8.4 or less.

[0027]

[18] An integrity test method described in any of

[14] to

[17] , wherein the porous hollow fiber membrane is a virus removal membrane.

[0028]

[19] An integrity test method according to any one of

[14] to

[18] , comprising a step of visually observing air bubbles arising from the porous hollow fiber membrane.

[0029]

[20] An integrity test method described in any of

[14] to

[18] , comprising a step of measuring the pressure fluctuation value of either the outer surface side space or the inner surface side space, or a step of measuring the amount of air inflow required to maintain the pressure in either one of the spaces constant. Effect of the Invention

[0030] According to the present invention, there is provided a porous hollow fiber membrane containing regenerated cellulose, which enables evaluation of high virus removal performance by a leak test, etc. Furthermore, according to the present invention, there is provided a method of integrity testing in which a leak test method is performed on the porous hollow fiber membrane. [Brief description of the drawings]

[0031] [Figure 1] 1 is a schematic diagram of a cross section of a porous hollow fiber membrane, showing the relationship between the inner diameter (R) and the membrane thickness (t) of the porous hollow fiber membrane. [Diagram 2] 1 is a graph used to determine the elastic limit pressure of the porous hollow fiber membrane of Example 1. [Diagram 3] 1 is an image of the porous hollow fiber membrane of Example 1 observed by a scanning microscope. [Figure 4] 1 is an image of a porous hollow fiber membrane according to Example 2 observed by a scanning microscope. [Diagram 5] 1 is an image of a porous hollow fiber membrane according to Example 3 observed by a scanning microscope. [Figure 6] 1 is an image of a porous hollow fiber membrane according to Example 4 observed by a scanning microscope. [Figure 7] 1 is a graph showing the correlation between the ratio (R / t) of the inner diameter (R) to the membrane thickness (t) of a porous hollow fiber membrane and the elastic limit pressure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] The present invention will be described in detail below with reference to specific embodiments (hereinafter, referred to as the present embodiments). However, the present invention is not limited to the following embodiments, and can be embodied in any form without departing from the spirit of the present invention.

[0033] The regenerated cellulose-containing porous hollow fiber membrane according to this embodiment will be described below.

[0034] The porous hollow fiber membrane according to this embodiment is a hollow membrane having a porous structure containing a large number of pores for permeating or capturing substances. The shape of the porous hollow fiber membrane is not particularly limited, but it can have a cylindrical continuous shape. In this specification, the surface of the porous hollow fiber membrane located inside the cylinder is referred to as the inner surface, and the surface located outside the cylinder is referred to as the outer surface.

[0035] The porous hollow fiber membrane according to the present embodiment is not particularly limited as long as it is a porous hollow fiber membrane containing regenerated cellulose. The regenerated cellulose is not particularly limited as long as it is cellulose that is regenerated by another chemical treatment after being shaped with a stock solution in which natural cellulose is dissolved by chemical treatment, and examples thereof include regenerated cellulose obtained by a method of preparing from a cuprammonium cellulose solution (cuprammonium method) or a method of preparing by saponifying cellulose acetate with an alkali (saponification method).

[0036] The porous hollow fiber membrane according to the present embodiment may contain components other than regenerated cellulose, and the regenerated cellulose may be partially modified. For example, regenerated cellulose in which the cellulose hydroxyl group is esterified or partially crosslinked may be exemplified. The surface of the porous hollow fiber membrane may be coated with a polymer film. Examples of polymers for coating include polyhydroxyethyl methacrylate, a copolymer of 2-hydroxyethyl methacrylate and acrylamide, polymethoxyethyl acrylate, a copolymer of 2-hydroxyethyl methacrylate and diethylaminoethyl methacrylate, a copolymer of 2-methacryloyloxyethyl phosphorylcholine and n-butyl methacrylate, a copolymer of 2-(N-3-sulfopropyl-N,N-dimethylammonium)ethyl methacrylate and n-butyl methacrylate, hydroxypropyl cellulose, polyvinylpyrrolidone, or a copolymer of vinylpyrrolidone and vinyl acetate.

[0037] The porous hollow fiber membrane according to the present embodiment is not particularly limited as long as it is a porous hollow fiber membrane that enables evaluation of high virus removal performance in an integrity test such as a leak test or enables detection of minute pinholes, but examples thereof include porous hollow fiber membranes that exhibit an elastic limit pressure of a specific value or more. Examples of the elastic limit pressure include 200 kPa or more, 210 kPa or more, 220 kPa or more, 230 kPa or more, 240 kPa or more, or 250 kPa or more. In addition, examples of other aspects of the elastic limit pressure include 215 kPa or more, 225 kPa or more, 235 kPa or more, 245 kPa or more, 255 kPa or more, 270 kPa or more, or 280 kPa or more. The upper limit of the elastic limit pressure is not particularly limited as long as it is a pressure that can be practically applied, but examples include 1000 kPa or less, 900 kPa or less, 800 kPa or less, 700 kPa or less, 600 kPa or less, 500 kPa or less, 450 kPa or less, 400 kPa or less, 350 kPa or less, or 300 kPa or less.

[0038] The elastic limit pressure is defined as the pressure at which the expansion observed by the change in the outer diameter of the hollow fiber membrane accompanying the increase in pressure when the air is applied from the inner surface side of the hollow fiber membrane deviates from a linear change. The deviation of the hollow fiber membrane expansion from a linear change occurs due to the plastic deformation of the hollow fiber membrane. In various inspections, filtration, and integrity tests such as leak tests in the manufacturing process of the porous hollow fiber membrane, it is preferable that there is no substantial change in the virus removal ability and water permeability of the porous membrane before and after the test, and it is preferable to select a pressure below the elastic limit as the pressure used for the test. The elastic limit pressure of the porous hollow fiber membrane according to this embodiment is measured in a state in which the porous hollow fiber membrane is wetted with water.

[0039] The leak test is a method to detect the presence or absence of large holes (pinholes) that are not in the original pore size distribution of a porous membrane. The relationship between the pinhole diameter at a temperature of 20°C, the test pressure, and the gas flow rate flowing out of the pinhole is given by the choke flow equation (1). Therefore, it is possible to calculate the pinhole diameter from the test pressure value, the gas flow rate flowing out of the pinhole, and equation (1). Q = 30πR 2 (P1+0.1) (1) (In the formula, Q: flow rate (mL / min), R: pinhole diameter (μm) at a temperature of 20°C, P1: test pressure (MPa))

[0040] It is preferable to construct a system that pressurizes the porous membrane with a higher pressure and observes small changes in flow rate in order to detect smaller pinholes. On the other hand, considering the amount of air diffusion in the original pore size distribution of the porous membrane and its detection accuracy, the air flow rate at which the presence of a pinhole can be determined in a leak test is, for example, in the range of 0.2 mL / min to 0.5 mL / min, and when the set pressure is 200 kPa, the smallest pinhole diameter that can be detected is in the range of 2.7 μm to 4.2 μm, when it is 250 kPa, it is in the range of 2.5 μm to 3.9 μm, and when it is 300 kPa, it is in the range of 2.3 μm to 3.6 μm.

[0041] The lower limit of the elastic limit pressure of the porous hollow fiber membrane according to this embodiment is 200 kPa or more, preferably 220 kPa or more, more preferably 250 kPa or more, and even more preferably 300 kPa or more, from the viewpoint of being able to detect pinholes smaller than about 3 μm in diameter by setting the pressure for pressurizing the porous hollow fiber membrane in a region greater than 98 kPa in a leak test. The upper limit of the elastic limit pressure of the porous hollow fiber membrane according to this embodiment is preferably a certain value or less from the viewpoint of the flexibility required when the porous hollow fiber membrane is used as a filter module, and is exemplified by 800 kPa or less, preferably 700 kPa or less, more preferably 600 kPa or less, even more preferably 500 kPa or less, and particularly preferably 400 kPa or less.

[0042] Setting the lower limit of the elastic limit pressure of the porous hollow fiber membrane of this embodiment to 200 kPa or more is also preferable from the viewpoint that the transmembrane pressure difference during filtration, which was approximately 98 kPa for a porous hollow fiber membrane made of regenerated cellulose in the prior art, can be set to more than 150 kPa.

[0043] Setting the transmembrane pressure difference during filtration high has the economic advantage of increasing the amount of processing per unit time, and also has the advantage that the higher the transmembrane pressure difference during filtration is set, the stronger the physical binding force of the virus to the membrane is, and the more reliable the virus capture is. The transmembrane pressure difference is preferably set to about 75% or less of the elastic limit pressure of the porous hollow fiber membrane according to this embodiment, and therefore more preferably the transmembrane pressure difference during filtration is 165 kPa or more, 188 kPa or more, or 225 kPa or more. Other examples of the transmembrane pressure difference during filtration include 150 kPa or more, 200 kPa or more, and 250 kPa or more. The upper limit of the transmembrane pressure during filtration is not particularly limited as long as it is a pressure that can be practically applied, and examples thereof include 1000 kPa or less, 900 kPa or less, 800 kPa or less, 700 kPa or less, 600 kPa or less, 500 kPa or less, 450 kPa or less, 400 kPa or less, 350 kPa or less, or 300 kPa or less. Under conditions where a particularly high filtration back pressure is not applied, the transmembrane pressure is treated as synonymous with the filtration pressure of low-pressure filtration. In addition, as a means for controlling the transmembrane pressure, the object to be filtered may be pumped so that the pressure applied to the porous hollow fiber membrane is constant, or the object to be filtered may be filtered at a constant speed within a range that does not exceed the elastic limit pressure of the porous hollow fiber membrane.

[0044] Here, the transmembrane pressure difference of the porous hollow fiber membrane according to the present embodiment refers to the pressure difference between the pressure on the inner surface side of the porous hollow fiber membrane and the pressure on the outer surface side of the porous hollow fiber membrane, and is exemplified by the value obtained by subtracting the pressure on the outer surface side of the porous hollow fiber membrane from the pressure on the inner surface side of the porous hollow fiber membrane.

[0045] The porous hollow fiber membrane according to this embodiment preferably has a ratio (R / t) of the inner diameter (R (μm)) to the membrane thickness (t (μm)) of 8.4 or less. As shown in Fig. 1, the inner diameter (R) and membrane thickness (t) are measured from a cross-sectional image of a hollow fiber in a dry state, where the inner diameter is the inner surface diameter of the hollow fiber and the membrane thickness is the vertical distance between the inner and outer surfaces of the hollow fiber. Hereinafter, unless otherwise specified, the inner diameter (R) and membrane thickness (t) are values ​​measured in a dry state.

[0046] Based on the profile of the porous hollow fiber membrane, the present inventors have found that there is a specific correlation between the ratio of the inner diameter (R) to the membrane thickness (t) of the porous hollow fiber membrane and the elastic limit pressure for a porous hollow fiber membrane containing regenerated cellulose suitable for filtering objects with particle diameters of slightly less than 20 nm to approximately 100 nm (see Examples 1 to 4 and Comparative Examples 1 to 2 described below, and FIG. 7). From the viewpoint of realizing an elastic limit pressure of 200 kPa or more for the porous hollow fiber membrane, the upper limit value of R / t is preferably 8.4 or less. In correspondence with the more preferable lower limit value of the elastic limit pressure described above, the more preferable range of R / t is 8.0 or less, and the even more preferable range is 7.7 or less. From the viewpoint of stably producing a hollow fiber shape and satisfying the balance between the supply flow rate and the permeation flow rate as a hollow fiber filtration membrane, the lower limit value of R / t is preferably 2.0, that is, the inner diameter is at least twice the membrane thickness.

[0047] The thickness of the porous membrane according to this embodiment is preferably in the range of 20 μm or more and 70 μm or less. From the viewpoint of ease of designing the area for capturing micro-substances by the sieve effect of the porous membrane, the thickness is preferably 20 μm or more. In addition, from the viewpoint of ease of setting the permeability of the porous membrane to a high level, the thickness is preferably 70 μm or less. The thickness of the porous membrane is more preferably in the range of 30 μm or more and 60 μm or less, and even more preferably in the range of 40 μm or more and 50 μm or less.

[0048] The porous hollow fiber membrane according to the present embodiment is preferably made of regenerated cellulose obtained by the cuprammonium method, from the viewpoint of achieving both the porous structure and excellent hydrophilicity required for a virus removal membrane. An example of a method for producing the porous hollow fiber membrane according to the present embodiment using the cuprammonium method is described below.

[0049] First, a spinning stock solution with a cellulose concentration of 6% to 8% by mass, an ammonia concentration of 4% to 5% by mass, and a copper concentration of 2% to 3% by mass, in which cellulose is dissolved in a cuprammonium solution, an internal coagulation liquid which is an aqueous solution with an acetone concentration of 30% to 50% by mass and an ammonia concentration of 0.5% to 1.0% by mass, and an external coagulation liquid which is an aqueous solution with an acetone concentration of 20% to 40% by mass and an ammonia concentration of 0.2% or less by mass, are prepared. The spinning stock solution may contain an inorganic salt such as sodium sulfate in the range of about 0.03% to 0.1% by mass in order to adjust the speed of microphase separation of the stock solution.

[0050] Next, it is preferable to discharge the spinning dope from the annular double spinneret at a rate of 2 mL / min to 5 mL / min, and at the same time, discharge the internal coagulation liquid from the central spinneret provided at the center of the annular double spinneret at a rate of 0.3 mL / min to 3.0 mL / min. For example, in order to obtain a hollow fiber inner diameter and membrane thickness that realize an elastic limit pressure exceeding 200 kPa, in order to make the membrane thickness of the porous hollow fiber membrane to be manufactured in a preferred range of 20 μm to 70 μm, it is more preferable to set the dope discharge rate to 2.5 mL / min to 4 mL / min and the internal coagulation liquid rate to 0.3 mL / min to 1.6 mL / min. A more preferable method is to set the internal coagulation liquid rate to 0.3 mL / min to 1.4 mL / min. The spinning dope and internal coagulation liquid discharged from the annular double spinneret are immediately immersed in the external coagulation liquid to coagulate the internal coagulation liquid and the external coagulation liquid, and then the membrane is wound up on a frame.

[0051] The immersion of the spinning dope and the internal coagulation liquid in the external coagulation liquid can be achieved by immersing the spinning dope and the internal coagulation liquid in the external coagulation liquid stored in a coagulation bath, by allowing the dope to flow down and drop together with the external coagulation liquid in a spinning funnel to proceed with coagulation, or by using a U-shaped capillary tube as a method using a spinning funnel. The method using a U-shaped capillary tube is preferred from the viewpoint of realizing a membrane structure having a high fine particle removal rate by suppressing elongation during the coagulation process.

[0052] From the viewpoint of forming the porous hollow fiber membrane according to this embodiment into a membrane structure that stably achieves the water permeability and virus removal performance described below, it is preferable to control the temperature of the external coagulation liquid at a predetermined temperature selected from the range of 25° C. to 45° C. A more preferable temperature range is 30° C. to 45° C., and an even more preferable range is 35° C. to 45° C.

[0053] The wound hollow fiber membrane is immersed in a 2% to 10% by mass dilute aqueous sulfuric acid solution and then washed with pure water to regenerate the cellulose. The moisture in the hollow fiber membrane is then replaced with an organic solvent such as methanol or ethanol. After that, both ends of the hollow fiber membrane bundle are fixed and the hollow fiber membrane is stretched by 1% to 8% and dried under reduced pressure at 30°C to 60°C and 5 kPa or less to obtain a dry hollow fiber membrane.

[0054] The filtration method according to this embodiment includes filtering a solution to be filtered using the porous hollow fiber membrane according to this embodiment.

[0055] In order to effectively capture fine particles in an aqueous solution, the porous hollow fiber membrane according to this embodiment is preferably used in a filtration method (internal pressure filtration method) in which the liquid flows from the inner surface side of the hollow fiber to the outer surface side, and from the viewpoint of realizing a high flow rate and suppressing clogging of the porous membrane, it is preferable that the pore size on the inner surface is larger than the pore size on the outer surface. Furthermore, from the viewpoint of improving the capture performance of fine particles and suppressing the influence of clogging, it is more preferable that the membrane has a gradient structure in which the pore size decreases from the inner surface side to the outer surface side, and further includes a homogeneous structure with little change in pore size in order to capture the fine particles to be removed. Here, the pore size refers to the size of the pore part in the image obtained by observing the inner surface, outer surface, or cross section of the hollow fiber membrane with an optical microscope or a scanning electron microscope, and the degree of difference by the comparison is preferably clear enough to be visible in the microscope image.

[0056] The porous hollow fiber membrane according to this embodiment can be used for removing viruses, which is one type of fine particle removal, and can be particularly suitably used as a membrane for removing parvovirus, which is classified as a small virus among viruses.

[0057] When the porous hollow fiber membrane according to this embodiment is used as a parvovirus removal membrane, the water permeability at a filtration pressure of 27 kPa and 37° C. is 10 L / (m 2 ·hr) or more 50L / (m 2 ·hr) or less is preferable.

[0058] Water permeability is the flow rate per unit time when water is filtered by internal pressure filtration, and a virus removal membrane designed to have a high water permeability can complete the virus removal process of biological products in a short time. On the other hand, water permeability is a measure of the overall average pore size of the porous hollow fiber membrane, and is designed according to the size of the virus particles to be removed. Therefore, from the perspective of ensuring the capture performance of parvoviruses with diameters of less than 20 nm, a water permeability of 10 L / (m 2 ·hr) or more 50L / (m 2 ·hr) or less, and 15 L / (m 2 ·hr) or more 45L / (m 2 The reason why the water permeability is specified here under the conditions of a filtration pressure of 27 kPa and 37°C is because this is a common condition for measuring the water permeability when calculating the average pore size (nm) of a porous membrane in this technical field.

[0059] The water permeability of the porous hollow fiber membrane according to this embodiment is 20 L / (m) under filtration conditions of a filtration pressure of 98 kPa and 25°C. 2 ·hr) or more 100L / (m 2 ·hr) or less is preferable, and 30L / (m 2 ·hr) or more 85L / (m 2 hr) or less is more preferable.

[0060] In addition, when the porous hollow fiber membrane according to the present embodiment is used as a parvovirus removal membrane, the bubble point is not particularly limited as long as it is 1.2 MPa or more. Here, the bubble point is a scale indicating the size of the maximum pore of the porous hollow fiber membrane. From the viewpoint of reliably capturing parvoviruses having a diameter of less than 20 nm, the lower limit of the bubble point is preferably 1.3 MPa or more, more preferably 1.4 MPa or more, and even more preferably 1.5 MPa or more. From the viewpoint of realizing the above-mentioned water permeability, the upper limit of the bubble point is preferably 2.4 MPa or less, more preferably 2.3 MPa or less, and even more preferably 2.2 MPa or less. The bubble point refers to the pressure at which the leaking gas flow rate is 2.4 mL / min when a test module is prepared by sealing one end of the porous hollow fiber membrane according to the present embodiment and pressurizing the other end with air or nitrogen while immersing the test module in a fluorine-based liquid having a low surface tension.

[0061] When the porous hollow fiber membrane according to the present embodiment is used as a virus removal membrane, the virus removal ability of the porous hollow fiber membrane according to the present embodiment is determined by the 50% tissue culture infectiousness (TCID 50 The viral removal rate is evaluated as the logarithmic reduction value (LRV), which is the logarithmic ratio of the viral removal rate (GFR / mL).

[0062] When the porous hollow fiber membrane according to this embodiment is used as a parvovirus removal membrane, it has a concentration of 6.0 TCID 50 / mL or more 8.0TCID 50 The original solution containing parvovirus of 1 / mL or less was placed under a transmembrane pressure of 196 kPa and 150 L / m 2 The parvovirus removal rate when filtered through a volume of 100 ml of the solution is preferably 4.0 or more. Taking into consideration the ability to accommodate a larger filtration volume and the effect on filtration pressure fluctuations, the parvovirus removal rate under the same conditions is more preferably 4.5 or more, and even more preferably 5.0 or more. The LRV is preferably measured using the virus-containing protein solution described in (5-A) in the method for measuring LRV described in (5) of the Examples described below.

[0063] When the porous hollow fiber membrane according to this embodiment is used as a virus removal membrane, the biological preparation contained in the solution to be purified is not particularly limited, but examples include immunoglobulins (polyclonal antibodies), albumin, blood coagulation factors, prothrombin complexes, culture media, monoclonal antibodies, antibody-drug conjugates, vaccines, recombinant proteins, viral vectors, DNA, and RNA.

[0064] The subject of purification of the porous hollow fiber membrane according to this embodiment may be a protein such as an antibody. The antibody may be a human antibody, or may be an antibody protein derived from a mammal other than human, such as a cow or a mouse. Alternatively, the antibody may be a chimeric antibody protein with human IgG, or a humanized antibody. A chimeric antibody with human IgG is an antibody in which the variable region is derived from an organism other than human, 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 an organism other than human, but the other framework region (FR) is derived from humans. Humanization further reduces immunogenicity compared to chimeric antibodies.

[0065] 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, depending on the structure of the constant region. However, the antibody to be purified by the porous hollow fiber membrane according to the embodiment 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. The subclass of the antibody to be purified by the porous hollow fiber membrane according to the embodiment may be any of the classes. 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 the antibody to be purified by the porous hollow fiber membrane according to the embodiment.

[0066] Furthermore, antibodies can also be classified according to origin. The antibody to be purified by the porous membrane hollow fiber membrane according to the embodiment may be any of natural human antibodies, recombinant human antibodies produced by gene recombination technology, monoclonal antibodies, and polyclonal antibodies. Among these antibodies, the antibody to be purified by the porous membrane hollow fiber membrane according to the embodiment is preferably a monoclonal antibody from the viewpoint of demand and importance as an antibody drug, but is not limited thereto.

[0067] Examples of the antibody include monoclonal or polyclonal antibodies including any of IgM, IgD, IgG, IgA, or IgE. The antibody may be derived from, for example, a plasma product or a cell culture medium. When obtaining the antibody by cell culture, animal cells or microorganisms can be used as the cells. The animal cells are not particularly limited in type, but include CHO cells, Sp2 / 0 cells, NS0 cells, Vero cells, PER.C6 cells, etc. The microorganisms are not particularly limited in type, but include Escherichia coli and yeast.

[0068] When the porous hollow fiber membrane according to the present embodiment is used as a membrane for removing viruses contained in a protein-containing solution to be purified by filtration, the protein-containing solution can be filtered with a high transmembrane pressure and a high virus removal rate. The transmembrane pressure is preferably set to about 75% or less of the elastic limit pressure of the porous hollow fiber membrane according to the present embodiment, and therefore, more preferable transmembrane pressure during filtration is 150 kPa or more, 165 kPa or more, 188 kPa or more, 200 kPa or more, 225 kPa or more, or 250 kPa or more. The upper limit of the transmembrane pressure is not particularly limited as long as it is a pressure that can be practically applied, and examples thereof include 1000 kPa or less, 900 kPa or less, 800 kPa or less, 700 kPa or less, 600 kPa or less, 500 kPa or less, 450 kPa or less, 400 kPa or less, 350 kPa or less, or 300 kPa or less.

[0069] The longer the time for which the porous hollow fiber membrane is subjected to filtration, the greater the advantage of the increased amount of filtration, and thus 30 minutes or more is an example, with 1 hour or more being preferred, 3 hours or more being more preferred, and 6 hours or more being even more preferred. The upper limit of the filtration time is not particularly limited, but examples include 7 days or less, 6 days or less, 5 days, 2 days or less, 4 days or less, and 3 days or less.

[0070] In addition, when the porous membrane hollow fiber membrane according to the present embodiment is used in the virus removal step, another purification step may be performed in the previous step, the subsequent step, or both. Examples of the equipment used in the other purification step include a protein A carrier, an ion exchange chromatography, a depth filter, an ultrafiltration membrane, a prefilter, and activated carbon.

[0071] As a further example of this embodiment, a method for testing the integrity of a membrane module filled with porous hollow fiber membranes will be described.

[0072] The membrane module filled with the porous hollow fiber membrane according to the present embodiment is composed of a cylindrical body, a lid, and a potting agent. The hollow fiber membrane housed inside the cylindrical body is joined to the cylindrical body at both ends by the potting agent, forming a space (hereinafter referred to as the "outer surface side space") that contacts the inner surface of the cylindrical body, the outer surface of the hollow fiber, and the outer surface of the hollow fiber surrounded by the surface of the potting agent, and the outer surface side space is connected to the outside world through a nozzle possessed by the cylindrical body. In the membrane module, two lids are joined so that both ends where the hollow fiber membrane and the cylindrical body are joined by the potting agent form a certain space, and a space (hereinafter referred to as the "inner surface side space") that contacts the inner surface of the lid, the inner surface of the hollow fiber, and the inner surface of the hollow fiber surrounded by the other surface of the potting agent is formed, and the inner surface side space is connected to the outside world through a nozzle possessed by the lid.

[0073] The membrane module filled with the porous hollow fiber membrane of this embodiment can be used for filtration by passing a liquid under pressure through a nozzle in the lid to move the liquid from the inner surface side space to the outer surface side space via the membrane, and recovering the liquid from the nozzle in the cylindrical body.

[0074] In the method for testing the integrity of a membrane module according to the present embodiment, as described above, the membrane module has two spaces, an outer surface side space in contact with the outer surface of the porous hollow fiber membrane and an inner surface side space in contact with the inner surface of the porous hollow fiber membrane, (1) filling the outer surface side space with a liquid; and (2) A step of pressurizing the inner surface side space with air so that the transmembrane pressure difference of the porous hollow fiber membrane is greater than 98 kPa and is within a range not exceeding the elastic limit pressure of the porous hollow fiber membrane.

[0075] In the method of testing the integrity of a membrane module according to this embodiment, the liquid may be filled into the space on the outer surface side of the membrane module through a nozzle on the cylindrical body of the membrane module, or through a nozzle on the lid body to fill the liquid in a manner similar to that used for filtration.

[0076] When filling the space on the outer surface side of the membrane module with liquid, it is more preferable to carry out the procedure shown below from the viewpoint of reducing the influence of microbubbles in the porous membrane.

[0077] The membrane module was placed upright, and a flow rate of 2 L / (m) was applied from the nozzle of the lower cover of the membrane module. 2 Next, the upper inner side of the two nozzles in the cylindrical body are opened to fill the space on the inner surface with liquid at a flow rate of 1 L / (m 2 The liquid is filled into the outer surface space by a filtering operation of about 10 minutes. Finally, the liquid in the inner surface space is discharged from the nozzle of the lower cover of the membrane module.

[0078] The liquid used in the membrane module integrity test method according to the present embodiment is not particularly limited as long as it does not change the membrane structure of the porous hollow fiber membrane according to the present embodiment, but it is preferable to use water, which is easy to replace with the liquid to be filtered, for the integrity test before or after use of the membrane module. If the water contains foreign matter or microair, it may block part of the structure of the porous hollow fiber membrane and affect the results of the integrity test, so it is preferable to use water that has been treated with an ultrafiltration membrane, reverse osmosis membrane, degassing membrane, or the like. On the other hand, from the viewpoint of performing a more accurate measurement, a fluorocarbon-based liquid with low surface tension may be used.

[0079] In the integrity test of the membrane module according to this embodiment, the pressure to which the air in the inner surface space is pressurized is a pressure at which the transmembrane pressure difference of the porous hollow fiber membrane is greater than 98 kPa and is equal to or less than the elastic limit pressure of the porous hollow fiber.

[0080] In the integrity test, pinholes in the porous hollow fiber membrane can be detected with high accuracy by increasing the pressure for pressurizing the air in the space on the inner surface side. However, pressurizing the porous hollow fiber membrane with a pressure exceeding the elastic limit pressure causes plastic deformation in the porous hollow fiber membrane, so it is preferable to test at or below the elastic limit pressure, more preferably at about 85% or less of the elastic limit pressure, and even more preferably at about 75% or less.

[0081] In the integrity test of parvovirus removal membranes, it is important to determine a parvovirus removal rate of 4.0 or more. The allowable pinhole size of the porous hollow fiber membrane in the membrane module is experimentally determined according to the membrane area, and is 0.001 m 2 About 3μm, 0.01m 2 At 0.1m, it is about 6.5μm. 2 At 12.5μm, 1m 2 It is about 33 μm.

[0082] On the other hand, as mentioned above, there is a limit to the accuracy of pinhole determination in integrity testers that use leak tests to measure pressure fluctuations and flow rates. When the set pressure is 200 kPa, the smallest pinhole diameter that can be detected is in the range of 2.7 μm to 4.2 μm, when it is 250 kPa, the range is 2.5 μm to 3.9 μm, and when it is 300 kPa, the range is 2.3 μm to 3.6 μm. Therefore, when performing integrity testing on a membrane module with a small area, it is preferable to set the pressure to 200 kPa or more.

[0083] As a method for determining the integrity test method of the membrane module according to this embodiment, it is preferable to select one from a method of visually observing air bubbles generated from the porous hollow fiber membrane, a method of measuring the pressure fluctuation value of one of the two spaces of the membrane module, or a method of measuring the amount of air inflow required to maintain the pressure in one of the spaces constant.

[0084] The visual observation method involves visually observing continuous air bubbles that are generated when there are defects such as pinholes in the porous hollow fibers when the inner surface space of a membrane module, in which the outer surface space is filled with liquid, is pressurized with air from a nozzle on one of the lids.

[0085] Although it is a qualitative method, the method of measuring air flow rate described later cannot provide a highly accurate judgment of 0.001 m due to equipment limitations. 2 or 0.01m 2 This is a particularly effective means for a membrane module having a small membrane area.

[0086] In order to determine whether a membrane module having a small membrane area has no defects in the porous hollow fibers and has achieved a parvovirus removal rate of 4.0 or more due to the designed pore size distribution, it is preferable to adopt a method in which the inner surface side space is pressurized with a set pressure of at least 150 kPa or more, and then visually observes whether continuous air bubbles are not generated in the outer surface side space for 30 seconds, preferably 60 seconds.

[0087] Among the methods for determining the integrity test of the membrane module according to this embodiment, a method for measuring the pressure fluctuation value of either the outer surface side space or the inner surface side space of the membrane module, or a method for measuring the amount of air inflow required to maintain the pressure in either one of the spaces constant will be described.

[0088] In these methods, the membrane module is arranged in an upright position, the inner surface side space of the membrane module is pressurized with air from a nozzle on the upper cover of the membrane module, and a circuit is formed to discharge the air leaking from the upper nozzle of the cylindrical body, and a regulator for adjusting the pressure, a flow meter, and a pressure sensor are arranged on the pressurizing side, and a flow meter is arranged on the discharge side according to each method. Also, a container may be arranged so as to communicate with the nozzle on the cover below the membrane module in order to reduce the effect of liquid remaining in the porous membrane during measurement.

[0089] The pressure change and air flow rate change when the membrane module is pressurized measured by these methods are a combination of the change due to the amount of air diffusion inside the porous hollow fiber membrane and the change due to defects such as pinholes in the porous hollow fiber. For the integrity test, it is necessary to measure in advance the change caused by air diffusion due to the pore size distribution of the original porous membrane, and to set a margin to prevent erroneous judgment. Therefore, the judgment value (threshold value) of the pressure fluctuation value or flow rate fluctuation value for judging whether defects such as pinholes in the porous hollow fiber have occurred is experimentally obtained and is not particularly limited. For example, the threshold value can be appropriately set by performing a leak test on multiple (e.g., nine) membrane modules and taking into account the average value and deviation of the pressure fluctuation value or flow rate fluctuation value. EXAMPLES

[0090] The present invention will be described in more detail below with reference to examples. However, the present invention is not limited to the following examples, and can be carried out in any form without departing from the spirit of the present invention.

[0091] (1) Method for measuring the elastic limit pressure of a porous hollow fiber membrane A measurement module is prepared by sealing one end of a 50 mm long porous hollow membrane with a hardening liquid resin such as urethane resin so that air does not leak out, and inserting the other end into a microcoupler (MC-04PH, manufactured by Nitto Kohki Co., Ltd.) and gluing it with a hardening liquid resin such as urethane resin so that the hollow part is not filled. A pressure device equipped with a microcoupler (MC-10SM, manufactured by Nitto Kohki Co., Ltd.) is separately prepared so that a pressure control valve, a pressure gauge, and the microcoupler of the measurement module can be connected to the piping for supplying compressed air. The measurement module is connected to the pressure device while immersed in water, and compressed air is supplied to the hollow part by increasing the pressure at 20 kPa intervals. The outer diameter of the hollow fiber is measured with a dimension measuring instrument (LS-9006M, manufactured by Keyence Co., Ltd.). The outer diameter change rate (%) at each measurement pressure is calculated using the following formula, and a graph is created with the measurement pressure (kPa) on the X axis and the outer diameter change rate (%) on the Y axis. Outer diameter change rate (%) = (D / D0-1) x 100 (In the formula, D: outer diameter (μm) at each pressure, D0: initial outer diameter value (μm) in the non-pressurized state)

[0092] Next, find the regression line equation (Y = aX) that passes through the origin using five measured values ​​at 20 kPa intervals from 20 kPa to 100 kPa, and add 1 to the right-hand side of that equation, which signifies a 1% increase in the rate of change in outer diameter, to derive the equation (Y = aX + 1). Add the line based on the derived equation to the graph above, and among the plotted pressures that do not exceed the rate of change in outer diameter on the line, the highest pressure is determined as the elastic limit pressure of the measurement module.

[0093] The test is carried out on six or more measurement modules, and the average value is regarded as the elastic limit pressure of the porous hollow fiber membrane.

[0094] (2) Measurement method for inner diameter and film thickness A cross-sectional slice of the porous hollow fiber membrane was prepared, and an image was taken at 200x magnification using a microscope (Keyence Corporation, model VHX-5000). The membrane thickness of the hollow fiber cross section on the image was measured at at least 20 points around the entire circumference, and the average value was used as the measured membrane thickness.

[0095] The area of ​​the hollow portion of the cross section of the hollow fiber in the image is determined by image processing, and the inner diameter is calculated as the diameter when the area is approximated as a circle.

[0096] (3) Method for measuring water permeability of porous hollow fiber membrane A measurement module is prepared by bundling 10 porous hollow fiber membranes, attaching a polyethylene tube that can be connected to a water permeability measuring device to one end of the bundle with adhesive, and adjusting and sealing the other end of the hollow fiber so that the effective length is 16 cm.

[0097] The permeability measuring device is equipped with a mechanism for discharging water at a constant pressure from a conduit section to which the polyethylene tube of the measuring module can be connected, a mechanism for quantifying the amount of discharged liquid with high precision, a mechanism for measuring the time it takes to determine the amount of discharged liquid, a bathtub in which the measuring module is immersed, and a mechanism for regulating the temperature of the discharged water and bath water.

[0098] The measurement module is immersed in a water bath at 37°C, and the duct of the water permeability measuring device is connected to the polyethylene tube of the measurement module, and the time required to pass 1 mL of water at 37°C at 27 kPa is measured. The membrane area is calculated based on the inner diameter (μm) measurement results of the porous hollow fiber membrane prepared under the same conditions as the porous hollow fiber membrane of the measurement module, and the measured value of the time required to pass 1 mL of water. 2 , water permeability per hour (L / (m 2 hr) is calculated.

[0099] The test is carried out on three or more evaluation modules, and the average value is regarded as the water permeability of the porous hollow fiber membrane.

[0100] (4) Method for measuring the bubble point of a porous hollow fiber membrane A test module (effective length 8 cm) was created in which one end of the porous hollow fiber membrane was sealed and the other end was fixed to a metal coupler with urethane resin so that it could be pressurized with air or nitrogen. A tube was attached to the test module, and 3M Novec 7200 high-performance liquid (trademark, manufactured by 3M Japan Ltd.) was poured into the tube to immerse the porous hollow fiber membrane in the liquid.

[0101] The bubble point measurement device is equipped with a pressure adjustment mechanism that applies pressure to the inner surface side of the porous hollow fiber membrane through a metal coupler, allowing the pressure to be gradually increased, and a pressure display mechanism, and is also equipped with a flow meter that can measure the gas flow rate flowing out of the tube of the test module.

[0102] The metal coupler of the test module is attached to the end of the pressurizing mechanism, and the line of the flow measurement mechanism is attached to the end of the tube of the test module, and the pressure (MPa) is detected when the leaking gas flow rate is 2.4 mL / min when the pressure is gradually increased. The test is performed on three or more test modules, and the average value is the bubble point value.

[0103] (5) Method for measuring virus LRV in porous hollow fiber membrane Using a known technique, the small membrane module shown in FIG. 1 of JP 2013-17990 A was 2 The membrane area is created as follows.

[0104] The solution to be filtered is prepared by the method described in (5-A) or (5-B) below.

[0105] (5-A) To prepare a virus-containing protein solution, first, a polyclonal antibody (human IgG) (Venoglobulin-IH, manufactured by Venesys) is diluted with water for injection (Otsuka Pharmaceutical) to obtain an antibody solution with an antibody concentration of 1 mg / mL. The salt concentration is adjusted to 0.1 mol / L using a 1 mol / L NaCl aqueous solution. The hydrogen ion exponent (pH) is then adjusted to 4.0 using 0.1 mol / L HCl or 0.1 mol / L NaOH to obtain a protein solution. 1.0 vol% porcine parvovirus (PPV, Animal Biological Products Association) is added to the obtained protein solution and thoroughly stirred to obtain a virus-containing protein solution.

[0106] (5-B) As a virus-containing solution, an aqueous solution of 0.2% porcine parvovirus (PPV, type VR742, purchased from the American Type Culture Collection (ATCC)) was added to an aqueous solution of pH 4.5, 0.02 mol / L acetic acid, and 0.1 mol / L NaCl to prepare an aqueous solution.

[0107] Prepared 0.001m 2 Using a small membrane module and the virus-containing protein solution or the virus-containing solution, a filtration volume of 150 L / m was obtained by dead-end internal pressure filtration using the virus-containing protein solution (5-A). 2 When using the virus-containing solution (5-B), the filtration volume is 5 L / m 2 The filtration pressure is selected according to the elastic limit pressure of the porous hollow fiber membrane, and the appropriate pressure is 98 kPa for porous hollow fibers whose elastic limit pressure is less than 200 kPa, and 196 kPa for porous hollow fibers whose elastic limit pressure exceeds 200 kPa.

[0108] Next, to measure the viral infectivity, 3% BenchMark Fetal Bovine Serum (trademark, manufactured by Gemini Bio-Products) and 1% PENICILLIN STREPTOMYCIN SOL (trademark, manufactured by Life Technologies Corporation) in Dulbecco's Modified Eagle Medium (1X), liquid + 4.5 g / L D-Glucose +L-Glutamine - Sodium Pyruvate (trademark, manufactured by Life Technologies Corporation, hereafter referred to as D-MEM) solution (hereafter referred to as 3% FBS / D-MEM) were prepared after inactivation by heating in a water bath at 56°C for 30 minutes. The original solution and the filtrate were each diluted 10 times and 10 times with 3% FBS / D-MEM. 2 Double, 10 3 Double, 10 4 Double and 10 5 Dilute twice.

[0109] Next, PK-13 cells (No. CRL-6489, purchased from ATCC) were diluted with 3% FBS / D-MEM to a cell concentration of 2.0 × 10 5Prepare a diluted cell suspension (cells / mL) and dispense 100 μL into each of the ten wells of a 96-well round-bottom cell culture plate. In addition, dispense 100 μL each of the original filtrate and its dilution, and the filtrate and its dilution, prepared for every eight wells. Then, culture the cells at 37°C in a 5% carbon dioxide atmosphere for 10 days.

[0110] The 50% tissue culture infection value (TCID 50 ) is measured.

[0111] In other words, chicken preserved blood (trademark, manufactured by Japan Bio-Test Laboratories Co., Ltd.) was diluted 5-fold with PBS(-) adjusted solution of Dulbecco's PBS(-) Powder (trademark, manufactured by Nissui Pharmaceutical Co., Ltd.), and then centrifuged at 2500 rpm, 4°C, for 5 minutes. The supernatant was removed by suction, and the resulting precipitate was again diluted 200-fold with PBS(-) adjusted solution. 100 μL of the diluted solution was dispensed into all wells of a cell culture plate. After leaving the plate to stand for 2 hours, the adsorption of red blood cells to the surface of the cell tissue was observed to evaluate the virus infection. The proportion of virus infection was confirmed for the original filtrate, the filtrate, and each diluted solution, and the infectivity titer (TCID 50 Calculate the concentration (μg / mL).

[0112] The logarithmic reduction ratio (LRV) of a virus is LRV=log 10 (C0 / C F ) where C0 is the infectious titer (TCID 50 / mL), C F is the infectivity value of the filtrate after filtration through a virus removal membrane (TCID 50 / mL).

[0113] 0.001m 2 The filtration rate of the virus-containing protein solution through the membrane module is 150 L / m 2 The time it takes for the membrane area to reach 2 , filtration volume per hour (L / (m 2hr)).

[0114] (6) 0.001 m 2 Measuring method for water permeability of membrane module 0.001m 2 A membrane module was prepared, and pure water was filtered through an ultrafiltration membrane at a temperature of 25°C and a transmembrane pressure difference of 98 kPa for 10 minutes using the internal pressure filtration and dead-end method. The filtrate was weighed and the membrane area was calculated as 1 m. 2 , water permeability per hour (L / (m 2 hr)).

[0115] (7) 0.001 m 2 How to measure colloidal gold LRV in membrane modules A solution containing colloidal gold with a particle size of approximately 20 nm, AGP-HA20 (trademark, Asahi Kasei Medical Corporation), was diluted with distilled water for injection (Otsuka Pharmaceutical Co., Ltd.) and a 0.27% by mass aqueous solution of SDS (sodium lauryl sulfate), and the absorbance at a wavelength of 526 nm, as measured using an ultraviolet-visible spectrophotometer (Shimadzu Corporation, model UV-2450), was adjusted to 1.00 to prepare a base solution of colloidal gold solution to be filtered.

[0116] 0.001m 2 A membrane module was prepared, and the colloidal gold solution was used for internal pressure filtration, using the dead-end method at a temperature of 25°C, a transmembrane pressure difference of 25 kPa, and a filtration volume of 2 L / m. 2 Filtration was carried out under the conditions of 0.5 L / m 2 to 2.0 L / m 2 The filtrate is sampled.

[0117] Using an ultraviolet-visible spectrophotometer (Shimadzu Corporation, model UV-2450), the absorbance of the original solution and the filtrate at a wavelength of 526 nm was measured, and the LRV was calculated as 10 The logarithmic removal rate (LRV) of colloidal gold particles was calculated from the formula (A / B), where A is the absorbance of the original solution and B is the absorbance of the filtrate.

[0118] [Example 1] Cotton linter (average molecular weight 1.44 × 10 5) was dissolved in a cuprammonium solution prepared by a known method, and the solution was filtered and degassed to prepare a spinning solution containing 7.5% by mass of cellulose, 4.4% by mass of ammonia, and 2.7% by mass of copper. An aqueous solution containing 38% by mass of acetone and 0.65% by mass of ammonia was prepared as an internal coagulation liquid, and an aqueous solution containing 28% by mass of acetone was prepared as an external coagulation liquid.

[0119] The spinning dope (central spinneret) and the internal coagulation liquid (outer spinneret) prepared using a circular double spinneret were discharged at 3.78 mL / min and 0.69 mL / min, respectively, and introduced into the external coagulation liquid flowing at 140 mL / min in a U-shaped funnel capillary tube with a diameter of 7 mm to form a hollow fiber membrane, which was then wound up in water at a winding speed (spinning speed) of 10 m / min. The wound hollow fiber was regenerated in a 3% by mass sulfuric acid aqueous solution to regenerate the cellulose of the hollow fiber membrane, and further washed with water. The moisture of the obtained hollow fiber membrane bundle was replaced with ethanol, and then the bundle was fixed at both ends and stretched by 3.5%, and vacuum dried under conditions of 40 ° C and 3 kPa to obtain the porous hollow fiber membrane of Example 1.

[0120] The elastic limit pressure, inner diameter (R), membrane thickness (t), water permeability, and bubble point of the obtained porous hollow fiber membrane according to Example 1 were measured by the above-mentioned various measuring methods, and the results are shown in Table 1. In addition, a graph created to derive the elastic limit pressure is shown in Figure 2.

[0121] A cross-section of the obtained porous hollow fiber membrane was prepared by freeze-fracturing, and a photograph of the cross-section was taken using a scanning electron microscope (Hitachi High-Tech Corporation, Model S-4700) at an accelerating voltage of 1.0 kV and a magnification of 2000 times. The photograph is shown in Figure 3.

[0122] Next, using the porous hollow fiber membrane of Example 1, a small membrane module similar to that shown in FIG. 1 of JP 2013-17990 A was fabricated by a known technique using a 0.001 m 2 The membrane area was 0.001 m 2 A membrane module was used, and the virus LRV (using the virus-containing protein solution described in (5-A) above), water permeability, and gold colloid LRV were measured. The results are shown in Table 1.

[0123] Furthermore, to confirm that the performance of the porous hollow fiber had not changed when the integrity test was performed, the obtained 0.001 m 2 With the outer surface area of ​​the porous hollow fiber membrane of the membrane module filled with pure water, the inner surface side of the porous hollow fiber membrane was pressurized with air at 250 kPa for 10 minutes, and then the water permeation rate and gold colloid LRV were measured. To show the difference between the results obtained without applying pressure and those obtained before and after pressurization, the ratios of the water permeation rate and gold colloid LRV before and after pressurization are shown in Table 1.

[0124] The reason why the inventors chose to evaluate colloidal gold removal performance as an alternative method to evaluate virus removal performance is that virus evaluation methods have a measurement limit that depends on the virus concentration in the solution, and furthermore, virus solutions contain many non-infectious particles, so that in order to determine subtle differences in membrane structure, it is preferable to evaluate the removal ability of colloidal gold particles.

[0125] [Examples 2 to 4 and Comparative Examples 1 and 2] In comparison with Example 1, the manufacturing conditions of the porous hollow fiber membrane, i.e., the discharge amount of the spinning dope, the acetone concentration of the internal coagulation liquid, the ammonia concentration of the internal coagulation liquid, the discharge amount of the internal coagulation liquid, the acetone concentration of the external coagulation liquid, the ammonia concentration of the external coagulation liquid, and the flow rate of the external coagulation liquid, were changed to the conditions shown in Table 1, and the porous hollow fiber membranes of Examples 2 to 4 and Comparative Examples 1 and 2 were manufactured, respectively.

[0126] The elastic limit pressure, inner diameter (R), membrane thickness (t), water permeability, and bubble point of the obtained porous hollow fiber membranes of Examples 2 to 4 and Comparative Examples 1 and 2 were measured, and the results were also compared with those of Example 1. 2 A membrane module was created and the results of virus LRV (using the virus-containing protein solution described in (5-A) above), water permeability evaluation, and gold colloid particle removal evaluation were performed, and are shown in Table 1. The filtration rate of the virus-containing protein solution using the porous hollow fiber membrane of Example 2 was 145 LMH. The filtration rate of the virus-containing protein solution using the porous hollow fiber membrane of Comparative Example 1 was 73 LMH.

[0127] Photographs of cross-sectional pieces of the porous hollow fiber membranes of Examples 2 to 4 observed in the same manner as in Example 1 are shown in Figs. 4 to 6, respectively.

[0128] 7 shows a graph in which the elastic limit pressure is plotted against the ratio (R / t) of the inner diameter (R) to the membrane thickness (t) of the porous hollow fiber membranes according to Examples 1 to 4 and Comparative Examples 1 and 2. It was confirmed that there is a high correlation between the ratio (R / t) of the inner diameter (R) to the membrane thickness (t) of the porous hollow fiber membrane and the elastic limit pressure.

[0129] The porous hollow fiber membranes of Examples 1 to 4 exhibited an elastic limit pressure of 200 kPa or more, and had a ratio (R / t) of the inner diameter (R) to the membrane thickness (t) of 8.4 or less. The parvovirus LRV at 196 kPa filtration (using the virus-containing protein solution described in the above (5-A)) was a high value of 4.5 or more.

[0130] 0.001m 2 The performance change of the porous hollow fiber membrane when a pressure load of 250 kPa was applied for 10 minutes in the membrane module was an increase of about 10% in the water permeation rate and a decrease of about 0% to 5% in the gold colloid LRV. This is considered to be due to the average pore size being slightly enlarged by the pressure load, resulting in a slight increase in the water permeation rate and a slight decrease in the gold colloid removal performance. However, this level of change is within the acceptable range of performance change due to the integrity test. Therefore, it can be determined that the integrity test can be applied to the porous hollow fiber membrane according to the embodiment at a setting of 250 kPa. In addition, in Examples 1 to 4, it was confirmed that the ratio showing the change in gold colloid LRV before and after the pressure load decreased below 1 in accordance with the decrease in the elastic limit pressure of the porous hollow fiber membrane, that is, the gold colloid LRV decreased after the pressure load. A comparison between Example 3 and Example 4 showed that the set pressure for the integrity test is more preferably about 80% or less of the elastic limit pressure of the porous hollow fiber membrane.

[0131] On the other hand, the porous hollow fiber membranes according to Comparative Examples 1 and 2 have an elastic limit pressure of less than 200 kPa and a ratio (R / t) of the inner diameter (R) to the membrane thickness (t) of more than 8.4. The porous hollow fiber membranes according to Comparative Examples 1 and 2 have a preferable water permeability and parvovirus LRV (using the virus-containing protein solution described in (5-A) above), but the permeability is less than 0.001 m 2 The quality changes before and after the membrane module was pressurized at 250 kPa for 10 minutes were problematic for practical use.

[0132] That is, in Comparative Example 1, although the change in water permeability was within the acceptable range of +11%, the gold colloid LRV showed a higher performance after the pressure load was applied. Although not bound by theory, the cause is presumed to be that the pores were blocked due to localized plastic deformation caused by the pressure. If the virus removal ability of the porous hollow fiber membrane is evaluated higher than it actually is due to the pressure applied during the integrity test, this can cause the problem of a membrane that actually fails the integrity test being erroneously judged to pass.

[0133] On the other hand, in Comparative Example 2, the water permeability after pressure loading increased by about 40%, and the gold colloid LRV decreased by 10%. Therefore, it is considered that the average pore size was clearly enlarged due to plastic deformation. The 40% increase in water permeability due to pressure application during the integrity test is an unacceptable change in the quality of the porous hollow fiber membrane before and after the integrity test, and the integrity test is not valid.

[0134] Therefore, an integrity test in which a load of 200 kPa or more is applied to the porous hollow fiber membranes of Comparative Examples 1 and 2 cannot be applied.

[0135] [Example 5] From the outside of the porous hollow fiber membrane of Example 2, an excimer laser processing machine (manufactured by Sumitomo Heavy Industries, Ltd., model INDEX-800, wavelength 243 nm, rated output 80 W, repetition frequency 100 Hz, pulse energy 400 mJ) was used to perform laser processing with a spot diameter of 12 μm and a fluence of 2.1 J / cm. 2The irradiation was performed under the condition of 150 shots, and pinholes with a diameter of approximately 3 μm were created on the surface of the porous hollow fiber.

[0136] 0.001 m according to Example 2 2 In preparing the membrane module, about 12 to 13 porous hollow fiber membranes were used. In Example 5, the 0.001 m porous hollow fiber membranes according to Example 5 were prepared in the same manner as in Example 2, except that one of the membranes was a porous hollow fiber membrane having a 3 μm diameter pinhole prepared as described above. 2 A membrane module was constructed.

[0137] 0.001 m according to Example 5 2 When the space on the outer surface side of the porous hollow fiber membrane of the membrane module was filled with water and the space on the inner surface side of the porous hollow fiber membrane was pressurized to 216 kPa, the generation of continuous bubbles was visually confirmed in about 30 seconds. 2 Similarly, when the space on the outer surface side of the porous hollow fiber membrane of the membrane module was filled with water and the space on the inner surface side of the porous hollow fiber membrane was pressurized to 216 kPa, no air bubbles were observed even after 60 seconds. Since the elastic limit pressure of the porous hollow fiber membrane of Example 2 is 360 kPa, 216 kPa was selected as the test pressure with a margin of 100 kPa or more.

[0138] 0.001 m according to Example 5 2 The parvovirus LRV of the membrane module (using the virus-containing solution described in (5-B) above) was measured by the method described in the above-mentioned "Method for measuring virus LRV of porous hollow fiber membrane," and the LRV was calculated to be 4.2.

[0139] From the results of this example, the 0.001 m 2 It was confirmed that the integrity test of the membrane module by visual leak inspection can be passed if no continuous bubbles are generated under pressurized conditions of 216 kPa for 60 seconds.

[0140] [Comparative Example 3] 0.001 m according to Example 5 2When the space on the outer surface side of the porous hollow fiber membrane of the membrane module was filled with water and the space on the inner surface side of the porous hollow fiber membrane was pressurized to 98 kPa, no air bubbles were observed even after 60 seconds.

[0141] [Example 6] Using the porous hollow fiber membrane of Example 2, a membrane with a membrane area of ​​0.1 m similar to that shown in FIG. 4 of JP 2010-259992 A was prepared by a known technique. 2 A membrane module was created.

[0142] 0.1 m according to Example 6 2 The membrane module was connected to a Planova leak tester (trademark, Asahi Kasei Medical Co., Ltd., model PLT-AM10) so that the space on the inner surface side of the porous hollow fiber membrane could be pressurized while the outer surface side space of the porous hollow fiber membrane was filled with water. A leak test was performed on nine membrane module samples with a pressure setting of 196 kPa and a measurement time of 30 seconds, and the average pressure fluctuation value, which was the measurement result, was 43.6 Pa, with a deviation of 8.7 Pa.

[0143] The Planova leak tester (trademark, manufactured by Asahi Kasei Medical, model PLT-AM10) is a device that measures the pressure increase in the space on the outer surface side of a porous hollow fiber membrane when the space on the inner surface side of the porous hollow fiber membrane is held at a constant pressure.

[0144] In addition, the same excimer laser processing method as in Example 5 was used to prepare the porous hollow fiber membrane of Example 2 in which pinholes of diameters of 3, 6, 9, 12, 15, 18, and 21 μm were formed, and 0.1 m3 tubes each including one porous hollow fiber membrane of each pinhole size were prepared. 2 A membrane module was created.

[0145] 0.1m containing one porous hollow fiber membrane of various pinhole sizes 2 For the membrane module, the pinhole diameter required to realize parvovirus LRV4 or higher was evaluated to be 12.5 μm or less based on the relationship between pinhole size and parvovirus LRV (using the virus-containing solution described in (5-B) above).2 A leak test was carried out on the membrane module using a Planova leak tester (trademark, manufactured by Asahi Kasei Medical Co., Ltd., model PLT-AM10) in the same manner as described above, and the pressure fluctuation value was 3450 Pa. This value was the same as that of a normal 0.1 m hollow fiber that does not contain pinholes. 2 This was sufficiently high compared to the average pressure fluctuation value of 43.6 Pa for the membrane module. Therefore, by setting an appropriate threshold value for the pressure fluctuation value, it is possible to use a leak tester for the integrity test and 2 It was shown that it is possible to determine whether a membrane module has performance equal to or lower than that of the parvovirus LRV4.

[0146] [Example 7] 0.1 m according to Example 6 2 The membrane module was connected to a Palltronic Flowstar (trademark, manufactured by Pall, Type-IV, measured value displayed to two decimal places, measurement range 0.1-1000 mL / min) so that the space on the outer surface side of the porous hollow fiber membrane could be pressurized while the space on the inner surface side of the porous hollow fiber membrane was filled with water. A leak test was carried out on nine membrane module samples with a pressure setting of 196 kPa and a measurement time of 15 minutes, and the average value of the air flow rate fluctuation value, which is the measurement result, was 0.105 mL / min, with a deviation of 0.030 mL / min.

[0147] The Palltronic Flowstar (trademark, manufactured by Pall, Type-IV) is a device equipped with a mechanism for supplying air to compensate for the pressure loss caused by diffusion into the membrane when the space on the inner surface side of a porous hollow fiber membrane is maintained at a constant pressure, and the flow rate of the supplied air is measured by a flow meter.

[0148] In the same device, a 0.1 m3 tube containing one porous hollow fiber membrane with a pinhole size of about 12 μm in diameter prepared in Example 6 was 2 When a leak test was performed on the membrane module in the same manner as described above, the measured air flow rate fluctuation was 4.35 mL / min. This value was the same as that of a normal 0.1 m 2This was sufficiently high compared to the average air flow rate fluctuation value of the membrane module, 0.105 mL / min. Therefore, by setting an appropriate threshold value for the air flow rate fluctuation value taking into account the average value and deviation, it was possible to use the same device for integrity testing and obtain a value of 0.1 m 2 It was shown that it is possible to determine whether a membrane module has performance equal to or lower than that of the parvovirus LRV4.

[0149] [Example 8] 0.1 m according to Example 6 2 The membrane module was connected to a Sartocheck (trademark, manufactured by Sartorius, Type-4Plus, measurement value displayed to one decimal place, measurement range 0.1 to 3000 mL / min) so that the space on the outer surface side of the porous hollow fiber membrane could be pressurized while the space on the inner surface side of the porous hollow fiber membrane was filled with water. A leak test was performed on nine membrane module samples with a pressure setting of 196 kPa and a measurement time of 15 minutes, and the measurement results showed that the average value of the air flow rate fluctuation value was 0.24 mL / min, with a deviation of 0.05 mL / min.

[0150] Sartocheck (trademark, manufactured by Sartorius, Type-4Plus) is an instrument that measures the pressure reduction due to diffusion into the membrane when the space on the inner surface side of a porous hollow fiber membrane is held at a constant pressure, and converts the reduced pressure into a diffusion flow rate from information on the volume of the space on the inner surface side.

[0151] In the same device, a 0.1 m3 tube containing one porous hollow fiber membrane with a pinhole size of about 12 μm in diameter prepared in Example 6 was 2 When a leak test was performed on the membrane module in the same manner as described above, the measured air flow rate fluctuation was 4.5 mL / min. This value was the same as that of a normal 0.1 m hollow fiber module that does not contain pinholes. 2 This was sufficiently high compared to the average air flow rate fluctuation value of the membrane module, 0.24 mL / min. Therefore, by setting an appropriate threshold value for the air flow rate fluctuation value taking into account the average value and deviation, it was possible to use the same device for integrity testing and obtain a value of 0.1 m 2 It was shown that it is possible to determine whether a membrane module has performance equal to or lower than that of the parvovirus LRV4.

[0152] [Comparative Example 4] The 0.1 m 2 The air flow rate fluctuation measurements were performed in the same manner using nine samples of membrane modules, except that the pressure setting was changed from 196 kPa to 98 kPa. As a result, the air flow rate fluctuation value was less than 0.10 mL / min in the device of Example 7, and the air flow rate fluctuation value was 0.0 mL / min or 0.1 mL / min in the device of Example 8. The measurement results, which were less than 0.1 mL / min, the lower measurement limit of each device, did not confirm that the measurement of a normal membrane module had been performed correctly, and the measurement of the 0.1 m sample including the hollow fiber in which a pinhole had been formed was not performed. 2 Since it was not possible to establish an appropriate threshold between the measured values ​​of the membrane module and the pressure, it was shown that it was inappropriate to use both devices at 98 kPa.

[0153] [Example 9] 0.001 m according to Example 2 2 For the measurement of parvovirus LRV (using the virus-containing protein solution described above in (5-A)) using a membrane module, 150 L / m 2 The filtrate obtained after filtration is 150L / m 2 After filtration, release the pressure for 3 hours and then increase the pressure further to 15L / m 2 The parvovirus LRV was measured by the same method, except that the filtrate was obtained by filtering the sample. The evaluation results of all six evaluation modules were determined to be parvovirus LRV of 5.3 or more.

[0154] [Example 10] Six evaluation modules were measured in the same manner as in Example 9 (using the virus-containing protein solution described in (5-A) above), except that the filtration pressure was changed from 196 kPa to 150 kPa. As a result, the parvovirus LRV was determined to be 5.3 or more in all cases.

[0155] [Example 11] Six evaluation modules were measured in the same manner as in Example 9 (using the virus-containing protein solution described in (5-A) above), except that the filtration pressure was changed from 196 kPa to 98 kPa. As a result, five of the six modules were determined to have a parvovirus LRV of 5.3 or more, while one module was determined to have a parvovirus LRV of 4.1. [Table 1] [Industrial Applicability]

[0156] The present invention is suitable as a porous hollow fiber membrane containing regenerated cellulose, and enables an integrity test method in which a leak test method is performed alone on the porous hollow fiber membrane.

Claims

1. A porous hollow fiber membrane comprising regenerated cellulose, The elastic limit pressure is 200 kPa or more, The ratio (R / t) of the inner diameter (R) to the membrane thickness (t) of the porous hollow fiber membrane is 4.8 or more and 7.7 or less, The thickness (t) is in the range of 40.5 μm or more and 49.6 μm or less, The bubble point is 1.2 MPa or more, Used to remove viruses, The parvovirus clearance rate (LRV) is 4.0 or more. Porous hollow fiber membrane.

2. The porous hollow fiber membrane according to claim 1, wherein the inner diameter (R) is 239.9 μm or more and 313.0 μm or less.

3. 3. The porous hollow fiber membrane according to claim 1, wherein the regenerated cellulose is regenerated by a cuprammonium method.

4. 4. The porous hollow fiber membrane according to claim 1, wherein the pore size at the inner surface of the porous hollow fiber membrane is larger than the pore size at the outer surface.

5. The porous hollow fiber membrane according to claim 1 , having a gradient structure in which the pore size decreases from the inner surface side to the outer surface side of the porous hollow fiber membrane.

6. The water permeability at filtration pressure of 27 kPa and 37°C is 10 L / (m 2 ・hr) or more 50L / (m 2 6. The porous hollow fiber membrane according to claim 1 , wherein the thermal expansion coefficient is 0.05-0.25 s or less.

7. A method for filtering a liquid containing a biological agent using a porous hollow fiber membrane described in any one of claims 1 to 6, wherein the transmembrane pressure difference of the porous hollow fiber membrane during filtration is 150 kPa or more.

8. A complete test method for a membrane module filled with porous hollow fiber membranes containing regenerated cellulose, the porous hollow fiber membranes having an elastic limit pressure of 200 kPa or more, comprising: The membrane module has an outer surface side space in contact with the outer surface of the porous hollow fiber membrane and an inner surface side space in contact with the inner surface of the porous hollow fiber membrane, Filling the outer surface side space with a liquid; Pressurizing the inner surface side space with air so that the transmembrane pressure difference of the porous hollow fiber membrane is greater than 98 kPa and is equal to or less than the elastic limit pressure of the porous hollow fiber membrane; Including, The ratio (R / t) of the inner diameter (R) to the membrane thickness (t) of the porous hollow fiber membrane is 4.8 or more and 7.7 or less, The thickness (t) is in the range of 40.5 μm or more and 49.6 μm or less, The bubble point of the porous hollow fiber membrane is 1.2 MPa or more, The porous hollow fiber membrane is used for removing viruses, The parvovirus removal rate (LRV) of the porous hollow fiber membrane is 4.0 or more. Integrity test methods.

9. The method for testing integrity according to claim 8, further comprising a step of visually observing air bubbles arising from the porous hollow fiber membrane.

10. 9. The integrity test method according to claim 8, further comprising a step of measuring a pressure fluctuation value of either the outer surface side space or the inner surface side space, or a step of measuring an amount of air inflow required to keep the pressure of either one of the spaces constant.

11. 9. The method of claim 8, wherein the inner diameter (R) is 239.9 μm or more and 313.0 μm or less.

12. The integrity test method described in any one of claims 8 to 11, wherein the water permeability through the porous hollow fiber membrane at a filtration pressure of 27 kPa and 37°C is 10 L / (m2·hr) or more and 50 L / (m2·hr) or less.

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