Porous hollow fiber membrane and integrity test method

Abstract

A porous hollow fiber membrane containing regenerated cellulose, the elastic limit pressure of which is 200 kPa or more.

Description

Technical Field

[0001] This invention relates to porous hollow fiber membranes containing regenerated cellulose, and to a method for testing the integrity of membrane modules filled with the porous membrane. Background Technology

[0002] For biological agents such as plasma fractionation preparations derived from human blood and biopharmaceuticals, as a countermeasure to improve their safety against viruses, a virus removal / inactivation process is introduced into their manufacturing process. Among these, virus removal methods using porous membrane filtration are an effective way to reduce viruses without denaturing useful proteins. Porous membranes containing regenerated cellulose are widely used for virus removal in various biological agents due to their excellent hydrophilicity and low protein adsorption. (See, for example, Patent Documents 1 and 2.)

[0003] For virus removal methods that utilize filtration with porous membranes, in order to ensure the safety of the manufactured drugs, it is necessary to conduct integrity tests on the membrane components to confirm that the virus removal membrane functions effectively in the virus removal process.

[0004] When large pores, such as pinholes with a diameter of approximately 100 μm, are generated in a porous membrane beyond its inherent pore size distribution, a decrease in virus removal cannot be confirmed when evaluating the membrane using fine particles as a substitute for the virus. In contrast, a known method for testing the integrity of a membrane module with an average pore size of 35 nm, formed from copper ammonia regenerated cellulose, uses a test pressure of 1 kgf / cm². 2 During a leak test at (98 kPa), the reduction in virus removal performance can be evaluated (see, for example, Patent Document 3).

[0005] On the other hand, methods for integrity testing of membrane modules include leak tests and diffusion tests to determine the permissible pinhole diameter for the desired virus removal performance and to confirm the absence of defects larger than that pinhole diameter. For virus removal membranes aimed at removing tiny viruses with a diameter of approximately 20 nm, the test pressure in leak tests and similar tests needs to be set high because the pinholes that reduce virus removal performance are so small. However, integrity testing using this method is limited to porous membranes formed from polyvinylidene fluoride, polysulfone-based synthetic polymers, etc.

[0006] Existing technical documents

[0007] Patent documents

[0008] Patent Document 1: International Publication No. 2015 / 156401

[0009] Patent Document 2: International Publication No. 2017 / 170874

[0010] Patent Document 3: Japanese Patent Application Publication No. 7-132215 Summary of the Invention

[0011] The problem the invention aims to solve

[0012] One object of the present invention is to provide a porous hollow fiber membrane containing regenerated cellulose that can be evaluated for high fine particle removal performance using methods such as leakage testing. Another object of the present invention is to provide an integrity testing method for membrane modules containing this porous hollow fiber membrane using a leakage test.

[0013] Solution for solving the problem

[0014] As mentioned above, in leak tests and similar applications, virus removal membranes designed to remove tiny viruses with a diameter of approximately 20 nm require high test pressures to improve the detection accuracy of these pinholes, as the pinholes that reduce virus removal performance are extremely small. However, integrity tests using this method are limited to porous membranes made of polyvinylidene fluoride (PVDF) or polysulfone-based synthetic polymers. For regenerated cellulose membranes, setting the test pressure too high to improve pinhole detection accuracy presents a problem: the regenerated cellulose membrane may not be able to withstand the test pressure.

[0015] In order to solve the above-mentioned problems, the inventors conducted repeated and in-depth research and found that by setting the elastic limit pressure of the porous hollow fiber membrane containing regenerated cellulose to a specific value or higher, and by providing such a porous hollow fiber membrane containing regenerated cellulose, the detection accuracy of micro-pinholes in the porous hollow fiber membrane containing regenerated cellulose is improved in the integrity test method of the leakage test method using the membrane module containing the porous hollow fiber membrane, thereby completing the present invention.

[0016] That is, the detailed contents of the present invention are as follows.

[0017] [1] A porous hollow fiber membrane containing regenerated cellulose, wherein the elastic limit pressure of the porous hollow fiber membrane is above 200 kPa.

[0018] [2] According to the porous hollow fiber membrane described in [1], the ratio (R / t) of the inner diameter (R) of the porous hollow fiber membrane to the membrane thickness (t) is 8.4 or less.

[0019] [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 more than 20 μm and less than 70 μm.

[0020] [4] The porous hollow fiber membrane according to any one of [1] to [3], wherein the regenerated cellulose is regenerated cellulose obtained by the cuprammonium method.

[0021] [5] The porous hollow fiber membrane according to any one of [1] to [4], wherein the pore size of the inner surface of the porous hollow fiber membrane is larger than the pore size of the outer surface.

[0022] [6] The porous hollow fiber membrane according to any one of [1] to [5] has an inclined structure in which the pore size decreases from the inner surface side to the outer surface side of the porous hollow fiber membrane.

[0023] [7] The porous hollow fiber membrane according to any one of [1] to [6], wherein the water permeability at a filtration pressure of 27 kPa and a temperature of 37°C is 10 L / (m³). 2 •hr) or more and 50L / (m 2 (hr) and below.

[0024] [8] The porous hollow fiber membrane according to any one of [1] to [7], wherein the bubble point is 1.2 MPa or higher.

[0025] [9] The porous hollow fiber membrane according to any one of [1] to [8] is used for virus removal.

[0026]

[10] The porous hollow fiber membrane according to [9] has a parvovirus removal rate (LRV) of 4.0 or higher.

[0027]

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

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

[0028]

[12] According to the filtration method described in

[11] , the liquid containing biological agents 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.

[0029]

[13] The filtering method described in

[11] or

[12] is a filtering method for virus removal.

[0030]

[14] An integrity test method, which is an integrity test method for a membrane module filled with the porous hollow fiber membrane described in any one of [1] to

[10] ,

[0031] The membrane module has an outer surface side space that contacts the outer surface of the porous hollow fiber membrane, and an inner surface side space that contacts the inner surface of the porous hollow fiber membrane.

[0032] The method includes: filling the space on the outer surface side with liquid; and

[0033] The space on the inner surface is pressurized by air in a manner where the intermembrane pressure difference of the porous hollow fiber membrane is greater than 98 kPa and the pressure is below the elastic limit pressure of the porous hollow fiber membrane.

[0034]

[15] An integrity test method, which is a method for testing the integrity of a membrane module filled with a porous hollow fiber membrane of regenerated cellulose.

[0035] The membrane module has an outer surface side space that contacts the outer surface of the porous hollow fiber membrane, and an inner surface side space that contacts the inner surface of the porous hollow fiber membrane.

[0036] The method involves pressurizing the inner surface space with a pressure that is 98 kPa or more between the membranes of the porous hollow fiber membrane and below the elastic limit pressure of the porous hollow fiber membrane.

[0037]

[16] According to the integrity test method described in

[14] or

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

[0038]

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

[14] to

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

[0039]

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

[14] to

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

[0040]

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

[14] to

[18] includes a step of visually observing the bubbles generated by the porous hollow fiber membrane.

[0041]

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

[14] to

[18] includes: a step of measuring the pressure variation value of any one of the outer surface side space and the inner surface side space; or a step of measuring the amount of air inflow required to keep the pressure of any one space constant.

[0042] The effects of the invention

[0043] According to the present invention, a porous hollow fiber membrane containing regenerated cellulose is provided, which can be evaluated for high virus removal performance using methods such as leak testing. Furthermore, according to the present invention, a method for conducting an integrity test using the leak testing method is provided for this porous hollow fiber membrane. Attached Figure Description

[0044] Figure 1 This is a schematic diagram of the cross-section of a porous hollow fiber membrane. It shows the relationship between the inner diameter (R) and the membrane thickness (t) of the porous hollow fiber membrane.

[0045] Figure 2 This is a graph showing the determination of the elastic limit pressure of the porous hollow fiber membrane used in Example 1.

[0046] Figure 3 The image shows the porous hollow fiber membrane of Example 1, obtained using a scanning microscope.

[0047] Figure 4 The image shown is an observation image of the porous hollow fiber membrane of Example 2 obtained using a scanning microscope.

[0048] Figure 5 The image shown is an observation image of the porous hollow fiber membrane of Example 3 obtained using a scanning microscope.

[0049] Figure 6 The image shown is an observation image of the porous hollow fiber membrane of Example 4 obtained using a scanning microscope.

[0050] Figure 7 A graph showing the relationship between the ratio of the inner diameter (R) to the membrane thickness (t) of a porous hollow fiber membrane (R / t) and the elastic limit pressure. Detailed Implementation

[0051] The present invention will now be described in detail with reference to specific embodiments (hereinafter referred to as "this embodiment"). However, the present invention is not limited to this embodiment and can be implemented in any manner without departing from the spirit of the present invention.

[0052] The porous hollow fiber membrane containing regenerated cellulose of this embodiment will be described.

[0053] The porous hollow fiber membrane of this embodiment is a hollow membrane with a porous structure containing many fine pores, used for the permeation or capture of substances. The shape of the porous hollow fiber membrane is not particularly limited, and it can have a continuous cylindrical shape. In this specification, the surface located inside the cylinder of the porous hollow fiber membrane is described as the inner surface, and the surface located outside the cylinder is described as the outer surface.

[0054] The porous hollow fiber membrane of this embodiment is not particularly limited to a porous hollow fiber membrane containing regenerated cellulose. As for the regenerated cellulose, it is not particularly limited to cellulose obtained by exfoliating a solution of natural cellulose obtained by chemical treatment and then regenerating it by other chemical treatments. Examples of regenerated cellulose obtained by a method of preparation using a cuprammonium cellulose solution (cuprammonium method) or by a method of preparation using alkali to saponify cellulose acetate (saponification method).

[0055] The porous hollow fiber membrane of this embodiment may contain components other than regenerated cellulose, or a portion of the regenerated cellulose may be modified. Examples include regenerated cellulose modified by esterification of cellulose hydroxyl groups or partially cross-linked regenerated cellulose. Furthermore, the surface of the porous hollow fiber membrane may be coated with a polymer coating. Examples of polymers used for coating include polyhydroxyethyl methacrylate, copolymers of 2-hydroxyethyl methacrylate and acrylamide, polymethoxyethyl methacrylate, copolymers of 2-hydroxyethyl methacrylate and diethylaminoethyl methacrylate, copolymers of 2-methacryloyloxyethyl phosphorylcholine and n-butyl methacrylate, copolymers of 2-(N-3-sulfopropyl-N,N-dimethylammonium)ethyl methacrylate and n-butyl methacrylate, hydroxypropyl cellulose, polyvinylpyrrolidone, or copolymers of vinylpyrrolidone and vinyl acetate.

[0056] The porous hollow fiber membrane of this embodiment is not particularly limited to any type that can be evaluated for high virus removal performance in integrity tests such as leak tests, or that can detect tiny pinholes. Examples of porous hollow fiber membranes exhibiting an elastic limit pressure exceeding a specific value can be shown. Examples of this 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. Other examples of 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 if it is the pressure that can actually be applied. 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.

[0057] The elastic limit pressure is defined as the pressure at which the expansion deviates from a linear change, observed as a result of the change in the outer diameter of the hollow fiber membrane due to the increase in pressure caused by air pressurization from the inner surface of the membrane. This deviation from a linear expansion is caused by the plastic deformation of the hollow fiber membrane. In integrity tests, such as inspections, filtration tests, and leak tests, during the manufacturing process of the porous hollow fiber membrane, it is preferable that there is no substantial change in the virus removal and water permeability of the porous membrane before and after the test, and the pressure used in the test is preferably selected to be below the elastic limit. It should be noted that the elastic limit pressure of the porous hollow fiber membrane in this embodiment is measured when the porous hollow fiber membrane is wetted with water.

[0058] Leakage testing is a method for detecting the presence or absence of large pores (pinholes) outside the original pore size distribution of a porous membrane. The relationship between the pinhole diameter, test pressure, and gas flow rate from the pinhole at 20°C is provided by equation (1) for choked flow. Therefore, the pinhole diameter can be calculated from the test pressure value, the gas flow rate from the pinhole value, and equation (1).

[0059] Q = 30πR 2 (P1+0.1) (1)

[0060] (Where, Q: flow rate (mL / min), R: pinhole diameter at 20℃ (μm), P1: test pressure (MPa))

[0061] A system that pressurizes a porous membrane with higher pressure and constructs a system for observing small flow rate changes is preferred because it can detect even smaller pinholes. On the other hand, considering the amount of air diffusion in the inherent pore size distribution of the porous membrane and its detection accuracy, in a leak test, the air flow rate at which pinholes can be detected is, for example, in the range of 0.2 mL / min to 0.5 mL / min. At a pressure of 200 kPa, the smallest pinhole diameter that can be detected is in the range of 2.7 μm to 4.2 μm, at 250 kPa it is in the range of 2.5 μm to 3.9 μm, and at 300 kPa it is in the range of 2.3 μm to 3.6 μm.

[0062] From the viewpoint that setting the pressure applied to the porous hollow fiber membrane in a leak test to a range greater than 98 kPa enables the detection of pinholes smaller than approximately 3 μm in diameter, the lower limit of the elastic limit pressure of the porous hollow fiber membrane in 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 the required flexibility when forming the porous hollow fiber membrane into a filter assembly, the upper limit of the elastic limit pressure of the porous hollow fiber membrane in this embodiment is preferably a certain value or less, for example, 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.

[0063] The lower limit of the elastic limit pressure of the porous hollow fiber membrane in this embodiment is set to 200 kPa or more. From the viewpoint that the intermembrane pressure difference during filtration of the porous hollow fiber membrane formed by regenerated cellulose in the prior art is about 98 kPa, it is preferred to set it to more than 150 kPa.

[0064] Setting a high intermembrane pressure differential during filtration offers economic advantages, including increased throughput per unit time. Furthermore, for virus removal membranes, a higher intermembrane pressure differential during filtration results in greater physical confinement of the virus to the membrane, thereby improving the reliability of virus capture. The intermembrane pressure differential is preferably set to approximately 75% or less of the elastic limit pressure of the porous hollow fiber membrane in this embodiment. Therefore, more preferably, the intermembrane pressure differential during filtration is 165 kPa or more, 188 kPa or more, or 225 kPa or more. Other examples of intermembrane pressure differentials during filtration include 150 kPa or more, 200 kPa or more, and 250 kPa or more. The upper limit of the intermembrane pressure difference during filtration is not specifically limited if it is a practically applicable pressure. 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. The intermembrane pressure difference is treated synonymously with the filtration pressure of low-pressure filtration when no particularly high filtration discharge pressure is applied. Furthermore, as a means of controlling the intermembrane pressure difference, the filtration material can be pressed through a fixed pressure applied to the porous hollow fiber membrane, or the filtration material can be filtered at a fixed speed within the elastic limit pressure range of the porous hollow fiber membrane.

[0065] Here, the intermembrane pressure difference of the porous hollow fiber membrane in this 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. An example can be shown is 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.

[0066] In this embodiment, the porous hollow fiber membrane preferably has an inner diameter (R(μm)) to membrane thickness (t(μm)) ratio (R / t) of 8.4 or less. Figure 1 As shown, the inner diameter (R) and film thickness (t) are determined from cross-sectional images obtained by cutting the hollow fiber in a dry state into circular slices. The inner diameter is the diameter of the inner surface of the hollow fiber, and the film thickness is the vertical distance between the inner and outer surfaces of the hollow fiber. Unless otherwise specified, the inner diameter (R) and film thickness (t) refer to the values ​​measured in the dry state.

[0067] Based on the curves of porous hollow fiber membranes, the inventors discovered that for porous hollow fiber membranes containing regenerated cellulose suitable for filtering objects with particle sizes of less than 20 nm to approximately 100 nm, the ratio of the inner diameter (R) of the porous hollow fiber membrane to the membrane thickness (t), R / t, has a specific correlation with the elastic limit pressure (refer to Examples 1-4 and Comparative Examples 1-2 described below). Figure 7 From the viewpoint of achieving an elastic limit pressure of 200 kPa or higher for porous hollow fiber membranes, the upper limit value of R / t is preferably 8.4 or lower. Corresponding to a more preferred lower limit value of the above-mentioned elastic limit pressure, a more preferred range of R / t is 8.0 or lower, and a further preferred range is 7.7 or lower. From the viewpoint of stably producing hollow fiber shapes and, in addition, satisfying the balance between supply flow and permeate flow as a hollow fiber filter membrane, the lower limit value of R / t is preferably 2.0, that is, more than twice the inner diameter relative to the membrane thickness.

[0068] The thickness of the porous membrane in this embodiment is preferably in the range of 20 μm or more and 70 μm or less. From the viewpoint of ease of designing areas that utilize the sieving effect of the porous membrane to capture minute particles, a membrane thickness of 20 μm or more is preferred. Furthermore, from the viewpoint of ease of setting high permeability of the porous membrane, a membrane thickness of 70 μm or less is preferred. More preferably, the membrane thickness is in the range of 30 μm or more and 60 μm or less, and even more preferably, it is in the range of 40 μm or more and 50 μm or less.

[0069] The porous hollow fiber membrane of this embodiment is preferably made of regenerated cellulose obtained by the cuprammonium process, considering that it can balance the porous structure required for virus removal membranes with excellent hydrophilicity. An example of the manufacturing method of the porous hollow fiber membrane of this embodiment using the cuprammonium process will be described below.

[0070] First, prepare a spinning solution with a cellulose concentration of 6%–8% by mass, an ammonia concentration of 4%–5% by mass, and a copper concentration of 2%–3% by mass, obtained by dissolving cellulose in a copper ammonia solution. Also prepare an aqueous solution with an acetone concentration of 30%–50% by mass and an ammonia concentration of 0.5%–1.0% by mass (the internal coagulation solution) and an aqueous solution with an acetone concentration of 20%–40% by mass and an ammonia concentration below 0.2% by mass (the external coagulation solution). From the perspective of adjusting the rate of microphase separation in the spinning solution, inorganic salts such as sodium sulfate can be present in the range of approximately 0.03%–0.1% by mass.

[0071] Next, the spinning solution is preferably ejected from the annular double-layer spinneret at a rate of 2 mL / min to 5 mL / min, while the internal coagulation solution is ejected from the central spinning outlet located in the center of the annular double-layer 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 achieves an elastic limit pressure exceeding 200 kPa, and to ensure that the membrane thickness of the manufactured porous hollow fiber membrane is within the preferred range of 20 μm to 70 μm, it is more preferable to set the spinning solution ejection rate to be 2.5 mL / min or more and 4 mL / min or less, and the internal coagulation solution rate to be within the range of 0.3 mL / min or more and 1.6 mL / min or less. A further preferred method is to keep the internal coagulation solution rate within the range of 0.3 mL / min or more and 1.4 mL / min or less. The spinning solution and internal coagulation solution ejected from the annular double-layer spinneret are immediately immersed in the external coagulation solution. After the internal and external coagulation solutions solidify, the film is wound up using a yarn frame.

[0072] Impregnation of the spinning solution and internal coagulation solution with the external coagulation solution can be exemplified by methods such as impregnating the spinning solution and internal coagulation solution with the external coagulation solution stored in the coagulation bath; coagulation occurring simultaneously as the external coagulation solution flows down and falls within a spinning funnel; and a method using a U-shaped capillary tube, similarly employing a spinning funnel. From the viewpoint of achieving a membrane structure with a high fine particle removal rate through stretching suppression during the coagulation process, the method using a U-shaped capillary tube is preferred.

[0073] From the viewpoint of forming the porous hollow fiber membrane of this embodiment into a membrane structure that stably achieves the water permeability and virus removal performance described later, the temperature of the external coagulation liquid is preferably controlled within a predetermined temperature range of 25°C or higher and 45°C or lower. A more preferred temperature range is 30°C or higher and 45°C or lower, and a further preferred range is 35°C or higher and 45°C or lower.

[0074] The wound hollow fiber membrane is immersed in a 2% to 10% by mass dilute sulfuric acid aqueous solution, followed by washing with pure water to regenerate the cellulose. Then, the water in the hollow fiber membrane is replaced by organic solvents such as methanol and ethanol. The two ends of the hollow fiber membrane bundle are fixed, and the hollow fiber membrane is stretched by 1% to 8%. It is then dried under reduced pressure at 30°C to 60°C and below 5 kPa to obtain a dried hollow fiber membrane.

[0075] The filtration method of this embodiment includes filtering the solution of the object to be filtered using the porous hollow fiber membrane of this embodiment.

[0076] In order to effectively capture fine particles in an aqueous solution, the porous hollow fiber membrane of this embodiment is preferably used in a filtration method (internal pressure filtration) in which the liquid flows from the inner surface side of the hollow fiber to the outer surface side. From the viewpoint of achieving high flow rates and suppressing porous membrane clogging, it is preferable that the pore size of the inner surface is larger than that of the outer surface. Furthermore, from the viewpoint of improving the fine particle capture performance and suppressing the effects of clogging, it is more preferable to have an inclined structure in which the pore size decreases from the inner surface side to the outer surface side, and also to include a uniform structure with small pore size variation in order to capture the fine particles to be removed. Here, pore size refers to the size of the pore portion in an image obtained by observing the inner surface, outer surface, or cross-section of the hollow fiber membrane cut into a disc using an optical microscope or a scanning electron microscope. The degree of difference obtained by this comparison is preferably clear enough to be visible using a microscope image.

[0077] The porous hollow fiber membrane of this embodiment can be used for virus removal as one of the fine particle removal methods, and can be particularly suitable as a removal membrane for small viruses, which are classified as microviruses.

[0078] When using the porous hollow fiber membrane of this embodiment as a parvovirus removal membrane, the preferred water permeability at a filtration pressure of 27 kPa and a temperature of 37°C is 10 L / (m³). 2 ●hr) or higher and 50L / (m 2 ●hr) or less.

[0079] Water permeability refers to the flow rate per unit time when filtering water using an internal pressure filtration method. A virus removal membrane with a high water permeability design allows for the removal of viruses from biological agents 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, corresponding to the size of the virus particles targeted for removal. Therefore, from the viewpoint of more practically achieving the capture performance of tiny viruses with a diameter less than 20 nm, a permeability of 10 L / (m²) is more preferable. 2 ●hr) or higher and 50L / (m 2 ●hr) or less, further preferred 15L / (m2 ●hr) or higher and 45L / (m 2 ●hr) or less. Here, the water permeability is specified under the conditions of filtration pressure of 27 kPa and 37°C because it is usually used as the measurement condition for water permeability when calculating the average pore size (nm) of the porous membrane in this technical field.

[0080] If the water permeability of the porous hollow fiber membrane of this embodiment is expressed under filtration conditions of 98 kPa filtration pressure and 25°C, it is preferably 20 L / (m³). 2 ●hr) or higher and 100L / (m 2 ·hr) or less, more preferably 30L / (m 2 •hr) or above and 85L / (m 2 (hr) and below.

[0081] Furthermore, when using the porous hollow fiber membrane of this embodiment as a parvovirus removal membrane, there is no particular limitation if the bubble point is 1.2 MPa or higher. Here, bubble point refers to a scale representing the size of the largest pore in the porous hollow fiber membrane. From the viewpoint of making the capture of parvoviruses smaller than 20 nm in diameter more reliable, the lower limit of the bubble point is preferably 1.3 MPa or higher, more preferably 1.4 MPa or higher, and even more preferably 1.5 MPa or higher. Furthermore, from the viewpoint of achieving the aforementioned water permeability, the upper limit of the bubble point is preferably 2.4 MPa or lower, more preferably 2.3 MPa or lower, and even more preferably 2.2 MPa or lower. It should be noted that bubble point refers to the pressure at which the gas flow rate leaking out when a test assembly capable of sealing one end of the porous hollow fiber membrane of this embodiment and pressurizing the other end with air or nitrogen is pressurized while the test assembly is immersed in a fluorine-based liquid with low surface tension is 2.4 mL / min.

[0082] When using the porous hollow fiber membrane of this embodiment as a virus removal membrane, the virus removal performance of the porous hollow fiber membrane of this embodiment is compared with that of the stock solution containing virus and the filtrate at 50% of the total TCID (Total Tissue Culture Infection Value). 50 The logarithm of the ratio of ( / mL) to the virus removal rate (LRV) is used to evaluate the virus removal rate.

[0083] When using the porous hollow fiber membrane of this embodiment as a parvovirus removal membrane, 6.0 TCID... 50 / mL or higher and 8.0 TCID 50 Stock solutions containing parvovirus at concentrations below 1 / mL were prepared with a membrane pressure difference of 196 kPa and a flow rate of 150 L / m. 2The parvovirus removal rate during mass filtration is preferably 4.0 or higher. Furthermore, considering the handling of large filtration volumes and the impact of filtration pressure variations, the parvovirus removal rate under the same conditions is more preferably 4.5 or higher, and even more preferably 5.0 or higher. This LRV is preferably measured using the protein solution containing the virus described in (5-A) in the LRV determination method described in Example (5) below.

[0084] When using the porous hollow fiber membrane of this embodiment as a virus removal membrane, the biological agents contained in the solution to be purified are not particularly limited, and examples include immunoglobulins (polyclonal antibodies), albumin, blood coagulation factors, prothrombin complex, culture medium, monoclonal antibodies, antibody-drug complexes, vaccines, recombinant proteins, viral vectors, DNA, and RNA.

[0085] The porous hollow fiber membrane of this embodiment can be used to purify proteins such as antibodies. The antibodies can be human antibodies or antibody proteins derived from mammals other than humans, such as cattle and mice. Alternatively, the antibodies can be chimeric antibody proteins with human IgG or humanized antibodies. Chimeric antibodies with human IgG refer to antibodies whose variable region is derived from organisms other than mice, but whose other constant regions are replaced with immunoglobulins derived from humans. Humanized antibodies, on the other hand, refer to antibodies in which the complementarity-determining region (CDR) of the variable region is derived from organisms other than humans, but whose other framework regions (FR) are derived from humans. Compared to chimeric antibodies, humanized antibodies exhibit further reduced immunogenicity.

[0086] The type (isotype) and subtype of antibodies are not particularly limited. For example, antibodies are classified into five types—IgG, IgA, IgM, IgD, and IgE—based on the different structures of their constant regions. However, the antibody targeted for purification using the porous hollow fiber membrane of this embodiment can be any of these five types. Furthermore, human antibodies include four subtypes of IgG (IgG1 to IgG4) and two subtypes of IgA (IgA1 and IgA2). The subtype of antibody targeted for purification using the porous hollow fiber membrane of this embodiment can be any. It should be noted that antibody-related proteins, such as Fc fusion proteins formed by binding proteins in the Fc region, can also contain the antibody targeted for purification using the porous hollow fiber membrane of this embodiment.

[0087] Furthermore, antibodies can also be classified according to their origin. The antibodies used for purification in the porous hollow fiber membrane of the embodiment can be any one of natural human antibodies, recombinant human antibodies manufactured using gene recombination technology, monoclonal antibodies, or polyclonal antibodies. Among these antibodies, monoclonal antibodies are suitable from the viewpoint of their necessity and importance in antibody medicine, but are not limited to this.

[0088] Examples of antibodies include monoclonal or polyclonal antibodies containing any one of IgM, IgD, IgG, IgA, or IgE. Furthermore, antibodies can be derived from plasma products or cell culture media. When antibodies are obtained through cell culture, animal cells or microorganisms can be used as cells. There are no particular limitations on the type of animal cell, but examples include CHO cells, Sp2 / 0 cells, NSO cells, Vero cells, and PER.C6 cells. There are no particular limitations on the type of microorganism, but examples include Escherichia coli and yeast.

[0089] When using the porous hollow fiber membrane of this embodiment as a membrane for removing viruses contained in a protein-containing solution that will be purified by filtration, the protein-containing solution can be filtered with a high intermembrane pressure difference and a high virus removal rate. The intermembrane pressure difference is preferably set to about 75% or less of the elastic limit pressure of the porous hollow fiber membrane of this embodiment. Therefore, more preferred intermembrane pressure differences during filtration include 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 intermembrane pressure difference is not particularly limited if it is a practically applicable pressure, and 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.

[0090] It has the advantage that the longer the filtration time of the porous hollow fiber membrane is set, the greater the filtration volume. Therefore, examples include 30 minutes or more, preferably 1 hour or more, more preferably 3 hours or more, and even more preferably 6 hours or more. There is no particular limit to the upper limit of the filtration time, and examples include 7 days or less, 6 days or less, 5 days or less, 4 days or less, and 3 days or less.

[0091] Furthermore, when the porous hollow fiber membrane of this embodiment is used in the virus removal process, other purification processes can be performed in the pre-process, post-process, or both. Examples of apparatus used in other purification processes include protein A carriers, ion exchange chromatography, depth filtration, ultrafiltration membranes, pre-filters, and activated carbon.

[0092] As a further example of this embodiment, a method for testing the integrity of a membrane module filled with a porous hollow fiber membrane will be described.

[0093] The membrane module filled with the porous hollow fiber membrane of this embodiment is composed of a cylindrical body, a cover, and a potting compound. The hollow fiber membrane, housed inside the cylindrical body, is bonded to the cylindrical body at both ends using the potting compound, forming a space (hereinafter referred to as the "outer surface space") that is surrounded by the inner surface of the cylindrical body, the outer surface of the hollow fiber, and the surface of the potting compound, and communicates with the outside of the hollow fiber. The outer surface space communicates with the outside through nozzles provided by the cylindrical body. For the membrane module, two cover bodies are attached to the two ends of the hollow fiber membrane and the cylindrical body, respectively, in a manner that forms a certain space, forming a space (hereinafter referred to as the "inner surface space") that is surrounded by the inner surface of the cover, the inner surface of the hollow fiber, and the other surface of the potting compound, and communicates with the inside of the hollow fiber. The inner surface space communicates with the outside through nozzles provided by the cover.

[0094] The membrane module filled with the porous hollow fiber membrane of this embodiment can be used for filtration by pressurizing and passing liquid through the nozzle of the cover body, causing the liquid to move from the inner surface side space to the outer surface side space via the membrane, and then recovering the liquid through the nozzle of the cylindrical body.

[0095] The membrane module integrity test method of this embodiment, as described above, has two spaces: an outer surface side space that contacts the outer surface of the porous hollow fiber membrane, and an inner surface side space that contacts the inner surface of the porous hollow fiber membrane.

[0096] The method includes:

[0097] (1) The process of filling the space on the outer surface with liquid; and

[0098] (2) A process of pressurizing the inner surface space with air in such a way that the intermembrane pressure difference of the porous hollow fiber membrane is greater than 98 kPa and the pressure is below the elastic limit pressure of the porous hollow fiber membrane.

[0099] In the membrane module integrity test method of this embodiment, the method of filling liquid into the outer surface side space of the membrane module can be to fill liquid through the nozzle of the cylindrical body of the membrane module, or to add liquid through the nozzle of the cover body, or to fill liquid using the same method as the filtration operation.

[0100] When filling the outer surface space of the membrane module with liquid, from the viewpoint of reducing the influence of microbubbles in the porous membrane, it is more preferable to perform the process by the steps shown below.

[0101] Configured with the membrane module in an upright position, the flow rate is 2 L / (m³) from the nozzles on the lower cover of the membrane module.2 The space is filled with liquid from the inner surface side for approximately 1 minute. Then, the upper inner side of the two nozzles on the cylindrical body is opened, allowing liquid to flow through at a flow rate of 1 L / (m³). 2 The filtration process, lasting approximately one minute, fills the outer surface space with liquid. Finally, the liquid in the inner surface space is discharged through the nozzles of the lower cover of the membrane module.

[0102] The liquid used in the membrane module integrity test method of this embodiment is not particularly limited as long as the membrane structure of the porous hollow fiber membrane of this embodiment does not change. For integrity tests before or after use of the membrane module, water that is easy to replace with the liquid to be filtered is preferred. If the water contains foreign matter or micro-air, it may block part of the structure of the porous hollow fiber membrane and affect the results of the integrity test. Therefore, water treated with ultrafiltration membranes, reverse osmosis membranes, or degassing membranes is preferred. On the other hand, from the viewpoint of performing measurements with higher accuracy, Freon-based liquids with low surface tension can be used.

[0103] In the integrity test of the membrane module in this embodiment, the air in the inner surface space is pressurized to a pressure that is greater than 98 kPa between the membranes of the porous hollow fiber membrane and below the elastic limit pressure of the porous hollow fiber membrane.

[0104] In the integrity test, pinholes in the porous hollow fiber membrane can be detected with good accuracy by pressurizing the air in the space on the inner surface. However, pressurizing to a pressure exceeding the elastic limit pressure of the porous hollow fiber membrane will cause plastic deformation of the membrane. Therefore, it is preferable to conduct the test below or less than the elastic limit pressure, more preferably below about 85% of the elastic limit pressure, and even more preferably below about 75%.

[0105] In the parvovirus removal membrane integrity test, a parvovirus removal rate of 4.0% or higher is crucial. Therefore, the permissible pinhole size of the porous hollow fiber membrane in the membrane module is determined based on the membrane area experiment. For 0.001 μm... 2 For approximately 3μm, for 0.01m 2 For approximately 6.5 μm, for 0.1 m 2 For example, it is about 12.5 μm, and for 1 m 2 It is approximately 33 μm.

[0106] On the other hand, for integrity testing machines that use pressure variation and flow rate measurements for leakage tests, as mentioned above, there are limits to the accuracy of pinhole detection. When the pressure is set at 200 kPa, the smallest pinhole diameter that can be detected is in the range of 2.7 μm or more and 4.2 μm or less, for 250 kPa it is in the range of 2.5 μm or more and 3.9 μm or less, and for 300 kPa it is in the range of 2.3 μm or more and 3.6 μm or less. In integrity tests of small-area membrane modules, it is preferable to set the pressure to 200 kPa or more.

[0107] As a method for determining the integrity of the membrane module in this embodiment, it is preferable to choose one of the following: visually observing the bubbles generated by the porous hollow fiber membrane; measuring the pressure variation value of any one of the two spaces in the membrane module; or measuring the amount of air inflow required to keep the pressure of any one space constant.

[0108] The visual observation method involves using air to pressurize the inner surface space of a membrane assembly whose outer surface space is filled with liquid, and then visually observing the continuous bubbles generated when defects such as pinholes exist in the porous hollow fibers.

[0109] Although it is a qualitative method, when using the airflow measurement method described later, the equipment cannot perform a high-precision determination of 0.001m. 2 0.01m 2 For membrane modules with small membrane area, this is a particularly effective method.

[0110] For membrane modules with small membrane area, in order to determine that the porous hollow fibers are free of defects and to achieve a small virus removal rate of 4.0 or higher through the pore size distribution as designed, the following method is preferred: visually observe for 30 seconds, preferably 60 seconds, when the inner surface side space is pressurized at a set pressure of at least 150 kPa, and no continuous bubbles are generated in the outer surface side space.

[0111] In the integrity test method of the membrane module in this embodiment, a method for determining the pressure variation value of any one of the outer surface side space and inner surface side space of the membrane module, or a method for determining the air inflow rate required to keep the pressure of any one space constant will be described.

[0112] In these methods, the membrane module is configured with its upright position. Air is used to pressurize the space on the inner surface side of the membrane module through nozzles on the top cover, forming a loop that allows leaked air to exit through nozzles on the upper side of the cylindrical body. Corresponding to each method, a pressure regulator, flow meter, and pressure sensor are installed on the pressurization side, and a flow meter is installed on the outlet side. Furthermore, to mitigate the influence of liquid remaining in the porous membrane during measurement, the container can be configured to communicate with nozzles on the cover below the membrane module.

[0113] The pressure and airflow changes measured by these methods when the membrane module is pressurized are a combination of changes caused by air diffusion within the porous hollow fiber membrane and changes caused by defects such as pinholes in the porous hollow fiber. For integrity testing, it is necessary to predict the changes caused by air diffusion due to the inherent pore size distribution of the porous membrane, and then set limits to prevent erroneous judgments. Therefore, experimentally determined threshold values ​​(thresholds) for judging whether defects such as pinholes in the porous hollow fiber have occurred are not particularly limited. For example, this threshold can be appropriately set by conducting leak tests on multiple (e.g., nine) membrane modules, taking into account the average and deviation of the pressure or flow changes.

[0114] Example

[0115] The following examples illustrate the invention in more detail. However, the invention is not limited to these examples and can be implemented in any manner without departing from its spirit.

[0116] (1) Method for determining the elastic limit pressure of porous hollow fiber membranes

[0117] One end of a 50mm long porous hollow membrane is sealed with a curable liquid resin such as polyurethane to prevent air leakage. The other end is then bonded and fixed with a curable liquid resin such as polyurethane to prevent the hollow portion from being filled, while the membrane is inserted into a micro-connector (Made by Nitto Koki Co., Ltd., MC-04PH). This prepares a testing component. Additionally, a pressurizing device equipped with a micro-connector (Made by Nitto Koki Co., Ltd., MC-10SM) is prepared, which can be connected to a pressure regulating valve, pressure gauge, and testing component via a compressed air supply pipe. The testing component is connected to the pressurizing device while immersed in water. Compressed air is supplied to the hollow portion by increasing the pressure at 20kPa intervals. The outer diameter of the hollow fiber is measured using a dimensional measuring instrument (Made by KEYENCE CORPORATION, model LS-9006M). The outer diameter change rate (%) caused by each measurement pressure is calculated using the following formula, and a graph is created with the X-axis representing the measurement pressure (kPa) and the Y-axis representing the outer diameter change rate (%).

[0118] Outer diameter change rate (%) = (D / D0-1)×100

[0119] (Where, D: outer diameter under various pressures (μm), D0: initial outer diameter under no pressure (μm))

[0120] Next, using five measured values ​​at 20 kPa intervals from 20 kPa to 100 kPa, a regression line formula (Y = aX) passing through the origin was derived. Then, by adding 1 to the right side of this formula, representing a 1% change in outer diameter, a new formula (Y = aX + 1) was obtained. This derived line was added to the above graph, and the highest pressure among the pressures on the curves that do not exceed the rate of change in outer diameter of the line was set as the elastic limit pressure of the component being measured.

[0121] The test was conducted on six or more components, and their average value was set as the elastic limit pressure of the porous hollow fiber membrane.

[0122] (2) Methods for determining inner diameter and film thickness

[0123] Cross-sectional sections of porous hollow fiber membranes were prepared and photographed at 200x magnification using a microscope (KEYENCE CORPORATION, model VHX-5000). The membrane thickness was measured at least 20 points along the entire circumference of the image and the average value was taken as the measured membrane thickness.

[0124] For the inner diameter, the area of ​​the hollow part of the hollow fiber cross-section in the same image is obtained through image processing, and the diameter is calculated as the diameter when approximating a circle.

[0125] (3) Method for determining the water permeability of porous hollow fiber membranes

[0126] Ten porous hollow fiber membranes are bundled together. A polyethylene tube that can be connected to a water permeability testing machine is installed at one end using an adhesive. The other end of the hollow fiber is adjusted and sealed to form an effective length of 16 cm, preparing the testing assembly.

[0127] The water permeability testing device includes a mechanism for spraying water at a constant pressure through a conduit section made of polyethylene that can be connected to the testing components; a mechanism for accurately quantifying the amount of sprayed liquid; a mechanism for measuring the quantitative time of the sprayed liquid amount; a bath for immersing the testing components; and a mechanism for regulating the temperature of the sprayed water and the bath water.

[0128] The testing module was immersed in a 37°C water bath. The time required for 1 mL of 37°C water to flow through the polyethylene tube of the testing module to the water permeability testing machine was measured at 27 kPa. The filter membrane area was calculated based on the measured value of the time for 1 mL of water to flow through the porous hollow fiber membrane manufactured under the same conditions as the testing module, using the inner diameter (μm) of the porous hollow fiber membrane. The membrane area per m² was then calculated. 2 1 hour water permeability (L / (m)) 2 ·hr).

[0129] The test was conducted on at least three evaluation components, and their average value was set as the water permeability of the porous hollow fiber membrane.

[0130] (4) Method for determining the bubble point of porous hollow fiber membranes

[0131] One end of the porous hollow fiber membrane was sealed, and the other end was fixed to a metal connector using polyurethane resin pressurized with air or nitrogen to create a test assembly (effective length 8 cm). The test assembly was installed in a tube, and 3M Novec 7200 high-performance liquid (trademark, manufactured by 3M Japan Limited) was injected into the tube to immerse the porous hollow fiber membrane in the liquid.

[0132] The bubble point measuring device has a pressure adjustment mechanism that pressurizes the inner surface of the porous hollow fiber membrane through a metal connector and can slowly increase the pressure; and a pressure display mechanism, and a flow meter that can measure the flow rate of gas flowing out of the tube of the test component.

[0133] A pressurizing mechanism was installed at the end of the metal connector of the test assembly, and a flow measuring mechanism was installed at the end of the tube of the test assembly. The pressure (MPa) at which the leaking gas flow rate was 2.4 mL / min during slow pressurization was detected. The test was conducted on at least three test assemblies, and the average value was taken as the bubble point value.

[0134] (5) Determination method of LRV virus in porous hollow fiber membranes

[0135] Using known techniques at 0.001m 2 The membrane area is as described in Japanese Patent Application Publication No. 2013-17990. Figure 1 The small membrane module described in the document.

[0136] The solution to be filtered is prepared using the method described in (5-A) or (5-B) below.

[0137] (5-A) Preparation of the virus-containing protein solution: First, a polyclonal antibody (human IgG) (Venom Globulin-IH, manufactured by Benesis Corporation) was diluted with water for injection (Otsuka Pharmaceutical) to achieve an antibody concentration of 1 mg / mL, thus obtaining an antibody solution. Next, the salt concentration was adjusted to 0.1 mol / L using a 1 mol / L NaCl aqueous solution. Then, the pH was adjusted to 4.0 using 0.1 mol / L HCl or 0.1 mol / L NaOH, thus setting it as a protein solution. 1.0 vol% porcine parvovirus (PPV, Animal Biological Preparations Association) was added to the obtained protein solution, and the mixture was stirred thoroughly to obtain a virus-containing protein solution.

[0138] As a (5-B) virus-containing solution, 0.2% porcine parvovirus (PPV, type VR742, purchased from the American Institute for the Preservation of Cultured Cells (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.

[0139] Use the prepared 0.001m 2 Small membrane modules and the aforementioned virus-containing protein solution or virus-containing solution are filtered using a dead-end internal pressure filtration method with the aforementioned virus-containing protein solution (5-A) until a filtration capacity of 150 L / m³ is achieved. 2 Up to this point, when using the above-mentioned virus-containing solution (5-B), filter until a filtration rate of 5 L / m³ is achieved. 2 The filtrate is obtained by proceeding to the next step. The filtration pressure is selected based on the elastic limit pressure of the porous hollow fiber membrane. For porous hollow fibers with an elastic limit pressure less than 200 kPa, 98 kPa is set as the adaptive pressure. For porous hollow fibers with an elastic limit pressure greater than 200 kPa, 196 kPa is set as the adaptive pressure.

[0140] Next, to determine the viral infection titer, a solution of Dulbecco's Modified Eagle Medium (1X), liquid + 4.5 g / L D-Glucose + L-Glutamine-Sodium Pyruvate (hereinafter D-MEM), which had been inactivated by heating in a 56°C water bath for 30 minutes, was prepared (hereinafter, 3% FBS / D-MEM). The solution consisted of 3% Benchmark Fetal Bovine Serum (trademark, manufactured by Gemini Bio-Products), 1% Penicillin Streptomatine Sol (trademark, manufactured by Life Technologies Corporation), and D-MEM. Separate portions of the stock solution and filtrate were taken and diluted 10-fold with 3% FBS / D-MEM. 2 times, 10 3 times, 10 4 Multiplied by 10 5 times.

[0141] Next, PK-13 cells (No. CRL-6489, purchased from ATCC) were diluted with 3% FBS / D-MEM to create a cell concentration of 2.0 × 10⁻⁶ cells / year. 5 A diluted cell suspension (cells / mL) was dispensed into each of the 10 wells of a 96-well round-bottom cell culture plate (100 μL per well). Additionally, 100 μL of the prepared stock solution and its dilution, and 100 μL of the filtrate and its dilution were dispensed into each of the 8 wells. Cells were then cultured for 10 days at 37°C under a 5% carbon dioxide atmosphere.

[0142] For cells cultured for 10 days, a 50% TCID dose was determined using the erythrocyte adsorption method (refer to the General Introduction to Virology, edited by the National Institute for Preventive Health, p. 173). 50 The determination of ).

[0143] Specifically, the method is as follows: Chicken blood (trademark, manufactured by NIPPON BIOTEST LABO) was diluted 5-fold with PBS (-) conditioning solution of DulbeccoPBS (-) powder (trademark, manufactured by Nissui Pharmaceutical Co., Ltd.), and centrifuged at 2500 rpm, 4°C, for 5 minutes. The supernatant was removed by aspiration, and the resulting precipitate was diluted again with PBS (-) conditioning solution to 200-fold. This diluted solution was dispensed into all wells of a cell culture plate (100 μL each). After standing for 2 hours, the adsorption of red blood cells onto the cell tissue surface was observed to evaluate viral infection. The ratio of confirmed viral infection to the filtrate, filtrate, and each diluted solution was used to calculate the infection titer (TCID) using the Spearman-Karber formula. 50 / mL).

[0144] The logarithmic removal rate (LRV) of the virus is expressed as LRV = log 10 (C0 / C F Calculated here, C0 represents the infection titer (TCID) of the filtrate. 50 / mL), C F This indicates the infection titer (TCID) of the filtrate after filtration using a virus removal membrane. 50 / mL).

[0145] For using 0.001m 2 The filtration rate of the virus-containing protein solution in the membrane module was measured until a filtration capacity of 150 L / m³ was reached. 2 The time up to that point, as per membrane area 1m 2 Filtration capacity per hour (L / (m)) 2 ·hr)) calculation.

[0146] (6) 0.001m 2 Method for determining the water permeability of membrane modules

[0147] Prepare 0.001m 2 The membrane module filters pure water that has passed through the ultrafiltration membrane using internal pressure filtration and a dead-end method at a temperature of 25°C, an intermembrane pressure difference of 98 kPa, and for 10 minutes. The filtrate is metered, and the filtrate is used as the filtrate per 1m² membrane area. 2 1 hour water permeability (L / (m)) 2 ·hr)) calculation.

[0148] (7) 0.001m 2 Method for determining the LRV of colloidal gold in membrane modules

[0149] A solution containing colloidal gold with a particle size of approximately 20 nm, AGP-HA20 (trademark, manufactured by Asahi Kasei Medical Co., Ltd.), was diluted with distilled water for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.) and 0.27% SDS (sodium lauryl sulfate) aqueous solution. The solution was adjusted so that the absorbance at a wavelength of 526 nm, measured using a UV / Vis spectrophotometer (manufactured by Shimadzu Corporation, model UV-2450), was 1.00. This was used to prepare the colloidal gold solution stock solution.

[0150] Prepare 0.001m 2 The membrane module, using the prepared colloidal gold solution, was subjected to internal pressure filtration, dead-end filtration at a temperature of 25°C, an intermembrane pressure difference of 25 kPa, and a filtration rate of 2 L / m³. 2 Filtration was carried out under the following conditions, for 0.5L / m 2 ~2.0L / m 2 The filtrate was sampled.

[0151] Using a UV / Vis spectrophotometer (Shimadzu UV-2450), the absorbance at 526 nm was measured for both the filtrate and the stock solution. The result was calculated using LRV = log0. 10 The logarithmic removal rate (LRV) of colloidal gold particles is calculated using the formula (A / B). In the formula, A represents the absorbance of the original filtrate, and B represents the absorbance of the filtrate.

[0152] [Example 1]

[0153] The cotton lint (average molecular weight 1.44 × 10⁻⁶) 5 The cellulose is dissolved in a copper-ammonia solution prepared by a known method, 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 is prepared as the internal coagulation solution, and an aqueous solution containing 28% by mass of acetone is prepared as the external coagulation solution.

[0154] Using a ring-shaped double-layer spinneret, the prepared spinning solution (central spinning outlet) and the inner coagulation solution (outer spinning outlet) were ejected at 3.78 mL / min and 0.69 mL / min, respectively, and introduced into the outer coagulation solution flowing at 140 mL / min in a 7 mm diameter U-shaped funnel-shaped capillary tube to form a hollow fiber membrane. The membrane was then wound in water at a winding speed (spinning speed) of 10 m / min. The wound hollow fibers were regenerated in a 3% (w / w) sulfuric acid aqueous solution to regenerate the cellulose in the hollow fiber membrane, followed by further washing. The water in the resulting hollow fiber membrane bundle was replaced with ethanol, and then both ends of the bundle were fixed. The bundle was then vacuum-dried at 40°C and 3 kPa with a stretch of 3.5% to obtain the porous hollow fiber membrane of Example 1.

[0155] For the porous hollow fiber membrane of Example 1, the results obtained by measuring the elastic limit pressure, inner diameter (R), membrane thickness (t), water permeability, and bubble point using the various measurement methods described above are shown in Table 1. Additionally, a graph was created to derive the elastic limit pressure as shown in... Figure 2 As shown.

[0156] The porous hollow fiber membrane prepared by the cryo-cutting method was cut into circular slices. The resulting images were obtained using a scanning electron microscope (Hitachi High-Technologies Corporation, model S-4700) at an accelerating voltage of 1.0 kV and a magnification of 2000x. Figure 3 As shown.

[0157] Next, using the porous hollow fiber membrane from Example 1, and employing known techniques, a 0.001m... 2 The membrane area is manufactured in accordance with Japanese Patent Application Publication No. 2013-17990. Figure 1 Similar small membrane modules, forming 0.001m 2 The results of measuring viral LRV (using the viral protein solution described in (5-A) above), water permeability, and colloidal gold LRV of the membrane module are shown in Table 1.

[0158] Furthermore, to confirm that the properties of the porous hollow fiber do not change during the integrity test, the obtained 0.001m 2 With the outer surface of the porous hollow fiber membrane of the membrane module filled with pure water, the inner surface of the porous hollow fiber membrane was pressurized with air at 250 kPa for 10 minutes. The water permeability and colloidal gold LRV were measured to obtain one result. The other result was obtained without the aforementioned pressurization. The ratio of water permeability and colloidal gold LRV before and after pressurization to represent the difference between the two results is shown in Table 1.

[0159] Here, as an alternative method for evaluating virus removal performance, the inventors chose to evaluate the removal performance of colloidal gold because virus evaluation methods have a detection limit corresponding to the virus concentration in the solution, and the virus solution contains many non-infectious particles, etc. In order to determine the subtle differences in membrane structure, the removal performance of colloidal gold particles is preferred.

[0160] [Examples 2-4, and Comparative Examples 1 and 2]

[0161] Compared to Example 1, the spinning solution ejection rate, internal coagulation liquid acetone concentration, internal coagulation liquid ammonia concentration, internal coagulation liquid ejection rate, external coagulation liquid acetone concentration, external coagulation liquid ammonia concentration, and external coagulation liquid flow rate of the porous hollow fiber membrane were changed to the conditions shown in Table 1, and porous hollow fiber membranes of Examples 2 to 4 and Comparative Examples 1 and 2 were manufactured respectively.

[0162] For the porous hollow fiber membranes obtained in Examples 2-4 and Comparative Examples 1 and 2, the elastic limit pressure, inner diameter (R), membrane thickness (t), water permeability, and bubble point were measured, and 0.001m membranes were prepared using the same method as in Example 1. 2 The results of membrane module testing, viral LRV (using the viral protein solution described in (5-A) above), water permeability evaluation, and colloidal gold particle removal evaluation are shown in Table 1. The filtration rate of the viral protein solution using the porous hollow fiber membrane of Example 2 was 145 LMH. The filtration rate of the viral protein solution using the porous hollow fiber membrane of Comparative Example 1 was 73 LMH.

[0163] The photographs obtained by observing the porous hollow fiber membranes of Examples 2-4 cut into circular slices using the same method as in Example 1 are shown below. Figures 4-6 As shown.

[0164] In addition, a graph showing the elastic limit pressure was plotted for the ratio (R / t) of the inner diameter (R) to the membrane thickness (t) of the porous hollow fiber membranes of Examples 1-4 and Comparative Examples 1 and 2. Figure 7 As shown, a high correlation was confirmed between the ratio of the inner diameter (R) to the membrane thickness (t) (R / t) of the porous hollow fiber membrane and the elastic limit pressure.

[0165] The porous hollow fiber membranes of Examples 1-4 exhibit an elastic limit pressure of over 200 kPa, and the ratio of their inner diameter (R) to membrane thickness (t) (R / t) is 8.4 or less. They can achieve a high value of over 4.5 for filtering parvovirus LRV (using the protein solution containing the virus described in (5-A) above) at 196 kPa.

[0166] As a result of utilizing 0.001m 2The performance changes of the porous hollow fiber membrane under a 250 kPa, 10-minute pressurization load on the membrane module showed an increase of approximately 10% in water permeability and a decrease of approximately 0% to 5% in colloidal gold LRV. This is attributed to a slight increase in average pore size and a slight increase in water permeability due to the pressurization load, resulting in a slight decrease in colloidal gold removal performance. However, this degree of change is within the permissible range for performance changes caused by integrity testing. Therefore, it can be determined that the porous hollow fiber membrane of the examples is suitable for integrity testing at a setting of 250 kPa. Furthermore, in Examples 1 to 4, it was confirmed that the ratio of the change in colloidal gold LRV before and after pressurization load tends to decrease to less than 1, corresponding to a decrease in the elastic limit pressure of the porous hollow fiber membrane, i.e., a tendency for the colloidal gold LRV to decrease after pressurization load. A comparison of Examples 3 and 4 shows 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.

[0167] On the other hand, the porous hollow fiber membranes of Comparative Examples 1 and 2 have an elastic limit pressure of less than 200 kPa and an inner diameter (R) to membrane thickness (t) ratio (R / t) exceeding 8.4. Although the porous hollow fiber membranes of Comparative Examples 1 and 2 have preferred water permeability and parvovirus LRV (using the virus-containing protein solution described in (5-A) above), by using 0.001 m... 2 The quality changes of the membrane module before and after being subjected to a 250 kPa, 10-minute pressurization load are problematic changes in practical use.

[0168] That is, in Comparative Example 1, although the change in permeability was within the acceptable range of +11%, the colloidal gold LRV exhibited higher performance after being subjected to pressure. While the reason is not theoretically limited, it is presumed that localized plastic deformation caused by pressure led to the closure of the pores. Because the virus removal capacity of porous hollow fiber membranes is evaluated as higher than it should be due to pressure during the integrity test, there is a possibility that a non-compliant case might be mistakenly judged as compliant during the integrity test.

[0169] On the other hand, in Comparative Example 2, the permeability increased by about 40% and the LRV of the colloidal gold decreased by 10% after the pressure load was applied. Therefore, it is believed that there was a significant increase in the average pore size due to plastic deformation. Since the 40% increase in permeability caused by the pressure during the integrity test is an unacceptable change in the quality of the porous hollow fiber membrane before and after the integrity test, the integrity test is invalid.

[0170] Therefore, the porous hollow fiber membranes of Comparative Examples 1 and 2 cannot be subjected to integrity tests with loads exceeding 200 kPa.

[0171] [Example 5]

[0172] From the outside of the porous hollow fiber membrane of Example 2, an excimer laser processing machine (Sumitomo Heavy Industries, Ltd., model INDEX-800, wavelength 243nm, rated output power 80W, repetition rate 100Hz, pulse energy 400mJ) was used, with a spot diameter of 12μm and a flux of 2.1J / cm². 2 Irradiation with 150 irradiations was used to process pinholes with a diameter of approximately 3 μm on the surface of porous hollow fibers.

[0173] Prepared as 0.001m in Example 2 2 When assembling the membrane module, approximately 12 to 13 porous hollow fiber membranes are used. In Example 5, one of the membranes used is a porous hollow fiber membrane with 3 μm diameter pinholes prepared as described above, and the rest is prepared using the same method as in Example 2 to form the 0.001m membrane of Example 5. 2 Membrane module.

[0174] To Example 5, 0.001m 2 The outer surface space of the porous hollow fiber membrane in the membrane module was filled with water, and the inner surface space of the porous hollow fiber membrane was pressurized to 216 kPa. After approximately 30 seconds, the generation of continuous bubbles could be visually confirmed. For the 0.001 m... 2 Similarly, water was filled into the outer surface space of the porous hollow fiber membrane, and the inner surface space of the porous hollow fiber membrane was pressurized at 216 kPa. Even after more than 60 seconds, no bubble formation could be confirmed. It should be noted that the elastic limit pressure of the porous hollow fiber membrane in Example 2 is 360 kPa; therefore, 216 kPa was chosen as the test pressure, providing a margin of at least 100 kPa.

[0175] For Example 5, 0.001m 2 The parvovirus LRV of the membrane module (using the virus-containing solution described in (5-B) above) was measured using the method described in the "Method for Determining Virus LRV of Porous Hollow Fiber Membrane" above, and the LRV was calculated to be 4.2.

[0176] The results of this embodiment confirm that the 0.001m in Example 1 is... 2 The integrity test of the membrane module using visual leak inspection can be set as qualified if no continuous bubbles are generated under a pressure of 216 kPa for 60 seconds.

[0177] [Comparative Example 3]

[0178] To Example 5, 0.001m 2The outer surface space of the porous hollow fiber membrane of the membrane module was filled with water, and the inner surface space of the porous hollow fiber membrane was pressurized at 98 kPa. As a result, even after more than 60 seconds, the generation of bubbles could not be confirmed.

[0179] [Example 6]

[0180] Using the porous hollow fiber membrane of Example 2, a product similar to that disclosed in Japanese Patent Application Publication No. 2010-259992 was manufactured using known techniques. Figure 4 A similar membrane area of ​​0.1m 2 Membrane modules.

[0181] In Example 6, 0.1m 2 With the outer surface space of the porous hollow fiber membrane of the membrane module filled with water, the inner surface space of the porous hollow fiber membrane can be pressurized and connected to a Planova leak tester (trademark, Asahi Kasei Medical Co., Ltd., model PLT-AM10). Leakage tests were conducted on nine membrane module samples under a pressure setting of 196 kPa and a measurement time of 30 seconds. The average pressure variation value obtained was 43.6 Pa, with a deviation of 8.7 Pa.

[0182] The Planova leak tester (trademark, manufactured by Asahi Kasei Medical Co., Ltd, model PLT-AM10) is a device for measuring the pressure increase of the outer surface space of a porous hollow fiber membrane while maintaining a constant pressure on the inner surface space of the membrane.

[0183] In addition, using the same excimer laser processing method as in Example 5, pinholes with diameters of 3, 6, 9, 12, 15, 18, and 21 μm were formed in the porous hollow fiber membrane of Example 2 to prepare hollow fibers, thus producing one 0.1m porous hollow fiber membrane containing various pinhole sizes. 2 Membrane modules.

[0184] For a 0.1m porous hollow fiber membrane containing various pinhole sizes 2 The membrane module was evaluated based on the relationship between pinhole size and parvovirus LRV (using the virus-containing solution described in (5-B) above), achieving a pinhole diameter of 12.5 μm or less for parvovirus LRV4 and above. For a 0.1 m porous hollow fiber membrane containing pinholes approximately 12 μm in diameter... 2The membrane module was subjected to a leak test using a Planova leak tester (brand name: Asahi Kasei Medical Co., Ltd., model: PLT-AM10) using the same method described above. The resulting pressure variation was 3450 Pa. This value is relative to the normal 0.1 m... 2 The average pressure variation of the membrane module, 43.6 Pa, is considered sufficiently high. Therefore, it is demonstrated that by setting an appropriate threshold for the pressure variation, the leak tester can be used for integrity testing, determining a leakage rate of 0.1 m. 2 Does the membrane module have performance for parvovirus LRV4 and below?

[0185] [Example 7]

[0186] In Example 6, 0.1m 2 With the outer surface space of the porous hollow fiber membrane module filled with water, the inner surface space of the porous hollow fiber membrane can be pressurized and connected to a Palltronic Flowstar (trademark, manufactured by Pall Corporation, Type-IV, indicating two decimal places for the measured value, measurement range 0.1–1000 mL / min). Leakage tests were conducted on nine membrane module samples under a pressure setting of 196 kPa and a measurement time of 15 minutes. The average airflow variation obtained was 0.105 mL / min, with a deviation of 0.030 mL / min.

[0187] The Palltronic Flowstar (trademark, manufactured by Pall Corporation, Type-IV) is a device that provides air to replenish the pressure reduced due to diffusion of the membrane while maintaining the inner surface space of a porous hollow fiber membrane at a constant pressure, and uses a flow meter to measure the flow rate of the supplied air.

[0188] In this Palltronic Flowstar device, for one 0.1m porous hollow fiber membrane containing pinholes with a diameter of approximately 12 μm prepared in Example 6... 2 The membrane module was subjected to a leak test using the same method described above, and the resulting airflow variation was 4.35 mL / min. This value is relative to the normal 0.1 m of a membrane module without pinhole hollow fibers. 2 The average airflow variation of the membrane module, 0.105 mL / min, is considered sufficiently high. Therefore, it is shown that by appropriately setting a threshold that takes into account both the average and deviation of the airflow variation, the Palltronic Flowstar device can be used for integrity testing and to determine the 0.1 m... 2 Does the membrane module have performance for parvovirus LRV4 and below?

[0189] [Example 8]

[0190] In Example 6, 0.1m 2 With the outer surface space of the porous hollow fiber membrane of the membrane module filled with water, the inner surface space of the porous hollow fiber membrane can be pressurized and connected to a Sartocheck (trademark, manufactured by Sartorius, Type-4 Plus, indicating one decimal place for the measured value, measurement range 0.1–3000 mL / min). Leakage tests were conducted on nine membrane module samples under a pressure setting of 196 kPa and a measurement time of 15 minutes. The average airflow variation obtained was 0.24 mL / min, with a deviation of 0.05 mL / min.

[0191] Sartocheck (trademark, manufactured by Sartorius, Type-4 Plus) is a device for measuring the pressure reduction due to diffusion of a porous hollow fiber membrane when the inner surface space is kept at a constant pressure. The device converts the aforementioned pressure reduction into diffusion flow rate from information about the volume of the inner surface space.

[0192] In this Sartocheck device, for one 0.1m porous hollow fiber membrane containing pinholes with a diameter of approximately 12 μm prepared in Example 6... 2 The membrane module was subjected to a leak test using the same method described above, and the resulting airflow variation was 4.5 mL / min. This value is relative to the normal 0.1 m of a membrane module without pinhole hollow fibers. 2 The average airflow variation of the membrane module, 0.24 mL / min, is considered sufficiently high. Therefore, it is shown that by appropriately setting a threshold that takes into account both the average and deviation of the airflow variation, the Sartocheck device can be used for integrity testing to determine the value of 0.1 mL / min. 2 Does the membrane module have performance for parvovirus LRV4 and below?

[0193] [Comparative Example 4]

[0194] In Examples 7 and 8, the 0.1m sample from Example 6 was used. 2The airflow variation values ​​of nine samples of the membrane module were measured by changing the pressure setting from 196 kPa to 98 kPa, while maintaining the same method. The results showed that the airflow variation value was less than 0.10 mL / min when using the apparatus of Example 7, and 0.0 mL / min or 0.1 mL / min when using the apparatus of Example 8. Since the measurement results are less than 0.1 mL / min, which is the lower limit of measurement for each apparatus, it cannot be confirmed that normal membrane module measurements can be correctly performed. Furthermore, it is not possible to measure the airflow variation of a membrane module containing hollow fibers with pinholes. 2 Appropriate thresholds should be set between the measured values ​​of the membrane modules, therefore using two devices under conditions of 98 kPa is inappropriate.

[0195] [Example 9]

[0196] For the 0.001m used in Example 2 2 The determination of parvovirus LRV (using the protein solution containing the virus described in (5-A) above) in the membrane module will yield 150 L / m 2 The filtrate after filtration was changed to 150 L / m³. 2 After filtration, pressure is released for 3 hours, followed by pressurization to 15 L / m³. 2 The filtrate was obtained by filtration, and parvovirus LRV was determined using the same method. The evaluation results of all six evaluation components were determined to be parvovirus LRV ≥ 5.3.

[0197] [Example 10]

[0198] For the determination of parvovirus LRV (using the protein solution containing the virus described in (5-A) above) in Example 9, the filtration pressure was set to 150 kPa instead of 196 kPa, and the same method was used to measure the six evaluation components. The results showed that all samples had a parvovirus LRV of 5.3 or higher.

[0199] [Example 11]

[0200] For the determination of parvovirus LRV (using the protein solution containing the virus described in (5-A) above) in Example 9, the filtration pressure was set to 98 kPa instead of 196 kPa, and the same method was used to measure the six evaluation components. As a result, five of the six components were determined to have a parvovirus LRV of 5.3 or higher, while the parvovirus LRV of one component was determined to be 4.1.

[0201] [Table 1]

[0202]

[0203] Industrial availability

[0204] The present invention is suitable as a porous hollow fiber membrane containing regenerated cellulose, and the method for performing an integrity test using a leakage test method alone is applicable to the porous hollow fiber membrane.

Claims

1. A porous hollow fiber membrane containing regenerated cellulose, wherein the elastic limit pressure of the porous hollow fiber membrane is above 200 kPa. The ratio of the inner diameter R to the membrane thickness t of the porous hollow fiber membrane, R / t, is greater than 4.8 and less than 7.

7. The film thickness t is in the range of 40.5 μm or more and 49.6 μm or less. The bubble point of porous hollow fiber membranes is above 1.2 MPa. Porous hollow fiber membranes are used for virus removal. The parvovirus removal rate (LRV) is above 4.

0.

2. The porous hollow fiber membrane according to claim 1, wherein, The elastic limit pressure of the porous hollow fiber membrane is above 220 kPa and below 800 kPa.

3. The porous hollow fiber membrane according to claim 1 or 2, wherein, The elastic limit pressure of the porous hollow fiber membrane is above 250 kPa and below 500 kPa.

4. The porous hollow fiber membrane according to claim 1, wherein, The method for determining the elastic limit pressure of the porous hollow fiber membrane is as follows: One end of a 50mm long porous hollow fiber membrane is sealed with curable liquid resin in a way that prevents air leakage. The other end is then bonded and fixed with curable liquid resin while inserted into a micro-connector in a way that does not fill the hollow part, thus preparing a testing component. A pressurization device is prepared, which is equipped with a micro-connector that can be connected to a pressure regulating valve, a pressure gauge, and the testing component via a compressed air supply pipe. The testing component is connected to the pressurization device while immersed in water, and compressed air is supplied to the hollow part by increasing the pressure at 20kPa intervals. The outer diameter of the porous hollow fiber membrane at this time is measured using a dimensional measuring instrument. The percentage change in outer diameter caused by each measurement pressure is calculated using the following formula, and a graph is generated with the X-axis set to the measurement pressure in kPa and the Y-axis set to the percentage change in outer diameter. Outer diameter change rate % = (D / D0-1) × 100 In the formula, D: outer diameter under various pressures, in μm; D0: initial outer diameter under no pressure, in μm; Next, using five measured values ​​at 20 kPa intervals from 20 kPa to 100 kPa, the regression line formula passing through the origin is obtained: Y = aX. The formula is derived by adding 1 to the right side of this formula, which means an additional 1% of the outer diameter change rate: Y = aX + 1. The straight line based on the derived formula is added to the above figure, and the highest pressure among the pressures of the curves that will not exceed the outer diameter change rate of the straight line is set as the elastic limit pressure of the component used for measurement. The test was conducted on six or more components, and their average value was set as the elastic limit pressure of the porous hollow fiber membrane.

5. The porous hollow fiber membrane according to claim 1, wherein, The regenerated cellulose is obtained using the copper ammonia process.

6. The porous hollow fiber membrane according to claim 1, wherein, The pore size on the inner surface of this porous hollow fiber membrane is larger than that on the outer surface.

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

8. The porous hollow fiber membrane according to claim 1, wherein, The water permeability at a filtration pressure of 27 kPa and a temperature of 37℃ is 10 L / (m³). 2 •hr) or more and 50L / (m 2 (hr) and below.

9. The porous hollow fiber membrane according to claim 1, wherein, The water permeability at a filtration pressure of 27 kPa and a temperature of 37℃ is 15 L / (m³). 2 •hr) or above and 45L / (m 2 (hr) and below.

10. The porous hollow fiber membrane according to claim 1, wherein, The bubble point is above 1.4 MPa and below 2.4 MPa.

11. The porous hollow fiber membrane according to claim 1, wherein, The bubble point is above 1.5 MPa and below 2.3 MPa.

12. The porous hollow fiber membrane according to claim 1, used for the removal of parvoviruses.

13. The porous hollow fiber membrane according to claim 12, wherein, The parvovirus removal rate (LRV) is above 4.

5.

14. The porous hollow fiber membrane according to claim 12, wherein, The parvovirus removal rate (LRV) is above 5.

0.

15. A filtration method for filtering a liquid containing a biological agent using a porous hollow fiber membrane according to any one of claims 1 to 14, wherein the intermembrane pressure difference of the porous hollow fiber membrane during filtration is 150 kPa or more.

16. The filtering method according to claim 15, wherein, The liquid containing biological agents contains at least one of immunoglobulins, albumin, blood clotting factors, prothrombin complex, culture medium, antibody-drug complex, vaccine, recombinant protein, viral vector, DNA, and RNA.

17. The filtering method according to claim 16, wherein, The liquid containing the biological agent contains at least one of polyclonal antibodies and monoclonal antibodies.

18. The filtration method according to claim 15 or 16, wherein it is a filtration method for virus removal.

19. An integrity testing method for a membrane module filled with the porous hollow fiber membrane according to any one of claims 1 to 14. The membrane assembly has an outer surface side space that contacts the outer surface of the porous hollow fiber membrane, and an inner surface side space that contacts the inner surface of the porous hollow fiber membrane. The method includes: Fill the space on the outer surface side with liquid; and The inner surface space is pressurized by air in a manner where the intermembrane pressure difference of the porous hollow fiber membrane is greater than 98 kPa and the pressure is below the elastic limit pressure of the porous hollow fiber membrane.

20. The integrity test method according to claim 19, comprising the step of visually observing the bubbles generated by the porous hollow fiber membrane.

21. The integrity testing method according to claim 19 or 20, comprising: The process of measuring the pressure variation value of either the outer surface side space or the inner surface side space; or the process of measuring the amount of air inflow required to keep the pressure of either space constant.

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