Method for analyzing the performance of virus removal filters

Abstract

This invention provides a method for comparing the performance of a virus removal filter by filtering a colloidal solution containing gold nanoparticles using a virus removal filter, without using valuable bio-derived raw materials, and by comparative analysis of the filtration behavior and / or the filtrate. [Solution] A method for comparing and analyzing the performance of virus removal filters, comprising a preparation step of preparing two or more filters as filters, and a filtration step of passing a colloidal solution containing gold nanoparticles through each of the two or more filters in an amount greater than 0.5 L per 1 m2 of filter to obtain a filtrate.

Description

[Technical Field]

[0001] This invention relates to a method for analyzing the performance of a virus removal filter. [Background technology]

[0002] Biologically derived raw materials are raw materials derived from humans and / or other living organisms (excluding plants) that can be used in products such as pharmaceuticals, quasi-drugs, cosmetics, medical devices, and / or regenerative medicine. When manufacturing pharmaceuticals, etc., necessary measures are stipulated for biologically derived raw materials to ensure the quality, efficacy, and safety of the pharmaceuticals, etc. For example, in Japan, "Standards for Biologically Derived Raw Materials" have been established (Non-Patent Literature 1).

[0003] Bio-derived raw materials are natural polymers mainly produced by the cells of living organisms, and these natural polymers are called biomacromolecules. Because bio-derived raw materials are derived from living organisms, there are concerns that they may be affected by pathogens such as viruses. For example, in plasma-derived products that use proteins purified from human plasma, and in biological products that use biomacromolecules expressed through culture by adding bovine serum, etc., as active ingredients, there are concerns that infectious agents such as viruses may be introduced during the manufacturing process. Therefore, when manufacturing products using biological raw materials, it is necessary to include a process for inactivating or removing viruses and other pathogens.

[0004] Examples of processes for inactivating viruses include heat treatment, organic solvent / surfactant treatment (S / D treatment), chemical treatment, pH treatment, irradiation, photosensitizer, and UV treatment (Non-Patent Literature 2).

[0005] Examples of virus removal processes include precipitation, chromatography, and filtration methods (Non-Patent Literature 2). These methods allow for the physical removal of viruses without chemical alteration of biomolecules. Among these, filtration using virus removal filters has attracted attention in recent years. As materials for the filter membranes of virus removal filters, natural materials such as cellulose, or synthetic polymers such as polyvinylidene fluoride (PVDF), polyethersulfone (PES), polysulfone (PSU), or polyethylene (PE) are known. The pore size of these filter membranes is 10 to 70 nm, and filter types include hollow fiber type, pleated type, or flat membrane type.

[0006] Filtration using virus removal filters must be performed under the operating conditions recommended by the filter manufacturer. For example, each filter has an upper limit set for the pressure range of the intermembrane differential pressure.

[0007] In filtration methods using virus removal filters, if the molecular weight of the biopolymers in the biopolymer solution is small, a virus removal filter with a pore size that allows the biopolymers to pass through but not the viruses is used. However, one major problem is that the virus removal filter can become clogged, making filtration difficult or impossible. Therefore, it is necessary to select the virus removal filter to be used considering the size of the biopolymers. Depending on the size of the biopolymers to be allowed to pass through, separation of the biopolymers and viruses may become impossible. Furthermore, clogging can occur due to trace amounts of aggregates or other biopolymers interfering in the biopolymer solution.

[0008] Some virus removal filters are designed to remove even small viruses, such as non-enveloped viruses. Examples of such filters include Planova® 15N (Asahi Kasei Medical Corporation), Planova® 20N (Asahi Kasei Medical Corporation), Planova® S20N (Asahi Kasei Medical Corporation), Planova® Bio-EX (Asahi Kasei Medical Corporation), Ultipore VF-DV20 (Nippon Pall Co., Ltd.), Viasolve NFP (Merck KGaA), and Viasolve Pro (Merck KGaA).

[0009] Furthermore, Non-Patent Document 3 describes filtering a prion protein solution using a virus removal filter with a pore size of 15 nm. Non-Patent Document 3 also states that the above filter exhibits a certain clearance index for abnormal prion proteins that have become fine particles, and can remove a certain amount, thereby reducing the risk of infection.

[0010] However, simply filtering biomolecules with a virus removal filter is insufficient as a safety measure against viruses, and a virus clearance test is conducted to confirm the virus removal performance of the virus removal filter. The virus clearance test is a test that evaluates the virus inactivation and / or removal ability required during process development and when applying for drug approval in order to manage the safety of pharmaceuticals that apply biotechnology, and is performed in a state that simulates the actual process. By conducting a virus clearance test, the virus inactivation ability and removal ability in the virus inactivation or removal process can be quantitatively evaluated (Non-Patent Literature 4). The virus clearance index R obtained by the virus clearance test is generally expressed as a logarithm of the amount of virus reduced from the raw material by performing the virus inactivation or removal process. Specifically, it is expressed by the following formula. R = log((V1 × T1) / (V2 × T2)) In the formula, R is the logarithmic reduction rate, V1 is the volume of the sample before processing, T1 is the viral titer of the sample before processing, V2 is the volume of the sample after processing, and T2 is the viral titer of the sample after processing. The viral clearance index R is also called RF (reduction factor) or LRV (log reduction value).

[0011] The viral clearance index of Planova®, a virus removal filter, increases in proportion to the size of the virus particle. For example, Non-Patent Literature 5 describes the viral clearance index of PPV (porcine parvovirus, size 18-24 nm). It states that when Planova® 35N (average pore size 35 ± 2 nm) is used, the viral clearance index is less than 1.0, and when Planova® 20N (average pore size 19 ± 2 nm) and Planova® 15N (average pore size 15 ± 2 nm) are used, the viral clearance index is 4.0 or higher. It also states that when these filters are used, viruses can be removed and separated by the filtration principle of size exclusion (sieving by size), without being affected by physicochemical properties such as adsorption, and that as long as the size of the virus meets the conditions of the filtration membrane, it is possible to remove viruses even if unknown viruses are present.

[0012] Therefore, by using a virus removal filter with the smallest possible pore size while ensuring the filterability of biomolecules, it is possible to remove known small-diameter viruses as well as unknown viruses and other pathogens.

[0013] The pore size of the virus removal filter is measured, for example, by the water flow method (Non-Patent Literature 6). However, the pore size of many virus removal filters is not disclosed by the manufacturer. In this case, the filter user must measure the pore size, but this measurement is not easy. It is not easy for users to compare the performance of multiple types of filters, and for users to compare and select a virus removal filter and set the analysis conditions, numerous experiments using the valuable biomolecular samples that will actually be used are required.

[0014] Furthermore, after filtering the biopolymer solution through a virus removal filter, it is sometimes necessary to confirm that the used filter is not damaged. For example, one method is to visually check the filter for leaks or damage. Another method is to check the integrity of the filter by passing a colloidal solution containing gold nanoparticles through the used filter and measuring the difference in absorbance between the colloidal solution containing gold nanoparticles and its filtrate.

[0015] For example, Non-Patent Document 7 describes performing a Gold Particle Test (GPT) on a virus removal filter after it has been used for filtering biomolecules. This test is a destructive test to confirm that there is no change in the performance of the virus removal filter after use by washing off any proteins remaining on the filter after use, and then passing a colloidal solution containing gold nanoparticles through the washed filter. The colloidal solution containing gold nanoparticles is also called an integrity test solution. Non-Patent Document 7 describes using AGP-HA35, AGP-HA20, and AGP-HA15 as integrity test solutions for Planova® 35N (average pore size 35±2nm), Planova® 20N (average pore size 19±2nm), and Planova® 15N (average pore size 15±2nm), respectively.

[0016] Patent Document 1 describes an automatic measuring device and its control method used for a test (also referred to as a integrity test or a gold colloid removal test) to confirm that there is no change in the performance of a virus removal filter when a colloidal solution containing gold nanoparticles is passed through. In Patent Document 1, the absorbance (A) of the colloidal solution containing gold nanoparticles, the absorbance (B) of the filtrate of the filter, and the absorbance (C) of water are measured, and the common logarithm ratio Log 10 (A / (B - C)) is obtained. If the obtained value is within a predetermined range, the virus removal performance of the virus removal filter after use is determined to be qualified.

[0017] Regarding the above-mentioned index of the common logarithm ratio, it is described in Non-Patent Document 7. The index of the common logarithm ratio means the value (AGP LRV determination reference value) at which the virus removal performance is determined to be qualified when AGP-HA35, AGP-HA20, and AGP-HA15 are passed through Planova (trademark) 35N (average pore size 35 ± 2 nm), Planova (trademark) 20N (average pore size 19 ± 2 nm), and Planova (trademark) 15N (average pore size 15 ± 2 nm) respectively as integrity test solutions after being used for filtering biopolymers. The above values are described as ≧1.84, ≧1.40, and ≧2.03 respectively. The setting of these AGP LRV determination reference values is explained as follows. That is, for Planova (trademark) 35N, it corresponds to a logarithmic removal rate of Japanese encephalitis virus (size about 50 nm) of 4.5 or more. For Planova (trademark) 20N, it corresponds to a logarithmic removal rate of porcine parvovirus (size 18 - 24 nm) of 3.0 or more. And for Planova (trademark) 15N, it corresponds to a logarithmic removal rate of poliovirus (size 25 - 30 nm) of 4.0 or more.

[0018] Non-Patent Document 8, Non-Patent Document 9, and Non-Patent Document 10 show the details of the respective integrity test solutions AGP-HA35, AGP-HA20, and AGP-HA15. These integrity test solutions contain colloidal solutions containing gold nanoparticles. These integrity test solutions are composed of a concentrated solution containing gold nanoparticles and sodium lauryl sulfate (SDS) powder, which is a surfactant. When using the integrity test solution, an aqueous solution of 0.27% SDS is prepared, and the obtained SDS solution and the concentrated solution are mixed at a ratio of 9:1. The solution at the time of use is a colloidal solution containing 0.27% SDS, 0.25% polyvinylpyrrolidone (PVP), and gold nanoparticles at the final concentration. These non-patent documents show that the integrity test solution is passed through filters after use having various membrane areas, and a filtrate amount corresponding to 0.5 L / m 2 is obtained. Based on the information shown in these non-patent documents, Table 1 shows a summary of the characteristics of each integrity test solution at the time of use. As shown in Table 1, the average particle diameter of the gold nanoparticles in AGP-HA20 and AGP-HA15 is about 20 nm, suggesting that AGP-HA20 contains smaller-diameter gold nanoparticles. Also, it can be seen that the wavelength at which absorbance is observed shifts to the longer wavelength side as the average particle diameter of the gold nanoparticles increases.

[0019]

Table 1

[0020] Patent Document 2 describes a colloidal solution of metal particles or metal compound particles used in a gold colloid removal test. An example is shown in which a surfactant or chelating agent, as well as a water-soluble high molecular weight dispersant containing N groups, is used with a colloidal solution containing gold nanoparticles. When the membrane material of the virus removal filter is made of cellulose, an example is described in which either a surfactant or a chelating agent, or a combination of both, is used with the colloidal solution. When the membrane material of the virus removal filter is made of synthetic polymer, an example is described in which a chelating agent is used without a surfactant. Table 8 of Patent Document 2 states that when the material of the porous filter membrane is hydrophilic PVDF instead of cellulose, the recovery rate of the colloidal solution containing gold nanoparticles is improved by using sodium polyacrylate, sodium acrylate-sodium methacrylate copolymer, or ethylenediaminetetraacetic acid (EDTA) instead of SDS.

[0021] Gold nanoparticles are particles ranging in size from a few nanometers to about 100 nanometers and are used in sensors, nanocarriers, cosmetics, and other applications. Colloidal solutions of gold nanoparticles are negatively charged in aqueous solvents, exhibiting high affinity for proteins, and are therefore also used as biomarkers.

[0022] A typical characteristic of gold citrate colloids, to which citric acid has been added as a stabilizer, is that their maximum absorption wavelength differs depending on the particle size of the prepared product. For example, the Tanaka Precious Metals Group Industrial Business Global Site (https: / / tanaka-preciousmetals.com / jp / products / detail / gold-colloids / ) states that in colloidal solutions of gold nanoparticles with particle sizes in the range of 5 to 150 nm, the particle size and the maximum absorption wavelength correspond in the wavelength range of approximately 500 to 600 nm.

[0023] The particle size of gold nanoparticles in a colloidal solution containing gold nanoparticles can be measured, for example, by dynamic light scattering. For instance, Shimadzu Techno-Research Corporation (https: / / www.shimadzu-techno.co.jp / technical / tes / colloidal_gold_zeta.html) presents an example of analysis of commercially available gold colloid with a diameter of 20 nm using dynamic light scattering.

[0024] Size exclusion chromatography (SEC) is a separation analysis method that elutes molecules in order from largest to smallest based on differences in size, and can separate molecules based on particle size. In a review article on the analysis of gold nanoparticles by SEC (Non-Patent Literature 11), it was pointed out that a major challenge in the SEC analysis of gold nanoparticles is that the gold nanoparticles adsorb to the analytical column packing material, and problems such as incomplete recovery of the analyte are pointed out. Furthermore, as a prior art in Non-Patent Literature 11, 10 is used as the mobile phase for the SEC analysis of gold citrate nanoparticles. -3 Examples are provided of using aqueous solutions of surfactants such as mol / L sodium citrate and SDS, or water. [Prior art documents] [Patent Documents]

[0025] [Patent Document 1] Japanese Patent Publication No. 2008-149205 [Patent Document 2] International Publication No. 2005 / 007328 [Non-patent literature]

[0026] [Non-Patent Document 1] Ministry of Health, Labour and Welfare Notification No. 37 / Established February 28, 2018 [Non-Patent Document 2] Biopharmaceutical Handbook, 4th Edition, 2020, Jiho Co., Ltd. [Non-Patent Document 3] Modern Media. 2013 59(9):231-237 [Non-Patent Document 4] Viral safety evaluation of biotechnology-derived pharmaceuticals manufactured using human or animal cell lines (ICH-Q5A) [Non-Patent Document 5] Planova Filter Catalog, Asahi Kasei Medical Corporation, Bioprocess Division, TAE31002J-4.1 [Non-Patent Document 6] Planova Virus Removal Filter Product Specification Sheet, Asahi Kasei Medical Corporation, Bioprocess Division, TAE33015-2.0 [Non-Patent Document 7] Integrity Test Operating Standards Manual: Planova 15N, 20N, and 35N Filters; Gold Colloid Removal Test (GPT) using Asahi Kasei Integrity Test Solution Kit; Asahi Kasei Medical Corporation, Bioprocess Division; TAS33031J-4.0 [Non-Patent Document 8] Validation Report, Asahi Kasei Integrity Test Solution Kit AGP-HA35, Asahi Kasei Medical Corporation, Bioprocess Division, TAE34020J-3.1 [Non-Patent Document 9] Validation Report, Asahi Kasei Integrity Test Solution Kit AGP-HA20, Asahi Kasei Medical Corporation, Bioprocess Division, TAE34017J-3.1 [Non-Patent Document 10] Validation Report, Asahi Kasei Integrity Test Solution Kit AGP-HA15, Asahi Kasei Medical Corporation, Bioprocess Division, TAE34019J-3.1 [Non-Patent Document 11] Pitkanen L, Striegel AM. Size-exclusion chromatography of metal nanoparticles and quantum dots. Trends Analyt Chem. 2016 Jun;80:311-320. [Overview of the project] [Problems that the invention aims to solve]

[0027] As described above, the filtration of biopolymers using virus removal filters and the virus removal performance of said filters have been evaluated by actually using the biopolymer solution being filtered. However, only non-uniform and insufficient information has been disclosed regarding the pore size or allowable load of the virus removal filters, requiring extensive consideration when selecting a virus removal filter. Valuable biopolymer samples have been used in this consideration.

[0028] Therefore, the object of the present invention is to provide a method for easily comparing and analyzing the pore size and allowable load of a virus removal filter without using a biopolymer sample. [Means for solving the problem]

[0029] As a result of repeated studies to solve the above problems, the present inventors have found that it is possible to filter a virus removal filter using a colloidal solution containing gold nanoparticles without using valuable bio-derived raw materials, and to compare the performance of the filter by comparative analysis of the filtration behavior and / or the filtrate, thereby completing the present invention.

[0030] Therefore, this disclosure provides the following: [1] The preparation step involves preparing two or more filters as filters, and Using each of the two or more filters mentioned above, a colloidal solution containing gold nanoparticles is filtered through a filter 1 m 2 Filtration process: Passing a liquid through at a volume greater than 0.5 L per unit area to obtain the filtrate. A method for comparing and analyzing the performance of virus removal filters, including [specific components / features]. [2] The filter is a filter that has not been used for filtering biomolecules, as described in [1] for the comparative analysis method. [3] The comparative analysis method according to [1] or [2], wherein the average particle size of the gold nanoparticles is 17 nm or larger. [4] 1m filter 2 A comparative analysis method described in any one of [1] to [3], wherein a colloidal solution of 1 L or more is passed through each volume. [5] The comparative analysis method described in any one of [1] to [4] is performed by maintaining a constant flow rate of the colloidal solution. [6] The comparative analysis method described in any one of [1] to [5] is performed by maintaining a constant pressure of the colloidal liquid loaded onto the filter. [7] The comparative analysis method described in any one of [1] to [6] states that the maximum absorbance of the colloidal solution in the wavelength range of 500 to 550 nm is 0.9 or higher. [8] The process involves measuring the absorbance of each of the obtained filtrates to light of a specific wavelength, and A process of comparing the measured maximum absorbance of the filtrate when using two or more filters, and comparing the average pore size of the two or more filters. Includes, The comparative analysis method according to any one of [1] to [7], wherein the filtration step is performed by keeping the flow rate of the colloidal liquid constant, or by keeping the pressure of the colloidal liquid loaded onto the filter constant. [9] In the filtration step, a step of measuring the absorbance of the colloidal solution before filtration and the absorbance of the filtrate in response to light of a specific wavelength, A step of determining the difference between the absorbance of the colloidal solution before filtration and the maximum absorbance of the filtrate when using two or more filters, and A step of comparing the difference in absorbance when using two or more filters, and comparing the difference in the gold nanoparticle capture ability of the two or more filters. Includes, The comparative analysis method according to any one of [1] to [7], wherein the filtration step is performed by keeping the flow rate of the colloidal liquid constant, or by keeping the pressure of the colloidal liquid loaded onto the filter constant.

[10] The filtration process includes measuring the intermembrane pressure of the filter, determining the change in the measured intermembrane pressure, and A step of comparing the amount of change when using each of two or more filters, and comparing the loading capacity of gold nanoparticles of the two or more filters. Includes, The filtration process is carried out by maintaining a constant flow rate of the colloidal liquid. A comparative analysis method described in any one of [1] to [9].

[11] The filtration process involves measuring the amount of liquid passing through the filter and determining the change in the measured amount of liquid passing through, and A step of comparing the amount of change when using each of two or more filters, and comparing the loading capacity of gold nanoparticles of the two or more filters. Includes, The filtration process is carried out by maintaining a constant pressure of the colloidal liquid loaded onto the filter. A comparative analysis method described in any one of [1] to [9].

[12] A step to measure the particle size of gold nanoparticles contained in the filtrate obtained in the above filtration step, and thereby compare and analyze the pore sizes of two or more filters. A comparative analysis method described in any one of [1] to

[11] , including the above.

[13] A process to compare and analyze the pore size of filters by measuring the absorbance of the filtrate obtained in the filtration step to light of a variable wavelength, and comparing the particle size of gold nanoparticles in the filtrates of two or more filters based on the maximum absorption wavelength of each filtrate. The comparative analysis method described in

[12] , including the method described in

[12] .

[14] The filtrate obtained in the above filtration step is subjected to size exclusion chromatography to measure the elution time of gold nanoparticles contained in the filtrate, and the pore size of the filters is compared by comparing the particle size of the gold nanoparticles in the filtrates of two or more filters based on the elution times of the gold nanoparticles in each filtrate. including, The comparative analysis method described in

[12] or

[13] .

[15] The mobile phase of the aforementioned size exclusion chromatography analysis contains a compound having a carboxyl group or a salt thereof, and the pH of the mobile phase is in the range of 3 to 7. The comparative analysis method described in

[14] .

[16] The mobile phase comprises at least one of citric acid, ethylenediaminetetraacetic acid, and salts thereof. The comparative analysis method described in

[15] .

[17] In the mobile phase, the concentration of the compound having a carboxyl group is less than 100 mmol per liter of the mobile phase. The comparative analysis method described in

[15] or

[16] .

[18] In the mobile phase, the concentration of the compound having a carboxyl group is greater than 10 mmol per 1 L of mobile phase. A comparative analysis method described in any one of

[15] to

[17] .

[19] A process to compare and analyze the pore size of filters by measuring the particle size of gold nanoparticles contained in the filtrate obtained in the above filtration step by dynamic light scattering analysis, and by comparing the particle size of gold nanoparticles in the filtrate of two or more filters. A comparative analysis method described in any one of

[12] to

[18] , including the above. [Effects of the Invention]

[0031] In this invention, without using valuable bio-derived raw materials, a colloidal solution containing gold nanoparticles is filtered through a virus removal filter, and the performance of the filter can be compared by comparative analysis of the filtration behavior and / or the filtrate. [Brief explanation of the drawing]

[0032] [Figure 1A] Figure 1A is a graph showing the relationship between the filtration volume (L / m2) of the colloidal solution containing gold nanoparticles in Example 1 and the absorbance (mAbs.) of the filtrate at a wavelength of 530 nm. [Figure 1B]Figure 1B is a graph showing the relationship between the amount of colloidal solution containing gold nanoparticles in Example 1 filtered (L / m2) and the change in the differential pressure across the filter membrane (kPa). [Figure 2A] Figure 2A is a graph showing the relationship between the filtration volume (L / m2) of the colloidal solution containing gold nanoparticles in Example 2 and the absorbance (mAbs.) of the filtrate at a wavelength of 526 nm. [Figure 2B] Figure 2B is a graph showing the relationship between the amount of colloidal solution containing gold nanoparticles in Example 2 filtered (L / m2) and the change in the differential pressure across the filter membrane (kPa). [Figure 2C] Figure 2C is a graph showing the relationship between wavelength (nm) and absorbance (Abs.) of the colloidal solution containing gold nanoparticles used in Example 2. [Figure 3A] Figure 3A is a graph showing the relationship between the filtration volume (L / m2) of the colloidal solution containing gold nanoparticles in Example 3 and the absorbance (mAbs.) of the filtrate at a wavelength of 526 nm. [Figure 3B] Figure 3B is a graph showing the relationship between the amount of colloidal solution containing gold nanoparticles in Example 3 filtered (L / m2) and the change in the differential pressure across the filter membrane (kPa). [Figure 3C] Figure 3C is a graph showing the relationship between wavelength (nm) and absorbance (Abs.) of the colloidal solution containing gold nanoparticles used in Example 3. [Figure 4] Figure 4 is a graph showing the chromatogram obtained by analyzing the colloidal solution containing gold nanoparticles in Example 3. [Figure 5] Figure 5 is a graph showing chromatograms obtained by analyzing the colloidal solutions containing gold nanoparticles from Examples 2 and 3. [Modes for carrying out the invention]

[0033] The following describes the analysis method used in this disclosure, but this disclosure is not limited to this method.

[0034] The comparative analysis method for the performance of the virus removal filters in this disclosure is as follows: The preparation step involves preparing two or more filters as filters, and Using each of the two or more filters mentioned above, a colloidal solution containing gold nanoparticles is filtered through a filter 1 m 2 Filtration process: Passing a liquid through at a volume greater than 0.5 L per unit area to obtain the filtrate. Includes.

[0035] In this disclosure, a colloidal solution containing gold nanoparticles is used in the comparative study of virus removal filters. Therefore, this disclosure does not use any valuable biologically derived target substances. By passing the colloidal solution containing gold nanoparticles through two or more virus removal filters and comparing them, the removal performance and / or loading capacity of viruses of a similar size to the gold nanoparticles can be compared among two or more virus removal filters.

[0036] The relative removal performance of virus removal filters can be determined, for example, by comparing specific physical properties in multiple filtrates, such as by comparing the average pore size of the filters. These multiple filtrates are obtained using multiple filters, and the relative removal performance between filters can be determined by comparing the magnitude of absorbance at specific wavelengths and / or by analyzing the particle size of gold nanoparticles, etc.

[0037] The load capacity of a virus removal filter can be determined, for example, by preparing two or more filters and comparing the degree of clogging of each filter. Specifically, the load capacity can be determined by comparing the fluctuations in the differential pressure between filter membranes obtained by filtering under a constant flow rate, or by comparing the fluctuations in flow rate obtained by filtering under a constant pressure.

[0038] (preparation process) The term "filter" here refers to a virus removal filter.

[0039] Two or more filters means any number of filters, and is not particularly limited. For example, it could be 2 to 6 filters, 2 to 4 filters, 2 to 3 filters, or even just 2 filters.

[0040] Preferably, the average pore size of one or more of the two or more filters is known. Here, the known average pore size refers to the value listed in the catalog of the virus removal filter, etc.

[0041] The comparative analysis method disclosed herein includes comparing results from two or more filters, but filtration with each filter does not need to be performed simultaneously. For example, when comparing results from two filters, it is possible to compare results obtained from one filter in the past with results newly obtained from the other filter.

[0042] The above-mentioned filter is preferably a filter that has not been used for filtering biomolecules. Hereinafter, the above-mentioned filter may be referred to as an "unused filter" or "filter".

[0043] The material of the filter membrane is not particularly limited as long as it can be used as a virus removal filter, but examples include those composed of cellulose, synthetic polymers (for example, hydrophilic polyvinylidene fluoride (PVDF), polyethersulfone (PES), polysulfone (PSU), or polyethylene (PE)), etc.

[0044] Examples of filters made from cellulose include Planova 15N (average pore size 15±2nm), Planova 20N (average pore size 19±2nm), Planova 35N (average pore size 35±2nm), and Planova® S20N (all manufactured by Asahi Kasei Medical Corporation).

[0045] Examples of filters composed of synthetic polymers include Planova BioEX (hydrophilic PVDF) and Viasorb Pro (PES).

[0046] In one embodiment, two or more filters are made of the same material.

[0047] In one embodiment, two or more filters are made of different materials.

[0048] The membrane area of the filter is not particularly limited. For example, it can be 0.0003 to 4.0 m 2 Specifically, it can be 0.0003 m 2 or 0.001 m 2 The membrane area of the filter refers to the area of the filter portion in contact with the colloidal liquid being passed through.

[0049] The filter is not particularly limited. For example, it can have a shape such as a hollow fiber type, a pleated type, or a flat membrane type.

[0050] (Filtration step) The liquid passing is at a rate greater than 0.5 L per 1 m 2 of the filter, preferably 1 L or more, more preferably 10 L or more, and even more preferably 40 L or more. The liquid passing can be, for example, 1000 L or less, or even 100 L or less.

[0051] In one embodiment, the filtration step can be performed by keeping the flow rate of the colloidal liquid constant. That is, the colloidal liquid supplied to the filter can be of a constant amount. Keeping the flow rate constant means, for example, that a predetermined amount of colloidal liquid passes through the same amount of filter per unit time.

[0052] The flow rate of the colloidal liquid is, for example, 0.01 L to 10 L / min / m 2 Specifically, it is 0.1 L to 1 L / min / m 2

[0053] ​In one embodiment, the filtration process can be carried out by maintaining a constant pressure of the colloidal liquid loaded onto the filter.

[0054] A colloidal solution is a liquid containing gold nanoparticles. A gold colloid refers to a colloidal solution in which gold nanoparticles (gold microparticles) are dispersed in a liquid.

[0055] The particle size of the gold nanoparticles may be, for example, in the range of 1 to 300 nm or in the range of 5 to 150 nm. The lower limit of the particle size may be, for example, 5 nm or more, specifically 10 nm or more, more specifically 15 nm or more, and the upper limit may be, for example, 100 nm or less, specifically 50 nm or less, more specifically 40 nm or less, and even more specifically 24 nm or less. Having the above particle sizes allows the gold nanoparticles to be stably dispersed in a colloidal solution. Having the above particle sizes allows them to be captured by a virus removal filter. Furthermore, having the above particle sizes allows for the evaluation of viruses of a similar size to the gold nanoparticles. The average size of a virus is 100 nm or less.

[0056] In one embodiment, the particle size of the gold nanoparticles is 17 nm or larger.

[0057] The average particle size of the gold nanoparticles is preferably the average particle size of the target material (i.e., the substance filtered by the filter) or a value in the vicinity of that average particle size. The vicinity value may be, for example, ±2 to 9 nm of the average particle size of the target material. The target material refers to the material that is removed by the filter, and examples include viruses.

[0058] The particle size of gold nanoparticles can be measured by dynamic light scattering analysis in one embodiment.

[0059] In one embodiment, the particle size of gold nanoparticles can be evaluated by measuring the maximum absorption wavelength.

[0060] In one embodiment, the particle size of gold nanoparticles can be evaluated by size exclusion chromatography (SEC).

[0061] The variation rate of the particle size distribution of gold nanoparticles can be, for example, 30% or less, specifically 10% or less.

[0062] Examples of colloidal solutions containing gold nanoparticles include Asahi Kasei Medical's Integrity Test Solution Kits AGP-HA15 and AGP-HA20, which are readily available.

[0063] The colloidal solution may contain gold nanoparticles in an amount of, for example, 0.0001 to 0.1 mass%, more specifically 0.001 to 0.08 mass%, and more specifically 0.002 to 0.07 mass%.

[0064] The maximum absorbance of the colloidal solution is preferably 0.9 or higher for light with wavelengths in the range of 500 to 550 nm. When a colloid containing gold nanoparticles is passed through a filter, and many gold nanoparticles pass through the filter during this filtration, i.e., when the filtrate contains many gold nanoparticles, the absorbance value of the filtrate increases.

[0065] Absorbance measurements are performed using a Shimadzu SPD-10Avp UV-VIS detector and a Runtime Instruments Rec-Pro recorder when continuously measuring the absorbance of the filtrate in a filtration apparatus. When measuring the absorbance of a sample of the filtrate, a JASCO V-750 UV-Vis spectrophotometer is used.

[0066] The pH of the colloidal solution may be in the range of 4 to 11, for example, and more specifically, in the range of 4 to 6. This allows the gold nanoparticles to be stably dispersed.

[0067] The colloidal solution preferably contains a solvent along with gold nanoparticles. Water is a suitable solvent.

[0068] The colloidal solution may further contain other compounds. Examples of other compounds include surfactants, chelating agents, water-soluble polymer dispersants, and pH adjusters.

[0069] As the surfactant, anionic surfactants and nonionic surfactants can be used. Colloids containing gold nanoparticles have adhesive properties to certain substances, but by adding a surfactant, the adhesion between the gold nanoparticles and the specific substance can be inhibited. Here, the specific substance is, for example, a biopolymer such as protein and / or a membrane material.

[0070] Examples of anionic surfactants include dodecyl sulfate (SDS) or its salts. The salts are not particularly limited, but examples include lithium salts and sodium salts.

[0071] Examples of nonionic surfactants include Triton X-100, Tween20, and Tween80.

[0072] The surfactant may be present in the colloidal solution in an amount of, for example, 0.001 to 7.0% by mass, more specifically 0.01 to 3.0% by mass, or more specifically 0.05 to 2.0% by mass. This can further suppress the inhibition of adhesion to proteins and / or adsorption to the membrane material.

[0073] Examples of chelating agents include tripolyphosphate, polyacrylic acid, polyacrylic acid copolymers, ethylenediaminetetraacetic acid (EDTA), citric acid, or salts thereof, with ethylenediaminetetraacetic acid being a more specific example. The salts are not particularly limited, but examples include sodium salts and potassium salts. For example, sodium tripolyphosphate, sodium polyacrylate, sodium polyacrylate copolymer, sodium ethylenediaminetetraacetic acid salt, sodium citrate, and especially disodium ethylenediaminetetraacetate salt.

[0074] The chelating agent may be present in the colloidal solution in an amount of, for example, 0.001 to 7.0% by mass, more specifically 0.05 to 5.0% by mass, or more specifically 0.5 to 3.0% by mass.

[0075] Examples of water-soluble polymer dispersants include polyvinylpyrrolidone (PVP) or polyvinylpyrrolidone copolymers. Adding a water-soluble polymer dispersant can suppress the adhesion of gold nanoparticles to the hollow fiber membrane filter.

[0076] The water-soluble polymer dispersant may be present in the colloidal solution in an amount of, for example, 0.001 to 10% by weight, more specifically 0.01 to 5% by weight, or more specifically 0.1 to 3% by weight.

[0077] The pH adjusting agent is not particularly limited as long as it can adjust the pH, but for example, dilute hydrochloric acid or sodium hydroxide solution can be used.

[0078] In one embodiment, when the filter membrane material is composed of cellulose, the colloidal solution contains at least one of a surfactant and a chelating agent.

[0079] In one embodiment, when the filter membrane material is composed of a synthetic polymer system, the colloidal solution contains a chelating agent. In this case, it is preferable that the colloidal solution does not contain a surfactant. This can improve the recovery rate of gold nanoparticles. For example, when the filter is made of hydrophilic PVDF, sodium acrylate, sodium acrylate-sodium methacrylate copolymer, ethylenediaminetetraacetic acid (EDTA), or a salt thereof may be used without containing a surfactant.

[0080] The maximum absorbance of the colloidal solution containing gold nanoparticles is preferably 0.9 or higher at a certain wavelength within the range of 500 to 550 nm. There is no particular upper limit to the maximum absorbance of the filtered filtrate of the colloidal solution.

[0081] In one embodiment, The process involves measuring the absorbance of each of the obtained filtrates to light of a specific wavelength, and A process of comparing the measured maximum absorbance of the filtrate when using two or more filters, and comparing the average pore size of the two or more filters. Includes, The filtration process is carried out by maintaining a constant flow rate of the colloidal liquid, or by maintaining a constant pressure of the colloidal liquid loaded onto the filter.

[0082] Examples of specific wavelengths of light include light with wavelengths in the range of 520-550 nm, which is near the maximum absorption wavelength of the colloidal solution containing gold nanoparticles. Specifically, examples include light with wavelengths in the range of 526 nm or 530 nm. Absorbance can be measured in the same manner as described above.

[0083] The maximum absorbance of the filtrate is the maximum value of the absorbance of the filtrate in response to light of a certain wavelength.

[0084] "Load capacity" refers to the amount of liquid that can pass through the filter under the recommended operating conditions set by the filter manufacturer. When filtering by maintaining a constant flow rate, clogging can increase the differential pressure across the filter membrane. In this case, it refers to the amount of test liquid that can be supplied at or below the upper limit of the pressure range set by the filter manufacturer. Alternatively, when filtering by maintaining a constant pressure of the test liquid, clogging of the filter can reduce the flow rate of the filtrate. In this case, it refers to the amount of test liquid from which the filtrate can be recovered without clogging the filter.

[0085] In one embodiment, In the filtration step, a step of measuring the absorbance of the colloidal solution before filtration and the absorbance of the filtrate in response to light of a specific wavelength, A step of determining the difference between the absorbance of the colloidal solution before filtration and the maximum absorbance of the filtrate when using two or more filters, and A step of comparing the difference in absorbance when using two or more filters, and comparing the difference in the gold nanoparticle capture ability of the two or more filters. Includes, The filtration process is carried out by maintaining a constant flow rate of the colloidal liquid, or by maintaining a constant pressure of the colloidal liquid loaded onto the filter.

[0086] The light of the specific wavelengths mentioned above is synonymous with the above. Absorbance can be measured in the same manner as described above.

[0087] In one embodiment, The filtration process includes measuring the intermembrane pressure of the filter, determining the change in the measured intermembrane pressure, and A step of comparing the amount of change when using each of two or more filters, and comparing the loading capacity of gold nanoparticles of the two or more filters. Includes, The filtration process is carried out by maintaining a constant flow rate of the colloidal liquid.

[0088] Intramembrane pressure differential refers to the difference between the pressure exerted on the filter from the colloidal liquid side and the pressure exerted on the filter from the filtrate side.

[0089] The change in intermembrane differential pressure refers to the increase or decrease in intermembrane differential pressure per unit area per unit time during filtration.

[0090] In this embodiment, clogging and other issues can be prevented by comparing the amount of change in the differential pressure between the membranes of the filter.

[0091] In one embodiment, The filtration process involves measuring the amount of liquid passing through the filter and determining the change in the measured amount of liquid passing through, and A step of comparing the amount of change when using each of two or more filters, and comparing the loading capacity of gold nanoparticles of the two or more filters. Includes, The filtration process is carried out by maintaining a constant pressure of the colloidal liquid loaded onto the filter.

[0092] The change in liquid flow rate refers to the increase or decrease in the liquid flow rate per unit area per unit time during filtration.

[0093] In this embodiment, clogging and other problems can be prevented by comparing the change in the amount of liquid passing through the filter.

[0094] In one embodiment, A step to measure the particle size of gold nanoparticles contained in the filtrate obtained in the above filtration step, and thereby compare and analyze the pore sizes of two or more filters. Includes.

[0095] The particle size of gold nanoparticles in the colloidal solution before filtration and the particle size of gold nanoparticles in the filtrate can be determined. This allows for comparison of the pore sizes between two or more filters.

[0096] In one embodiment, The process includes measuring the absorbance of the filtrate obtained in the filtration step to light of a variable wavelength, and comparing and analyzing the pore size of the filters by comparing the particle size of the gold nanoparticles in the filtrates of two or more filters based on the maximum absorption wavelengths of the filtrates of each filter.

[0097] Tunable wavelength light refers to light obtained, for example, by varying its wavelength. An example of a tunable wavelength range is 500-550 nm.

[0098] The maximum absorption wavelength is the wavelength at which the absorbance is greatest among the absorbances for light of a variable wavelength. When comparing the maximum absorption wavelengths of colloidal solutions containing gold nanoparticles, if the maximum absorption wavelength is relatively large (long), it is considered that the particle size of the gold nanoparticles is larger than that of solutions with a smaller (shorter) maximum absorption wavelength. When measuring the maximum absorption wavelengths of filtrates of colloidal solutions containing multiple gold nanoparticles, the relative magnitudes (lengths and lengths) of the maximum absorption wavelengths correspond to the relative particle sizes of the gold nanoparticles contained in each filtrate.

[0099] In one embodiment, The filtrate obtained in the above filtration step is subjected to size exclusion chromatography to measure the elution time of gold nanoparticles contained in the filtrate, and the pore size of the filters is compared by comparing the particle size of the gold nanoparticles in the filtrates of two or more filters based on the elution times of the gold nanoparticles in each filtrate. Includes.

[0100] Size exclusion chromatography (SEC) or size exclusion high-performance liquid chromatography (SE-HPLC) is a separation and analysis method that elutes molecules in order from largest to smallest based on their size. It allows for separation based on particle size. SEC includes SE-HPLC.

[0101] The particle size of the gold nanoparticles in the filtrate obtained by passing a colloidal solution containing gold nanoparticles through a filter can be determined by analyzing the filtrate using SE-HPLC. A Shimadzu LC-10A system can be used as the SE-HPLC instrument.

[0102] SEC or SE-HPLC has a stationary phase and a mobile phase.

[0103] The stationary phase is a column packed with a size exclusion chromatography packing material. The packing material can be made of synthetic polymers or silica gel, and a column packed with silica gel packing material may be used, for example, the G3000SWXL and G4000SWXL (both manufactured by Tosoh Corporation).

[0104] The mobile phase preferably comprises a compound having a carboxyl group or a salt thereof, more preferably comprising at least one of acetic acid, citric acid, ethylenediaminetetraacetic acid, and salts thereof, and specifically may comprise sodium citrate or disodium ethylenediaminetetraacetic acid (EDTA-2Na).

[0105] The mobile phase contains a solvent along with a compound having a carboxyl group or a salt thereof. The solvent can be any solvent that dissolves the compound having a carboxyl group or a salt thereof, for example, water.

[0106] The concentration of the carboxyl group compound or its salt contained in the mobile phase is not particularly limited, but for example, it may be less than 100 mmol per 1 L of mobile phase and greater than 10 mmol. Specifically, it is in the range of 20 to 40 mmol / L. With the above configuration, the elution of gold nanoparticles by SE-HPLC is improved.

[0107] The mobile phase may further contain pH adjusters and the like.

[0108] Examples of pH adjusting agents include dilute hydrochloric acid or sodium hydroxide solution.

[0109] The pH of the mobile phase is preferably in the range of 3 to 7, more preferably in the range of 4 to 6, for example, 5.0. With the above configuration, gold nanoparticles contained in the filtrate can be eluted relatively well.

[0110] The flow rate in the mobile phase may vary depending on the inner diameter of the stationary phase column, but may be, for example, 0.01 to 2.0 mL / min, and preferably 0.2 to 1.0 mL / min. Note that flow rate refers to the volume flowing per minute.

[0111] A process to compare and analyze the pore size of filters by measuring the particle size of gold nanoparticles contained in the filtrate obtained in the above filtration step by dynamic light scattering analysis, and by comparing the particle size of gold nanoparticles in the filtrate of two or more filters. It may include.

[0112] Dynamic light scattering analysis is a common method and can be performed using photon correlation spectroscopy. Normally, when a liquid containing gold nanoparticles is passed through a filter, the filter captures gold nanoparticles larger than the filter's average pore size, allowing only those smaller than the filter's average pore size to pass through. Therefore, the gold nanoparticles in the filtrate obtained by passing a colloidal liquid containing gold nanoparticles through a filter are considered to have a particle size smaller than the filter's average pore size. Consequently, by measuring or comparing the particle size of the gold nanoparticles in the filtrate, particularly the largest particle size of the gold nanoparticles, the pore size of the filter or its relative size can be inferred.

[0113] The above describes the method for analyzing the performance of the virus removal filter in this disclosure, but this disclosure is not limited to what is stated above. [Examples]

[0114] The present invention will be described in detail below with reference to examples, but the present invention is not limited in any way to these examples.

[0115] [Gold colloid solution] As colloidal solutions containing gold nanoparticles, Asahi Kasei Medical's Integrity Test Solution Kits AGP-HA15 and AGP-HA20 were used. The properties of AGP-HA15 and AGP-HA20 are shown in Table 1 above.

[0116] Each colloidal solution containing gold nanoparticles was prepared by mixing 1 volume of the concentrated solution containing gold nanoparticles from the product with 9 volumes of solvent. The absorbance of each prepared colloidal solution was measured at each wavelength using a JASCO V-750 UV-Vis spectrophotometer, and it was confirmed that the values ​​were within the ranges shown in Table 1. Each colloidal solution is thought to contain gold nanoparticles with the average particle size and standard deviation shown in Table 1.

[0117] [Example 1]: Filtration of colloidal solution AGP-HA15 containing gold nanoparticles through an unused filter. Membrane area 0.001m 2A colloidal solution AGP-HA15 containing gold nanoparticles is added to Planova 15N (average pore size 15±2nm), Planova 20N (average pore size 19±2nm), and Planova 35N (average pore size 35±2nm) at a rate of approximately 0.2 L / min / m². 2 Each was passed through at a constant flow rate. The differential pressure between the membranes of the filter, the volume of filtrate, and the absorbance of the filtrate at a wavelength of 530 nm were monitored. As shown in Table 1, the average particle size of the gold nanoparticles contained in AGP-HA15 is approximately 20-24 nm, with a standard deviation of 3-5 nm.

[0118] [Regarding filtration] Filtration channel: Formed using a silicone tube. Fluid delivery was performed using a Peristaltic Bio Mini Pump AC-2120 manufactured by ATTO Corporation.

[0119] [Measurement of intermembrane pressure differential] Krone KDM-30 digital pressure gauges were installed before (colloidal liquid side) and after (filtrate side) the filter to continuously measure the pressure before and after the filter and determine the differential pressure across the filter membrane.

[0120] [Measurement of absorbance] The absorbance of the filtrate at a wavelength of 530 nm was continuously monitored using a Shimadzu SPD-10Avp UV-VIS detector and a Runtime Instruments Rec-Pro recorder.

[0121] [Measurement of flow rate] The filtrate was continuously weighed and collected using an electronic balance EJ-610 and weighing data logger AD-1688 manufactured by A&D Co., Ltd. A filtrate volume of 1.0 g was considered equivalent to 1.0 mL.

[0122] Here, the AGP LRV judgment criterion value for AGP-HA15 is ≥2.03. As mentioned above, the AGP LRV judgment criterion value is the clearance index of gold nanoparticles in which the virus removal performance is judged to be satisfactory, and can be calculated by measuring the absorbance of the colloidal solution containing gold nanoparticles, the filtrate of its filter, and water.

[0123] The results are shown in Table 2. As mentioned above, the flow rate when performing a gold colloid removal test on a used filter is generally 0.5 L / m². 2 Therefore, the membrane area is 0.001 m². 2 The liquid flow rate in the filter test was 0.5 mL. In Example 1, the filtration volume (liquid flow rate) was 100 mL or more in all cases, regardless of which filter was used. It can be seen that the liquid flow rate in this example is far greater than the liquid flow rate in the gold colloid removal test applied to filters after they have been used to filter biopolymers.

[0124] [Table 2]

[0125] As shown in Table 2, when filtered using Planova 35N, the absorbance of the filtrate was as high as that of the colloidal solution subjected to filtration. In other words, it was considered that the colloidal solution containing gold nanoparticles almost completely passed through this filter. Furthermore, the intermembrane pressure did not increase.

[0126] Among the three types of filters, using Planova 15N resulted in a lower maximum absorbance of the filtrate compared to using Planova 35N and Planova 20N. This suggests that Planova 15N has the smallest pore size among the three types of filters used in Example 1, which is consistent with the pore size relationships of filters disclosed by the filter manufacturer.

[0127] Furthermore, the change in intermembrane pressure across the three types of filters did not change significantly with the Planova 35N. When comparing filtration with Planova 20N and Planova 15N using roughly the same amount of material, the change in intermembrane pressure was greater with Planova 15N. From these results, it was concluded that the loading capacity of the colloidal solution containing gold nanoparticles was smallest with Planova 15N among the three filters of the same membrane area tested.

[0128] As described above, the colloidal solution AGP-HA15 containing gold nanoparticles used in Example 1 is a solution for a gold colloid removal test to confirm that there is no change in the performance of Planova 15N after use in filtering biopolymers, and the AGP LRV judgment criterion value for determining that the virus removal performance is acceptable is ≥2.03. In Example 1, when a large amount of this colloidal solution containing gold nanoparticles was passed through an unused Planova 15N, its AGP LRV was 2.11, which shows that it has sufficient virus removal performance even when an excessive amount of colloidal solution is passed through it.

[0129] The results of Example 1 are shown in Figures 1A and 1B. Figure 1A shows the flow rate (filtration rate) of the colloidal solution AGP-HA15 containing gold nanoparticles (L / m³) on the horizontal axis. 2 Figure 1B shows the absorbance (mAbs.) of the filtrate from each filter at a wavelength of 530 nm on the vertical axis. The horizontal axis of Figure 1B shows the volume (filtration volume) (L / m³) of the colloidal solution AGP-HA15 containing gold nanoparticles. 2 The vertical axis of the graph shows the change in the differential pressure between membranes of each filter (kPa). The average particle size of the gold nanoparticles in the colloidal solution AGP-HA15 containing gold nanoparticles is 20-24 nm. "mAbs." and "Abs." are abbreviations for absorbance.

[0130] As shown in Figure 1A, when filtering was performed using Planova 35N (average pore size 35±2 nm), the absorbance of the filtrate increased immediately after the start of filtration, suggesting that many gold nanoparticles were passing through without being captured by the filter. Furthermore, as shown in Figure 1B, the change in the intermembrane pressure difference of Planova 35N was small.

[0131] As shown in Figure 1A, when filtering with Planova 15N (average pore size 15±2 nm), the absorbance of the filtrate was the lowest among the three filters compared. Among the three filters compared, Planova 15N has the smallest pore size according to the catalog specifications, and this was also suggested by the low absorbance of the filtrate. As shown in Figure 1B, the change in intermembrane pressure was the largest among the three filters compared with Planova 15N. Given that the upper limit of the recommended operating pressure range for this filter is 98 kPa, it was expected that filtering a larger amount of colloidal liquid containing gold nanoparticles would reach this upper limit the fastest. Therefore, Planova 15N was considered to have the smallest load capacity among the three filters compared.

[0132] As shown in Figure 1A, when filtered using Planova 20N (average pore size 19±2 nm), the absorbance of the filtrate was greater than that of Planova 15N and less than that of Planova 35N. The pore size of the three filters compared corresponded to the absorbance of the filtrate. As shown in Figure 1B, the change in intermembrane pressure for Planova 20N was smaller than that for Planova 15N, suggesting that Planova 20N has a larger load capacity than Planova 15N.

[0133] [Example 2]: Filtration of colloidal solution AGP-HA20 containing gold nanoparticles through an unused filter. Membrane area 0.001m 2 Planova 15N (average pore size 15±2nm), Planova 20N (average pore size 19±2nm), and Planova 35N (average pore size 35±2nm) are mixed with a colloidal solution AGP-HA20 containing gold nanoparticles at a rate of approximately 0.2 L / min / m². 2 The liquids were passed through each filter at a constant flow rate. The differential pressure between the membranes of the filter, the volume of filtrate, and the absorbance of the filtrate at a wavelength of 526 nm were monitored. As shown in Table 1, the average particle size of the gold nanoparticles contained in AGP-HA20 is approximately 17-24 nm, with a standard deviation of 2-5 nm.

[0134] The results are shown in Table 3. As mentioned above, the flow rate when performing a gold colloid removal test on a used filter is generally 0.5 L / m³. 2Therefore, the membrane area is 0.001 m². 2 The liquid flow rate in the filter test is 0.5 mL. It can be seen that the filtration volume (liquid flow rate) in Example 2 is far greater than the liquid flow rate in the gold colloid removal test applied to filters after they have been used to filter biomolecules, regardless of which filter is used.

[0135] [Table 3]

[0136] As shown in Table 3, when filtered using Planova 35N, the absorbance of the filtrate was as high as that of the colloidal solution subjected to filtration. In other words, it was considered that the colloidal solution containing gold nanoparticles almost completely passed through this filter. Furthermore, the intermembrane pressure did not increase. The filtration experiment using Planova 35N was terminated when the liquid flow rate exceeded 50 mL.

[0137] Among the three types of filters, the maximum absorbance of the filtrate was lower when filtered using Planova 15N than when filtered using Planova 35N and Planova 20N. This suggests that Planova 15N has the smallest pore size among the three types of filters used in Example 2, which is consistent with the pore size relationships of filters disclosed by the filter manufacturer.

[0138] The change in intermembrane pressure across the three types of filters did not change significantly with Planova 35N. However, when comparing filtration with Planova 20N and Planova 15N using the same amount of material, the change in intermembrane pressure was greater with Planova 15N. Based on these results, it was concluded that the loading capacity of the colloidal solution containing gold nanoparticles was smallest with Planova 15N among the three filters of the same membrane area tested.

[0139] As described above, the colloidal solution AGP-HA20 containing gold nanoparticles used in Example 2 is a solution for a gold colloid removal test to confirm that there is no change in the performance of Planova 20N after use in filtering biopolymers, and the AGP LRV judgment criterion value for determining that the virus removal performance is acceptable is ≥1.40. In Example 2, when a large amount of this colloidal solution containing gold nanoparticles was passed through an unused Planova 20N, its AGP LRV was 1.45, which shows that it has sufficient virus removal performance even when an excessive amount of colloidal solution is passed through it.

[0140] The results of Example 2 are shown in Figures 2A, 2B, and 2C. Figure 2A shows the flow rate (filtration rate) of the colloidal solution AGP-HA20 containing gold nanoparticles (L / m³) on the horizontal axis. 2 Figure 2B shows the absorbance (mAbs.) of the filtrate from each filter at a wavelength of 526 nm on the vertical axis. The horizontal axis of Figure 2B shows the flow rate (filtration rate) (L / m³) of the colloidal solution AGP-HA20 containing gold nanoparticles. 2 Figure 2C shows the change in intermembrane pressure (kPa) of each filter on the vertical axis. Figure 2C shows the wavelength (nm) on the horizontal axis and the absorbance (Abs.) of the colloidal solution AGP-HA20 containing gold nanoparticles on the vertical axis. The average particle size of the gold nanoparticles in the colloidal solution AGP-HA20 is 17-24 nm.

[0141] The same results as those obtained in Figures 1A and 1B were obtained in Figures 2A and 2B.

[0142] Furthermore, in Example 2, a colloidal solution containing gold nanoparticles with a smaller particle size than in Example 1 was used, resulting in a larger absorbance of the filtrate and a greater change in the intermembrane pressure difference compared to Example 1. From this, it can be concluded that the filter is able to remove and separate materials by the filtration principle of size exclusion (screening by size).

[0143] Figure 2C shows the visible region absorption spectrum of the colloidal solution AGP-HA20 containing gold nanoparticles that was filtered in Example 2. When measured with a spectrophotometer capable of variable measurement wavelength, the maximum absorption wavelength was 526 nm. From Table 1 above, it is considered that the absorbance of AGP-HA20 is controlled at a wavelength of 526 nm, and that it exhibits the maximum absorption wavelength as specified. Note that the above colloidal solution contains SDS and PVP along with the gold nanoparticles. 〔measurement〕 A UV-Vis spectrophotometer V-750 manufactured by JASCO Corporation was used to measure water as a control in the wavelength range of 360 to 600 nm.

[0144] Although the tests in Examples 1 and 2 were conducted under conditions of a constant flow rate, the same can be understood under conditions where the pressure of the colloidal liquid loaded onto the filter is kept constant. That is, the load capacity can be compared and analyzed not only by comparing the change in the intermembrane pressure, but also by the change in the flow rate of the filtrate obtained by filtration. Filter clogging can be evaluated by the degree of increase in intermembrane pressure when the flow rate is kept constant, and by the degree of decrease in the flow rate of the filtrate when the pressure of the colloidal liquid loaded onto the filter is kept constant.

[0145] [Example 3]: Filtration of a colloidal solution containing gold nanoparticles and EDTA through an unused filter. Planova 15N, Planova 20N, Planova 35N, filter A (pore size unknown), filter B (pore size unknown), and filter C (pore size unknown) were prepared. The characteristics of the filters used are shown in Table 4.

[0146] [Table 4]

[0147] In Example 3, a colloidal solution containing the chelating agent EDTA-2Na and gold nanoparticles was prepared. This colloidal solution was prepared by mixing 1 volume of Asahi Kasei Integrity Test Solution AGP-HA20 (concentrated solution, containing SDS) with 9 volumes of a pH 5 solution containing 0.02 mol / L of EDTA-2Na. As a result, the SDS concentration in the diluted colloidal solution containing gold nanoparticles was reduced to 0.027%. The above colloidal solution can be applied at a rate of approximately 0.2 L / min / m². 2 The liquid was passed through each filter listed in Table 4 at a constant flow rate. The differential pressure between the membranes of the filters, the volume of filtrate, and the absorbance of the filtrate at a wavelength of 526 nm were monitored. From Table 1, the average particle size of the gold nanoparticles contained in AGP-HA20 is estimated to be around 17-24 nm, with a standard deviation of 2-5 nm. The results are shown in Table 5.

[0148] [Table 5]

[0149] As shown in Table 5, when filtered using Planova 35N, the absorbance of the filtrate was as high as that of the colloidal solution subjected to filtration. In other words, it was considered that the colloidal solution containing gold nanoparticles almost completely passed through this filter. Furthermore, the intermembrane pressure did not increase. The filtration experiment using Planova 35N was terminated after approximately 40 mL of solution had been passed through.

[0150] Table 5 compares the results of the six types of filters. The maximum absorbance of the filtrate was as follows: filter B < filter C < Planova 15N < filter A < Planova 20N < Planova 35N. Within the experimental range, at the end of filtration, the absorbance of the filtrate using filter C was close to the absorbance of the filtrate using filter B. Furthermore, the filtration results using Planova 15N and Planova 20N showed that the absorbance of their filtrates was higher than the value in Example 2, suggesting that the filterability of the colloidal solution containing gold nanoparticles may have differed slightly depending on the composition of the colloidal solution, such as the presence of a chelating agent.

[0151] In filtration using filters with membrane materials other than cellulose (filters B and C), the absorbance of the filtrate was relatively lower than in the example of filtration using filters with membrane materials made of cellulose. This suggests that the filter membrane material may also affect the filterability of colloidal solutions containing gold nanoparticles. However, the difference in absorbance of these filtrates may also be influenced by differences in filter pore size.

[0152] The results of Example 3 are shown in Figures 3A, 3B, and 3C. Figure 3A shows the volume (filtration rate) of the colloidal solution containing gold nanoparticles and EDTA on the horizontal axis (L / m³). 2 Figure 3B shows the absorbance (mAbs.) of the filtrate from each filter at a wavelength of 526 nm on the vertical axis. The horizontal axis of Figure 3B shows the volume (filtration volume) (L / m³) of the colloidal solution containing gold nanoparticles and EDTA. 2 Figure 3C shows the change in intermembrane pressure (kPa) of each filter on the vertical axis. Figure 3C shows the wavelength (nm) on the horizontal axis and the absorbance (Abs.) of the colloidal solution containing gold nanoparticles and EDTA on the vertical axis. This colloidal solution was prepared from a concentrated solution of the colloidal solution AGP-HA20 containing gold nanoparticles, and the average particle size of the gold nanoparticles in AGP-HA20 is 17-24 nm.

[0153] As shown in Figure 3A, the absorbance of the filtrates of Planova 35N, Planova 20N, and Planova 15N was Planova 35N > Planova 20N > Planova 15N, which corresponds to the pore size differences shown in the catalog.

[0154] As shown in Figure 3A, the absorbance of the filtrates obtained using filters A, B, and C increased in the order of filter B, filter C, and filter A. While the filterability of the colloidal solution containing gold nanoparticles may differ depending on the filter material, the differences in absorbance of these filtrates may also be influenced by the differences in filter pore size.

[0155] As shown in Figure 3B, the change in intermembrane pressure differed depending on the filter. Similar to Example 1, the change in intermembrane pressure was Planova 15N > Planova 20N > Planova 35N. Furthermore, the increase in intermembrane pressure when using filter B was greater than when using filters A, C, Planova 20N, and Planova 35N.

[0156] Since the upper limit of the recommended operating pressure range differs for each filter, it is thought that the allowable load capacity of each virus removal filter can be estimated by extrapolating the pressure rise curve. For example, in this embodiment, the intermembrane differential pressure at the start of filtration of Planova 15N was approximately 30 kPa. From Table 5, the liquid flow rate was 120 L / m³. 2 At this point, the intermembrane pressure difference increased by 37.6 kPa. Assuming that the intermembrane pressure difference increases linearly over time during filtration up to the upper limit of the Planova 15N's recommended operating pressure range of 98 kPa, the allowable load of colloidal solution containing gold nanoparticles in the Planova 15N is 200 L / m³. 2 It was thought to be less than [a certain value].

[0157] Figure 3C shows the visible absorption spectrum of the colloidal solution containing gold nanoparticles and EDTA that was filtered in Example 3.

[0158] Comparing Figure 2C, which shows the absorption spectrum of a colloidal solution of gold nanoparticles without the chelating agent EDTA and containing a larger amount of SDS, with Figure 3C, which shows the absorption spectrum of a colloidal solution of gold nanoparticles containing the chelating agent EDTA and with a lower SDS content, the maximum absorption wavelength of both colloidal solutions was 526 nm. Furthermore, the absorbance at wavelengths of 500-550 nm was 0.9 or higher in both Figure 2C and Figure 3C. This suggests that, as long as there are no interfering substances, the colloidal solution containing gold nanoparticles can be well monitored at a constant wavelength within this range.

[0159] In Example 3, the maximum absorption wavelengths of each filtrate were determined. The results are shown in Table 6.

[0160] [Table 6]

[0161] Table 6 shows that the maximum absorption wavelengths for each fraction obtained in each filtration experiment were in the range of 520–530 nm. Furthermore, no significant changes in the maximum absorption wavelengths were observed due to differences in the filtration fractions of each filter.

[0162] In addition, for filtration fractions where the absorbance of the filtrate was relatively low, the maximum absorption wavelength could not be determined, and these are indicated as ND (Not Determined) in the table.

[0163] As mentioned above, it is thought that as the particle size of gold nanoparticles decreases, the maximum absorption wavelength shifts to the shorter wavelength side.

[0164] Table 6 shows that the maximum absorption wavelengths of the filtrate were Planova 35N > Planova 20N = Filter A > Planova 15N > Filter C = Filter B, which followed a similar trend to the relative absorbance relationships of the filtrates in Figure 3A.

[0165] [Example 4] Investigation of SE-HPLC measurement conditions As mentioned above, size exclusion high-performance liquid chromatography (SE-HPLC) is a separation and analysis method that elutes molecules in order from largest to smallest based on their size. We investigated whether it is possible to compare the particle sizes of gold nanoparticles in a colloidal solution containing gold nanoparticles by analyzing the filtered filtrate using SE-HPLC.

[0166] The analysis was performed using one of the two existing silica gel-based packing columns for aqueous polymer analysis shown in Table 7.

[0167] [Table 7]

[0168] Table 8 shows an example of analysis of molecular weight markers for protein analysis using column 1. [HPLC equipment] Shimadzu LC-10A system Flow rate: 0.5mL / min Column: Use column 1, 25°C Detection wavelength: 526nm Mobile phase: 50 mM sodium phosphate, 300 mM sodium chloride, 0.05% sodium azide (pH 5.5) After equilibration at a flow rate of 0.5 mL / min, Bio-Rad Gel Filtration Standard (1511901) was injected and monitored for 40 minutes.

[0169] [Table 8]

[0170] Regarding the relationship between protein molecular weight and size, for example, Veritas Corporation describes an example where antibodies were measured as particles (https: / / www.veritastk.co.jp / products / reference / faq / dynabeads_protein_a_immunoprecipitation_kit.html) as follows: "Antibodies are thought to be approximately 16 nm in length and 8 nm in width. Considering that bovine serum albumin (BSA), with a molecular weight of about 66 kDa, has a diameter of about 4 nm, it can be inferred that IgG is roughly the same size." In the SE-HPLC analysis shown in Table 8, the retention time at the exclusion limit was approximately 10 minutes, and the peak of protein aggregates eluted at 10.748 minutes. The IgG (γ-globulin) dimer and monomer eluted at 13.644 minutes and 15.936 minutes, respectively. Assuming that the size difference between the dimer and monomer is twice as large, and that this difference is 8-16 nm, a difference of 8-16 nm in size would result in a retention time difference of approximately 2.3 minutes, and a difference of 1-2 nm in size would result in a retention time difference of around 0.2 minutes.

[0171] It is known that colloidal solutions containing gold nanoparticles may precipitate under high salt concentrations (for example, Patent Document 2). When we attempted to analyze the colloidal solution containing gold nanoparticles using the mobile phase used in the SE-HPLC analysis of the protein used in Example 4, no elution occurred. It should be noted that adding inorganic salts such as sodium chloride to the mobile phase to suppress non-specific adsorption of proteins, etc., due to ionic interactions is a technique used in SE-HPLC analysis of proteins. Furthermore, when a colloidal solution containing gold nanoparticles was analyzed using the same column with a mobile phase containing an organic solvent such as 40% acetonitrile containing 0.05% trifluoroacetic acid, no elution occurred. It should be noted that using a mobile phase containing an organic solvent such as 40% acetonitrile containing 0.05% trifluoroacetic acid is a technique used in SE-HPLC analysis of polypeptides.

[0172] However, when a mobile phase with a pH of 5 was prepared using a salt of a compound containing a carboxyl group (e.g., acetic acid, citric acid, EDTA), and a colloidal solution containing gold nanoparticles was injected and analyzed, it was found that the gold nanoparticles eluted.

[0173] [Example 5] SE-HPLC analysis 1 This example shows the results of injecting a fixed amount of the colloidal solution containing EDTA and gold nanoparticles from Example 3 into an HPLC instrument for analysis.

[0174] [HPLC equipment] Shimadzu LC-10A system Flow rate: 0.5mL / min Column: Use column 1, 25°C Detection wavelength: 526nm Mobile phase: 10-100 mmol / L sodium citrate, pH 5

[0175] Figure 4 shows a superimposed chromatogram obtained by changing the citrate concentration of the mobile phase. Specifically, studies were conducted with sodium citrate solutions of 10 mmol / L, 20 mmol / L, 40 mmol / L, and 100 mmol / L. The pH of the sodium citrate solution was 5 at this time.

[0176] As shown in Figure 4, a peak of gold nanoparticles, originating from the injected sample and detectable at a wavelength of 526 nm, appeared at a retention time of approximately 10 minutes. When a fixed amount of colloidal solution containing gold nanoparticles was injected and analyzed, the peak was larger when the mobile phase citrate concentration was 20 mmol / L and 40 mmol / L compared to when it was 10 mmol / L. On the other hand, when the mobile phase citrate concentration was 100 mmol / L, the peak was smaller than when it was 20 mmol / L and 40 mmol / L. From this, it was found that a mobile phase citrate concentration of approximately 20-40 mmol / L is optimal for SE-HPLC analysis of colloidal solutions containing gold nanoparticles.

[0177] [Example 6] SE-HPLC analysis 2 A colloidal solution containing SDS and gold nanoparticles (colloidal solution of Example 2) or a colloidal solution with reduced SDS concentration containing EDTA-2Na and gold nanoparticles (colloidal solution of Example 3) was injected three times for analysis. The following HPLC instrument was used.

[0178] [HPLC equipment] Shimadzu LC-10A system Flow rate: 0.5mL / min Column: Use column 1, 25°C Detection wavelength: 526nm Mobile phase: 30 mmol / L sodium citrate and 0.05% polyethylene glycol 6000, pH 5

[0179] Figure 5 shows a superimposed graph of the results obtained from Examples 2 and 3. From the measurement results, the peak amount obtained from the analysis of the colloidal solution containing SDS and gold nanoparticles (Example 2) was relatively small. The colloidal solution with a lower SDS content and containing EDTA and gold nanoparticles (Example 3) showed a larger peak amount from SE-HPLC analysis. As shown in Figure 5, adding synthetic polymer additives such as polyethylene glycol to the mobile phase did not interfere with the analysis.

[0180] [Example 7] SE-HPLC analysis 3 The colloidal solution containing gold nanoparticles used in Example 3, and the filtered filtrate of the colloidal solution containing gold nanoparticles obtained in Example 3, were subjected to SE-HPLC analysis using column 1. The analytical conditions were the same as in Example 6.

[0181] The results are shown in Table 9. Note that "SD" stands for standard deviation.

[0182] [Table 9]

[0183] As shown in Table 9, no significant difference in the retention time of the elution peak was observed among the fractional portions of the filtrate from each filter. Comparing the average retention times of the filtrates from each filter, the order was: tested colloidal solution < filtrate passed through Planova 35N < filtrate passed through Planova 20N = filtrate passed through filter A ≤ filtrate passed through Planova 15N < filtrate passed through filter C < filtrate passed through filter B. The difference in retention times was small, which was thought to be due to separation near the exclusion limit of this column. The relationship between the magnitudes of the retention times followed a similar trend to the relationship between the absorbances of the filtrates shown in Figure 3A, and the length of the maximum absorption wavelengths of the filtrates shown in Table 6.

[0184] As described above, by passing the colloidal solution containing gold nanoparticles through each filter and determining the magnitude of the maximum absorbance of the filtrate, as well as its maximum absorption wavelength, information regarding the particle size of gold nanoparticles in the filtrate was obtained. Furthermore, by comparing the retention times of the elution peaks of the colloidal solution containing gold nanoparticles using SE-HPLC analysis, information regarding the particle size of gold nanoparticles in the filtrate was obtained.

[0185] [Example 8] SE-HPLC analysis 4 For SE-HPLC analysis, column 2 (G4000SWXL) was used, and the same mobile phase as in Example 7 was used to analyze the colloidal solution containing gold nanoparticles from Example 3 and the filtrate from the filter obtained in Example 3.

[0186] Table 10 shows the holding times.

[0187] [Table 10]

[0188] The retention times of the colloidal solution containing gold nanoparticles, as determined by SE-HPLC analysis, were generally slower compared to the results in Example 7, and the differences in retention times between filtrate samples were also larger. This was thought to be due to the use of column 2, which has a wider range of molecular weight cutoff than column 1.

[0189] [Example 9] SE-HPLC analysis 5 A 10-100 mmol / L EDTA-2Na solution with pH 5 was used as the mobile phase. After equilibrating column 1 (G3000SWXL), a fixed amount of colloidal solution containing EDTA and gold nanoparticles was injected.

[0190] At a retention time of approximately 10 minutes, a peak of gold nanoparticles originating from the injected sample and detectable at a wavelength of 526 nm appeared. The amount of this peak indicated that a mobile phase concentration of EDTA·2Na of approximately 20–40 mmol / L was optimal. Furthermore, at lower or higher concentrations of EDTA salt, the gold nanoparticle peak became smaller, exhibiting a similar trend to SE-HPLC analysis using citrate as the mobile phase.

[0191] Furthermore, adding synthetic polymer additives such as polyethylene glycol to the mobile phase did not affect the analytical results.

[0192] [Example 10] SE-HPLC analysis 6 Using 30 mmol / L EDTA·2Na, 0.05% polyethylene glycol 6000, and pH 5 as the mobile phase, the colloidal solution containing gold nanoparticles used in Example 3 and the filtered filtrate of the colloidal solution containing gold nanoparticles obtained in Example 3 were subjected to SE-HPLC analysis using column 2. The analytical conditions were the same as in Example 9.

[0193] The results are shown in Table 11.

[0194] [Table 11]

[0195] Table 11 shows that comparing the retention times of the filtrates through each filter, the relationship is as follows: the colloidal solution tested < filtrate passed through Planova 35N < filtrate passed through Planova 20N < filtrate passed through filter A < filtrate passed through Planova 15N. This relationship is similar to the relationship between the absorbances of the filtrates shown in Figure 3A, and also similar to the relationship between the maximum absorption wavelengths of the filtrates shown in Table 6. Furthermore, similar results were observed even when the type of SE-HPLC column and mobile phase were changed.

[0196] It is easy to imagine that the particle size of gold nanoparticles contained in the filtered filtrate of a colloidal solution containing gold nanoparticles can be measured, for example, by dynamic light scattering analysis. [Industrial applicability]

[0197] By applying the analysis method of the present invention to various virus removal filters, including filters with unknown pore sizes, it is possible to determine the relative sizes of the pore sizes of the filters with unknown pore sizes. Furthermore, by applying the analysis method of the present invention to various virus removal filters, it is possible to compare their load capacities. The present invention can be used as an analysis method during the initial evaluation in the selection of virus removal filters.

Claims

1. The preparation step involves preparing two or more filters as filters, and Using each of the two or more filters mentioned above, a colloidal liquid containing gold nanoparticles is filtered through a filter 1 m ２ Filtration process: Pass a liquid through at a volume greater than 0.5 L per unit to obtain the filtrate. A comparative analysis method for the performance of virus removal filters, including [specific component].

2. The comparative analysis method according to claim 1, wherein the filter is a filter that has not been used for filtering biomolecules.

3. The comparative analysis method according to claim 1, wherein the average particle size of the gold nanoparticles is 17 nm or more.

4. 1m filter ２ The comparative analysis method according to claim 1, wherein a colloidal solution of 1 liter or more is passed through each volume.

5. The comparative analysis method according to claim 1, wherein the flow of the colloidal liquid is performed by maintaining a constant flow rate.

6. The comparative analysis method according to claim 1, wherein the liquid is passed through by maintaining a constant pressure of the colloidal liquid loaded onto the filter.

7. The comparative analysis method according to claim 1, wherein the maximum absorbance of the colloidal solution in the wavelength range of 500 to 550 nm is 0.9 or higher.

8. A step of measuring the absorbance of each of the obtained filtrates to light of a specific wavelength, and A process of comparing the measured maximum absorbance of the filtrate when using two or more filters, and comparing the average pore size of the two or more filters. Includes, The comparative analysis method according to claim 1, wherein the filtration step is performed by keeping the flow rate of the colloidal liquid constant, or by keeping the pressure of the colloidal liquid loaded onto the filter constant.

9. In the filtration step, a step of measuring the absorbance of the colloidal solution before filtration and the absorbance of the filtrate in response to light of a specific wavelength, A step of determining the difference between the absorbance of the colloidal solution before filtration and the maximum absorbance of the filtrate when using two or more filters, and A step of comparing the difference in absorbance when using two or more filters, and comparing the difference in the gold nanoparticle capture ability of the two or more filters. Includes, The comparative analysis method according to claim 1, wherein the filtration step is performed by keeping the flow rate of the colloidal liquid constant, or by keeping the pressure of the colloidal liquid loaded onto the filter constant.

10. The filtration process includes measuring the intermembrane pressure of the filter, determining the change in the measured intermembrane pressure, and A step of comparing the amount of change when using two or more filters and comparing the loading capacity of gold nanoparticles of the two or more filters. Includes, The filtration process is carried out by maintaining a constant flow rate of the colloidal liquid. The comparative analysis method according to claim 1.

11. The filtration process involves measuring the amount of liquid passing through the filter and determining the change in the measured amount of liquid passing through, and A step of comparing the amount of change when using two or more filters and comparing the loading capacity of gold nanoparticles of the two or more filters. Includes, The filtration process is carried out by maintaining a constant pressure of the colloidal liquid loaded onto the filter. The comparative analysis method according to claim 1.

12. A step to measure the particle size of gold nanoparticles contained in the filtrate obtained in the filtration step, and thereby compare and analyze the pore sizes of two or more filters. The comparative analysis method according to claim 1, including the method described in claim 1.

13. A process to compare and analyze the pore size of filters by measuring the absorbance of the filtrate obtained in the filtration step to light of a variable wavelength, and comparing the particle size of gold nanoparticles in the filtrates of two or more filters based on the maximum absorption wavelength of the filtrates of each filter. The comparative analysis method according to claim 12, including the method described in claim 12.

14. The filtrate obtained in the above filtration step is subjected to size exclusion chromatography to measure the elution time of gold nanoparticles contained in the filtrate, and the pore size of the filters is compared by comparing the particle size of the gold nanoparticles in the filtrates of two or more filters based on the elution times of the gold nanoparticles in each filtrate. including, The comparative analysis method according to claim 12.

15. The mobile phase of the aforementioned size exclusion chromatography analysis contains a compound having a carboxyl group or a salt thereof, and the pH of the mobile phase is in the range of 3 to 7. The comparative analysis method according to claim 14.

16. The mobile phase comprises at least one of citric acid, ethylenediaminetetraacetic acid, and salts thereof. The comparative analysis method according to claim 15.

17. In the mobile phase, the concentration of the compound having a carboxyl group is less than 100 mmol per liter of the mobile phase. The comparative analysis method according to claim 15.

18. In the mobile phase, the concentration of the compound having a carboxyl group is greater than 10 mmol per liter of the mobile phase. The comparative analysis method according to claim 15.

19. A process to compare and analyze the pore size of filters by measuring the particle size of gold nanoparticles contained in the filtrate obtained in the above filtration step by dynamic light scattering analysis, and by comparing the particle size of gold nanoparticles in the filtrate of two or more filters. The comparative analysis method according to claim 12, including the method described in claim 12.

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