Method for filtering a protein solution containing fibrinogen

Abstract

To provide a method for filtering fibrinogen-containing solutions that achieves both productivity and safety. [Solution] A method for filtering a protein solution containing fibrinogen, comprising a protein solution preparation step for preparing the protein solution having an average particle size of 30 nm or less and a concentration of 0.7 mg / mL or more and 4.5 mg / mL or less, and a water permeability of 45 L / hr·m 2 ·bar or more 200L / hr·m 2 A method comprising: a virus removal step, which is the step of filtering the protein solution using a virus removal filter containing a microporous membrane with a bar of less than or equal to bar.

Description

[Technical Field]

[0001] The present invention relates to a method for filtering a protein solution containing fibrinogen. [Background technology]

[0002] Plasma-derived products such as albumin, immunoglobulins, and fibrinogen are valuable biological products obtained from human blood. In recent years, the demand for immunoglobulins and fibrinogen has been increasing due to their applicability to various indications and diseases, as well as the challenges in manufacturing these products from animal cells. Plasma-derived products are generally obtained by 1) thawing frozen human plasma at low temperatures, treating it with ethanol, and recovering it by centrifugation to precipitate the target protein; 2) purifying the recovered protein by chromatography or other methods; and 3) filtering the purified protein through a virus removal filter.

[0003] Since the introduction of albumin preparations in 1942 and immunoglobulin preparations in 1943, plasma-derived products have been widely used worldwide. However, infections caused by viruses contained in plasma-derived products were reported until the 1980s. Currently, to mitigate the risk of viral infection, the manufacture of biological products is required to incorporate multiple virus inactivation and removal processes. Virus inactivation processes include acid treatment, heat treatment, solvent / detergent (S / D) treatment, and virus removal filtration. Acid treatment, heat treatment, and S / D treatment are not effective against all viruses and carry the risk of protein denaturation and yield loss, while virus removal filtration based on size exclusion is effective against all viruses and does not induce a decrease in recovery rate due to protein denaturation, etc. Therefore, virus removal membranes are widely used in virus inactivation processes and greatly contribute to the safety of biological products.

[0004] The primary performance requirement for a virus removal membrane is safety. Safety means preventing the contamination of plasma-derived products and biopharmaceuticals with pathogenic substances such as viruses. Non-patent document 1 states that the target clearance (LRV) for mouse microviruses and porcine parvoviruses in a virus removal membrane should be 4.

[0005] The second performance requirement for a virus removal membrane is productivity. Productivity refers to the efficient recovery of the protein formulation. Fibrinogen is a large protein (average molecular weight 340 kDa) found in plasma, and its structure consists of three pairs of polypeptide chains, giving it a linear structure (equivalent to 9 x 50 nm). It also readily associates and aggregates in protein solutions. When a protein solution containing such fibrinogen is passed through a filter to remove viruses, the filter easily becomes clogged. On the other hand, if a filter with a large pore size is used as the microporous membrane constituting the filter to suppress clogging, the virus leaks out, creating a problem.

[0006] As research to solve such problems, Patent Document 1 discloses a method for filtering a fibrinogen solution using SV4™ manufactured by Pall Corporation and Planova 20N™ manufactured by Asahi-Kasei Medical Corporation. Patent Document 2 discloses a method for filtering a fibrinogen solution using Pall's DV20™. Patent Document 3 discloses a method for filtering a fibrinogen solution using Planova 15N (trademark) manufactured by Asahi-Kasei Medical Co., Ltd. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] International Publication No. 2020128385 [Patent Document 2] Patent No. 4272562 [Patent Document 3] Patent No. 4495404 [Non-patent literature]

[0008] [Non-Patent Document 1] ICHQ5A [Overview of the Initiative] [Problems that the invention aims to solve]

[0009] However, Patent Document 1 (International Publication No. 2020128385) describes filtering a fibrinogen solution using Pall's SV4™ filter. While this method recovers fibrinogen with high productivity, the high pore size prevents the viral clearance (LRV) from reaching the target of 4 or higher set by Non-Patent Document 1 (ICHQ5A). Furthermore, while Planova 20N™ from Asahi-Kasei Medical is used to filter the fibrinogen solution, there is room for improvement in preventing clogging by fibrinogen and in achieving highly productive fibrinogen recovery.

[0010] In Patent Document 2 (International Publication No. 4272562), a fibrinogen solution is filtered using Pall's DV20™, but the FLUX is low, and highly productive fibrinogen recovery has not been achieved. Patent Document 3 (International Publication No. 4495404) describes filtering a fibrinogen solution using Planova 15N™ manufactured by Asahi-Kasei Medical, but there is room for improvement in increasing FLUX and recovering fibrinogen with higher productivity.

[0011] Further, as reported in the above Patent Documents 1, 2 and 3, it has been reported that the filterability of a protein solution can be improved to a certain extent by, for example, adding arginine to the protein solution. However, merely adding arginine may not improve the filterability in some cases, and it is important to bring the filtration solution into a state that suppresses clogging of the membrane. Heretofore, however, there have been no reports confirming a state of the filtration solution that suppresses clogging of the membrane.

[0012] In view of the above circumstances, an object of the present invention is to efficiently remove viruses while suppressing clogging during filtration of a solution containing fibrinogen which is a large protein, that is, to provide a method for filtering a solution containing fibrinogen that achieves both productivity and safety. [Means for Solving the Problems]

[0013] As a result of intensive studies to solve the above problems, the present inventors have found that, in particular, by combining a protein solution having an average particle diameter and a concentration within predetermined numerical ranges with a step of filtering the protein solution using a predetermined virus removal filter, the protein solution can be recovered with both productivity and safety, and the present invention has been completed. That is, the present invention is as follows. [1] A method for filtering a protein solution containing fibrinogen, comprising: a protein solution preparation step of preparing the protein solution having an average particle diameter of 30 nm or less and a concentration of 0.7 mg / mL or more and 4.5 mg / mL or less; and a virus removal step, which is a step of filtering the protein solution using a virus removal filter including a microporous membrane having a water permeability of 45 L / hr·m 2 ·bar or more and 200 L / hr·m 2 ·bar or less. The method. [2] The method according to [1], wherein the membrane material of the virus removal filter comprises cellulose. The method described in [1]. [3] In viral clearance tests, the logarithmic reduction rate of porcine parvovirus is 4 or higher. The method described in [1] or [2]. [4] The microporous membrane has an asymmetric structure and has a sparse layer on the filtration inlet side of the virus removal filter. The method described in any of [1] to [3]. [5] The protein solution contains arginine, The concentration of arginine relative to the total volume of the protein solution is between 150 mM and 1000 mM. The method described in any of [1] to [4]. [6] The protein solution contains sodium chloride, The concentration of sodium chloride relative to the total volume of the protein solution is between 50 mM and 500 mM. The method described in any of [1] to [5]. [7] The protein solution contains sodium citrate, The concentration of sodium citrate relative to the total volume of the protein solution is between 10 mM and 300 mM. The method described in any of [1] to [6]. [8] The aforementioned microporous membrane is a hollow fiber membrane. The method described in any of [1] to [7]. [Effects of the Invention]

[0014] According to the present invention, it is possible to provide a method for filtering a fibrinogen-containing protein solution that efficiently removes viruses while suppressing clogging during filtration of a solution containing fibrinogen, a large protein, that is, a method that achieves both productivity and safety. [Modes for carrying out the invention]

[0015] Embodiments of the present invention are described below. However, these embodiments should not be understood as limiting the present invention. Various alternative embodiments, examples, and operational techniques should become apparent to those skilled in the art from this disclosure. It should be understood that the present invention encompasses various embodiments and the like that are not described herein.

[0016] 1. Method for filtering a protein solution containing fibrinogen The method for filtering a fibrinogen-containing protein solution according to this embodiment is a method for filtering a fibrinogen-containing protein solution comprising a protein solution preparation step of preparing the protein solution having an average particle size of 30 nm or less and a concentration of 0.7 mg / mL or more and 4.5 mg / mL or less, and a water permeability of 45 L / hr·m 2 ·bar or more 200L / hr·m 2 The method includes a virus removal step, which is the step of filtering the protein solution using a virus removal filter containing a microporous membrane with a bar of less than or equal to bar. Furthermore, the protein filtration method of this embodiment may include other steps, as long as they do not hinder the effects of the present invention.

[0017] The mechanism by which the method for filtering a fibrinogen-containing protein solution in this embodiment is superior in productivity and safety is not particularly limited, but can be considered as follows. The average particle size of the protein solution in this embodiment is measured by dynamic light scattering, which is a method for measuring the average diffusion coefficient of particles in a solution. A large average particle size calculated by dynamic light scattering means a small average diffusion coefficient, and a small average particle size means a large average diffusion coefficient. Furthermore, when the substance contained in the solution is the same, if the average particle size measured by dynamic light scattering changes, it means that the average diffusion coefficient has changed. If the average diffusion coefficient increases, it means that the intermolecular interactions of the particles in the solution have been suppressed, and conversely, if the average diffusion coefficient decreases, it means that the intermolecular interactions of the particles in the solution have been suppressed. In other words, a large average particle size measured by dynamic light scattering indicates that intermolecular interactions are at work between particles, promoting association and aggregation, which can cause membrane clogging when the solution is filtered, making it difficult to recover highly productive proteins. Therefore, it is believed that if the average particle size of the particles in the protein solution, as measured by dynamic light scattering, is within a certain range, association and aggregation based on intermolecular interactions are suppressed, preventing membrane clogging when the protein solution is filtered, and enabling the recovery of highly productive proteins.

[0018] 1.1. Protein Solution Preparation Process The protein solution preparation step of this embodiment is a step of preparing a protein solution having an average particle size of 30 nm or less and a concentration of 0.7 mg / mL or more and 4.5 mg / mL or less.

[0019] <Concentration adjustment process, average particle size adjustment process> The protein solution preparation step of this embodiment may include a concentration adjustment step to adjust the concentration of the protein solution to 0.7 mg / mL or more and 4.5 mg / mL or less, and an average particle size adjustment step to adjust the average particle size to 30 nm or less. If the concentration adjustment step and the average particle size adjustment step are included, they may be performed simultaneously or separately.

[0020] <Purification process> Furthermore, from the viewpoint of superior productivity, the protein solution preparation step of this embodiment preferably includes a purification step before and / or after the concentration adjustment step and the average particle size adjustment step, in which fibrinogen associations and aggregates in the fibrinogen-containing protein solution are removed from the filtered solution.

[0021] The purification process is not particularly limited, but examples include size removal, chromatographic removal, and pretreatment with pre-filtration.

[0022] For size removal, there are no particular limitations, but for example, conventionally known filtration membranes can be used. Such filtration membranes are not particularly limited, but for example, membranes with a nominal pore size of 0.1 to 0.22 μm such as Merck's Millex™, or Asahi-Kasei Medical's Planova 35N™, Planova 75N™, etc. For chromatographic removal, conventional methods can be used, and there are no particular limitations, but for example, hydrophobic interaction chromatography, ion exchange chromatography, etc. For pretreatment with a prefilter, conventional methods can also be used, and there are no particular limitations, but for example, methods using Sartorius' Virosart Max™ and Merck's Viresolve Pro Shield™, etc.

[0023] The solution temperature in the concentration adjustment step, average particle size adjustment step, and purification step is preferably 40°C or lower. This is because lowering the solution temperature to 40°C or lower tends to prevent fibrinogen from denaturing and becoming inactive. The lower limit of the temperature is not particularly limited, but for example, it may be 25°C, 30°C, or 35°C.

[0024] <Average particle size> The average particle size of the fibrinogen-containing protein solution is 30 nm or less, preferably 20 nm to 29 nm, more preferably 20 nm to 28 nm, and even more preferably 20 nm to 26 nm.

[0025] By reducing the average particle size of the fibrinogen-containing protein solution to 30 nm or less, the association and aggregation of proteins can be suppressed, preventing clogging during filtration and enabling highly productive protein recovery.

[0026] The average particle size according to this embodiment is measured using dynamic light scattering. Specifically, measurements were performed using Marvern's Zetasizer ZSP at pH 7.0, a measurement temperature of 25°C, equilibration for 120 seconds, 3 measurements, and a detection angle of 173 degrees (backscatter). The average particle size was then calculated by cumulant analysis.

[0027] <Concentration> Furthermore, the concentration of the protein solution containing fibrinogen is 0.7 mg / mL or more and 4.5 mg / mL or less, preferably 1.0 mg / mL or more and 4.0 mg / mL or less, more preferably 1.5 mg / mL or more and 3.5 mg / mL or less, and even more preferably 1.5 mg / mL or more and 2.5 mg / mL or less.

[0028] By maintaining a fibrinogen-containing protein solution concentration of 0.7 mg / mL or higher, it is possible to prevent the protein concentration in the solution from becoming too low, thereby achieving highly productive protein recovery. On the other hand, by setting an upper limit on the concentration of the fibrinogen-containing protein solution, it is possible to prevent the association and aggregation of proteins. A fibrinogen-containing protein solution concentration of 4.5 mg / mL or lower provides excellent inhibition of protein association and aggregation, enabling highly productive protein recovery.

[0029] <Arginine> From the viewpoint of superior productivity, the protein solution of this embodiment contains arginine, and the concentration of arginine relative to the total amount of the protein solution is preferably 150 mM or more and 1000 mM or less, more preferably 200 mM or more and 800 mM or less, even more preferably 250 mM or more and 600 mM or less, and even more preferably 300 mM or more and 500 mM or less.

[0030] Although not bound by theory, adding arginine to the fibrinogen-containing protein solution tends to reduce the average particle size because arginine coordinates between fibrinogen molecules, suppressing their proximity. This further inhibits the association and aggregation of fibrinogen molecules. Furthermore, it is expected to assist in the refolding of reversibly deformed aggregates and convert them back into monomers, thus reducing the average particle size of the fibrinogen-containing protein solution. Arginine concentrations higher than 150 mM tend to be more effective in inhibiting the association and aggregation of fibrinogen molecules, resulting in higher productivity. Additionally, arginine concentrations below 1000 mM tend to shorten the time required to remove arginine for formulation after the virus removal process, further improving productivity.

[0031] <Sodium Chloride> From the viewpoint of superior productivity, the protein solution of this embodiment contains sodium chloride, and the concentration of sodium chloride relative to the total amount of the protein solution is preferably 50 mM or more and 500 mM or less, more preferably 50 mM or more and 400 mM or less, even more preferably 50 mM or more and 200 mM or less, and even more preferably 50 mM or more and 150 mM or less.

[0032] <Sodium citrate> From the viewpoint of superior productivity, the protein solution of this embodiment contains sodium citrate, and the concentration of sodium citrate relative to the total amount of the protein solution is preferably 10 mM to 300 mM, more preferably 10 mM to 200 mM, and even more preferably 10 mM to 100 mM.

[0033] 2. Virus removal process The virus removal step of this embodiment is a step of filtering the protein solution using a virus removal filter that includes a microporous membrane containing cellulose as the membrane material and having a logarithmic reduction rate of 4 or more for porcine parvovirus in a virus clearance test, and the permeability of the virus removal filter is 45 L / hr·m 2 ·bar or more 200L / hr·m 2 It is less than or equal to bar.

[0034] Furthermore, the temperature of the virus removal process is preferably 40°C or lower. This is to prevent fibrinogen from denaturing and becoming inactive. The lower limit of the temperature is not particularly limited, but for example, it may be 25°C, 30°C, or 35°C.

[0035] <Selection of virus removal membrane> In the virus removal step of this embodiment, the protein solution is filtered using a virus removal filter that includes a microporous membrane containing cellulose as the membrane material and exhibiting a logarithmic reduction rate of 4 or more for parvovirus in a virus clearance test.

[0036] Because the cellulose included as a membrane material is hydrophilic, it is less prone to protein adsorption, membrane contamination, and clogging. The cellulose mentioned above is not particularly limited, but examples include regenerated cellulose, natural cellulose, and cellulose acetate. The method for producing regenerated cellulose is not particularly limited, but examples include producing it from a copper ammonia cellulose solution or producing it by saponifying cellulose acetate with an alkali.

[0037] The microporous membrane of the virus removal filter may be a hollow fiber membrane or may be in the form of a flat membrane. Among these, from the viewpoint of a large filtration area and excellent productivity, the microporous membrane of the present embodiment is preferably a hollow fiber membrane.

[0038] In the virus removal step of the present embodiment, when filtering a protein solution containing fibrinogen with a hollow fiber membrane, from the viewpoint of enabling effective capture of viruses in the solution, it is preferable to employ a method of performing filtration in the direction in which the solution flows from the inner surface side to the outer surface side of the hollow fiber (internal pressure filtration method).

[0039] The thickness of the microporous membrane included in the virus removal filter is not particularly limited, and is, for example, 30 μm or more and 45 μm or less in a dry state. When the thickness is 30 μm or more, the pressure resistance of the membrane tends to be maintained, and when the thickness is 45 μm or less, it is possible to further prevent the membrane resistance from increasing and the FLUX from decreasing, and it tends to make filter molding easier.

[0040] The water permeability of the virus removal filter is 45 L / hr·m 2 ·bar or more and 200 L / hr·m 2 ·bar or less, preferably 50 L / hr·m 2 ·bar or more and 160 L / hr·m 2 ·bar or less, and it is further preferred that the water permeability of the virus removal filter is 55 L / hr·m 2 ·bar or more and 80 L / hr·m 2 ·bar or less. When the water permeability is 45 L / hr·m 2 ·bar or more, it is possible to further prevent the FLUX from decreasing and the productivity of protein solution recovery from lowering, and when the water permeability is 200 L / hr·m 2 ·bar or less, the average pore diameter of the membrane tends to increase, which tends to further prevent the virus removal rate from decreasing and the safety of the preparation from lowering.

[0041] The water permeability in the present embodiment refers to a case where the effective membrane area of the filtration membrane is 1 cm2 The virus removal filter, assembled to achieve this configuration, is subjected to constant-pressure dead-end filtration at 1.0 bar, and the amount of pure water filtered at 25°C is measured, representing the flow rate per unit pressure, per unit area, and per unit time. Permeability is a measure of the overall average pore size of the microporous membrane.

[0042] <Microporous membrane> In this embodiment, the microporous membrane has an asymmetric structure, and it is preferable that it has a sparse layer on the filtration inlet side of the virus removal filter, from the viewpoint of superior productivity and safety. Furthermore, it is even more preferable that it further includes a dense layer, which has a homogeneous structure with little change in pore size, in order to capture the viruses to be removed.

[0043] In this embodiment, an asymmetric structure refers to a structure in which a sparse layer is present on the filtration inlet side of the virus removal filter (hereinafter also referred to as the inlet surface) and a dense layer is present on the filtration outlet side (hereinafter also referred to as the outlet surface).

[0044] Here, a sparse layer refers to a layer with a gradient structure in which the average pore diameter gradually increases from the surface near the exit towards the inlet. A dense layer is a layer with a homogeneous structure in which the pore diameter changes little, and its specific definition is described below.

[0045] The presence of a sparse layer and a dense layer in the microporous membrane of this embodiment can be determined by the following method. First, a cross-section of the microporous membrane of this embodiment is photographed using a scanning electron microscope (SEM). Specifically, for example, the magnification is set to 50,000x, and a field of view is set horizontally with respect to the film thickness direction at any point in the cross-section of the microporous membrane. After photographing one field of view, the field of view is moved horizontally with respect to the film thickness direction, and the next field of view is photographed. This photography operation is repeated to take photographs of the membrane cross-section without gaps, and the obtained photographs are combined to obtain a single photograph of the membrane cross-section. In this photograph of the cross-section, the average pore diameter in the range of (2 μm in the circumferential or planar direction of the membrane) × (1 μm from the outer surface toward the inner surface) is calculated, and the gradient structure of the membrane cross-section is quantified at 1 μm intervals from the outlet surface toward the inlet surface. This quantification allows us to confirm that the microporous membrane has a sparse layer in which the average pore diameter increases from the outer surface towards the inner surface. Here, the proportion of the sparse layer to the total film thickness is not particularly limited, but for example, it may be equivalent to 10% or more of the total film thickness, 20% or more, or 30% or more. The upper limit is not particularly limited, but it may be 50%.

[0046] Here, for a structure to be said to have a sparse layer, it is sufficient to confirm that the average pore diameter tends to increase from the surface near the exit toward the inlet, and it does not mean that the average pore diameter is smallest in the 1 μm range from the surface near the exit toward the inlet, and that the average pore diameter is largeest in the 1 μm range from the inlet toward the surface near the exit.

[0047] The average pore diameter is calculated using an image analysis method. Specifically, Image-pro plus from MediaCyberbetics is used to perform binarization of pores and solid areas. Pores and solid areas are identified based on brightness, and any unidentified areas or noise are corrected using a freehand tool. Edges that form the contour of pores and porous structures observed deep within pores are identified as pores. After binarization, the area value of each pore is assumed to be a perfect circle, and the diameter of the pore is calculated. This is performed for every pore, and the average pore diameter is calculated for each 1 μm × 2 μm area. Pores that are interrupted at the edge of the field of view are also counted. In this embodiment, a 1 μm × 2 μm area where the average pore diameter is calculated to be 50 nm or less is defined as a dense layer.

[0048] Here, the proportion of the dense layer to the total film thickness is not particularly limited, but for example, it may be equivalent to 90% or less of the total film thickness, 80% or less, or 70% or less. The lower limit is not particularly limited, but it may be 50%.

[0049] By forming a porous layer on the filtration inlet side, the flowability of the protein solution at the filtration inlet side is improved, and clogging tends to be further suppressed. The protein solution that passes through the porous layer moves to the dense layer located inside the microporous membrane. The dense layer may be a layer with a consistent microporous structure, or it may be a layer with variations in pore size within it.

[0050] If there is variation in the pore size of the dense layer, the layer may have a continuous pore size gradient structure, or it may have an inflection point of pore size change within it. However, it is preferable to have an asymmetric structure in which the pore size gradient is located on the filtration inlet side and the dense layer is located from the center of the membrane to near the outermost layer on the filtration outlet side.

[0051] The above-mentioned sparse and dense layers can be produced by conventionally known methods. Specifically, although not particularly limited, for example, as disclosed in Patent Document 4, when forming a film from a copper ammonia cellulose solution, a sparse layer can be formed by increasing the ammonia concentration of the internal solution compared to the film-forming stock solution, and a dense layer with a narrow pore size distribution and thickness can be formed by lowering the concentration of non-solvents in the internal and external solutions.

[0052] <Virus clearance test> The clearance of porcine parvovirus by the microporous membrane contained in the virus removal filter is preferably 4 or higher as a logarithmic reduction rate (hereinafter also referred to as LRV), more preferably 5 or higher, and even more preferably 6 or higher. In this embodiment, the logarithmic reduction rate is the ratio of the amount of virus after the virus removal process to the amount of virus before the virus removal process, expressed logarithmically, and can be expressed by the following formula (1). • Logarithmic decrease rate (%) = log 10 (Amount of virus after virus removal process / Amount of virus before virus removal process) ... Equation (1) [Examples]

[0053] The present invention will be described in detail below with reference to examples, but the present invention is not limited to the following examples. The test methods shown in the examples are as follows.

[0054] (Water permeability measurement) Effective film area is 1 cm 2 The filter, assembled to achieve the desired result, was subjected to constant-pressure dead-end filtration at 1.0 bar to measure the amount of pure water filtered at 25°C, and the permeability was calculated based on the filtration time.

[0055] <Protein Solution Preparation Process> (Fibrinogen-containing protein solution filtration) A protein solution containing fibrinogen (Sigma-Aldrich, catalog number F3879) was dissolved in 50 mM citrate buffer at pH 7 containing 200 mM sodium chloride. Ammonium sulfate was then added in small amounts to obtain a protein solution containing 80% ammonium sulfate. The solution was further cooled on ice, and the supernatant was removed by centrifugation. The precipitate was then dissolved in 50 mM citrate buffer at pH 7 containing 50 mM sodium chloride, and filtered using Merck Millex 0.2 μm and 0.1 μm filters. The filtrate was dialyzed using the same solution as the external solution with a Thermo Fisher Slide-A-Lyzer Dialysis Cassette MWCO20k. The resulting solution was passed through a Cytiva HiTrap SP HP and then through a Cytiva HiTrap Phenyl HP for purification. The protein solution was treated using adsorbent membranes, Merck Millex 0.2 μm and 0.1 μm, and hydrophobic ion exchange chromatography systems, Cytiva HiTrap SP HP and HiTrap Phenyl HP, to remove aggregates and adjust the average particle size to 30 nm or less. Subsequently, the solution was prepared so that the pH of the fibrinogen-containing protein solution was 7, and the fibrinogen concentration, citrate concentration, arginine concentration, and sodium chloride concentration were all at predetermined concentrations. <Purification process> The solution prepared above was filtered using Merck Millex 0.2 μm and 0.1 μm filters at a dead end under a constant pressure of 0.5 bar. This solution was then filtered again at a dead end under a constant pressure of 1.0 bar for 180 minutes. <Average particle size measurement> The average particle size according to this embodiment is measured using dynamic light scattering. Specifically, measurements were performed using Marvern's Zetasizer ZSP at pH 7.0, a measurement temperature of 25°C, equilibration for 120 seconds, 3 measurements, and a detection angle of 173 degrees (backscatter). The average particle size was then calculated by cumulant analysis.

[0056] <Virus Removal Process> (Pig parvovirus clearance measurement) The protein solution containing fibrinogen prepared as described above was spiked with a 0.35% by volume solution of porcine parpovirus (hereinafter simply referred to as PPV) to obtain a filtrate. The prepared filtrate was filtered at a constant pressure of 1.0 bar or 2.0 bar for 180 minutes using a dead end filter.

[0057] The table shows "Cumulative fibrinogen treatment volume over 180 minutes" (g / m²). 2 The productivity evaluation index ( / 3hr) was calculated based on the amount of filtrate recovered, the fibrinogen concentration of the filtrate, and the membrane area of ​​the filter over 180 minutes under the above conditions. The cumulative fibrinogen treatment amount over 180 minutes was 100 g / m². 2 Cases with a filtration pressure of 3hr / bar or higher were defined as examples, and cases with a value lower than that were defined as comparative examples. That is, when the filtration pressure is 1.0 bar, the value is 100 g / m³. 2 At 3 hours and a filtration pressure of 2.0 bar, the reading is 200 g / m³. 2 This can be converted to 3 hours.

[0058] The logarithmic reduction rate is an indicator of safety, and the Titer (TCID50 value) of the filtrate was measured using a viral assay. The viral clearance of PPV was calculated using the formula LRV = Log(TCID50) / mL (filtrate) - Log(TCID50) / mL (filtrate). Cases with a logarithmic reduction rate of 4 or higher were designated as examples, and cases with a rate less than 4 were designated as comparative examples.

[0059] (Example 1) A protein solution containing fibrinogen (Sigma-Aldrich) was dissolved in a 50 mM citrate buffer at pH 7 containing 200 mM sodium chloride. Ammonium sulfate was then added in small amounts to obtain a protein solution containing 80% ammonium sulfate. The solution was further cooled on ice, and the supernatant was removed by centrifugation. The precipitate was then dissolved in a 50 mM citrate buffer at pH 7 containing 50 mM sodium chloride and filtered using Merck Millex 0.2 μm and 0.1 μm filters. The filtrate was dialyzed using the same solution as the external solution with a Thermo Fisher Slide-A-Lyzer Dialysis Cassette MWCO20k. The resulting solution was passed through a Cytiva HiTrap SP HP and then through a Cytiva HiTrap Phenyl HP for purification. Subsequently, a solution was prepared with a fibrinogen concentration of 2 mg / mL, a citrate concentration of 50 mM, an arginine concentration of 400 mM, a sodium chloride concentration of 150 mM, and a pH of 7. Subsequently, the mixture was filtered using Merck Millex 0.2 μm and 0.1 μm filters. Further filtration using Sartorius Virosart Max filters yielded treatment solution 1.

[0060] Processing solution 1 was filtered using a Planova 20N at a dead end under a constant pressure of 1.0 bar for 180 minutes. The results are shown in Table 1. From Table 1, it was confirmed that Example 1 is a filtration method for fibrinogen-containing protein solutions that balances productivity and safety.

[0061] For Examples 2-9 and Comparative Examples 2-6, the virus removal process described above was performed on treatment solutions 2-9 and 12-16 according to the conditions in Tables 1 and 2, using the same procedure as for treatment solution 1 described above, except for the conditions in Tables 1 and 2. The results are shown in Tables 1 and 2.

[0062] In Example 10, treatment solution 1 was filtered using a Planova S20N at a dead end under a constant pressure of 2.0 bar for 180 minutes. The results are shown in Table 1.

[0063] [Table 1]

[0064] [Table 2]

[0065] In Comparative Example 1, the solution was prepared to have the same composition as in Example 1, except that the purification described above was not performed. Then, it was filtered at a constant pressure of 0.5 bar with a dead end using Merck Millex 0.2 μm and 0.1 μm filters, and then filtered again at a constant pressure of 1.0 bar for 180 minutes with a dead end using a Planova 20N filter. The results are shown in Tables 1 and 2.

[0066] Tables 1 and 2 confirm that in Examples 2 to 9, the filtration of the fibrinogen-containing protein solution achieved both productivity and safety.

[0067] In Comparative Example 1, the average particle size was greater than 30 nm, causing clogging during filtration, and therefore, the protein solution containing fibrinogen could not be filtered with high productivity.

[0068] In Comparative Example 2, the concentration of the fibrinogen-containing protein solution was less than 0.7 mg / mL, making it impossible to filter the fibrinogen-containing protein solution with high productivity.

[0069] In Comparative Example 3, the concentration of the fibrinogen-containing protein solution was greater than 4.5 mg / mL, causing clogging during filtration. As a result, the fibrinogen-containing protein solution could not be filtered with high productivity.

[0070] In Comparative Example 4, the average particle size was over 30 nm, causing clogging during filtration, and therefore, the protein solution containing fibrinogen could not be filtered with high productivity.

[0071] In Comparative Example 5, the water permeability of the virus removal filter was 45 L / hr·m. 2 Because the pore size was less than bar and the pore diameter was small, it was not possible to filter the fibrinogen-containing protein solution with high productivity.

[0072] In Comparative Example 6, the water permeability of the virus removal filter was 200 L / hr·m. 2 Due to its large pore size and high safety profile, it was not possible to filter out protein solutions containing fibrinogen. [Industrial applicability]

[0073] The present invention provides a filtration method capable of removing viruses from a protein solution containing fibrinogen, and can be used in the production of plasma-derived products containing fibrinogen.

Claims

1. A method for filtering a protein solution containing fibrinogen, A protein solution preparation step for preparing the protein solution having an average particle size of 30 nm or less and a concentration of 0.7 mg / mL or more and 4.5 mg / mL or less, The water permeability is 45 L / hr·m. 2 ・Over bar 200L / hr・m 2 - A virus removal step which includes filtering the protein solution using a virus removal filter containing a microporous membrane with a bar of 0. method.

2. The aforementioned virus removal filter contains cellulose as its membrane material. The method according to claim 1.

3. In viral clearance tests, the logarithmic reduction rate of porcine parvovirus is 4 or higher. The method according to claim 1.

4. The microporous membrane has an asymmetric structure and has a sparse layer on the filtration inlet side of the virus removal filter. The method according to claim 1.

5. The protein solution contains arginine, The concentration of arginine relative to the total volume of the protein solution is 150 mM or more and 1000 mM or less. The method according to claim 1.

6. The protein solution contains sodium chloride, The concentration of sodium chloride relative to the total amount of the protein solution is 50 mM or more and 500 mM or less. The method according to claim 1.

7. The protein solution contains sodium citrate, The concentration of sodium citrate relative to the total volume of the protein solution is 10 mM or more and 300 mM or less. The method according to claim 1.

8. The method according to claim 1, wherein the microporous membrane is a hollow fiber membrane.

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