Method for removing viruses from protein solutions

Abstract

Provided is a method for deactivating or removing viruses in a protein solution, the method including a step in which radiation treatment and virus removal membrane treatment or SD treatment are integrated and the conditions of these treatments are optimized to be mutually complementary. More specifically, the method includes: (a) a step in which a protein solution is treated by being irradiated with radiation so that the removal coefficient (LRV) for viruses with a virus particle diameter of less than 33 nm in the protein solution is at least 1.00; and (b1) a step in which viruses are removed from the treated protein solution by a virus removal membrane with an LRV of at least 4.00 for bacteriophage PP7, or (b2) a step in which the treated protein solution is brought into contact with a liquid mixture (SD liquid mixture) of an organic solvent and a surfactant and viruses are deactivated by organic solvent / surfactant treatment (SD treatment) so that the LRV for envelope viruses in the protein solution is at least 2.00. Also provided is a method for manufacturing a protein solution in / from which viruses have been deactivated / removed, the method including the abovementioned steps.

Description

[Technical Field]

[0001] This invention relates to a method for removing viruses from a protein solution, and a method for producing a protein solution from which the virus has been removed. [Background technology]

[0002] The manufacturing processes for plasma-derived products and biopharmaceuticals incorporate a virus inactivation and / or removal process (hereinafter abbreviated as "virus inactivation / removal") from the standpoint of safety and stability of the products. Generally, virus inactivation conditions are designed to satisfy both conditions that prevent protein denaturation and conditions that inactivate the virus. Therefore, if all viruses are to be effectively inactivated, the amount of protein denaturation increases, which would prevent the achievement of the formulation objective or worsen profitability.

[0003] Furthermore, proteins such as coagulation factors have low resistance to physicochemical stress. Therefore, if virus inactivation conditions are set within a range where unstable proteins do not undergo denaturation, problems arise such as the inactivation effect being observed only on some viruses. As can be understood from the above, virus inactivation and removal technology is, in a sense, an imperfect technology, and it is extremely difficult to completely inactivate and remove all viruses while avoiding protein denaturation with a single method. In the manufacture of biopharmaceuticals, it is common to simply combine multiple virus inactivation and removal processes.

[0004] Currently, methods such as liquid / drying heat treatment, organic solvent / surfactant treatment (SD treatment), low pH treatment, and virus removal membrane treatment are being introduced as virus inactivation and removal processes in the manufacturing processes of biopharmaceuticals and the like (Patent Documents 1-3 and Non-Patent Document 1).

[0005] The liquid / drying heat treatment and SD treatment described above are known to be effective inactivation treatments for enveloped viruses such as human immunodeficiency virus (HIV) and hepatitis C virus (HCV). However, non-enveloped viruses such as human parvovirus B19 (hereinafter abbreviated as "B19") and hepatitis A virus (hereinafter abbreviated as "HAV") are resistant to the above-mentioned virus inactivation treatments, and there is a problem that the same level of effectiveness against enveloped viruses cannot be expected.

[0006] The aforementioned virus removal membrane treatment has the advantage of being able to remove viruses based solely on size, regardless of the chemical or thermal properties of the target material, because the separation operation is performed according to the size of the particles. For this reason, in recent years, membrane filtration using virus removal membranes has been commonly adopted from the perspective of preventing contamination by non-enveloped viruses and other pathogens.

[0007] Examples of non-enveloped parvoviruses include B19, mouse microvirus (hereinafter abbreviated as "MVM"), and porcine parvovirus (hereinafter abbreviated as "PPV"). B19 is a linear single-stranded DNA virus belonging to the Parvoviridae family, and its size is approximately 23-28 nm (ICTV, https: / / talk.ictvonline.org / ictv-reports / ictv_online_report / ssdna-viruses / w / parvoviridae). In the past, there have been cases of contamination with rodent-derived MVM during cell culture in the manufacture of biopharmaceuticals, and this is recognized as a risk in the above-mentioned manufacturing processes. Membrane filtration using virus removal membranes is widely used as an effective method for removing parvoviruses and other viruses. The basic principle of this membrane filtration method is achieved by setting conditions in which the target protein can pass through the virus removal membrane, but the virus cannot, depending on the pore size of the virus removal membrane used.

[0008] The smallest non-enveloped virus currently known is porcine circovirus (hereinafter sometimes abbreviated as "PCV"), with a particle size of approximately 15-25 nm (ICTV, https: / / talk.ictvonline.org / ictv-reports / ictv_online_report / ssdna-viruses / w / circoviridae). PCV belongs to the Family Circoviridae and is classified into categories such as PCV1 and PCV2. PCV is one of the viruses with a high risk of contamination in the manufacturing process of biopharmaceuticals, and cases of contamination have been reported in the past. PCV is resistant to low pH treatment and heat treatment, and even with a virus removal membrane having a pore size of 15 nm, it is difficult to capture the virus in solutions containing PCV. This presents a problem in that virus removal membranes introduced in the manufacturing processes of many biopharmaceuticals are insufficient to remove PCV. Furthermore, although treatments using ion exchange resins, for example, are also performed, it is difficult to say that sufficient effects are obtained.

[0009] Currently, the optimization of virus inactivation and removal conditions in the manufacturing processes of plasma-derived products and biopharmaceuticals is designed for each step where implementation is desired, and each step is characterized based on the guidelines of the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH). Furthermore, the evaluation of the overall virus inactivation and removal capacity of the biopharmaceutical manufacturing process is performed by implementing multiple individually optimized virus inactivation and removal steps, and evaluating the inactivation and removal capacity of each step and their sum (Non-Patent Literature 1).

[0010] Due to the evaluation methods described above, methods that possess superior virus inactivation and removal capabilities other than those mentioned above, but have characteristics such as a narrow or biased range of conditions under which they can be introduced into the manufacturing process of biopharmaceuticals, etc., are difficult to introduce into such manufacturing processes. Taking radiation (e.g., ultraviolet rays, electron beams, gamma rays, etc.) as an example, the mechanism of virus inactivation by radiation irradiation is to damage the viral nucleic acid with the energy of electromagnetic waves or electron beams, and it has the characteristic that the smaller the genome size (i.e., the smaller the particle size of the virus), the lower the energy required for inactivation (Non-Patent Literature 2). Therefore, the higher the irradiation energy, the more easily proteins are denatured, and under irradiation conditions of radiation (e.g., ultraviolet rays, electron beams, gamma rays, etc.) that can achieve inactivation of large viruses, protein denaturation is significant, and it is considered to be very difficult to introduce them into the manufacturing process of biopharmaceuticals, etc. Furthermore, the irradiation dose of radiation (e.g., ultraviolet rays, electron beams, gamma rays, etc.) can differ depending on the type of detector even under the same conditions, so the difficulty in standardizing the irradiation dose is also considered to be an obstacle to introduction. [Prior art documents] [Patent Documents]

[0011] [Patent Document 1] Japanese Patent Publication No. 2006-151840 [Patent Document 2] International Publication No. 2010 / 109920 [Patent Document 3] International Publication No. 2018 / 030437 [Non-patent literature]

[0012] [Non-Patent Document 1] Caroline Goussen et al., BioDrugs (2007) 31:251-261 [Non-Patent Document 2] Wang J et al., Vox Sang 2004 May; 86(4):230-238 [Overview of the project] [Problems that the invention aims to solve]

[0013] The object of the present invention is to provide a method for inactivating and removing viruses in a protein solution, which includes a step of integrating radiation irradiation treatment and virus removal membrane treatment or SD treatment and optimizing the conditions thereof, and a method for producing a protein solution from which viruses have been inactivated and removed, which includes the above step. [Means for solving the problem]

[0014] The inventors of this invention have diligently studied how to introduce irradiation treatment, which has excellent virus inactivation and removal capabilities but is considered extremely difficult to introduce into the manufacturing process of biopharmaceuticals and the like, into such processes. As a result of these studies, the inventors focused on the characteristics of the process, which center on virus inactivation / removal and protein denaturation, and conceived of integrating (i) a method of virus inactivation by irradiation treatment and (ii) a method of virus removal by virus removal membrane treatment or a method of virus inactivation by SD treatment, in order to design mutually complementary virus inactivation and removal conditions. This idea is extremely difficult to conceive using existing methods such as conducting process characteristic evaluations for each virus inactivation and removal process that is desired to be introduced into the manufacturing process. Furthermore, no reports have been found to date that have designed a process using such an idea and evaluated its virus inactivation and removal capabilities. Furthermore, since it is difficult to measure the absolute dose of radiation, especially ultraviolet radiation, we conceived the idea of ​​standardizing the dose by focusing on the change in absorbance of a specific compound and using this change in absorbance as an indicator to calibrate measuring instruments. Based on this idea, further research led to the design of a virus inactivation and removal process that integrates and optimizes the above two methods, making it possible to introduce it into the manufacturing process of biopharmaceuticals, etc., thus completing the present invention.

[0015] In other words, the present invention is as follows: [1] A method for removing a virus from a protein solution, the following: (a) A step of irradiating a protein solution with radiation so that the removal coefficient (a logarithmic reduction in the virus, hereinafter referred to as "LRV") for viruses with a particle size of less than 33 nm in the protein solution is 1.00 or more, and (b1) The process of removing the virus from the treated protein solution using a virus removal membrane having an LRV of 4.00 or higher against bacteriophage PP7, or (b2) The process of contacting the treated protein solution with a mixture of organic solvent and surfactant (SD mixture) and inactivating the virus by organic solvent-surfactant treatment (SD treatment) so that the LRV against enveloped viruses in the protein solution becomes 2.00 or higher. Methods that include... [1-1](a) is a step of irradiating a protein solution with radiation so that the removal coefficient for viruses with a particle size of 30 nm or less in the protein solution is 1.00 or more, the method according to [1]. [2] The method according to [1] or [1-1], wherein the radiation is ultraviolet or gamma rays. [3] The method according to [2], wherein the radiation is ultraviolet light. [4] The ultraviolet radiation is 32 mJ / cm 2 ~256 mJ / cm 2 The method described in [3], which is irradiated within the range. [5] The method according to [2], wherein the radiation is gamma rays. [6] The method according to [5], wherein the gamma rays are irradiated in the range of 5 kGy to 50 kGy. [7] The method according to any one of [1] to [6], wherein the virus removal membrane having an LRV of 4.00 or higher against bacteriophage PP7 is a virus removal membrane with a pore size of 20 nm or less. [8] The method according to [7], wherein the virus removal membrane with a pore size of 20 nm or less is a virus removal membrane with a pore size of 15 nm or more. [8-1] The method according to any one of [1] to [6], wherein the virus removal membrane has a pore size of less than 33 nm and an LRV of 4.00 or higher against bacteriophage PP7. [8-2] The method according to [8-1], wherein the pore size of the virus removal membrane is 13 to 33 nm. [8-3] The method according to [8-1], wherein the pore size of the virus removal membrane is 15 to 33 nm. [8-4] The method according to [8-1], wherein the pore size of the virus removal membrane is 17 to 33 nm. [8-5] The method according to [8-1], wherein the pore size of the virus removal membrane is 17 to 30 nm. [8-6] The method according to [8-1], wherein the pore size of the virus removal membrane is 17 to 21 nm. [8-7] The method according to [8-1], wherein the pore size of the virus removal membrane is 19 to 21 nm. [8-8] The method according to [8-1], wherein the pore size of the virus removal membrane is 19 to 20 nm. [9] The method according to [7] or [8], wherein the virus removal membrane is a virus removal membrane with a pore size of approximately 20 nm.

[10] A method for producing a protein solution from which the virus has been removed, the following: (a) A step of irradiating a protein solution with radiation so that the removal coefficient (LRV) for viruses with a particle size of less than 33 nm in the protein solution becomes 1.00 or more, and (b1) A step of filtering the treated protein solution through a virus removal membrane having an LRV of 4.00 or higher against bacteriophage PP7, or (b2) The process of bringing the treated protein solution into contact with the SD mixture and performing SD treatment so that the LRV against enveloped viruses in the protein solution becomes 2.00 or higher. Methods that include... The method according to

[10] , wherein [10-1](a) is a step of irradiating a protein solution with radiation so that the removal coefficient for viruses with a particle size of 30 nm or less in the protein solution is 1.00 or more. The method according to [1] or [1-1], wherein the LRV in

[11] (a) is 2.00 or greater. The method according to [1] or [1-1], wherein the LRV in

[12] (a) is 4.00 or greater. The method according to [1], [1-1], or

[12] , wherein the LRV in

[13] (b2) is 4.00 or higher. The method according to

[10] or [10-1], wherein the LRV in

[14] (a) is 2.00 or greater. The method according to

[10] or [10-1], wherein the LRV in

[15] (a) is 4.00 or greater. The method according to

[10] , [10-1], or

[15] , wherein the LRV in

[16] (b2) is 4.00 or higher. [Effects of the Invention]

[0016] According to the present invention, it is possible to provide a method for removing viruses from a protein solution, which includes a step of integrating virus inactivation by radiation treatment and virus removal by virus removal membrane treatment or virus inactivation by SD treatment, and optimizing the conditions for these steps, and a method for producing a virus-removed protein solution, which includes the said step. Furthermore, in this process, viruses with a particle size of 30 nm or less (e.g., parvovirus, circovirus) are effectively inactivated by radiation (e.g., ultraviolet light, gamma rays), so even when using a virus removal membrane with a pore size of 15 nm or more, an extremely high LRV can be achieved synergistically. In another embodiment, in this process, viruses with a particle size of less than 33 nm (e.g., parvovirus, circovirus) are effectively inactivated by radiation (e.g., ultraviolet light, gamma rays), so even when using a virus removal membrane with a pore size of 13 nm or more, an extremely high LRV can be achieved synergistically. Moreover, in this virus inactivation and removal process, a larger pore size than that of conventionally used virus removal membranes can be employed, thus increasing the yield of the desired protein. In addition, the radiation treatment used in this virus inactivation and removal process does not require the addition of any drugs other than buffers and stabilizers, and there is no need to add steps such as the removal of added drugs in subsequent processes, thus contributing to the simplification of the manufacturing process. [Brief explanation of the drawing]

[0017] [Figure 1] Figure 1 shows the relationship between the IgG denaturation rate (polymer formation) when a 2.5% (25 mg / ml) IgG solution (0.1 M glycine, pH 3.0~6.8) is irradiated with ultraviolet C (UVC) light (0~1,024 mJ / cm2). [Figure 2] Figure 2 shows the relationship between the IgG denaturation rate (polymer formation) when a 5% (50 mg / ml) IgG solution (5% sorbitol, pH 4.1) is irradiated with UVC (0-1,024 mJ / cm2). [Figure 3] Figure 3 shows the relationship of inactivation when B19 in PBS is irradiated with UVC (0-512 mJ / cm2). [Figure 4] Figure 4 shows the relationship of inactivation when MVM in a 2.5% (25 mg / ml) IgG solution (0.1 M glycine, pH 4.1) or PBS is irradiated with UVC (0-512 mJ / cm2). [Figure 5] Figure 5 shows the relationship of inactivation when bovine viral diarrhea virus (hereinafter abbreviated as "BVDV" or "BVD") in a 2.5% (25 mg / ml) IgG solution (0.1 M glycine, pH 4.1) or PBS is irradiated with UVC (0-512 mJ / cm2). [Figure 6] Figure 6 shows the relationship between coagulation activity and protein denaturation rate when fibrinogen (hereinafter abbreviated as "Fib") (containing 0.5% sodium chloride and 1.6% sodium citrate as stabilizers) concentration is 3.9 mg / ml (E280: 5.8, E1% 280: 15-16) irradiated with UVC (0-768 mJ / cm2). [Figure 7] Figure 7 shows the relationship between B19 inactivation when B19 in a solution with a Fib concentration of 3.3 to 13.3 mg / ml (E280: 5 to 20, extinction coefficient (E1% 280: 15 to 16)) is irradiated with UVC (0 to 384 mJ / cm2). [Figure 8]Figure 8 shows the relationship between B19 inactivation when B19 in a solution or PBS with a Fib concentration of 0.004 to 3.6 mg / ml (E280: 0.0054 to 5.4, extinction coefficient (E1% 280: 15 to 16)) is irradiated with UVC (0 to 384 mJ / cm2). [Figure 9] Figure 9 shows the relationship between PPV inactivation when PPV in a solution or PBS with a Fib concentration of 0.04 to 3.6 mg / ml (E280: 0.054 to 5.4, extinction coefficient (E1% 280: 15 to 16)) is irradiated with UVC (0 to 384 mJ / cm2). [Figure 10] Figure 10 shows the relationship between coagulation activity and protein denaturation rate when thrombin (hereinafter abbreviated as "Thr") (containing 0.34% sodium chloride, 0.23% sodium citrate, and 0.27% calcium chloride as stabilizers) is irradiated with UVC (0-768 mJ / cm2) at a concentration of 2.48 mg / ml (E280: 5.3, extinction coefficient (E1% 280: 21.4)). [Figure 11] Figure 11 shows the relationship between B19 inactivation when B19 in a solution with a Thr concentration of 0.47 to 4.7 mg / ml (E280: 1 to 10, extinction coefficient (E1% 280: 21.4)) is irradiated with UVC (0 to 384 mJ / cm2). [Figure 12] Figure 12 shows the relationship between PPV inactivation when PPV in a solution with a Thr concentration of 0.47 to 4.7 mg / ml (E280: 1 to 10, extinction coefficient (E1% 280: 21.4)) is irradiated with UVC (0 to 384 mJ / cm2). [Figure 13] Figure 13 shows the relationship between protein denaturation and irradiation with gamma rays after freeze-drying Fib (containing sodium citrate and L-alginate as stabilizers and excipients). [Figure 14] Figure 14 shows the relationship between protein denaturation and irradiation with gamma rays after freeze-drying Thr (containing D-mannitol, sodium citrate, and L-alginate as stabilizers and excipients). [Figure 15]Figure 15 shows the relationship of protein denaturation when albumin (hereinafter abbreviated as "Alb") (containing a) sodium citrate, sodium chloride, and b) D-mannitol as stabilizers and excipients) is freeze-dried and irradiated with gamma rays. [Figure 16] Figure 16 shows the relationship of B19 inactivation when the above-mentioned Fib, Thr, Alb (a), and Alb (b), which contain B19, are freeze-dried and irradiated with gamma rays. [Figure 17] Figure 17 is a diagram for estimating the UVC irradiation dose (mJ / cm2) from the absorbance (E352) of 1% sodium iodide (NaI). [Modes for carrying out the invention]

[0018] 1. Method for removing viruses from protein solutions according to the present invention The present invention provides a method for inactivating and removing viruses from a protein solution, and the following: (a) A step of irradiating a protein solution with radiation so that the LRV for viruses with a particle size of 30 nm or less in the protein solution is 1.00 or higher, preferably 2.00 or higher, more preferably 3.00 or higher, and even more preferably 4.00 or higher, or (a') A step of irradiating a protein solution with radiation so that the LRV for viruses with a particle size of less than 33 nm in the protein solution is 1.00 or higher, preferably 2.00 or higher, more preferably 3.00 or higher, and even more preferably 4.00 or higher, and (b1) A step of filtering the treated protein solution through a virus removal membrane having an LRV of 4.00 or higher against bacteriophage PP7, or (b2) The process of bringing the treated protein solution into contact with the SD mixture and performing SD treatment so that the LRV against enveloped viruses in the protein solution becomes 2.00 or higher. This method includes [something].

[0019] In this specification, inactivation and removal of viruses in a protein solution includes not only bringing the protein solution to a state where no viruses are present, but also reducing the amount of virus present in the protein solution to a certain level or less that does not cause harm to health, and rendering infectious viruses present in the protein non-infectious (so-called virus inactivation).

[0020] In this specification, the protein solution is not particularly limited as long as it contains proteins that pass through a virus removal membrane when filtered through a membrane having an LRV of 4.00 or higher against bacteriophage PP7, and is a solution that may contain viruses. In particular, solutions derived from animal components, including human, or genes are highly likely to contain viruses, and by using them as protein solutions in the virus removal method of the present invention, a virus-free protein solution can be efficiently provided.

[0021] Examples of protein solutions that may contain viruses include, for example, solutions containing peptides, proteins, and animal-derived components as active ingredients, produced using biotechnology such as genetic engineering and cell culture, which are raw materials for biopharmaceuticals. Furthermore, examples include culture media containing animal-derived components used for cell culture, and enzymes such as animal-derived trypsin.

[0022] Furthermore, examples of protein solutions that may contain viruses include bodily fluids of humans or animals. Specifically, these may include, for example, blood, plasma, serum, saliva, sweat, urine, lymph, enzymes, tissue fluid, or solutions obtained by purifying these tissues or bodily fluids as raw materials, or solutions that contain bodily fluids. Another example is the raw material for plasma-derived products obtained by purification from plasma. Examples of plasma-derived products include immunoglobulin preparations, albumin preparations, and blood coagulation factor preparations. In particular, examples of blood coagulation factor preparations include blood coagulation factor VIII preparations, blood coagulation factor IX preparations, fibrinogen preparations, and antithrombin III preparations. The protein solution used in the virus removal method of the present invention may, for example, be prepared immediately before use, be thawed after being frozen, or be freeze-dried and then dissolved again in a desired solution.

[0023] Specific proteins include, for example, antibodies (e.g., polyclonal antibodies, monoclonal antibodies (e.g., IgG, IgM, IgA, IgD, IgE), etc.), hematopoietic factors (erythropoietin, thrombopoietin, etc.), proteins involved in blood coagulation (fibrinolytic factors (tissue plasminogen activator, prourokinase, thrombomodulin, etc.), blood coagulation factors (antithrombin, protein C, blood coagulation factor VII, blood coagulation factor VIII, blood coagulation factor IX, blood coagulation factor X, blood coagulation factor XI, blood coagulation factor XII, prothrombin complex, thrombin, fibrinogen, etc.)), plasma proteins (albumin, globulin, etc.), hormones (gonadotropins, thyroid-stimulating hormone, etc.), growth factors (epidermal growth factor (EGF), hepatocyte growth factor (HGF), keratinocyte growth factor, activin, bone morphogenetic factor, etc.), stem cell factors (SCF) (G- Examples include CSF, M-CSF, etc., cytokines (interferon α, interferon β, interferon γ, interleukin 2, interleukin 4, interleukin 5, interleukin 6, interleukin 10, interleukin 11, soluble interleukin 4 receptor, tumor necrosis factor α, etc.), nucleases (Dnasel, etc.), enzymes (galactosidase, α-glucosidase, glucocerebrosidase, etc.), proteins such as hemoglobin and transferrin, and partial fragments of these proteins, and unstable proteins with biological activity. In particular, from the viewpoint of balancing manufacturing conditions that can inactivate or remove viruses and maintain the quality and activity of proteins with economic efficiency, polyclonal antibodies, monoclonal antibodies, proteins involved in blood coagulation, plasma proteins, and unstable proteins with biological activity are preferred, and IgG, IgM, IgA, IgD, IgE, thrombin, fibrinogen, antithrombin, and albumin are more preferred.

[0024] Furthermore, in this specification, the protein contained in the protein solution may be a protein or a partial fragment of a protein to which a radioisotope, a low molecular weight drug, a high molecular weight drug, or a different protein or partial fragment of a protein has been chemically or genetically engineered to such a protein or partial fragment of a protein.

[0025] Examples of the antibodies mentioned above include human antibodies, human-type chimeric antibodies, and human-type complementarity-determining region-implanted antibodies, as well as fragments of these antibodies. Antibodies consist of a variable region (V region) and a constant region (C region), with the heavy chain variable region being called VH, the light chain variable region VL, the heavy chain constant region CH, and the light chain constant region CL.

[0026] A human chimeric antibody is an antibody composed of VH and VL of an antibody from a non-human animal and CH and CL of a human antibody. To produce a human chimeric transplant antibody, first, cDNA encoding the VH and VL of an antibody from a non-human mammal is designed and constructed. Next, the cDNAs are inserted into an animal cell expression vector containing cDNA encoding the CH and CL of a human antibody to construct a human chimeric transplant antibody expression vector. The antibody can then be expressed and produced by introducing this constructed vector into animal cells. The CH of the human-type chimeric antibody is not particularly limited as long as it belongs to human immunoglobulin (hereinafter sometimes abbreviated as "hIg"), but the hIgG class is preferred, and any of the subclasses belonging to the hIgG class, such as hIgG1, hIgG2, hIgG3, and hIgG4, can be used. Furthermore, the CL of the human-type chimeric antibody is not particularly limited as long as it belongs to hIg, and the κ class or λ class can be used. Mammals other than humans include, for example, mice, rats, hamsters, rabbits, etc.

[0027] Antibody fragments include peptides containing Fab, F(ab')2, Fab', scFv, diabody, dsFv, and CDR. Fab is an antibody fragment with an antigen-binding activity and a molecular weight of approximately 50,000, obtained by treating an IgG-type antibody molecule with the protease papain (cleaved at the 224th amino acid residue of the H chain), in which approximately half of the N-terminal side of the H chain and the entire L chain are linked by a disulfide bond. Fab can be produced by treating an antibody with the protease papain, or by inserting the DNA encoding the Fab of the antibody into a prokaryotic or eukaryotic expression vector and introducing the vector into a prokaryote or eukaryote.

[0028] The protein concentration in the protein solution used in the virus removal method of the present invention is not particularly limited, as long as it is a concentration that can be filtered by a virus removal membrane having an LRV of 4.00 or higher for bacteriophage PP7, preferably a virus removal membrane with a pore size of 15 nm to 20 nm, and within the range of the predetermined radiation irradiation dose used in the virus removal method of the present invention described later, an LRV of 1.00 or higher, preferably 2.00 or higher, more preferably 3.00 or higher, and even more preferably 4.00 or higher for viruses with a virus particle size of 30 nm or less can be achieved. In another embodiment, the protein concentration in the protein solution used in the virus removal method of the present invention is not particularly limited as long as it is a concentration that can be filtered by a virus removal membrane having an LRV of 4.00 or more for bacteriophage PP7, preferably a virus removal membrane with a pore size of 13 nm to 33 nm, and within the range of a predetermined radiation dose used in the virus removal method of the present invention described later, an LRV of 1.00 or more, preferably 2.00 or more, more preferably 3.00 or more, and even more preferably 4.00 or more for viruses with a particle size of less than 33 nm can be achieved. Specific protein concentrations (including upper limits) include, for example, 30 w / v% or less, 25 w / v% or less, 20 w / v% or less, 15 w / v% or less, 10 w / v% or less, 5.0 w / v% or less, 2.5 w / v% or less, 1.0 w / v% or less, 0.7 w / v% or less, and 0.5 w / v% or less. Specific protein concentrations (including lower limits) include, for example, 0.001 w / v% or more, 0.01 w / v% or more, 0.1 w / v% or more, 0.2 w / v% or more, and 0.3 w / v% or more.

[0029] The viruses that may be contained in the protein solution are not particularly limited, but include B19, MVM, PPV, bovine parvovirus (BPV), canine parvovirus (hereinafter abbreviated as "CPV"), poliovirus, PCV, HAV, and hepatitis E virus (hereinafter abbreviated as "HEV").

[0030] Of the viruses exemplified above, parvovirus has been reported in the plasma-derived products field in the past as a suspected case of infection caused by parvovirus B19, and guidelines on the viral safety of plasma-derived products have been issued by EMEA (European Medicines Agency, EMA / CHMP / BWP / 706271 / 2010). Furthermore, in the biopharmaceutical field, there have been cases of contamination of the biopharmaceutical manufacturing process due to the contamination of rodent-derived MVMs with CHO cells (hamster-derived), and guidelines on the viral safety assessment of biopharmaceuticals made using animal cells (ICH Topic Q5A) have been issued.

[0031] Furthermore, among the viruses exemplified above, PCV is the smallest non-enveloped virus currently known, with a particle size of approximately 15-25 nm (ICTV, https: / / talk.ictvonline.org / ictv-reports / ictv_online_report / ssdna-viruses / w / circoviridae). PCV belongs to the Family Circoviridae and is classified into PCV1, PCV2, etc. PCV is also one of the viruses with a high risk of contamination in the manufacturing process of biopharmaceuticals, and actual cases of contamination have been reported. PCV is resistant to low pH treatment and heat treatment, and it is difficult to capture and remove PCV even with a virus removal membrane having an average pore size of 15 nm. As described later, the virus inactivation and removal method of the present invention uses radiation doses that can effectively inactivate parvoviruses having a particle size larger than PCV (e.g., LRV≧1.0, LRV≧2.0, LRV≧4.0, LRV≧6.0, etc.), and therefore can similarly inactivate circoviruses with a particle size smaller than that.

[0032] Furthermore, among the viruses exemplified above, there is a possibility that some viruses, such as HAV (27-30 nm), poliovirus (30 nm), and HEV (32 nm) of the Picornaviridae family, and viruses with larger particle sizes than these, may not be partially inactivated by the radiation dose used in the virus inactivation method of the present invention. However, these viruses can be effectively filtered by a virus removal membrane having an LRV of 4.00 or higher for bacteriophage PP7, which is used in the virus removal method of the present invention.

[0033] The protein solution potentially containing a virus used in the virus removal method of the present invention may contain, in addition to the above-mentioned protein and virus, one or more components selected from the group consisting of amino acids, inorganic salts, buffer components, surfactants, and sugars. These components may be added to the protein solution potentially containing a virus before step (a) or (a') included in the virus removal method of the present invention, or they may be added between step (a) or (a') and step (b1) or (b2).

[0034] Among amino acids, basic amino acids include arginine, histidine, guanidine, lysine, or their derivatives, or salts thereof. Preferably, arginine, histidine, lysine, or their derivatives, or salts thereof, and more preferably, arginine or its derivatives, or salts thereof. Examples of neutral amino acids include glycine, its derivatives, or salts thereof. Examples of acidic amino acids include aspartic acid, its derivatives, or salts thereof.

[0035] Inorganic salts may include NaCl, CaCl2, buffer salts, etc. As buffer components, acetate buffer, citrate buffer, phosphate buffer, phosphate-buffered saline (PBS), Tris-HCl buffer, glycine buffer, etc. may be used. The concentrations of inorganic salts and buffer components may be appropriately determined by methods known to the public.

[0036] Examples of surfactants include nonionic surfactants such as Tween20®, Tween80®, Triton X100®, NP-40®, and Pluronic F-127®, which may be included in the protein solution at a concentration of 0.01 to 5 wt%.

[0037] The sugars are not particularly limited, but include monosaccharides, disaccharides, trisaccharides, oligosaccharides, polysaccharides, sugar alcohols, etc. Specifically, glucose, mannose, galactose, fructose, sorbose, maltose, sucrose, sorbitol, mannitol, dextran, alginic acid, and their hydrochloride salts may be included in the protein solution in an amount of 1 to 10 wt%, preferably 1 to 5 wt%, of one or more types.

[0038] The temperature of the protein solution that may contain viruses may be in the range of, for example, 1°C to 40°C, preferably 4°C to 35°C, throughout the entire process of the virus removal method of the present invention, from the viewpoint of preventing protein denaturation. The temperature affects the viscosity of the protein solution and the Flux during filtration using the virus removal membrane described later, and therefore depends on the temperature stability of the protein itself, but may be in the range of 20°C to 35°C.

[0039] The pH of a protein solution that may contain a virus is not particularly limited as long as the protein in the solution does not denature, but for example, it is between pH 3.0 and pH 8.0. More specifically, if the protein in the solution is polyclonal IgG, pH 4.1 to pH 7.2 is preferred, and if it is monoclonal IgG, the optimal pH will vary depending on the PI value of the IgG molecule.

[0040] The radiation used in step (a) or (a') of the virus removal method of the present invention includes both ionizing and non-ionizing radiation, specifically, examples of which include ultraviolet rays, alpha rays, beta rays, gamma rays, X-rays, electron beams, neutrons, etc. The radiation can be obtained, for example, from radioactive isotopes such as cobalt-60, strontium-90, and cesium-137, or from an X-ray imaging device, an electron beam accelerator, and an ultraviolet (continuous) irradiation device, etc., and such devices may be commercially available.

[0041] Among the specific examples above, ultraviolet light includes UVC (100-280 nm), UVB (280-320 nm), and UVA (320-400 nm). In the virus removal method of the present invention, irradiation with UVC is preferred, more preferably with UVC in the range of 250-280 nm, and even more preferably with UVC around 254 nm. Low-pressure mercury lamps, LEDs, etc., may be used as the ultraviolet irradiation source, and the method is not limited to the irradiation source.

[0042] The radiation irradiation method in the virus removal method of the present invention is not particularly limited as long as it can be processed so that the LRV for viruses with a particle size of 30 nm or less in the protein solution subjected to irradiation is 1.00 or higher. Furthermore, while an LRV of 1.00 or higher, preferably 2.00 or higher, more preferably 3.00 or higher, and even more preferably 4.00 or higher can be said to indicate that the virus has been effectively removed, if desired, the processing may be carried out to achieve an LRV of, for example, 5.00 or higher, or 6.00 or higher. Furthermore, the irradiation method in another embodiment of the virus removal method of the present invention is not particularly limited as long as it can be processed so that the LRV for viruses with a particle size of less than 33 nm in the protein solution subjected to irradiation is 1.00 or higher. In addition, while it can be said that the virus has been effectively removed if the LRV is 1.00 or higher, preferably 2.00 or higher, more preferably 3.00 or higher, and even more preferably 4.00 or higher, if desired, the processing may be carried out to achieve an LRV of, for example, 5.00 or higher or 6.00 or higher. Specifically, for example, when the radiation being irradiated is ultraviolet light, one example of an irradiation method is to use a device that has a cylindrical rotating tube in which a thin, uniform protein solution is distributed, and the rotation is around the rotational axis of the cylinder. The principle of this device is that the flow rate of the protein solution is adjusted using a pump to inject it into the inner wall of the rotating tube so that the thickness of the solution film is such that a thin film (e.g., 0.1 mm) is formed in which UVC irradiation is effective, thereby diffusing uniformly into the inner wall, and ultraviolet light is irradiated toward the inner wall of the tube from a low-pressure mercury lamp located at the center of the tube. The amount of ultraviolet light irradiated to the protein in the protein solution increases or decreases depending on the time it takes for the solution to pass through the inner wall of the tube, and by changing the angle of the tube, the passage time also changes, and the irradiation amount can be changed to the desired amount. Another method is to place a coil-shaped tube made of ultraviolet-transmitting resin or quartz glass around an ultraviolet light source such as a low-pressure mercury lamp or a deep ultraviolet LED, and pass the protein solution through it with a pump. In this method, the liquid is stirred as the protein solution passes through the coil-shaped tube, so uniform irradiation is performed. Another method involves arranging ultraviolet light sources in a planar configuration and using a pump to pass a protein solution between two flat plates, each shaped with a UV-transmitting resin or quartz glass, to irradiate a thin film layer. The flat plates may have patterned bumps or grooves to ensure uniform flow velocity. By changing the flow velocity, the passage time can also be changed, allowing the irradiation dose to be adjusted to the desired amount.

[0043] The radiation dose used in the virus removal method of the present invention should be set according to the type of radiation used so that the LRV for viruses with a particle size of 30 nm or less in the protein solution subjected to irradiation becomes 1.00 or higher, preferably 2.00 or higher, more preferably 3.00 or higher, and even more preferably 4.00 or higher. In another aspect, the irradiation dose of the radiation used in the virus removal method of the present invention may be set so that, depending on the type of radiation used, the log reduction value (LRV) for viruses with a particle diameter of less than 33 nm in the protein solution to be irradiated is 1.00 or more, preferably 2.00 or more, more preferably 3.00 or more, and even more preferably 4.00 or more. If the LRV is 4.00 or more, it can be said that the virus has been effectively removed. If desired, the treatment may be performed so as to achieve an LRV of, for example, 5.00 or more or 6.00 or more. The LRV can be determined by the calculation formula described later.

[0044] For example, when using UVC, the irradiation dose is specifically 256 mJ / cm 2 or less, 192 mJ / cm 2 or less, 96 mJ / cm 2 or less. Also, specifically, it is 32 mJ / cm 2 or more, 64 mJ / cm 2 or more, 128 mJ / cm 2 or more. Also, the irradiation dose may be a combination within the above range (e.g., 32 mJ / cm 2 ~256 mJ / cm 2 , 64 mJ / cm 2 ~256 mJ / cm 2 , 128 mJ / cm 2 ~256 mJ / cm 2 , 32 mJ / cm 2 ~192 mJ / cm 2 , 64 mJ / cm 2 ~192 mJ / cm 2 , 128 mJ / cm 2 ~192 mJ / cm 2 etc.). More specifically, for a protein solution (pH 3.5 to 6.8) containing 10% w / v (100 mg / ml) or less of IgG antibody, when the formation rate of the polymer of IgG antibody after UVC irradiation is set to 2% or less, the maximum irradiation dose of UVC is preferably 256 mJ / cm 2 ~1024 mJ / cm 2 and more preferably 512 mJ / cm2 That is the case. More specifically, the Fib concentration is 3.9 mg / ml (E 280 : 5.8, E 1% 280 For a protein solution containing (15-16), the maximum irradiation dose, set to maintain 90% or more of the coagulation activity, is preferably 96 mJ / cm². 2 ~192 mJ / cm 2 , more preferably 192 mJ / cm 2 That is the case. More specifically, the Thr concentration is 2.48 mg / ml (E 280 : 5.3, E 1% 280 For a protein solution containing (21.4), the maximum irradiation dose set to maintain 90% or more of the coagulation activity is preferably 96 mJ / cm². 2 ~192 mJ / cm 2 , more preferably 192 mJ / cm 2 That is the case.

[0045] Furthermore, for example, the irradiation dose when using gamma rays is specifically 5 kGy or more, 10 kGy or more, 15 kGy or more, 20 kGy or more, 25 kGy or more, 30 kGy or more, 35 kGy or more, 40 kGy or more, 45 kGy or more, 50 kGy or more, and 60 kGy or more. Also, the irradiation dose for gamma rays is specifically 5 kGy to 50 kGy, 10 kGy to 50 kGy, and 25 kGy to 50 kGy.

[0046] In the virus removal method of the present invention, the radiation dose may be appropriately calibrated and validated depending on the type of radiation being irradiated in order to obtain accurate measurement values. Specifically, for example, when using UVC, there may be errors in the readings depending on the type of ultraviolet irradiometer used to measure UVC. Therefore, for example, the principle of an existing method for estimating UVC irradiation dose using the change in absorbance of NaI as an indicator may be applied to create a calibration curve for UVC irradiation dose, and this calibration curve may be used to perform calibration between measuring instruments. Since the calibration curve will differ depending on the processing conditions, the absorbance (A352) is measured under experimental conditions that reflect the actual processing conditions (especially the total energy amount and film thickness), and a calibration curve and formula for determining the irradiation dose are obtained. The formula for estimating the UVC irradiation dose using the absorbance of 1% NaI obtained in the experiment shown in Figure 17 is as follows. UVC irradiation dose (energy amount) (mJ / cm²) 2 ) = 10 (2×Log 10 [A352 nm]+2.6)

[0047] Calibration of UVC irradiation dose using this calibration curve does not determine the absolute value of UVC irradiation dose, but by setting a specific reference measuring instrument, calibration against that instrument becomes possible. Furthermore, since this calibration standardizes UVC irradiation dose regardless of the measuring instrument used, it is possible to define a range of UVC irradiation doses that can process a protein solution usable at an industrial level so that the LRV for viruses with a particle size of 30 nm or less is 1.00 or higher, preferably 2.00 or higher, more preferably 3.00 or higher, and even more preferably 4.00 or higher. Similarly, it is possible to define a range of UVC irradiation doses that can process a protein solution so that the LRV for viruses with a particle size of less than 33 nm is 1.00 or higher, preferably 2.00 or higher, more preferably 3.00 or higher, and even more preferably 4.00 or higher. Therefore, the above-mentioned UVC irradiation doses are values ​​of critical significance regardless of the measuring instrument used by employing this calibration.

[0048] The virus removal membrane used in step (b1) of the virus removal method of the present invention is not particularly limited as long as it is a virus removal membrane having an LRV of 4.00 or higher against bacteriophage PP7. By filtering with the virus removal membrane, viruses with a particle size of 30 nm or larger can be effectively removed from the protein solution. Similarly, by filtering with the virus removal membrane, viruses with a particle size of 33 nm or larger can be effectively removed from the protein solution. A virus removal membrane with an LRV of 4.00 or higher against bacteriophage PP7 is defined as a membrane that is coated with a solution containing bacteriophage PP7 at a rate of 50 l / m². 2 This is a membrane in which the LRV (Light Rate Value) is 4.00 or higher when loaded. As a solution containing bacteriophage PP7, the bacteriophage PP7 concentration is added to 1 mg / ml BSA solution (PBS buffer, pH 7.4) at 10 7 Use a solution with a pfu / ml concentration.

[0049] The shape of the virus removal membrane used is not particularly limited, but it may be a flat membrane or a hollow fiber membrane, for example. When using a flat membrane, filtration may be performed with a single membrane or with multiple membranes stacked together.

[0050] The solution is treated by passing a pH-adjusted solution through a virus removal membrane having an LRV of 4.00 or higher for bacteriophage PP7 and filtering it. The filtration method may be either a dead-end method or a cross-flow method. Before treatment with the virus removal membrane having an LRV of 4 or higher for bacteriophage PP7, the solution may be pre-treated with a membrane having a pore size larger than 20 nm.

[0051] The pore size of a virus removal membrane with an LRV of 4.00 or higher for bacteriophage PP7 is not limited as long as it can remove viruses with a particle size of less than 33 nm, but is usually 30 nm or less, preferably 21 nm or less, and more preferably 20 nm or less. The pore size is usually 13 nm or more, preferably 15 nm or more, more preferably 17 nm or more, and particularly preferably 19 nm or more. For example, possible pore sizes for the virus removal membrane include 13 nm to 33 nm, 15 nm to 33 nm, 17 nm to 33 nm, 13 nm to 30 nm, 15 nm to 30 nm, 17 nm to 30 nm, 13 nm to 21 nm, 15 nm to 21 nm, 17 nm to 21 nm, 19 nm to 21 nm, 13 nm to 20 nm, 15 nm to 20 nm, 17 nm to 20 nm, and 19 nm to 20 nm.

[0052] Specific examples of virus removal membranes with an LRV of 4.00 or higher against bacteriophage PP7 include Planova® 15N (manufactured by Asahi Kasei Medical) and Planova® 20N (manufactured by Asahi Kasei Medical) made from regenerated cellulose, Planova® BioEX (manufactured by Asahi Kasei Medical) and Pegasus SV4 (manufactured by Pall) made from hydrophilic PVDF, and Virosart CPV (manufactured by Sartorius) and Viresolve Pro (manufactured by Millipore) made from hydrophilic PES.

[0053] For virus removal membranes with an LRV of 4.00 or higher against bacteriophage PP7, if the virus removal membrane is made of regenerated cellulose, the preferred membrane can also be defined by the average pore size of the vacancies in the virus removal membrane. The average pore size of the vacancies in the virus removal membrane is, for example, 15 nm (e.g., Planova® 15N; 15±2 nm), 19 nm (e.g., Planova® 20N; 19±2 nm), and 20 nm (e.g., Pegasus® SV4).

[0054] The average pore size of the voids can be calculated using the following formula, with reference to the method described in International Publication No. 2015 / 156401. Average pore diameter (nm)=2×10 3 ×√(V·d·μ / P·A·Pr) Here, V is the water permeability (ml / min), d is the film thickness (μm), μ is the viscosity of water (cp), P is the pressure difference (mmHg), and A is the film area (cm²). 2 ), Pr indicates porosity (%). The water permeability is measured by bundling 10 threads together to create a module with an effective length of 16 cm. One end of the resulting module is closed, and a pressure of 200 mmHg is applied to the other end, allowing water to pass through at 37°C. The amount of water that passes through the membrane is measured as the water permeability. The film area is calculated by measuring the inner diameter in a dry state. The film thickness refers to the film thickness in a dry state. The porosity is calculated using the following formula. Porosity (%)=(1-ρa / ρp)×100 The apparent density ρa of the hollow fiber is calculated using the following formula, where ρp is the density of cellulose (g / cm³). 3 ) means. Apparent density of hollow fibers (g / cm³) 3 )=Wd / Vw=4Wd / πl(Do 2 -Di 2 ) Here, Wd is the oven-dry weight of the hollow fiber (g), and Vw is the apparent volume of the hollow fiber (cm³). 3 ) means that l is the length of the hollow fiber (cm), Do is the outer diameter of the hollow fiber (cm), and Di is the inner diameter of the hollow fiber (cm).

[0055] In this specification, LRV refers to the Log Reduction Value, which is the degree of virus reduction expressed on a logarithmic scale, and is also called the reduction coefficient. LRV is calculated using the following formula. LRV=Log[(V1×T1))] / [(V2×T2))] V1: Sample volume before the virus removal process T1: Virus load (titer) before the virus removal process. V2: Sample volume after virus removal process T2: Virus load (titer) after the virus removal process.

[0056] The viral load of bacteriophage PP7 can be measured using methods such as the plaque assay, although there are no particular limitations. The plaque assay can be performed according to the method described in Journal of Pharmaceutical Science and Technology 2008; Supplement Volume 62 No. S-4. A solution sample containing bacteriophage PP7 is serially diluted, and each sample is mixed with Pseudomonas aeruginosa, after which soft agar is added. The mixed solution is poured onto an agar plate, solidified, and then incubated at 37°C for 1 day. The next day, the number of plaques is measured visually, and the phage concentration pfu (plaque forming unit) / ml that was present in the original solution is calculated using the following formula. (Plaque count × Dilution factor) pfu / Sample volume (ml) pfu / ml

[0057] In step (b1) of the virus removal method of the present invention, filtration using a virus removal membrane can be confirmed by LRV as an indicator of removal of viruses with a virus particle size of 30 nm or less (e.g., B19, MVM, PPV, etc.).

[0058] LRV for viruses with a particle size of 30 nm or less (e.g., B19, MVM, PPV, etc.) can be determined by measuring the viral load of viruses with a particle size of 30 nm or less (e.g., B19, MVM, PPV, etc.) using the above formula. The amount of viruses with a particle size of 30 nm or less (e.g., B19, MVM, PPV, etc.) is not particularly limited, but can be measured by infectivity titer or viral nucleic acid amount. Infectivity titer is obtained by inoculating indicator cells with the virus, culturing them for a certain period, and measuring the degeneration of the cultured cells, as well as the viral antigen and nucleic acid (TCID). 50(e.g., plaque assay). Quantitative polymerase chain reaction (Q-PCR), which measures nucleic acid content, can be performed using methods that are already known. When measuring the LRV of viruses with a particle size of 30 nm or less (e.g., B19, MVM, PPV, etc.), if the LRV is 4.00 or higher, it can be determined that viruses with a particle size of 30 nm or less have been effectively removed. Generally, in performance evaluation of virus removal membranes, an LRV of 1.00 or higher is considered to indicate limited removal, 2.00 to 4.00 indicates moderate removal, and 4.00 or higher indicates effective removal. An LRV of 5.00 or higher means that the virus has been removed to less than 1 / 10^5, and an LRV of 6.00 or higher means that the virus has been removed to less than 1 / 10^6, indicating almost no virus leakage.

[0059] In step (b1) of the virus removal method of the present invention, filtration using a virus removal membrane can be confirmed by LRV as an indicator of removal of viruses with a particle size of less than 33 nm (e.g., B19, MVM, PPV, etc.).

[0060] LRV for viruses with a particle size of less than 33 nm (e.g., B19, MVM, PPV, etc.) can be determined by measuring the viral load of viruses with a particle size of less than 33 nm (e.g., B19, MVM, PPV, etc.) using the above formula. The amount of viruses with a particle size of less than 33 nm (e.g., B19, MVM, PPV, etc.) is not particularly limited, but can be measured by infectivity titer or viral nucleic acid amount. Infectivity titer is obtained by inoculating indicator cells with the virus, culturing them for a certain period, and measuring the degeneration of the cultured cells, as well as the viral antigen and nucleic acid (TCID). 50 (e.g., plaque assay). Quantitative polymerase chain reaction (Q-PCR), which measures nucleic acid content, can be performed using methods that are already known. When measuring the LRV of viruses with a particle size of less than 33 nm (e.g., B19, MVM, PPV, etc.), if the value is 4.00 or higher, it can be determined that viruses with a particle size of less than 33 nm have been effectively removed.

[0061] In the virus removal method of the present invention, the protein solution after irradiation treatment by step (a) or (a') applied to a virus removal membrane having an LRV of 4.00 or higher for bacteriophage PP7 is, for example, 50 to 100 l / m² as the amount of filtrate per unit area of ​​the virus removal membrane. 2 It can also be processed at 50-100 l / m 2 By processing in this manner, the solution can be efficiently filtered without causing clogging of the virus removal membrane with proteins or other substances. The virus removal membrane area is calculated as the area of ​​the filtration surface (primary surface). In the case of a flat membrane, it is the area calculated from the lengths of the two sides, and in the case of a hollow fiber membrane, it is the area of ​​the inside (inner surface) of the hollow fiber. The amount of filtrate can be adjusted by the filtration time under constant filtration pressure conditions.

[0062] The filtration pressure in step (b1) of the virus removal method of the present invention depends on the material of the virus removal membrane, but is kept within a range less than or equal to the pressure resistance of the membrane. For example, in the case of a virus removal membrane made of regenerated cellulose, it is 0.00 kgf / cm². 2 (0.0 kPa) ~ 1.00 kgf / cm² 2 It may be performed within the range of (9.8 × 10 kPa). In the case of a virus removal membrane made of hydrophilic PVDF or hydrophilic PES, 0.00 kgf / cm 2 (0.0 kPa) ~ 5.00 kgf / cm² 2 (4.9 × 10 2 It may be performed within the range of kPa.

[0063] The SD treatment in step (b2) of the virus inactivation method of the present invention is not particularly limited as long as it can be performed so that the LRV for enveloped viruses in the protein solution subjected to the SD treatment becomes 2.00 or higher. An LRV of 2.00 or higher indicates moderate inactivation, and an LRV of 4.00 or higher indicates effective virus inactivation. However, if desired, the treatment may be performed to achieve LRVs of, for example, 3.00 or higher, 4.00 or higher, 5.00 or higher, or 6.00 or higher. Furthermore, if the SD treatment is selected in step (b2), the virus removal membrane treatment of step (b1) described above may be performed separately in a subsequent step.

[0064] The SD mixture may be any combination of organic solvent and surfactant known in the field of organic solvents and surfactants that can chemically inactivate enveloped viruses, as long as the above LRV conditions are met. Examples of organic solvents include dialkyl or trialkyl phosphates having an alkyl group with 1 to 10 carbon atoms, with trialkyl phosphates having an alkyl group with 2 to 10 carbon atoms being preferred. Specifically, examples include tri-(n-butyl) phosphate (hereinafter abbreviated as "TNBP"), tri-(t-butyl) phosphate, tri-(n-hexyl) phosphate, tri-(2-ethylhexyl) phosphate, tri-(n-decyl) phosphate, and ethyl-di(n-butyl) phosphate.

[0065] Typically, surfactants used are those capable of dispersing fats at a concentration of 0.1 w / w% in a 0.01 g / ml solution at room temperature. Specifically, examples include polyoxyethylene derivatives of fatty acids, polyoxyethylene sorbitan fatty acid esters, oxyethylated alkylphenols, polyoxyethylene alcohols, polyoxyethylene oils, and polyoxyethylene oxypropylene fatty acids. More specifically, examples include polyoxyethylene derivatives of fatty acids such as Tween® 80 and Tween® 20, partial esters of sorbitol anhydride such as polysorbate 80, oxyethylated alkylphenols such as polyoxyethylene octylphenyl ether (Triton® X-100), sodium cholate, sodium deoxycholate, sulfobetaines such as N-dodecyl-N,N-dimethyl-2-ammonio-1-ethanesulfonate, and nonionic detergents such as octyl-β,D-glucopyranoside. Preferably, it is a nonionic oil-soluble aqueous detergent such as Tween® 80, Triton® X-100, or sodium cholate.

[0066] The above SD mixture may contain two or more organic solvents and / or surfactants independently. The SD mixture may also contain other additives, such as reducing agents, as needed to enhance its effects. Preferably, the SD mixture contains the above-mentioned organic solvent (S) and surfactant (D) in amounts such that the S / D (w / w ratio) is 1 to 20.

[0067] SD treatment can be carried out by contacting a protein solution with an SD mixture at a temperature of 0 to 40°C, preferably 4 to 25°C, and more preferably 7 to 12°C. The effect usually appears after a few minutes of contact, preferably between 10 minutes and 2 hours, and typically around 30 to 60 minutes. No further increase in effect can be expected by treating for longer than 2 hours, but to ensure the effect, the treatment may be performed for a longer period, for example, 6 hours or more.

[0068] 2. Method for producing a protein solution from which the virus of the present invention has been removed. The present invention provides a method for producing a protein solution from which a virus has been removed, and is described below: (a) A step of irradiating a protein solution with radiation so that the LRV for viruses with a particle size of 30 nm or less in the protein solution is 1.00 or higher, preferably 2.00 or higher, more preferably 3.00 or higher, and even more preferably 4.00 or higher, or (a') A step of irradiating a protein solution with radiation so that the LRV for viruses with a particle size of less than 33 nm in the protein solution is 1.00 or higher, preferably 2.00 or higher, more preferably 3.00 or higher, and even more preferably 4.00 or higher, and (b1) A step of filtering the treated protein solution through a virus removal membrane having an LRV of 4.00 or higher against bacteriophage PP7, or (b2) The process of bringing the treated protein solution into contact with the SD mixture and performing SD treatment so that the LRV against enveloped viruses in the protein solution becomes 2.00 or higher. This method includes [something]. With respect to the above steps (a) or (a') and steps (b1) and (b2), the contents described in "1. Method for removing viruses from protein solutions of the present invention" above may be applied.

[0069] Before step (a) or (a') of the method for producing a virus-free protein solution according to the present invention, or between step (a) or (a') and step (b1), prefiltration may be performed using a filter with a larger pore size than the virus removal membrane used in step (b1). Examples of filters with a larger pore size include Planova® 35N (manufactured by Asahi Kasei Medical Co., Ltd.), Planova® 75N (manufactured by Asahi Kasei Medical Co., Ltd.), 0.1 μm filters, 0.2 μm filters, etc.

[0070] Furthermore, after step (b1) of the method for producing a virus-free protein solution according to the present invention, a washing step may be added to wash out the proteins inside the membrane to the filtrate side by filtering a protein-free solution (washing solution) as appropriate.

[0071] Furthermore, one or more of the following treatments may be performed before step (a) or (a') of the method for producing a virus-free protein solution of the present invention, between step (a) or (a') and step (b1) or (b2), or after step (b1) or (b2): chromatography, virus removal membrane treatment, SD treatment, concentration treatment, and concentration / buffer exchange treatment. The virus removal membrane treatment may be carried out by referring to the content of step (b1) described in "1. Method for removing viruses from a protein solution of the present invention" above.

[0072] Examples of chromatographic treatments include column chromatography, in which ion exchange resins or gel filtration resins are packed into a column, and membrane chromatography, in which ion exchange groups are attached to the surface of a porous membrane. Examples of chromatographic separation modes include gel filtration chromatography, ion exchange chromatography (cation exchange: CEX, anion exchange: AEX), hydrophobic chromatography (HIC), affinity chromatography, metal chelate affinity chromatography, and hydroxyapatite chromatography. Chromatography combining ion exchange and hydrophobic interaction may also be used as the ligand for chromatography. Regarding SD treatment, the content of step (b2) described in "1. Method for removing viruses from protein solutions of the present invention" above may be applied.

[0073] The concentration process may be carried out using an ultrafiltration (UF) membrane according to a known method. Alternatively, it may be carried out by centrifugal concentration.

[0074] The buffer exchange process may be carried out simultaneously with concentration using an ultrafiltration membrane, according to a known method. Alternatively, it may be carried out by gel filtration or dialysis using a dialysis membrane.

[0075] After steps (b1, b2) of the method for producing a virus-free protein solution of the present invention, the virus-free protein solution may be purified, for example, by chromatography. The virus-free protein solution of the present invention may be further concentrated by UF treatment.

[0076] Furthermore, the virus-free protein solution obtained in step (b1) or (b2), its purified product, or its concentrate may be formulated in its original liquid composition, or it may be formulated after, for example, buffer exchange with a solvent of another composition (which may include removal of SD by a known method). Alternatively, the virus-free protein solution, its purified product, or its concentrate may be subjected to freeze-drying before being formulated.

[0077] In the formulation described above, the virus-free protein solution obtained by the manufacturing method of the present invention, or its freeze-dried product, can be mixed with one or more pharmacologically acceptable carriers by a method well known in the field of pharmaceutical technology to produce a pharmaceutical preparation or pharmaceutical composition.

[0078] Pharmacologically acceptable carriers include various organic or inorganic carrier substances commonly used as formulation materials. Specific examples include excipients, lubricants, binders, and disintegrants in solid formulations, and solvents, solubilizers, suspending agents, isotonic agents, buffers, and analgesics in liquid formulations. During formulation, preservatives, antioxidants, colorants, sweeteners, and other formulation additives may be used as needed.

[0079] Excipients include lactose, sucrose, D-mannitol, D-sorbitol, starch, pregelatinized starch, dextrin, crystalline cellulose, low-substituted hydroxypropyl cellulose, sodium carboxymethylcellulose, acacia gum, pullulan, soft anhydrous silicic acid, synthetic aluminum silicate, magnesium aluminometasilicate, xylitol, sorbitol, and erythritol. Examples of lubricants include magnesium stearate, calcium stearate, talc, colloidal silica, and polyethylene glycol 6000.

[0080] Examples of binders include pregelatinized starch, sucrose, gelatin, gum arabic, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, crystalline cellulose, sucrose, D-mannitol, trehalose, dextrin, pullulan, hydroxypropylcellulose, hydroxypropylmethylcellulose, and polyvinylpyrrolidone.

[0081] Examples of disintegrants include lactose, sucrose, starch, carboxymethylcellulose, carboxymethylcellulose calcium, croscarmellose sodium, carboxymethyl starch sodium, low-substituted hydroxypropylcellulose, soft anhydrous silicic acid, and calcium carbonate.

[0082] Examples of solvents include sterile water for injection, physiological saline, Ringer's solution, alcohol, propylene glycol, polyethylene glycol, sesame oil, corn oil, olive oil, and cottonseed oil. Examples of solubilizers include polyethylene glycol, propylene glycol, D-mannitol, trehalose, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate, sodium salicylate, and sodium acetate.

[0083] Examples of suspending agents include surfactants such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, and glyceryl monostearate; hydrophilic polymers such as polyvinyl alcohol, polyvinylpyrrolidone, sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, and hydroxypropylcellulose; and polysorbates and polyoxyethylene hydrogenated castor oil.

[0084] Examples of isotonic agents include sodium chloride, glycerin, D-mannitol, D-sorbitol, glucose, xylitol, and fructose. Examples of buffering agents include buffer solutions such as phosphates, acetates, carbonates, and citrates. Examples of pain relievers include propylene glycol, lidocaine hydrochloride, and benzyl alcohol.

[0085] Examples of preservatives include para-hydroxybenzoic acid esters, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, and sorbic acid. Examples of antioxidants include sulfites and ascorbic acid salts. Examples of colorants include water-soluble colored tar dyes (e.g., food colorants such as Red No. 2 and 3, Yellow No. 4 and 5, and Blue No. 1 and 2), insoluble lake dyes (e.g., aluminum salts of the aforementioned water-soluble food colorants), and natural pigments (e.g., β-carotene, chlorophyll, red iron oxide). Examples of sweeteners include sodium saccharin, dipotassium glycyrrhizinate, aspartame, and stevia.

[0086] Furthermore, the most effective route of administration should be used for treatment, and the drug can be administered orally, by injection, or transdermally. Oral preparations include tablets (including sublingual tablets and orally disintegrating agents), capsules (including soft capsules and microcapsules), powders, granules, lozenges, syrups, emulsions, and suspensions. Injectable preparations include intradermal injection, subcutaneous injection, intravenous injection, intramuscular injection, intrathecal injection, epidural injection, and local injection. Transdermal preparations include patches, ointments, and powders. These preparations may also be controlled-release formulations such as immediate-release or sustained-release formulations (e.g., sustained-release microcapsules).

[0087] The dosage or frequency of administration varies depending on the desired therapeutic effect, method of administration, duration of treatment, age, weight, etc., but the amount of active ingredient (protein) can usually be 10 μg / kg to 5,000 mg / kg per day for adults.

[0088] The present invention will be described below with reference to examples. However, the present invention is not limited to these examples. [Examples]

[0089] Example 1: Investigation of inactivation of viruses in the presence of IgG by UVC irradiation. The effects of UVC irradiation on IgG molecules were evaluated using intravenous human immunoglobulin (IVIG). The sample (IVIG) was added to a petri dish to form a thin film, and UVC irradiation was performed while shaking the dish. UVC irradiation dose (irradiation energy) was measured using a UVR-300 (Topcon Techno House Co., Ltd.) and other equipment.

[0090] When the above measurements were performed by irradiating 2.5% or 5% IgG in 0.1 M glycine buffer (pH 3.0-6.8) with UVC, the effect of differences in IgG concentration on IgG polymer formation was minimal. On the other hand, IgG polymers were formed in proportion to the amount of UVC irradiation, and it was shown that the degree of polymer formation also differed depending on the pH. Polyclonal IgG is known to have higher solubility at lower pH and to be most stable around pH 4.2, and similarly, polymer formation was minimal around pH 3.5-5.5 even with UVC irradiation (Figures 1 and 2). From the above studies, if the upper limit for polymer formation is 2%, the UVC irradiation should be 512 mJ / cm². 2 The conditions up to this point were considered irradiable, and it was shown that a pH of around 4.1 was the condition that best suppressed polymer formation in polyclonal IgG. Since the optimal pH for solubility of monoclonal IgG changes depending on the PI value of the IgG molecule, the optimal conditions differ for each IgG molecule.

[0091] In the virus inactivation experiment, B19, MVM, and BVDV were added to PBS and irradiated using a UVR-300 (Topcon Techno House) with varying UVC irradiation doses. The change in infectivity (degree of inactivation) due to irradiation was evaluated (Figures 3-5). The change in infectivity was measured using the TCID (Typical Cholesterol Irradiation Spectrum). 50 The infectious titer was calculated using a method and evaluated from the change in infectious titer of samples before and after virus inactivation. The vertical axis of the figure shows the index value of infectious titer. The LRV, which indicates the logarithmic reduction rate, is calculated from the value obtained by subtracting the index value of infectious titer after inactivation from the index value of infectious titer before inactivation. Furthermore, UVC irradiation was performed under IgG coexistence conditions by adding MVM and BVDV to 0.1 M glycine buffer (pH 4.1) containing 2.5% IgG, and changes in infectivity (degree of inactivation) were evaluated (Figures 4 and 5). Since the IgG used contained anti-B19 antibodies, B19 was not used in this experiment under IgG coexistence conditions. The characteristics of the viruses used are summarized in Table 1.

[0092] [Table 1]

[0093] Under PBS conditions, the MVM was 32 mJ / cm³. 2 The above UVC irradiation eliminated (inactivated) the infectivity of the viruses, and the LRV levels were >3.8 and >5.0, respectively. B19 was 64 mJ / cm². 2 The viral LRV values ​​after UVC irradiation were 7.8, 7.4, and 7.2, respectively, while BVDV did not show an inactivation effect at the irradiation doses evaluated in this study. An LRV of 4.00 or higher is considered effective for virus inactivation and removal, but even if it is below 4.00, a value of 2.00 or higher is considered moderate, a value of 1.00 or higher is considered limited, and a value of 1.00 or lower is considered ineffective. In addition, in the presence of IgG, the MVM was 256 mJ / cm². 2Infectivity disappeared with the above UVC irradiation levels (LRV: >3.8, >4.6), but the viral inactivation curves at lower irradiation levels differed depending on whether IgG was present or absent. This is because, although these viruses are inactivated by UVC irradiation, the presence of IgG stabilizes the virus, increasing the irradiation dose required for inactivation, specifically at 256 mJ / cm². 2 It was shown that the critical point was exceeded. On the other hand, BVDV, which has larger particles and genome size, showed a reading of 512 mJ / cm³ under both PBS and IgG coexistence conditions. 2 Even with this energy level, there was no significant change in infectivity (i.e., it was not inactivated) (Figures 3-5).

[0094] These results indicate that the primary factor influencing viral inactivation by UVC irradiation is the size of the viral particle (genome size). Besides the viral particle size, the type and concentration of coexisting proteins, as well as pH, can also be influential factors. For example, in a solution containing IgG as the target protein, the optimal conditions for viral inactivation and removal are: treatment with a virus removal membrane with a pore size of 20 nm, an IgG concentration of 10% or less, a pH of 4.1-5.5, and a UVC irradiation dose of 256 mJ / cm². 2 Alternatively, a virus removal membrane treatment with a pore size of less than 33 nm, IgG concentration of 10% or less, pH 4.1-5.5, and UVC irradiation dose of 256 mJ / cm². 2 It was shown that...

[0095] Example 2: Investigation of inactivation of viruses in the presence of fibrin by UVC irradiation. Fib (containing 0.5% sodium chloride and 1.6% sodium citrate as stabilizers) concentration is 3.9 mg / ml (E 280 : 5.8, E 1% 280The effect of UVC irradiation on Fib molecules was evaluated using the solutions from 15-16). In this experiment, the UVC energy was measured using a UVC-254 (manufactured by Custom Co., Ltd.), which shows a value approximately 1 / 3 lower than that of the UVR-300 (manufactured by Topcon Techno House Co., Ltd.) used in Example 1 (Table 2). Therefore, in this experiment, a correction value was used in which the measured value was tripled to match the measurement value obtained with the UVR-300 (manufactured by Topcon Techno House Co., Ltd.).

[0096] Since Fib is a protein with coagulation activity, its coagulation activity and polymer formation were evaluated. Upon UVC irradiation, the coagulation activity was 192 mJ / cm². 2 Although it remained at almost 100% until irradiation, the polymer level increased in proportion to the irradiation dose. The polymer was detected even before irradiation, but at 96 mJ / cm³ 2 With irradiation, the amount of polymer increased by 20% compared to before irradiation (Figure 6). However, polymer content is not an important indicator for purposes such as forming a fibrin membrane before administration; coagulation activity is the important indicator for UVC irradiation.

[0097] Next, B19 or PPV was added to Fib solutions of various concentrations, and the UVC irradiation dose was varied to evaluate the change in viral infectivity (degree of inactivation) in the same manner as in Example 1. The properties of the viruses used are shown in Table 1. In the study using B19, at a Fib concentration of 3.3 mg / ml (E 280 : 5, Absorption coefficient (E 1% 280 :15~16)), 120 mJ / cm 2 Upon irradiation, B19 in the solution was inactivated to the detection limit (LRV: >4.6). A higher protein concentration, Fib concentration of 6.7 mg / ml (E 280 : 10, Absorption coefficient (E 1% 280 In sections 15-16), to achieve an LRV of >4.0, 192 mJ / cm² is required. 2 Irradiation is required, with a Fib concentration of 13.3 mg / ml (E 280 : 20, Absorption coefficient (E 1% 280:15 - 16), the LRV could not reach >4.00 within the scope of this experiment (Figs. 7 and 8).

[0098] In the study using PPV, at a Fib concentration of 3.6 mg / ml (E 280 : 5.4, extinction coefficient (E 1% 280 :15 - 16), 192 mJ / cm 2 irradiation showed that PPV in the solution was inactivated to the detection limit and exceeded the critical point (Fig. 9). From this example, in the case of a solution containing Fib as the target protein, examples of optimal virus inactivation and removal conditions include virus removal membrane treatment with a pore size of 20 nm, Fib absorbance A280 = 10 or less, 192 mJ / cm 2 UVC irradiation, or virus removal membrane treatment with a pore size of less than 33 nm, Fib absorbance A280 = 10 or less, 192 mJ / cm 2 UVC irradiation were shown.

[0099] Example 3: Investigation of inactivation of viruses in the presence of Thr by UVC irradiation For Thr (containing 0.34% sodium chloride, 0.23% sodium citrate, and 0.27% calcium chloride as stabilizers) at a concentration of 2.48 mg / ml (E 280 : 5.3, E 1% 280 :21.4) solution, the effect of UVC irradiation on Thr molecules was evaluated. In this experiment, the UVC energy amount was measured using UVC - 254 (manufactured by Custom Co., Ltd.), but this measuring instrument showed a value approximately 1 / 3 lower than that of UVR - 300 (manufactured by Topcon Techno House Co., Ltd.) (Table 2). Therefore, in this experiment, a corrected value obtained by multiplying the measured value by 3 was used so that the measured value would be the value measured by UVR - 300 (manufactured by Topcon Techno House Co., Ltd.).

[0100] Since Thr is a protein with coagulation activity, coagulation activity and polymer formation were evaluated. The coagulation activity decreased according to the irradiation dose and decreased to almost 90% at 192 mJ / cm 2 The polymers also increased according to the irradiation dose and reached 192 mJ / cm 2It increased up to 3.8% (Figure 10). Next, B19 or PPV was added to Fib solutions diluted to various concentrations, irradiated while changing the UVC irradiation dose, and the change in viral infectivity (degree of inactivation) was evaluated in the same manner as in Example 1. The properties of the viruses used are as shown in Table 1.

[0101] B19 and PPV showed similar inactivation curves. The B19 virus in a solution with a Thr concentration of 4.67 mg / ml (E 280 : 10, E 1% 280 :21.4) was inactivated to the detection limit by irradiation of 192 mJ / cm 2 . For PPV, the condition to achieve LRV: 4.00, although not below the detection limit, was an irradiation dose of 96 mJ / cm 2 . Although these viruses are inactivated by UVC irradiation, the coexistence of Thr stabilizes the virus and increases the irradiation dose required for inactivation. However, if the Thr concentration is 4.67 mg / ml or less (E 280 : 10, E 1% 280 :21.4), it was shown to exceed the critical point at 192 mJ / cm 2 . If the Thr concentration is 2.34 mg / ml (E 280 : 5, E 1% 280 :21.4), it was shown to exceed the critical point at 96 mJ / cm 2 (Figures 11 and 12). From this example, although it is an example, in the case of a solution containing Thr as the target protein, as an example of optimal virus inactivation and removal conditions, virus removal membrane treatment with a pore size of 20 nm, Thr concentration of less than 4.67 mg / ml (E 280 : 10, E 1% 280 :21.4), UVC irradiation of 192 mJ / cm 2 (or 96 mJ / cm 2 ), or virus removal membrane treatment with a pore size of less than 33 nm, Thr concentration of less than 4.67 mg / ml (E 280 : 10, E 1% 280 :21.4), UVC irradiation of 192 mJ / cm 2(or 96 mJ / cm²) 2 It was shown that the irradiation was UVC.

[0102] Example 4: Investigation of inactivation of viruses by gamma irradiation in the presence of Fib, Thr, and Alb. Solutions containing 26.65 mg / ml of Fib (53.3 mg / vial after lyophilization, containing sodium citrate and L-alginate as stabilizers and excipients), 3.5 mg / ml of Thr (7.0 mg / vial after lyophilization, containing D-mannitol, sodium citrate, and L-alginate as stabilizers and excipients), and 10 mg / ml of Alb (20 mg / vial after lyophilization, containing a) sodium citrate and sodium chloride, and b) D-mannitol as stabilizers and excipients) were lyophilized, and the effects of gamma irradiation on each protein were evaluated.

[0103] The degree of degradation product formation of these proteins by gamma irradiation was evaluated by the peak area ratio obtained by SEC (HPLC) analysis. Fib polymer increased by 20% with 25 kGy irradiation compared to unirradiated samples. Thr polymer showed no significant change even with 50 kGy irradiation. Albumin increased by approximately 10-15% with 50 kGy irradiation (Figure 13-15). Next, samples that had been freeze-dried after the addition of B19 were irradiated with varying gamma doses, and the change in viral infectivity (degree of inactivation) was evaluated in the same manner as in Example 1. The properties of the viruses used are shown in Table 1.

[0104] In the presence of albumin, B19 was inactivated to below the detection limit (LRV: >4.4, >4.2) with 25 kGy irradiation. In the presence of throcytes (Thr), it was inactivated to below the detection limit (LRV: >3.8) with 50 kGy irradiation. Furthermore, in the presence of fibrinopropyl alcohol (Fib), it was inactivated to LRV: 4.6 with 25 kGy irradiation and to below the detection limit (LRV: >5.0) with 50 kGy irradiation (Figure 16). From this example, it was shown that an example of the optimal conditions for B19 inactivation by gamma irradiation is 25 kGy or higher in the presence of albumin, 50 kGy or higher in the presence of Thr, and 25 kGy or higher in the presence of Fib.

[0105] Example 5: Examination of calibration errors in measurement values ​​due to differences in UVC measuring instruments When performing virus inactivation by UVC irradiation, it is necessary to accurately measure the UVC irradiation dose (energy amount). However, our investigation revealed that, as shown in Table 2, the indicated values ​​may differ depending on the detector, even under the same conditions.

[0106] [Table 2]

[0107] From Table 2 above, it is clear that standardizing the virus inactivation conditions by UVC irradiation is difficult when the same detector is not used. To solve the above standardization problem, we attempted to standardize the UVC irradiation dose using 1% NaI, which is known to change in absorbance with UVC irradiation. As shown in Figure 17, by using NaI and adopting a calibration curve method, the desired standardization was achieved. [Industrial applicability]

[0108] This invention integrates virus inactivation by radiation treatment and virus removal by virus removal membrane treatment or virus inactivation by SD treatment, optimizing the conditions so that their characteristics complement each other. Therefore, it is useful for inactivating and removing viruses in protein solutions, and for producing protein solutions from which viruses have been inactivated and removed.

[0109] This application is based on Japanese Patent Application No. 2020-175610, the contents of which are entirely incorporated herein.

Claims

1. A method for removing viruses from a protein solution, the following: (a) A step of irradiating a protein solution with radiation so that the removal coefficient (LRV) for viruses with a particle size of less than 25 nm in the protein solution is 4.00 or more, wherein the radiation is ultraviolet light or gamma rays, and the ultraviolet light is irradiated in the range of 32 mJ / cm² to 96 mJ / cm² or the gamma rays are irradiated in the range of 5 kGy to 50 kGy, and (b1) A step of removing the virus from the treated protein solution using a virus removal membrane having an LRV of 4.00 or more against bacteriophage PP7, wherein the virus removal membrane is a virus removal membrane with a pore size of 17 to 21 nm. Methods that include...

2. The method of claim 1, wherein (a) is a step of irradiating a protein solution with radiation so that the removal coefficient (LRV) for viruses with a viral particle diameter of less than 21 nm in the protein solution is 4.00 or more.

3. The method according to claim 1, wherein the radiation is ultraviolet light and is irradiated in the range of 32 mJ / cm² to 64 mJ / cm².

4. The method according to claim 1, wherein the radiation is gamma rays and is irradiated in the range of 25 kGy to 50 kGy.

5. The method according to claim 1, wherein the protein is one or more selected from: polyclonal or monoclonal antibodies selected from IgG, IgM, IgA, IgD, and IgE; hematopoietic factors selected from erythropoietin and thrombopoietin; proteins involved in blood coagulation selected from fibrinolytic factors and blood coagulation factors; plasma proteins selected from albumin and globulin; growth factors selected from epidermal growth factor (EGF) and hepatocyte growth factor (HGF); stem cell factors selected from G-CSF and M-CSF; cytokines selected from interferon-γ and tumor necrosis factor-α; enzymes selected from DNase I and galactosidase; hemoglobin; transferrin; and modifications or partial fragments of these proteins.

6. A method for producing a protein solution from which the virus has been removed, the following: (a) A step of irradiating a protein solution with radiation so that the removal coefficient (LRV) for viruses with a particle size of less than 25 nm in the protein solution is 4.00 or more, wherein the radiation is ultraviolet light or gamma rays, and the ultraviolet light is irradiated in the range of 32 mJ / cm² to 96 mJ / cm² or the gamma rays are irradiated in the range of 5 kGy to 50 kGy, and (b1) A step of filtering the treated protein solution through a virus removal membrane having an LRV of 4.00 or more against bacteriophage PP7, wherein the virus removal membrane is a virus removal membrane with a pore size of 17 to 21 nm. Methods that include...

7. The method of claim 6, wherein (a) is a step of irradiating a protein solution with radiation so that the removal coefficient (LRV) for viruses with a viral particle diameter of less than 21 nm in the protein solution is 4.00 or more.

8. The radiation is ultraviolet light, and the concentration is 32 mJ / cm². 2 ~64 mJ / cm² 2 The method according to claim 6, wherein irradiation is performed within the range.

9. The method according to claim 6, wherein the radiation is gamma rays and is irradiated in the range of 25 kGy to 50 kGy.

10. The method according to claim 6, wherein the protein is one or more selected from polyclonal or monoclonal antibodies selected from IgG, IgM, IgA, IgD, and IgE; hematopoietic factors selected from erythropoietin and thrombopoietin; proteins involved in blood coagulation selected from fibrinolytic factors and blood coagulation factors; plasma proteins selected from albumin and globulin; growth factors selected from epidermal growth factor (EGF) and hepatocyte growth factor (HGF); stem cell factors selected from G-CSF and M-CSF; cytokines selected from interferon-γ and tumor necrosis factor-α; enzymes selected from DNase I and galactosidase; hemoglobin; transferrin; and modifications or partial fragments of these proteins.

Images