WO2022085606A1

WO2022085606A1 - Method for removing viruses in protein solution

Info

Publication number: WO2022085606A1
Application number: PCT/JP2021/038339
Authority: WO
Inventors: 幹弘柚木; 健浦山
Original assignee: 一般社団法人日本血液製剤機構
Priority date: 2020-10-19
Filing date: 2021-10-18
Publication date: 2022-04-28
Also published as: JPWO2022085606A1; WO2022085606A9

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

Virus removal method in protein solution

The present invention relates to a method for removing a virus in a protein solution and a method for producing a protein solution from which the virus has been removed.

In the manufacturing process of plasma fractionation preparations and biopharmacy, from the viewpoint of safety and stability of the preparations, virus inactivation and / or removal (hereinafter abbreviated as "virus inactivation / removal") steps are included. It has been incorporated. Generally, the virus inactivation condition is designed as long as both the condition that the protein is not denatured and the condition that the virus is inactivated are satisfied. Therefore, if an attempt is made to effectively inactivate all viruses, the amount of protein denatured increases, the purpose of formulation cannot be achieved, or profitability deteriorates.

In addition, proteins such as coagulation factors have low resistance to physicochemical loads. Therefore, if the virus inactivating condition is set in a range where the protein having low stability does not cause denaturation, there arises a problem that the inactivating effect is shown only to some viruses. As can be understood from the above, virus inactivation / removal techniques are, in a sense, incomplete techniques, and it is extremely difficult to completely inactivate and remove all viruses while avoiding protein denaturation with one method. It is difficult, and in the production of biopharmaceuticals, it is common to simply combine multiple virus inactivation / removal steps.

Currently, as virus inactivation / removal methods, liquid / dry heat treatment, organic solvent / surfactant treatment (SD treatment), low pH treatment, virus removal membrane treatment, etc. are the virus inactivation / removal steps for manufacturing biopharmaceuticals, etc. It has been introduced into the process (Patent Documents 1 to 3 and Non-Patent Document 1).

The above-mentioned liquid / dry heat treatment and SD treatment are known as effective inactivating treatments against enveloped viruses such as human immunodeficiency virus (HIV) and hepatitis C virus (HCV). However, non-enveloped viruses such as human parvovir B19 (hereinafter abbreviated as "B19") and hepatitis A virus (hereinafter abbreviated as "HAV") resist the above-mentioned virus inactivation treatment. It has sex and has the problem that it cannot be expected to have a certain degree of effect on enveloped viruses.

In the above-mentioned virus removing membrane treatment, the separation operation is performed according to the size of the particles, so that the virus is removed only according to the size regardless of the chemical or thermal properties of the object to be separated. There is an advantage that it can be done. Therefore, in recent years, a membrane filtration method using a virus removing membrane has been generally adopted from the viewpoint of contamination with non-enveloped viruses and the like.

Examples of parvovirus, which is a non-enveloped virus, 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 about 23 to 28 nm (ICTV, https://talk.ictvonline.org/ictv-reports/). ictv_online_report / ssdna-viruses / w / parvoviridae). In the past, in the production of biopharmacy and the like, cases of contamination with rodent-derived MVM have occurred during cell culture, and this is recognized as a risk in the above production. As an effective virus removing method for parvovirus and the like, a membrane filtration method using a virus removing membrane is widely used. The basic principle of the membrane filtration method is achieved by setting conditions under which the target protein passes through the virus-removing membrane but the virus cannot pass, depending on the pore size of the virus-removing membrane used.

The smallest non-enveloped virus currently known is porcine circovirus (hereinafter sometimes abbreviated as "PCV"), which has a particle size of about 15 to 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, for example, PCV1, PCV2 and the like. PCV is also one of the viruses with a high risk of being contaminated in the manufacturing process of biopharmacy and the like, and cases of contamination have been reported in the past. PCV is resistant to low pH treatment and heat treatment, and it is difficult to capture the virus in solutions containing PCV even using a virus removal membrane with a pore size of 15 nm, and many biopharmacy There is a problem that the virus removing membrane introduced in the manufacturing process of pharmaceutical products cannot be sufficiently removed. Further, for example, although treatment using an ion exchange resin or the like is performed, it cannot be said that a sufficient effect is obtained.

Currently, optimization of virus inactivation / removal conditions in the manufacturing process of plasma fractionated products and biopharmacy is designed for each process desired to be introduced, and is based on the guidelines of the International Council for Harmonization of Pharmaceutical Regulations (ICH). Characteristic evaluation is carried out for each process. In addition, in the evaluation of the virus inactivation / removal ability in the entire biopharmacy manufacturing process, a plurality of individually optimized virus inactivation / removal steps are carried out, and the inactivation and removal ability in each step and the total thereof are carried out. It is evaluated in (Non-Patent Document 1).

Due to the above-mentioned evaluation methods, etc., the conditions (band) that can be introduced into the manufacturing process of biopharmacy, etc. are narrow or biased, despite having excellent virus inactivation and removal ability other than the above. There is a problem that it is difficult to introduce a method having characteristics such as virus into the manufacturing process. Taking radiation (eg, ultraviolet rays, electron beams, γ-rays, etc.) as an example, first, the mechanism of virus inactivation by irradiation with radiation is to damage viral nucleic acids by energy such as electromagnetic waves and electron beams, and the genome. The smaller the size (that is, the smaller the particle size of the virus), the lower the energy required for inactivation (Non-Patent Document 2). Therefore, the larger the irradiation energy, the easier it is for proteins to denature, and under irradiation conditions of radiation (eg, ultraviolet rays, electron beams, γ-rays, etc.) that can achieve inactivation of large viruses, protein denaturation is remarkable, and biopharmaceuticals, etc. Is considered to be very difficult to introduce into the manufacturing process. In addition, the irradiation amount of radiation (eg, ultraviolet rays, electron beams, γ-rays, etc.) may differ depending on the type of detector even under the same conditions, so it is difficult to standardize the irradiation amount, which also hinders the introduction. It is thought that it is.

Japanese Unexamined Patent Publication No. 2006-151840 International Publication No. 2010/109920 International Publication No. 2018/030437

The subject of the present invention includes a method for inactivating / removing a virus in a protein solution, which comprises a step of integrating irradiation treatment with a virus removing membrane treatment or SD treatment to optimize those conditions, and the above steps. It is to provide a method for producing a protein solution in which a virus has been inactivated and removed.

The present inventors are keen to introduce the irradiation treatment, which is extremely difficult to introduce into the manufacturing process of biopharmacy, etc., in spite of its excellent virus inactivating / removing ability. Study was carried out. As a result of this study, the present inventors focused on the characteristics of the process centering on virus inactivation / removal and protein denaturation, and (i) a virus inactivation method by irradiation treatment and (ii) a virus. The idea was to design virus inactivation / removal conditions in a mutually complementary manner by integrating the virus removal method by removal membrane treatment or the virus inactivation method by SD treatment. It is extremely difficult to come up with this idea from the existing method of performing process characteristic evaluation for each virus inactivation / removal process that is desired to be introduced into the manufacturing process as described above. So far, no report has been found that the process was designed with such an idea and the virus inactivation / removal ability was evaluated. In addition, since it is difficult to measure the absolute irradiation amount with radiation, especially ultraviolet rays, the standardization of the irradiation amount also focuses on the change in the absorbance of a specific compound and uses the change in the absorbance as an index between measuring instruments. I came up with the idea of calibrating. Then, based on this idea, as a result of further research, we designed a virus inactivation / removal process that is suitable for the above two methods as a whole, which enables introduction into the manufacturing process of biopharmacy and the like. The present invention has been completed.

That is, the present invention is as follows.
[1] A method for removing a virus in a protein solution, which is as follows:
(A) By irradiating the protein solution with radiation, the removal coefficient for viruses with a virus particle size of less than 33 nm in the protein solution (the degree of decrease of the virus represented by a logarithm, hereinafter referred to as "LRV") is The step of treating to 1.00 or more, and (b1) the step of removing the virus from the treated protein solution with a virus removing membrane having an LRV of 4.00 or more for Bacterophage PP7, or (b2) the above-mentioned treatment. The protein solution is brought into contact with a mixed solution of an organic solvent and a surfactant (SD mixed solution), and the organic solvent / surfactant treatment (SD treatment) is performed so that the LRV against the enveloped virus in the protein solution becomes 2.00 or more. A method comprising the step of inactivating a virus.
[1-1] (a) is a step of irradiating the protein solution with radiation so that the removal coefficient for the virus having a virus particle diameter of 30 nm or less in the protein solution is 1.00 or more [1]. ] The method described in.
[2] The method according to [1] or [1-1], wherein the radiation is ultraviolet rays or γ-rays.
[3] The method according to [2], wherein the radiation is ultraviolet light.
[4] The method according to [3], wherein the ultraviolet rays are irradiated in the range of 32 mJ / cm ² to 256 mJ / cm ² .
[5] The method according to [2], wherein the radiation is gamma rays.
[6] The method according to [5], wherein the γ-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-removing membrane having an LRV of 4.00 or more for bacteriophage PP7 is a virus-removing membrane having a pore size of 20 nm or less.
[8] The method according to [7], wherein the virus removing membrane having a pore size of 20 nm or less is a virus removing membrane having a pore diameter of 15 nm or more.
[8-1] The method according to any one of [1] to [6], wherein the LRV for bacteriophage PP7 is 4.00 or more and the pore size of the virus removing membrane is less than 33 nm.
[8-2] The method according to [8-1], wherein the virus removing membrane has a pore size of 13 to 33 nm.
[8-3] The method according to [8-1], wherein the virus removing membrane has a pore size of 15 to 33 nm.
[8-4] The method according to [8-1], wherein the virus removing membrane has a pore size of 17 to 33 nm.
[8-5] The method according to [8-1], wherein the virus removing membrane has a pore size of 17 to 30 nm.
[8-6] The method according to [8-1], wherein the virus removing membrane has a pore size of 17 to 21 nm.
[8-7] The method according to [8-1], wherein the virus removing membrane has a pore size of 19 to 21 nm.
[8-8] The method according to [8-1], wherein the virus removing membrane has a pore size of 19 to 20 nm.
[9] The method according to [7] or [8], wherein the virus removing membrane is a virus removing membrane having a pore size of about 20 nm.
[10] A method for producing a protein solution from which a virus has been removed.
(A) The step of irradiating the protein solution with radiation so that the removal coefficient (LRV) for the virus having a virus particle diameter of less than 33 nm in the protein solution is 1.00 or more, and (b1) the treatment. The step of filtering the protein solution with a virus-removing membrane having an LRV of 4.00 or higher for Bacterophage PP7, or (b2) contacting the treated protein solution with the SD mixture and LRV against the enveloped virus in the protein solution. A method that includes a step of SD processing so that is 2.00 or higher.
[10-1] (a) is a step of irradiating the protein solution with radiation so that the removal coefficient for the virus having a virus particle diameter of 30 nm or less in the protein solution is 1.00 or more [10]. ] The method described in.
[11] The method according to [1] or [1-1], wherein the LRV in (a) is 2.00 or more.
[12] The method according to [1] or [1-1], wherein the LRV in (a) is 4.00 or more.
[13] The method according to [1], [1-1] or [12], wherein the LRV in (b2) is 4.00 or more.
[14] The method according to [10] or [10-1], wherein the LRV in (a) is 2.00 or more.
[15] The method according to [10] or [10-1], wherein the LRV in (a) is 4.00 or more.
[16] The method according to [10], [10-1] or [15], wherein the LRV in (b2) is 4.00 or more.

According to the present invention, a virus in a protein solution comprises a step of integrating virus inactivation by irradiation treatment and virus removal by virus removal membrane treatment or virus inactivation by SD treatment to optimize those conditions. It is possible to provide a removal method and a method for producing a protein solution from which a virus has been removed, which comprises the step.
In addition, in this step, viruses with a particle size of 30 nm or less (eg, parvovirus, circoviridae) are effectively inactivated by radiation (eg, ultraviolet rays, γ-rays), and therefore have a pore size of 15 nm or more. Even with a virus-removing membrane, extremely high LRV can be synergistically achieved. In another embodiment, in the step, viruses with a particle size of less than 33 nm (eg, parvovir, circoviridae) are effectively inactivated by radiation (eg, ultraviolet, γ-ray), resulting in a pore size of 13 nm or more. It is also possible to synergistically achieve extremely high LRV by using a virus-removing membrane having. Further, in the virus inactivation / removal step, a pore diameter larger than that of a virus removing membrane generally used in the past can be adopted, so that the yield of a desired protein can be further increased. Moreover, in the irradiation treatment used in the virus inactivation / removal step, it is not necessary to add a drug other than a buffer solution or a stabilizer, and a step such as removal of the added drug is performed in a subsequent step of the step. There is no need to add it, which can contribute to the simplification of the manufacturing process.

Figure 1 shows IgG denaturation (polymer) when a 2.5% (25 mg / ml) IgG solution (0.1 M glycine, pH 3.0 to 6.8) was irradiated with ultraviolet C waves (UVC) (0 to 1,024 mJ / cm ² ). It is a figure which shows the relationship of the (formation) rate. Figure 2 shows the relationship between the IgG denaturation (polymer formation) rates when a 5% (50 mg / ml) IgG solution (5% sorbitol, pH 4.1) was irradiated with UVC (0 to 1,024 mJ / cm ² ). It is a figure which shows. FIG. 3 is a diagram showing the relationship of inactivation when B19 in PBS is irradiated with UVC (0 to 512 mJ / cm ² ). Figure 4 shows the inactivation relationship when UVC is applied to MVM in 2.5% (25 mg / ml) IgG solution (0.1 M glycine, pH 4.1) or PBS (0 to 512 mJ / cm ² ). Is. Figure 5 shows Bovine viral diarrheavirus (hereinafter abbreviated as "BVDV" or "BVD") in 2.5% (25 mg / ml) IgG solution (0.1 M glycine, pH 4.1) or PBS irradiated with UVC (0). It is a figure which shows the relationship of inactivation at the time of ~ 512 mJ / cm ² ). Figure 6 shows the concentration of fibrinogen (hereinafter abbreviated as "Fib") (including 0.5% sodium chloride and 1.6% sodium citrate as stabilizers) by UVC irradiation (0 to 768 mJ / cm ² ) at 3.9 mg / It is a figure which shows the relationship between the coagulation activity and the protein denaturation rate at the time of ml (E ₂₈₀ : 5.8, E ^1% ₂₈₀ : 15-16). Figure 7 shows B19 in a solution with a Fib concentration of 3.3 to 13.3 mg / ml (E ₂₈₀ : 5 to 20, absorption coefficient (E ^1% ₂₈₀ : 15 to 16)) irradiated with UVC (0 to 384 mJ /). It is a figure which shows the relationship of B19 inactivation at the time of cm ² ). Figure 8 shows B19 in a solution or PBS with a Fib concentration of 0.004 to 3.6 mg / ml (E ₂₈₀ : 0.0054 to 5.4, extinction coefficient (E ^1% ₂₈₀ : 15 to 16)) irradiated with UVC (0 to 384). It is a figure which shows the relationship of B19 inactivation at the time of mJ / cm ² ). Figure 9 shows UVC irradiation (0 to 384) of PPV in a solution or PBS with a Fib concentration of 0.04 to 3.6 mg / ml (E ₂₈₀ : 0.054 to 5.4, extinction coefficient (E ^1% ₂₈₀ : 15 to 16)). It is a figure which shows the relationship of PPV inactivation at the time of mJ / cm ² ). Figure 10 shows trombin (hereinafter abbreviated as “Thr”) by UVC irradiation (0 to 768 mJ / cm ² ) (including 0.34% sodium chloride, 0.23% sodium citrate, and 0.27% calcium chloride as stabilizers). It is a figure which shows the relationship between the coagulation activity and the protein denaturation rate when the concentration is 2.48 mg / ml (E ₂₈₀ : 5.3, absorption coefficient (E ^1% ₂₈₀ : 21.4)). Figure 11 shows B19 in a solution with a Thr concentration of 0.47 to 4.7 mg / ml (E ₂₈₀ : 1 to 10, absorption coefficient (E ^1% ₂₈₀ : 21.4)) irradiated with UVC (0 to 384 mJ / cm ² ). It is a figure which shows the relationship of B19 inactivation at the time of). Figure 12 shows PPV in a solution with a Thr concentration of 0.47 to 4.7 mg / ml (E ₂₈₀ : 1 to 10, extinction coefficient (E ^1% ₂₈₀ : 21.4)) irradiated with UVC (0 to 384 mJ / cm ² ). It is a figure which shows the relationship of PPV inactivation at the time of). FIG. 13 is a diagram showing the relationship of protein denaturation when Fib (containing sodium citrate and L-alginate salt as stabilizers / excipients) is freeze-dried and irradiated with gamma rays. FIG. 14 is a diagram showing the relationship of protein denaturation when Thr (including D-mannitol, sodium citrate, and L-alginate salt as stabilizers / excipients) is freeze-dried and irradiated with gamma rays. .. In FIG. 15, albumin (hereinafter abbreviated as “Alb”) (including a) sodium citrate, sodium chloride, and b) D-mannitol as stabilizers / excipients was freeze-dried and irradiated with gamma rays. It is a figure which shows the relationship of the protein denaturation at the time. FIG. 16 is a diagram showing the relationship of B19 inactivation when the above Fib, Thr, Alb (a), and Alb (b) including B19 are freeze-dried and irradiated with gamma rays. FIG. 17 is a diagram for estimating the UVC irradiation dose (mJ / cm ² ) from the absorbance (E ₃₅₂ ) of 1% sodium iodide (NaI).

1. 1. Method for removing virus in protein solution of the present invention The method of the present invention is a method for inactivating / removing a virus in a protein solution, and the following:
(A) Irradiate the protein solution so that the LRV for viruses with a virus particle size of 30 nm or less in the protein solution is 1.00 or more, preferably 2.00 or more, more preferably 3.00 or more, and even more preferably 4.00 or more. The LRV for viruses with a virus particle size of less than 33 nm in the protein solution is 1.00 or more, preferably 2.00 or more, more preferably 3.00 or more, and further by irradiating the protein solution with radiation. A step of treating to preferably 4.00 or more, and (b1) a step of filtering the treated protein solution with a virus-removing membrane having an LRV of 4.00 or more for Bacterophage PP7, or (b2) the treated. It is a method comprising contacting a protein solution with an SD mixture and performing SD treatment so that the LRV against enveloped virus in the protein solution is 2.00 or more.

In the present specification, inactivation / removal of a virus in a protein solution means not only making the virus absent in the protein solution, but also causing a health hazard due to the amount of virus present in the protein solution. It includes not keeping the amount below a certain level and making the infectious virus present in the protein non-infectious (so-called virus inactivation).

In the present specification, the protein solution contains a protein that passes through the membrane when filtered through a virus-removing membrane having an LRV of 4.00 or more for bacteriophage PP7, and is particularly a solution that may contain a virus. Not limited. In particular, since a solution made from an animal-derived component including humans or a gene has a high possibility of containing a virus, a protein solution from which the virus has been removed can be used as a protein solution to be used in the virus removing method of the present invention. It can be provided efficiently.

Protein solutions that may contain viruses include, for example, peptides, proteins, and animal-derived components produced using biotechnology such as genetic engineering and cell culture, which are raw materials for biopharmacy, as active ingredients. Examples include solutions. Further, a medium containing an animal-derived component to be used for cell culture and an enzyme such as trypsin derived from an animal can also be mentioned.

In addition, examples of the protein solution that may contain a virus include body fluids of humans and animals. Specifically, for example, blood, plasma, serum, saliva, sweat, urine, lymph, enzyme, tissue fluid, etc., or a solution obtained by purifying these tissues, body fluids, etc. as raw materials may be used. It may be a solution containing the above. Further, for example, a raw material for a plasma fractionation product obtained by purifying from plasma can also be mentioned. Examples of the plasma fractionation product include immunoglobulin preparations, albumin preparations, blood coagulation factor preparations, and the like. In particular, blood coagulation factor preparations include blood coagulation factor VIII preparations, blood coagulation factor IX preparations, fibrinogen preparations, and anticoagulation factors. Examples thereof include a thrombin III preparation.
The protein solution to be used in the virus removal method of the present invention may be, for example, prepared at the time of use, thawed in cryopreserved state, or lyophilized in a desired solution again. But it may be.

Specific proteins include, for example, antibodies (eg, polyclonal antibodies, monoclonal antibodies (eg IgG, IgM, IgA, IgD, IgE), etc.), hematopoietic factors (erythropoetin, thrombopoetin, etc.), proteins involved in blood coagulation (line). Hydrolytic factors (tissue-type 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 protein (albumin, globulin, etc.), hormone (gonad stimulating hormone, thyroid stimulating hormone, etc.), growth factor (epithelial growth factor (EGF), hepatocellular proliferation) Factors (HGF), keratinocyte growth factors, activins, bone-forming factors, etc.), stem cell factors (SCF) (G-CSF, M-CSF, etc.), cytokines (interferon α, interferon β, interferon γ, interleukin 2, interleukin, etc.) 4, Interleukin 5, Interleukin 6, Interleukin 10, Interleukin 11, Soluble interleukin 4 receptor, Tumor necrosis factor α, etc.), Nucleic acid degrading enzyme (Dnasel, etc.), Enzyme (galactosidase, α-glucosidase, Glucocerebero) Examples thereof include proteins such as cedase), hemoglobin, and transferase, and partial fragments of these proteins, unstable proteins having biological activity, and the like. Among them, polyclonal antibodies, monoclonal antibodies, proteins involved in blood coagulation, plasma proteins, and unstable biological activities are exhibited from the viewpoint of achieving both viral inactivation or removal and production conditions that can maintain protein quality and activity and economic efficiency. Proteins and the like are preferable, and IgG, IgM, IgA, IgD, IgE, thrombin, fibrinogen, antithrompine, albumin and the like are more preferable.

Further, in the present specification, the protein contained in the protein solution is a protein as exemplified above or a fragment of the protein thereof, and is a radioactive isotope, a low molecular weight drug, a high molecular weight drug, or a different protein. It may be a protein in which a partial fragment of the protein or the like is chemically or genetically engineered, or a partial fragment of the protein.

Examples of the above-mentioned antibody include humanized antibodies such as human antibodies, human chimeric antibodies and human complementarity determining region transplanted antibodies, and antibody fragments thereof. An antibody is composed of a variable region (V region) and a constant region (C region). The heavy chain variable region is called VH, the light chain variable region is called VL, the heavy chain constant region is called CH, and the light chain constant region is called CL.

The human chimeric antibody refers to an antibody consisting of VH and VL of non-human animal antibodies and CH and CL of human antibodies. The human chimeric transplant antibody first designs and constructs a cDNA encoding VH and VL of a non-human mammalian antibody. Next, each of the above cDNAs is inserted into an expression vector for animal cells having a cDNA encoding CH and CL of a human antibody to construct a human chimeric transplanted antibody expression vector. The above antibody can be expressed and produced by introducing the constructed vector into animal cells.
The CH of the human chimeric antibody is not particularly limited as long as it belongs to human immunoglobulin (hereinafter, may be abbreviated as "hIg"), but hIgG class is preferable, and hIgG1 belonging to hIgG class, Any of the subclasses such as hIgG2, hIgG3 and hIgG4 can be used. The CL of the human chimeric antibody is not particularly limited as long as it belongs to hIg, and κ class or λ class can be used. Mammals other than humans are, for example, mice, rats, hamsters, rabbits and the like.

Examples of the antibody fragment include peptides containing Fab, F (ab') ₂ , Fab', scFv, diabody, dsFv and CDR. Fab is a fragment obtained by treating an IgG antibody molecule with the proteolytic enzyme papain (cleaved at the 224th amino acid residue of the H chain), and about half of the N-terminal side of the H chain and the entire L chain are It is an antibody fragment having an antigen-binding activity with a molecular weight of about 50,000 bound by a disulfide bond. Fab treats an antibody with the proteolytic enzyme papain or inserts the Fab-encoding DNA of the antibody into a prokaryotic or eukaryotic expression vector and inserts the vector into a prokaryotic or eukaryotic expression vector. It can be manufactured by introducing it into.

The protein concentration in the protein solution used for the virus removing method of the present invention is a concentration that can be filtered by a virus removing membrane having an LRV of 4.00 or more with respect to bacteriophage PP7, preferably a virus removing membrane having a pore size of 15 nm to 20 nm. In addition, the LRV for a virus having a virus particle diameter of 30 nm or less is 1.00 or more, preferably 2.00 or more, more preferably 3.00 or more, and further, within the range of the predetermined radiation irradiation amount used in the virus removal method of the present invention described later. Desirably, the concentration is not particularly limited as long as it can achieve 4.00 or more.
In another embodiment, the protein concentration in the protein solution used in the virus removal method of the present invention is filtered by a virus removal membrane having an LRV of 4.00 or more against bacteriophage PP7, preferably a virus removal membrane having a pore size of 13 nm to 33 nm. The LRV for viruses with a virus particle size of less than 33 nm is 1.00 or more, preferably 2.00 or more, more preferably within the range of the predetermined radiation dose used in the virus removal method of the present invention, which is a possible concentration and will be described later. Is not particularly limited as long as the concentration can achieve 3.00 or higher, more preferably 4.00 or higher.
Specific protein concentrations (including the upper limit) 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, 0.5 w / v% or less. The specific protein concentration (including the lower limit) is, 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, 0.3 w / v. % Or more.

The virus that can be contained in the protein solution is not particularly limited, but is B19, MVM, PPV, bovine parvovirus (BPV), canine parvovirus (hereinafter abbreviated as "CPV"), poliovirus, PCV, HAV, E type. Hepatitis virus (hereinafter abbreviated as "HEV") and the like can be mentioned.

Among the viruses exemplified above, parvovirus has been reported in the past to be infected with B19, which is one of the parvoviruses, in the field of plasma fractionation preparations, and the guideline on the virus safety of plasma-derived preparations is EMEA. (European Pharmaceutical Examination Agency, EMA / CHMP / BWP / 706271/2010). Furthermore, in the field of biopharmacy, there is an example of contamination of the biopharmacy manufacturing process due to contamination of rodent-derived MVM into CHO cells (derived from hamster), and the virus safety of biopharmacy made using animal cells. Guidelines for sexual evaluation (ICH Topic Q5A) have been issued.

Among the viruses exemplified above, PCV is the smallest non-enveloped virus currently known, and its particle size is about 15 to 25 nm (ICTV, https://talk.ictvonline.org). / ictv-reports / ictv_online_report / ssdna-viruses / w / circoviridae). PCV belongs to the circoviridae family (FamilyCircoviridae) and is classified into PCV1, PCV2, etc. PCV is also one of the viruses with a high risk of being contaminated in the manufacturing process of biopharmacy and the like, and cases of contaminated with it have been reported. PCV is resistant to low pH treatment and heat treatment, and it is difficult to capture and remove PCV even by using a virus removing membrane having an average pore size of 15 nm. In the virus inactivation / removal method of the present invention, as will be described later, parvovirus having a particle size larger than that of PCV is effectively inactivated (eg, LRV ≧ 1.0, LRV ≧ 2.0, LRV ≧ 4.0, LRV ≧ LRV ≧). Since the irradiation amount of radiation that can be 6.0 etc.) is used, circoviruses having a particle size or smaller can be inactivated in the same manner.

Furthermore, among the viruses exemplified above, viruses such as HAV (27 to 30 nm), poliovirus (30 nm), HEV (32 nm) of the picornavirus family, and viruses having a larger particle size than these, etc. However, there may be some viruses that are not partially inactivated by the amount of radiation used in the virus inactivation method of the present invention, but the LRV for the bacteriophage PP7 used in the virus removal method of the present invention is 4.00 or higher. It can be effectively filtered by a virus-removing membrane.

In addition to the above proteins and viruses, the protein solution that may contain the virus used in the virus removal method of the present invention is selected from the group consisting of amino acids, inorganic salts, buffer components, surfactants and saccharides. It may contain one or more components. The above components may be added to a protein solution containing a virus before the step (a) or (a') included in the virus removing method of the present invention, and the step (a) or (a') may be added. And may be added during step (b1) or (b2).

Among the amino acids, examples of the basic amino acid include arginine, histidine, guanidine, lysine or a derivative thereof, or a salt thereof, and preferably arginine, histidine, lysine or a derivative thereof, or a salt thereof. More preferably, it is arginine or a derivative thereof, or a salt thereof.
Examples of the neutral amino acid include glycine or a derivative thereof, or a salt thereof.
Examples of acidic amino acids include aspartic acid or derivatives thereof, or salts thereof.

The inorganic salt may contain NaCl, CaCl ₂ , a buffer salt and the like.
As the buffer solution component, acetate buffer solution, citrate buffer solution, phosphate buffer solution, phosphate buffered saline (PBS), Tris-HCl buffer solution, glycine buffer solution and the like may be used. The concentrations of the inorganic salt and the buffer solution component may be appropriately determined by a method known per se.

Examples of the surfactant include nonionic surfactants such as Tween20 (registered trademark), Tween80 (registered trademark), Triton X100 (registered trademark), NP-40 (registered trademark), and Pluronic F-127 (registered trademark). Is exemplified, and may be contained in a protein solution at a concentration of 0.01 to 5 wt%.

Examples of saccharides include, but are not limited to, monosaccharides, disaccharides, trisaccharides, oligosaccharides, polysaccharides, sugar alcohols and the like, and specific examples thereof include glucose, mannose, galactose, fructose, sorbitol, maltose and sucrose ( Sucrose), sorbitol, mannitol, dextran, alginic acid and a plurality of kinds thereof may be combined and contained in a protein solution in an amount of 1 to 10 wt%, preferably 1 to 5 wt%.

The temperature of the protein solution that may contain the virus is, for example, in the range of 1 ° C to 40 ° C, preferably 4 ° C to 35 ° C throughout the entire steps of the virus removal method of the present invention from the viewpoint of preventing protein denaturation. May be. The temperature affects the viscosity of the protein solution and the Flux during filtration using a virus-removing membrane, which will be described later. Therefore, it depends on the stability of the protein itself with respect to the temperature, but it is in the range of 20 ° C to 35 ° C. May be.

The pH of the protein solution that may contain the virus is not particularly limited as long as the protein contained in the solution does not cause denaturation, but is, for example, pH 3.0 to pH 8.0. More specifically, when the protein contained in the solution is polyclonal IgG, pH 4.1 to pH 7.2 is preferable, and in the case of monoclonal IgG, the optimum pH varies depending on the PI value of the IgG molecule.

The radiation used in the step (a) or (a') included in the virus removing method of the present invention includes both ionizing radiation and non-ionizing radiation, and specifically, for example, ultraviolet rays, α rays, β rays, and the like. Examples include γ-rays, X-rays, electron beams, and neutrons. Radiation can be obtained from radioactive isotopes such as cobalt-60, strontium-90, and cesium-137, or by an X-ray machine, an electron beam accelerator, an ultraviolet (continuous) irradiation device, etc., and the device is a commercially available product. May be.

Among the above specific examples, ultraviolet rays include UVC (100 to 280 nm), UVB (280 to 320 nm) and UVA (320 to 400 nm). In the virus removing method of the present invention, UVC irradiation is preferable, UVC irradiation in the range of 250 to 280 nm is more preferable, and UVC irradiation in the vicinity of 254 nm is more preferable. A low-pressure mercury lamp, LED, or the like may be used as the ultraviolet irradiation source, and the irradiation source is not limited.

The method of irradiating radiation in the virus removing method of the present invention is not particularly limited as long as it can be treated so that the LRV for a virus having a virus particle size of 30 nm or less in the protein solution to be irradiated is 1.00 or more. Further, if the LRV is 1.00 or more, preferably 2.00 or more, more preferably 3.00 or more, and further preferably 4.00 or more, it can be said that the virus is effectively removed, but if desired, for example, 5.00 or more, It may be processed so that LRV of 6.00 or higher can be achieved.
Further, the irradiation method of radiation in the virus removing method of the present invention according to another aspect is not particularly limited as long as it can be treated so that the LRV for a virus having a virus particle size of less than 33 nm in the protein solution to be irradiated is 1.00 or more. Further, if the LRV is 1.00 or more, preferably 2.00 or more, more preferably 3.00 or more, and further preferably 4.00 or more, it can be said that the virus is effectively removed, but if desired, for example, 5.00 or more, It may be processed so that LRV of 6.00 or higher can be achieved.
Specifically, for example, when the irradiated radiation is ultraviolet light, it has a cylindrical rotating cylinder in which the protein solution is thinly and uniformly distributed, and the rotation is about the rotation center axis of the cylinder. An irradiation method using a device such as the above can be mentioned. The principle of the device is to adjust the flow velocity of the protein solution using a pump so that the film thickness of the solution forms a thin film (for example, 0.1 mm) for which UVC irradiation is effective on the inner wall of the rotating cylinder. Then, it diffuses uniformly to the inner wall, and the low-pressure mercury lamp located in the center of the cylinder irradiates the inner wall of the cylinder with ultraviolet rays. The amount of ultraviolet rays irradiated to the protein in the protein solution is increased or decreased depending on the time that the solution passes through the inner wall of the cylinder, and the passage time is also changed by changing the angle of the cylinder, so that the irradiation amount can be changed to a desired amount. can. There is also a method in which a coiled tube made of a resin or quartz glass that transmits ultraviolet rays is placed around an ultraviolet ray source such as a low-pressure mercury lamp or a deep ultraviolet LED, and a protein solution is passed by a pump. In this method, the protein solution is agitated as it passes through the coiled tube, so that the irradiation is uniformly performed. In addition, there is also a method in which the ultraviolet source is arranged in a plane, and the protein solution is passed by a pump between two flat plates shaped with a resin or quartz glass that transmits ultraviolet rays on the irradiation surface to irradiate the thin film layer. be. The flat plate may be cut with irregularities or grooves patterned so that the flow velocity becomes uniform. Then, by changing the flow velocity, the transit time also changes, and the irradiation amount can be changed to a desired amount.

The amount of radiation used in the virus removal method of the present invention is such that the LRV for a virus having a virus particle size of 30 nm or less in the protein solution used for the irradiation is 1.00 or more, preferably 2.00 or more, depending on the type of radiation used. It may be set so that it can be processed so that it is more preferably 3.00 or more, and even more preferably 4.00 or more.
In another aspect, the irradiation amount of the radiation used in the virus removing method of the present invention has an LRV of 1.00 or more for a virus having a virus particle size of less than 33 nm in the protein solution used for the irradiation, depending on the type of radiation used. , Desirably 2.00 or more, more preferably 3.00 or more, and even more preferably 4.00 or more.
It can be said that the virus is effectively removed if the LRV is 4.00 or higher, but if desired, it may be processed so as to achieve an LRV of 5.00 or higher and 6.00 or higher, for example. The LRV can be obtained by the formula described later.

For example, when UVC is used, the irradiation amount is specifically 256 mJ / cm ² or less, 192 mJ / cm ² or less, and 96 mJ / cm ² or less. Specifically, it is 32 mJ / cm ² or more, 64 mJ / cm ² or more, and 128 mJ / cm ² or more. In addition, the irradiation amount is a combination of the above ranges (example: 32 mJ / cm ^2-256 mJ / cm ² , 64 mJ / cm ^2-256 mJ / cm ² , 128 mJ / cm ^2-256 mJ / cm ² , It may be 32 mJ / cm ² to 192 mJ / cm ² , 64 mJ / cm ² to 192 mJ / cm ² , 128 mJ / cm ² to 192 mJ / cm ² , etc.).
More specifically, for a protein solution (pH 3.5 to 6.8) containing an IgG antibody of 10% w / v (100 mg / ml) or less, the formation rate of the IgG antibody polymer after UVC irradiation is 2% or less. When set, the maximum UVC dose is preferably 256 mJ / cm ² to 1024 mJ / cm ² , more preferably 512 mJ / cm ² .
More specifically, the maximum when a protein solution containing a Fib concentration of 3.9 mg / ml (E ₂₈₀ : 5.8, E ^1% ₂₈₀ : 15-16) is set on the condition that the coagulation activity is maintained at 90% or more. The irradiation dose is preferably 96 mJ / cm ² to 192 mJ / cm ² , and more preferably 192 mJ / cm ² .
More specifically, for a protein solution containing a Thr concentration of 2.48 mg / ml (E ₂₈₀ : 5.3, E ^1% ₂₈₀ : 21.4), the maximum irradiation dose is set on the condition that the coagulation activity is maintained at 90% or more. Is preferably 96 mJ / cm ² to 192 mJ / cm ² , more preferably 192 mJ / cm ² .

Further, for example, when using γ-rays, the irradiation amount 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, 60 kGy or more. Specifically, the irradiation amount of γ-rays is 5 kGy to 50 kGy, 10 kGy to 50 kGy, and 25 kGy to 50 kGy.

The irradiation amount of radiation in the virus removing method of the present invention may be appropriately calibrated and validated according to the type of radiation to be irradiated in order to obtain an accurate measured value.
Specifically, for example, when UVC is used, the indicated value may have an error depending on the type of the ultraviolet illuminance meter used for measuring UVC. Therefore, for example, by applying the principle of the estimation method of UVC irradiation amount using the change in the absorbance of existing NaI as an index, a calibration curve related to the UVC irradiation amount is created, and the calibration curve is used between measuring instruments. Calibration may be performed. Since the calibration curve differs depending on the treatment conditions, the absorbance (A352) is measured under experimental conditions that reflect the actual treatment conditions (particularly the total energy amount and film thickness), and the calibration curve and formula for obtaining the irradiation dose are used. demand. The formula for estimating the UVC irradiation dose using the absorbance of 1% NaI obtained in the experiment of FIG. 17 is as follows.
UVC irradiation amount (energy amount) (mJ / cm ² ) = 10 ^{(2 x Log} ₁₀ ^{[A352 nm] +2.6)}

Calibration of the UVC irradiation amount using the calibration curve does not obtain the absolute value of the UVC irradiation amount, but if a specific reference measuring device is set, the calibration for the measuring device becomes possible. In addition, since the UVC irradiation dose can be standardized using any measuring device by the calibration, the LRV for viruses with a virus particle size of 30 nm or less in protein solutions that can be used at the industrial level is 1.00 or more, preferably 1.00 or more. It can define a range of UVC doses that can be treated to be 2.00 or higher, more preferably 3.00 or higher, and even more preferably 4.00 or higher. Similarly, the range of UVC irradiation that can be treated so that the LRV for viruses with a virus particle size of less than 33 nm in the protein solution is 1.00 or more, preferably 2.00 or more, more preferably 3.00 or more, and even more preferably 4.00 or more. Can be specified. Therefore, the above-mentioned UVC irradiation amount is a value having a critical significance even when any measuring device is used by using the calibration.

The virus removing membrane used in the step (b1) included in the virus removing method of the present invention is not particularly limited as long as it is a virus removing membrane having an LRV of 4.00 or more with respect to bacteriophage PP7. By filtering with the virus removing membrane, the virus having a virus particle diameter of more than 30 nm in the protein solution can be effectively removed. Similarly, by filtering with the virus removing membrane, the virus having a virus particle diameter of 33 nm or more in the protein solution can be effectively removed.
A virus-removing membrane having an LRV of 4.00 or more for bacteriophage PP7 is a membrane having an LRV of 4.00 or more when a solution containing bacteriophage PP7 is loaded at 50 l / m ² on the virus-removing membrane to be used. As the solution containing bacteriophage PP7, a 1 mg / ml BSA solution (PBS buffer, pH 7.4) with a bacteriophage PP7 concentration of 10 ⁷ pfu / ml is used.

The shape of the virus removing membrane to be used is not particularly limited, but may be, for example, a flat membrane or a hollow fiber membrane. When a flat film is used, filtration may be performed with one sheet, or a plurality of sheets may be stacked for filtration.

By filtering a pH-adjusted solution through a virus-removing membrane with an LRV of 4.00 or higher for bacteriophage PP7, the pH-adjusted solution is a virus-removing membrane with an LRV of 4.00 or higher for bacteriophage PP7. Is processed by. The filtration method may be the Dead end method or the Cross flow method. Before treatment with a virus-removing membrane having an LRV of 4 or more for bacteriophage PP7, it may be preliminarily treated with a membrane having a pore size larger than 20 nm.

The pore size of the virus removal membrane having an LRV of 4.00 or more for bacteriophage PP7 is not limited as long as it can remove viruses having 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. It is as follows. The pore diameter 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, the pore sizes of the virus removal membrane are 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, and 13 nm to 21. Examples include 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.

Specific examples of virus-removing membranes having an LRV of 4.00 or higher against bacterial offer PP7 are Planova (registered trademark) 15N (manufactured by Asahi Kasei Medical) and Planova (registered trademark) 20N (Asahi Kasei Medical) made of regenerated cellulose. (Manufactured by Asahi Kasei Medical), Planova (registered trademark) BioEX (manufactured by Asahi Kasei Medical) consisting of hydrophilized PVDF, Pegasus SV4 (manufactured by Pall), and Virosart CPV consisting of hydrophilized PES (manufactured by Asahi Kasei Medical). Sartorius) and Viresolve Pro (Millipore) and the like.

In the case of a virus-removing membrane made of regenerated cellulose as a virus-removing membrane having an LRV of 4.00 or more for bacteriophage PP7, a preferable membrane can be defined by the average pore size of the pores of the virus-removing membrane. The average pore diameter of the virus-removing membrane is, for example, 15 nm (eg, Planova® 15N; 15 ± 2nm), 19nm (eg, Planova® 20N; 19 ± 2nm), 20 nm (eg Pegasus® SV4).

The average pore diameter of the pores can be calculated by the following formula with reference to the method described in International Publication No. 2015/156401.
Average pore size (nm) = 2 × 10 ³ × √ (V ・ d ・ μ / P ・ A ・ Pr)
Here, V is the permeability (ml / min), d is the film thickness (μm), μ is the viscosity of water (cp), P is the pressure difference (mmHg), A is the membrane area (cm ² ), and Pr is empty. Indicates the porosity (%).
For the water permeability, bundle 10 threads, make a module so that it has an effective length of 16 cm, close one end of the obtained module, apply a pressure of 200 mmHg to the other end, and let water pass at 37 ° C. .. At this time, the amount of water that comes out through the membrane is measured as the amount of water permeation.
The film area is calculated by measuring the inner diameter in a dry state. Further, the film thickness means the film thickness in a dry state.
The porosity is calculated by the following formula.
Porosity (%) = (1-ρa / ρp) x 100
The apparent density ρa of the hollow fiber is calculated by the following formula, and ρp means the density of cellulose (g / cm ³ ).
Apparent density of hollow fibers (g / cm ³ ) = Wd / Vw = 4Wd / πl (Do ² -Di ² )
Here, Wd means the absolute dry weight of the hollow thread (g), Vw means the apparent volume of the hollow thread (cm ³ ), l means the length of the hollow thread (cm), and Do means the outer diameter of the hollow thread (cm). ), Di means the inner diameter (cm) of the hollow thread.

In the present specification, LRV means a virus removal coefficient (Log Reduction Value), is a logarithmic reduction degree of a virus, and is also referred to as a removal coefficient. LRV is calculated by the following formula.
LRV = Log [(V ₁ x T ₁ ))] / [(V ₂ x T ₂ ))]
V ₁ : Volume of sample before virus removal treatment process T ₁ : Virus amount (titer) before virus removal treatment process
V ₂ : Volume of sample after virus removal treatment process T ₂ : Virus amount (titer) after virus removal treatment process

The viral load of bacteriophage PP7 is not particularly limited, but can be measured by a plaque assay method or the like. The plaque assay can be performed according to the method described in the Journal of Pharmaceutical Science and Technology 2008; Supplement Volume 62 No. S-4. Solution samples containing bacteriophage PP7 are serially diluted, each sample is mixed with Pseudomonas aeruginosa, and then soft agar is added. Pour the mixed solution onto an agar plate, allow it to solidify, and then incubate at 37 ° C for 1 day. The next day, the number of plaques is measured with the naked eye, and the phage concentration pfu (plaque forming unit) / ml contained in the original solution is calculated by the following formula.
(Number of plaques x dilution ratio) pfu / sample amount (ml) pfu / ml

The filtration by the virus removing membrane in the step (b1) of the virus removing method of the present invention can be confirmed by LRV as an index for removing viruses (eg, B19, MVM, PPV, etc.) having a virus particle diameter of 30 nm or less.

For LRV for viruses with a virus particle diameter of 30 nm or less (eg, B19, MVM, PPV, etc.), the viral load of viruses with a virus particle diameter of 30 nm or less (eg, B19, MVM, PPV, etc.) is measured in the above formula. It can be obtained by. The amount of virus (eg, B19, MVM, PPV, etc.) having a virus particle diameter of 30 nm or less is not particularly limited, but can be measured by the infectious titer or the amount of viral nucleic acid. The infectious titer is obtained by inoculating the indicator cells with the virus and then culturing for a certain period of time to measure the degeneration of the cells after culturing and the viral antigens and nucleic acids (TCID ₅₀ method, plaque method, etc.). The Quantitative polymerase chain reaction (Q-PCR) for measuring the amount of nucleic acid can be carried out by a method known per se. When the LRV of a virus with a virus particle diameter of 30 nm or less (eg, B19, MVM, PPV, etc.) is measured, it can be judged that the virus with a virus particle diameter of 30 nm or less was effectively removed when the virus particle diameter was 4.00 or more. .. Generally, in the performance evaluation of virus removal membranes, if the LRV is 1.00 or more, it is limited, if it is 2.00 or more and 4.00 or less, it is moderate, and if it is 4.00 or more, the virus is effectively removed. If is 5.00 or more, the virus is removed to 1/5 or less of 10, and if the LRV is 6.00 or more, the virus is removed to 1/6 or less of 10 and most of the virus leaks. It is said that it is not.

The filtration by the virus removing membrane in the step (b1) of the virus removing method of the present invention can be confirmed by LRV as an index for removing viruses (eg, B19, MVM, PPV, etc.) having a virus particle size of less than 33 nm.

LRV for viruses with a virus particle size of less than 33 nm (eg, B19, MVM, PPV, etc.) measures the viral load of viruses with a virus particle size of less than 33 nm (eg, B19, MVM, PPV, etc.) in the above formula. It can be obtained by. The amount of virus having a virus particle size of less than 33 nm (eg, B19, MVM, PPV, etc.) is not particularly limited, but can be measured by the infectious titer or the amount of viral nucleic acid. The infectious titer is obtained by inoculating the indicator cells with the virus and then culturing for a certain period of time to measure the degeneration of the cells after culturing and the viral antigens and nucleic acids (TCID ₅₀ method, plaque method, etc.). The Quantitative polymerase chain reaction (Q-PCR) for measuring the amount of nucleic acid can be carried out by a method known per se. When the LRV of a virus with a virus particle size of less than 33 nm (eg, B19, MVM, PPV, etc.) is measured, it can be determined that the virus with a virus particle size of less than 33 nm was effectively removed when the virus particle size was 4.00 or more. ..

In the virus removal method of the present invention, the protein solution after the irradiation treatment according to the step (a) or (a'), which is applied to the virus removal membrane having an LRV of 4.00 or more for bacteriophage PP7, is per virus removal membrane area. The amount of the filtrate may be, for example, 50 to 100 l / m ² . By treating at 50 to 100 l / m ² , the solution can be efficiently filtered without causing clogging of the virus removing membrane with proteins or the like. The virus removal membrane area is calculated as the area of the filtration surface (primary side 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 inside (inner surface) of the hollow fiber. The amount of the filtrate can be adjusted by the filtration time in the filtration under a constant filtration pressure condition.

The filtration pressure in the step (b1) of the virus removing method of the present invention depends on the material of the virus removing membrane, but is within the range of the pressure resistance of the membrane or less. For example, in the case of a virus removing membrane made of regenerated cellulose, it may be carried out in the range of 0.00 kgf / cm ² (0.0 kPa) to 1.00 kgf / cm ² (9.8 × 10 kPa). In the case of a virus-removing membrane made of hydrophilized PVDF or hydrophilized PES, it may be carried out in the range of 0.00 kgf / cm ² (0.0 kPa) to 5.00 kgf / cm ² (4.9 × 10 ² kPa).

The SD treatment in the step (b2) included in the virus inactivating method of the present invention is not particularly limited as long as it can be treated so that the LRV against the enveloped virus in the protein solution used for the SD treatment is 2.00 or more. For LRV, it can be said that moderate inactivation is moderate if it is 2.00 or more, and virus is effectively inactivated if it is 4.00 or more, but if desired, for example, 3.00 or more, 4.00 or more, 5.00 or more, 6.00. It may be processed so that the above LRV can be achieved. Further, when the SD treatment is selected in the step (b2), the virus removing membrane treatment in the above-mentioned step (b1) may be separately performed in the subsequent steps.

The SD mixture can be any combination of organic solvent and surfactant known in the art of organic solvents and surfactants that can chemically inactivate enveloped viruses as long as the above LRV conditions can be achieved. There may be.
Examples of the organic solvent include dialkyl or trialkyl phosphate having an alkyl group having 1 to 10 carbon atoms, and among them, trialkyl phosphate having an alkyl group having 2 to 10 carbon atoms is preferable. Specifically, tri- (n-butyl) phosphate (hereinafter abbreviated as "TNBP"), tri- (t-butyl) phosphate, tri- (n-hexyl) phosphate, tri- (2-ethylhexyl) phosphate, Examples thereof include tri- (n-decyl) phosphate and ethyl-di (n-butyl) phosphate.

As a surfactant, one that can disperse fat by 0.1 w / w% in a solution of 0.01 g / ml at room temperature is usually used. Specific examples thereof include polyoxyethylene derivatives of fatty acids, polyoxyethylene sorbitan fatty acid esters, oxyethylated alkylphenols, polyoxyethylene alcohols, polyoxyethylene oils, polyoxyethylene oxypropylene fatty acids and the like. More specifically, for example, polyoxyethylene derivatives of fatty acids such as Tween (registered trademark) 80 and Tween (registered trademark) 20, partial esters of sorbitol anhydride such as polysorbate 80, and polyoxyethylene octylphenyl ether (Triton (Registered Trademark)). Oxyethylated alkylphenols such as registered trademark) X-100), sodium colalate, sodium deoxycholate, sulfobetaines such as N-dodecyl-N, N-dimethyl-2-ammonio-1-ethanesulfonate, octyl-β, Examples thereof include nonionic detergents such as D-glucopyranoside. Preferably, it is a nonionic oil-soluble aqueous detergent such as Tween® 80, Triton® X-100, sodium corate and the like.

The SD mixture may contain two or more organic solvents and / or surfactants independently. Further, the SD mixed solution may contain other additive components for promoting the effect, for example, a reducing agent, if necessary. The SD mixture preferably contains the above-mentioned organic solvent (S) and surfactant (D) in an amount such that the S / D (w / w ratio) is 1 to 20.

The SD treatment can be performed by contacting the protein solution and the SD mixture at a temperature of 0 to 40 ° C, preferably 4 to 25 ° C, more preferably 7 to 12 ° C. The contact usually shows its effect after a few minutes of contact, preferably 10 minutes or more and 2 hours or less, typically about 30 to 60 minutes. Although the effect cannot be expected to increase even if the treatment is performed for a time longer than 2 hours, the treatment may be performed for a longer period of time, for example, 6 hours or more in order to ensure the effect.

2. The method for producing a protein solution from which the virus has been removed according to the present invention The method for producing a protein solution from which the virus has been removed is the method for producing a protein solution from which the virus has been removed.
(A) Irradiate the protein solution so that the LRV for viruses with a virus particle size of 30 nm or less in the protein solution is 1.00 or more, preferably 2.00 or more, more preferably 3.00 or more, and even more preferably 4.00 or more. The LRV for viruses with a virus particle size of less than 33 nm in the protein solution is 1.00 or more, preferably 2.00 or more, more preferably 3.00 or more, and further by irradiating the protein solution with radiation. A step of treating to preferably 4.00 or more, and (b1) a step of filtering the treated protein solution with a virus-removing membrane having an LRV of 4.00 or more for Bacterophage PP7, or (b2) the treated. It is a method comprising contacting a protein solution with an SD mixture and performing SD treatment so that the LRV against enveloped virus in the protein solution is 2.00 or more.
With respect to the above steps (a) or (a') and steps (b1) and (b2), the contents described in the above "1. Method for removing virus in protein solution of the present invention" can be incorporated.

Before step (a) or (a') of the method for producing a protein solution from which the virus of the present invention has been removed, or between steps (a) or (a') and step (b1), in step (b1). Preliminary filtration may be performed with a filter having a pore size larger than that of the virus removing membrane used. Examples of the filter made of a film having a large pore size include Planova (registered trademark) 35N (manufactured by Asahi Kasei Medical Co., Ltd.), Planova (registered trademark) 75N (manufactured by Asahi Kasei Medical Co., Ltd.), 0.1 μm filter, 0.2 μm filter and the like. ..

Further, after the step (b1) of the method for producing a protein solution from which the virus of the present invention has been removed, the protein inside the membrane is washed out to the filtrate side by appropriately filtering the protein-free solution (washing solution). A cleaning step may be added.

Further, before step (a) or (a') of the method for producing a protein solution from which the virus of the present invention has been removed, between steps (a) or (a') and step (b1) or (b2), or After the step (b1) or (b2), any one or more of a chromatography treatment, a virus removing membrane treatment, an SD treatment, a concentration treatment and a concentration / buffer exchange treatment may be performed. For the virus removing membrane treatment, the content of the step (b1) described in "1. Method for removing virus in protein solution of the present invention" can be incorporated.

Examples of the chromatography treatment include column chromatography in which an ion exchange resin and a gel filtration resin are packed in a column, and membrane chromatography in which an ion exchange group is added to the surface of a porous membrane. The 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, hydroxyapatite. Chromatography and the like can be mentioned. Chromatography in which ion exchange and hydrophobic interaction are combined may be used as the ligand for chromatography. For the SD treatment, the content of the step (b2) described in "1. Method for removing virus in protein solution of the present invention" can be incorporated.

The concentration treatment may be carried out by a method using an ultrafiltration (UF) membrane according to a method known per se. Further, it may be carried out by centrifugal concentration.

The buffer solution exchange treatment may be carried out at the same time as concentration using an ultrafiltration membrane according to a method known per se. Further, it may be carried out by a gel filtration method or a dialysis method using a dialysis membrane.

After the steps (b1, b2) of the method for producing a protein solution from which the virus has been removed according to the present invention, the protein solution from which the virus has been removed may be purified, for example, by chromatographic treatment. The protein solution from which the virus of the present invention has been removed may be further concentrated by UF treatment.

In addition, the virus-removed protein solution obtained in step (b1) or (b2), a purified product thereof or a concentrate thereof may be formulated with the same liquid composition, or, for example, a solvent having another composition. And the buffer solution may be exchanged (may include removal of SD by a method known per se) and then formulated. Further, the protein solution from which the virus has been removed, a purified product thereof or a concentrate thereof may be freeze-dried and then formulated.

In the above-mentioned formulation, a protein solution from which the virus obtained by the production method of the present invention has been removed, or a lyophilized product thereof, which is pharmacologically acceptable, is one kind that is pharmacologically acceptable by a method well known in the technical field of pharmaceutical science. It can be mixed with the above carrier and produced as a pharmaceutical preparation or a pharmaceutical composition.

As the pharmacologically acceptable carrier, various conventional organic or inorganic carrier substances are used as the pharmaceutical material, and specific examples thereof include excipients, lubricants, binders, disintegrants, and liquids in solid formulations. Examples thereof include a solvent, a solubilizing agent, a suspending agent, an tonicity agent, a buffering agent, and a pain-relieving agent in the pharmaceutical product. When formulating, if necessary, pharmaceutical additives such as preservatives, antioxidants, colorants, and sweeteners may be used.

Excipients include lactose, sucrose, D-mannitol, D-sorbitol, starch, pregelatinized starch, dextrin, crystalline cellulose, low-substituted hydroxypropyl cellulose, sodium carboxymethyl cellulose, gum arabic, pullulan, soft anhydrous silicic acid, Examples thereof include synthetic aluminum silicate, magnesium aluminometasilicate, xylitol, sorbitol, and erythritol.
Examples of the lubricant include magnesium stearate, calcium stearate, talc, colloidal silica, polyethylene glycol 6000 and the like.

Binders include pregelatinized starch, sucrose, gelatin, gum arabic, methyl cellulose, carboxymethyl cellulose, sodium carboxymethyl cellulose, crystalline cellulose, sucrose, D-mannitol, trehalose, dextrin, pullulan, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyvinyl. Examples include pyrrolidone.

Examples of the disintegrant include lactose, sucrose, starch, carboxymethyl cellulose, carboxymethyl cellulose calcium, croscarmellose sodium, carboxymethyl starch sodium, low degree of substitution hydroxypropyl cellulose, soft anhydrous silicic acid, calcium carbonate and the like.

Examples of the solvent include water for injection, physiological saline, Ringer's solution, alcohol, propylene glycol, polyethylene glycol, sesame oil, corn oil, olive oil, cottonseed oil and the like.
Examples of the solubilizing agent include polyethylene glycol, propylene glycol, D-mannitol, trehalose, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate, sodium salicylate, sodium acetate and the like. ..

As the suspending agent, for example, a surfactant such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, glycerin monostearate; for example, polyvinyl alcohol, polyvinylpyrrolidone. , Hydrophilic polymers such as sodium carboxymethyl cellulose, methyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose; polysorbates, polyoxyethylene hydrogenated castor oil and the like.

Examples of the tonicity agent include sodium chloride, glycerin, D-mannitol, D-sorbitol, glucose, xylitol, fructose and the like.
Examples of the buffering agent include buffer solutions such as phosphates, acetates, carbonates and citrates.
Examples of the soothing agent include propylene glycol, lidocaine hydrochloride, benzyl alcohol and the like.

Examples of the preservative include paraoxybenzoic acid esters, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid and the like.
Examples of the antioxidant include sulfites, ascorbic acid salts and the like.
Coloring agents include water-soluble colored tar pigments (eg, edible pigments such as Edible Red Nos. 2 and 3, Edible Yellow Nos. 4 and 5, Edible Blue Nos. 1 and 2), and insoluble lake dyes (eg, said water-soluble). (Aluminum salt of food coloring), natural food coloring (eg β-carotene, chlorophyll, red iron oxide) and the like.
Examples of the sweetener include sodium saccharin, dipotassium glycyrrhizinate, aspartame, stevia and the like.

In addition, it is desirable to use the most effective route of administration for treatment, and it can be administered by oral preparation, injection or transdermal preparation. Examples of the oral preparation include tablets (including sublingual tablets and orally disintegrating agents), capsules (including soft capsules and microcapsules), powders, granules, troches, syrups, emulsions and suspensions. .. Examples of the injection include intradermal injection, subcutaneous injection, intravenous injection, intramuscular injection, intraspinal injection, epidural injection, and local injection. In addition, examples of the transdermal preparation include patches, ointments, and sprays. These formulations may be release controlled formulations such as immediate release formulations or sustained release formulations (eg, sustained release microcapsules).

The dose or frequency of administration varies depending on the target therapeutic effect, administration method, treatment period, age, body weight, etc., but the amount of active ingredient (protein) is usually 10 μg / kg to 5,000 mg / kg per day for adults. May be good.

Hereinafter, the present invention will be described by way of examples. However, the present invention is not limited to these examples.

Example 1: Examination of inactivation of virus in the presence of IgG by UVC irradiation The effect of UVC irradiation on IgG molecules was evaluated using an intravenous human immunoglobulin preparation (IVIG). UVC irradiation was performed while shaking with the sample (IVIG) added to the petri dish to form a thin film. The UVC irradiation amount (irradiation energy) was measured using UVR-300 (manufactured by Topcon Techno House) or the like.

When the above measurement was performed by irradiating 2.5% or 5% IgG in 0.1 M glycine buffer (pH 3.0 to 6.8) with UVC, the effect of the difference in IgG concentration on the formation of IgG polymer was slight. On the other hand, it was shown that a polymer of IgG molecule was formed according to the UVC irradiation dose, and the degree of polymer formation was different depending on the difference in pH. It is known that polyclonal IgG has high solubility at low pH and is most stable at around pH 4.2, but polymer formation was also low at around pH 3.5 to 5.5 even under UVC irradiation (Figs. 1 and 2). ). From the above examination, when the upper limit of polymer formation is 2%, UVC irradiation is a condition that can be irradiated up to 512 mJ / cm ² , and in polyclonal IgG, around pH 4.1 is a condition that further suppresses polymer formation. Was shown. Since the pH at which the solubility of monoclonal IgG becomes optimal changes depending on the PI value of the IgG molecule, the optimum conditions differ for each IgG molecule.

In the virus inactivation experiment, B19, MVM, and BVDV were added to PBS, and UVR-300 (manufactured by Topcon Techno House) was used to irradiate with different UVC irradiation doses, and the infectivity was changed by irradiation (inactivation). (Degree of) was evaluated (Figs. 3-5). The change in infectivity was evaluated by calculating the infectious titer by the TCID ₅₀ method and evaluating the change in the infectious titer of the sample before and after virus inactivation. The vertical axis of the figure shows the index value of the infectious titer. The LRV indicating the logarithmic reduction rate is calculated from the value obtained by subtracting the index value of the infectious value after inactivation from the index value of the infectious value before inactivation.
In addition, UVC irradiation was performed under IgG coexistence conditions in which MVM and BVDV were added to 0.1 M glycine buffer (pH 4.1) containing 2.5% IgG, and changes in infectivity (degree of inactivation) were evaluated (Fig. 4, Fig. 4, Five). Since the IgG used contained an anti-B19 antibody, B19 was not used in this experiment under IgG coexistence conditions. Table 1 summarizes the properties of the viruses used.

Under PBS conditions, MVM was infectious (inactivated) by UVC irradiation of 32 mJ / cm ² or higher, and virus LRV was> 3.8 and> 5.0, respectively. In B19, the viral LRVs at UVC irradiation of 64 mJ / cm ² or more were 7.8, 7.4, and 7.2, respectively, whereas BVDV showed no inactivating effect at the irradiation dose evaluated this time. If LRV is 4.00 or more, it is evaluated as effective virus inactivation / removal, but if it is less than 4.00, it is evaluated as moderate if it is 2.00 or more, if it is 1.00 or more, it is evaluated as limited, and if it is 1.00 or less. If it is evaluated as ineffective. In addition, in the coexistence of IgG, the infectivity of MVM disappeared by UVC irradiation of 256 mJ / cm ² or more (LRV:>3.8,> 4.6), but the virus inactivation curve at irradiation doses lower than that showed the presence of IgG. It was different when it did and when it did not exist. It was shown that although UVC irradiation inactivates these viruses, the coexistence of IgG stabilizes the viruses and increases the amount of irradiation required for inactivation, but exceeds the critical point at 256 mJ / cm ² . rice field. On the other hand, BVDV with larger particles and genome size did not have a significant change in infectivity (ie, not inactivated) even at an energy amount of 512 mJ / cm ² under both PBS and IgG coexistence conditions (Figs. 3-5). ..

From these results, it can be understood that the particle size (genome size) of the virus is important as a factor influencing virus inactivation by UVC irradiation. Other than the particle size of the virus, the type and concentration of the protein coexisting with the virus may be the factor, and the pH may be the factor. As an example, in the case of a solution containing IgG as a target protein, the optimum virus inactivation / removal conditions are virus removal membrane treatment with a pore size of 20 nm, IgG concentration of 10% or less, pH 4.1 to 5.5, and It was shown that the UVC irradiation dose was 256 mJ / cm ² , or the virus removal membrane treatment with a pore size of less than 33 nm, the IgG concentration was 10% or less, the pH was 4.1 to 5.5, and the UVC irradiation dose was 256 mJ / cm ² . ..

Example 2: Examination of inactivation of virus in coexistence with Fib 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 ) The effect of UVC irradiation on Fib molecules was evaluated using the solution of ^% ₂₈₀ : 15-16). In this experiment, the amount of UVC energy was measured using UVC-254 (manufactured by Custom Co., Ltd.), but the measuring instrument was about 1/3 lower than UVR-300 (manufactured by Topcon Techno House Co., Ltd.) used in Example 1. The values are shown (Table 2). Therefore, in this experiment, a correction value obtained by multiplying the measured value by 3 was used so as to be the same as the measured value with UVR-300 (manufactured by Topcon Techno House Co., Ltd.).

Since Fib is a protein with coagulation activity, its coagulation activity and polymer formation were evaluated. By UVC irradiation, the coagulation activity was maintained at almost 100% up to 192 mJ / cm ² , but the polymer increased according to the irradiation dose. The polymer was detected before irradiation, but the amount of polymer reached 20% after irradiation at 96 mJ / cm ² compared to before irradiation (Fig. 6). The polymer is not an important index for the purpose of forming a fibrin film before administration, and the coagulation activity is an important index in UVC irradiation.

Next, B19 or PPV was added to Fib solutions of various concentrations, and irradiation was performed at different UVC irradiation doses, and changes in virus infectivity (degree of inactivation) were evaluated in the same manner as in Example 1. The properties of the virus used are as shown in Table 1. In the study using B19, the detection limit of B19 in the solution was obtained by irradiation with Fib concentration 3.3 mg / ml (E ₂₈₀ : 5, extinction coefficient (E ^1% ₂₈₀ : 15-16)) and 120 mJ / cm ² . Inactivated until (LRV:> 4.6). At a higher protein concentration, Fib concentration 6.7 mg / ml (E ₂₈₀ : 10, extinction coefficient (E ^1% ₂₈₀ : 15-16)), irradiation with 192 mJ / cm ² is required to achieve LRV:> 4.0. At a Fib concentration of 13.3 mg / ml (E ₂₈₀ : 20, extinction coefficient (E ^1% ₂₈₀ : 15-16)), LRV:> 4.00 could not be achieved in the range of this experiment (Figs. 7 and 8). ).

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

Example 3: Examination of inactivation of virus in coexistence with Thr by UVC irradiation Thr (containing 0.34% sodium chloride, 0.23% sodium citrate, 0.27% calcium chloride as stabilizers) concentration is 2.48 mg / ml (E ₂₈₀ ) : 5.3, E ^1% ₂₈₀ : 21.4) was used to evaluate the effect of UVC irradiation on Thr molecules. In this experiment, the amount of UVC energy was measured using UVC-254 (manufactured by Custom Co., Ltd.), and the measuring instrument showed a value about 1/3 lower than that of UVR-300 (manufactured by Topcon Techno House Co., Ltd.) (Table). 2). Therefore, in this experiment, a correction value obtained by multiplying the measured value by 3 was used so as to be the measured value with UVR-300 (manufactured by Topcon Techno House Co., Ltd.).

Since Thr is a protein with coagulation activity, the coagulation activity and polymer formation were evaluated. The coagulation activity decreased with the irradiation dose at 192 mJ / cm ² , which decreased to almost 90%. The polymer also increased with irradiation dose and increased to 3.8% at 192 mJ / cm ² (Fig. 10). Next, B19 or PPV was added to the Fib solution diluted to various concentrations, and irradiation was performed at different UVC irradiation doses, and the change in virus infectivity (degree of inactivation) was evaluated in the same manner as in Example 1. .. The properties of the virus used are as shown in Table 1.

B19 and PPV drew an approximate inactivated curve. 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 at 192 mJ / cm ² (LRV:> 5.00). Regarding PPV, the condition for achieving LRV: 4.00, although not below the detection limit, was an irradiation dose of 96 mJ / cm ² . Although UVC irradiation inactivates these viruses, the coexistence of Thr stabilizes the virus and increases the amount of irradiation required for inactivation, but the Thr concentration is 4.67 mg / ml (E ₂₈₀ : 10, E ^1%). If the concentration is ₂₈₀ : 21.4) or less, it exceeds the critical point at 192 mJ / cm ² , and if the Thr concentration is 2.34 mg / ml (E ₂₈₀ : 5, E ^1% ₂₈₀ : 21.4), it exceeds the critical point at 96 mJ / cm ² . It was shown (Figs. 11 and 12). From this example, although it is an example, in the case of a solution containing Thr as a target protein, as an example of optimal virus inactivation / removal conditions, a virus removal membrane treatment having a pore size of 20 nm and a Thr concentration of 4.67 mg / ml (E ₂₈₀ : 10, E ^1% ₂₈₀ : 21.4) or less, UVC irradiation of 192 mJ / cm ² (or 96 mJ / cm ² ), or virus removal membrane treatment with pore size less than 33 nm, Thr concentration 4.67 mg / It was shown that the UVC irradiation was 192 mJ / cm ² (or 96 mJ / cm ² ) or less under ml (E ₂₈₀ : 10, E ^1% ₂₈₀ : 21.4).

Example 4: Examination of inactivation by gamma beam irradiation for viruses in the coexistence of Fib, Thr, and Alb 26.65 mg / ml as Fib (53.3 mg / vial after freeze-drying, sodium citrate and L as stabilizers / excipients -Contains alginate), 3.5 mg / ml as Thr (7.0 mg / vial after freeze-drying, contains D-mannitol, sodium citrate, L-arginate as stabilizers / excipients), Alb As a 10 mg / ml (20 mg / vial after freeze-drying, containing a) sodium citrate, sodium chloride, b) D-mannitol as stabilizers / excipients), freeze-dry each solution and irradiate with gamma rays. The effect on each protein was evaluated.

The degree of decomposition product formation of these proteins by gamma-ray irradiation was evaluated by the Peak area ratio by SEC (HPLC) analysis. The Fib polymer increased by 20% with 25 kGy irradiation compared with non-irradiation. The Thr polymer did not show a significant change even after irradiation with 50 kGy. Albumin increased by 10-15% after irradiation with 50 kGy (Fig. 13-15). Next, the sample freeze-dried after adding B19 was irradiated with different gamma doses, and the change in virus infectivity (degree of inactivation) was evaluated in the same manner as in Example 1. The properties of the virus used are as shown in Table 1.

B19 in the presence of albumin was inactivated to below the detection limit (LRV:> 4.4,> 4.2) by irradiation with 25 kGy, respectively. In the coexistence of Thr, it was inactivated to below the detection limit (LRV:> 3.8) by irradiation with 50 kGy. In the coexistence of Fib, 25 kGy irradiation was inactivated to LRV: 4.6, and 50 kGy irradiation was inactivated to below the detection limit (LRV:> 5.0) (Fig. 16). From this example, it was shown that as an example of the optimum conditions for inactivating B19 by gamma-ray irradiation, it is 25 kGy or more in the presence of albumin, 50 kGy or more in the presence of Thr, and 25 kGy or more in the presence of Fib.

Example 5: Examination of calibration of measurement value error due to difference in UVC measuring device When virus inactivation by UVC irradiation is performed, it is necessary to correctly measure UVC irradiation amount (energy amount). However, as a result of examination, as shown in Table 2, it was found that the indicated value may differ depending on the detector even under the same conditions.

From Table 2 above, it is understood that it is difficult to standardize virus inactivation conditions by UVC irradiation unless the same detector is used. In order to solve the above standardization problem, we tried to standardize the UVC irradiation dose using 1% NaI, which is known to change the absorbance by UVC irradiation. By using NaI and adopting the calibration curve method as shown in FIG. 17, the desired standardization could be achieved.

In the present invention, virus inactivation by irradiation treatment and virus removal by virus removal membrane treatment or virus inactivation by SD treatment are integrated, and their characteristics are complementarily optimized for the conditions. It is useful for inactivating / removing the virus in the solution and for producing a protein solution in which the virus has been inactivated / removed.

This application is based on Japanese Patent Application No. 2020-175610, the contents of which are all included in the present specification.

Claims

A method for removing viruses in protein solutions, such as:
(A) The step of irradiating the protein solution with radiation so that the removal coefficient (LRV) for the virus having a virus particle diameter of less than 33 nm in the protein solution is 1.00 or more, and (b1) the treatment. The step of removing the virus from the protein solution with a virus removing membrane having an LRV of 4.00 or more for Bacterophage PP7, or (b2) the treated protein solution is a mixture of an organic solvent and a surfactant (SD mixture). A method comprising a step of inactivating the virus by an organic solvent / surfactant treatment (SD treatment) so that the LRV against the enveloped virus in the protein solution is 2.00 or more.
(A) is the step of irradiating the protein solution with radiation so that the removal coefficient (LRV) for the virus having a virus particle diameter of 30 nm or less in the protein solution is 1.00 or more. Method.
The method according to claim 1 or 2, wherein the radiation is ultraviolet rays or γ-rays.
The method according to claim 3, wherein the radiation is ultraviolet rays.
The method according to claim 4, wherein the ultraviolet rays are irradiated in the range of 32 mJ / cm 2 to 256 mJ / cm 2 .
The method according to claim 3, wherein the radiation is γ-rays.
The method according to claim 6, wherein the γ-rays are irradiated in the range of 5 kGy to 50 kGy.
The method according to any one of claims 1 to 7, wherein the virus removing membrane having an LRV of 4.00 or more for bacteriophage PP7 is a virus removing membrane having a pore size of less than 33 nm.
The method according to claim 8, wherein the virus removing membrane is a virus removing membrane having a pore size of 13 nm or more and less than 33 nm.
The method according to claim 9, wherein the virus removing membrane is a virus removing membrane having a pore size of 15 nm or more and less than 20 nm.
The method according to claim 9, wherein the virus removing membrane is a virus removing membrane having a pore size of 17 to 21 nm.
A method for producing a protein solution from which a virus has been removed.
(A) The step of irradiating the protein solution with radiation so that the removal coefficient (LRV) for the virus having a virus particle diameter of less than 33 nm in the protein solution is 1.00 or more, and (b1) the treatment. The step of filtering the protein solution with a virus-removing membrane having an LRV of 4.00 or higher for Bacterophage PP7, or (b2) contacting the treated protein solution with the SD mixture and LRV against the enveloped virus in the protein solution. A method that includes a step of SD processing so that is 2.00 or higher.
(A) is the step of irradiating the protein solution with radiation so that the removal coefficient (LRV) for the virus having a virus particle diameter of 30 nm or less in the protein solution is 1.00 or more. Method.