JP2018082987A - Virus inactivation method and virus inactivation device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a virus inactivating method and a virus inactivating device capable of appropriately inactivating a virus mixed in a liquid containing a useful substance. SOLUTION: In a virus inactivating method, a liquid containing a virus is filled in a pressure vessel 10, a pressure medium is introduced, the virus is inactivated by the hydrostatic pressure of the liquid, and the inactivated liquid is pressured. Of the openings provided in the container 10, the discharge is discharged through the same opening (10b) as the opening through the pressure medium. The virus inactivating device 1 includes a pressure container 10 for inactivating a virus with hydrostatic pressure of a liquid, a supply pipe 20 for supplying a liquid containing a virus, and a pressure medium introducing pipe 30 for introducing a pressure medium. A discharge pipe 40 for discharging the inactivated liquid and valves V1, V2, V3 are provided, and the discharge pipe 40 communicates with the same opening (10b) as the opening with which the pressure medium introduction pipe 30 communicates. ing. [Selection diagram] Fig. 1

Description

  The present invention relates to a virus inactivation method and a virus inactivation apparatus that inactivate viruses mixed in biological products such as pharmaceuticals and foods and drinks.

  In the production process of various products such as pharmaceuticals, foods and drinks, and cosmetics, an operation for culturing cells may be performed. For example, in a biopharmaceutical manufacturing process, cells capable of producing a target useful substance are liquid-cultured using a culture tank or the like. And the useful substance produced by the cell is refine | purified from a culture solution, and the biopharmaceutical containing the component derived from a living organism is commercialized.

  Biological products such as biopharmaceuticals are highly likely to be contaminated with microorganisms and viruses during the production process, and the safety and quality of the products are impaired by the contamination. Therefore, in many cases, biological products are subjected to sterilization treatment or virus inactivation treatment during the production process. Examples of methods for sterilization and virus inactivation include heat treatment, acid treatment, ultraviolet treatment, and gamma ray treatment.

  Antibody drugs are a type of biopharmaceutical and have been actively developed in recent years. In general, an antibody drug is prepared by culturing animal cells having antibody-producing ability and purifying the antibody produced in the culture solution by affinity chromatography or the like. As the affinity ligand, protein A or the like having specific affinity for the antibody is used.

  In purification by affinity chromatography, the culture solution is passed through an affinity column carrying protein A or the like, and the antibody produced in the culture solution is selectively adsorbed to protein A or the like under neutral conditions. The adsorbed antibody is recovered by elution under acidic conditions after contaminants other than the antibody are washed and removed. In collecting the antibody, in many cases, an acidic eluent having a pH of about 3 or less is retained in the column for several hours. By such an operation, the virus inactivation treatment by acid treatment is performed in combination with the elution of the antibody.

When a culture solution containing a proteinaceous useful substance such as an antibody is subjected to a virus inactivation treatment, the useful substance is denatured by heat treatment or acid treatment, and the quality of the product is lowered. Accordingly, various studies have been made on conditions for reliably inactivating viruses while ensuring product quality. For example, Patent Document 1 discloses a technique for inactivating a virus by heating a solution of a protein preparation at + 45 ° C. to + 95 ° C. with a residence time of 0.1 to 20 seconds. Patent Document 2 discloses a method of irradiating UV-C having an intensity of 20 mW / cm ² for 1 minute or more as a method for inactivating the hepatitis C virus with less influence on proteins and the like.

  Conventionally, as a virus inactivation method, in addition to heat treatment, ultraviolet treatment, etc., microbubble treatment, chemical treatment, and the like have been studied. For example, Non-Patent Document 1 reports that oysters artificially incorporated with feline calicivirus were treated in ozone nanobubble water for 6 hours, and as a result, the oyster itself remained alive and the virus infectivity titer was reduced. Yes. In addition, Non-Patent Document 2 reports that as a result of allowing 5000 ppm or more of sodium hypochlorite to act on feline calicivirus for 1 minute, the virus activity was reduced below the detection limit.

  In the field of food and drink, a technique for sterilizing microorganisms that may be mixed into products by high-pressure treatment has been developed. For example, Non-Patent Document 3 discloses a processing system in which a liquid processing product filled in a processing chamber is pressurized through a pressure medium and sterilized by high-pressure processing.

Japanese Patent Laid-Open No. 06-065091 JP 2009-005589 A

Masayoshi Takahashi et al., “Latest Technology of Fine Bubbles”, NTS Corporation, November 2006, p43-49 J.C.Doultree, J.D.Druce, C.J.Birch, D.S.Bowden, J.A.Marshall, “Inactivation of feline calicivirus, a Norwalk virus surrogate”, The Journal of Hospital Infection (1999), Vol. 41, Issue 1, p51-57 "High-temperature / high-pressure application technology High-pressure food technology", [online], Kobe Steel, Ltd. [Search April 13, 2016], Internet (URL: http://www.kobelco.co.jp/products/ip) /technology/food.html)

  Conventionally used virus inactivation methods such as heat treatment, acid treatment, etc. are useful substances contained in the liquid to be treated even if the treatment time is shortened as disclosed in Patent Document 1. There is a problem that severe deterioration of the inevitable. Further, the ultraviolet ray treatment as disclosed in Patent Document 2 and the virus inactivation treatment by gamma ray treatment also have problems that denature useful substances or newly generate degradation products. For this reason, virus inactivation methods such as heat treatment, acid treatment, ultraviolet treatment, and gamma ray treatment contain useful substances that are easily denatured, such as antibodies, and the modification of useful substances and the generation of degradation products affect product quality. Not suitable for easy liquid handling.

  In addition, microbubble treatment as disclosed in Non-Patent Document 1 and virus inactivation treatment by chemical treatment as disclosed in Non-Patent Document 2 may also denature useful substances, There is a problem that new foreign matters such as ozone and hypochlorite are mixed. Ozone and hypochlorite used in treatment, and decomposition products generated by these decomposition processes must be removed during the manufacturing process, which complicates the manufacturing process and requires time for commercialization. It will be prolonged. Further, as disclosed in Non-Patent Document 3, a method of performing high-pressure treatment is also conceivable. However, when a liquid treated at high pressure is released, a strong shearing force is generated along with decompression, so that a useful substance is denatured. There is a high risk of losing.

  Then, an object of this invention is to provide the virus inactivation method and virus inactivation apparatus which can inactivate the virus mixed in the liquid in which the useful substance is contained appropriately.

  In order to solve the above-mentioned problems, the virus inactivation method according to the present invention fills a pressure vessel with a liquid containing virus, and introduces a pressurized pressure medium into the pressure vessel filled with the liquid. The virus is inactivated by the hydrostatic pressure of the liquid held in the pressure vessel, and the inactivated liquid held in the pressure vessel is the same as the opening of the pressure vessel through the pressure medium. The pressure vessel is discharged through the opening.

  Further, the virus inactivation apparatus according to the present invention includes a pressure vessel for inactivating virus with a hydrostatic pressure of liquid, a supply pipe for supplying a liquid containing virus to the pressure vessel, and the pressure vessel. A pressure medium introduction pipe for introducing a pressurized pressure medium, a discharge pipe for discharging the inactivated liquid held in the pressure container from the pressure container, and a first valve capable of opening and closing the supply pipe; A second valve that can open and close the pressure medium introduction pipe, and a third valve that can open and close the discharge pipe, and the discharge pipe is an opening of the pressure vessel through which the pressure medium introduction pipe communicates It communicates with the same opening.

  The virus inactivation method and the virus inactivation apparatus according to the present invention can appropriately inactivate a virus mixed in a liquid containing a useful substance.

It is a figure which shows typically the virus inactivation apparatus which concerns on one Embodiment of this invention. It is a figure which shows the process of filling the to-be-processed liquid of the virus inactivation method which concerns on one Embodiment of this invention. It is a figure which shows the process of introducing the pressure medium of the virus inactivation method which concerns on one Embodiment of this invention. It is a figure which shows the process inactivated by the hydrostatic pressure of the virus inactivation method which concerns on one Embodiment of this invention. It is a figure which shows the process of discharging | emitting the processed liquid of the virus inactivation method which concerns on one Embodiment of this invention. It is a figure which shows typically the virus inactivation apparatus which concerns on a 1st modification. It is a figure which shows the process of introducing the pressure medium of the virus inactivation method which concerns on a 1st modification. It is a figure which shows the process of discharging | emitting the processed liquid of the virus inactivation method which concerns on a 1st modification. It is a figure which shows typically the virus inactivation apparatus which concerns on a 2nd modification. It is a figure which shows the process of introducing the pressure medium of the virus inactivation method which concerns on a 2nd modification. It is a figure which shows the process of discharging | emitting the processed liquid of the virus inactivation method which concerns on a 2nd modification. It is a figure which shows typically the virus inactivation apparatus which concerns on a 3rd modification. It is a figure which shows the 1st Example of the virus inactivation apparatus which concerns on one Embodiment of this invention. It is a figure which shows the 2nd Example of the virus inactivation apparatus which concerns on one Embodiment of this invention.

  Hereinafter, a virus inactivation method and a virus inactivation apparatus according to an embodiment of the present invention will be described with reference to the drawings. In addition, about the structure which is common in each following figure, the same code | symbol is attached | subjected and the overlapping description is abbreviate | omitted.

FIG. 1 is a diagram schematically showing a virus inactivation apparatus according to an embodiment of the present invention.
As shown in FIG. 1, the virus inactivation apparatus 1 includes a pressure vessel 10, a supply pipe 20, a pressure medium introduction pipe 30, a discharge pipe 40, a supply pipe valve (first valve) V1, and a pressure medium introduction. A pipe valve (second valve) V2, a discharge pipe valve (third valve) V3, and a high-pressure pump P1 are provided.

  A virus inactivation apparatus 1 shown in FIG. 1 is an apparatus that inactivates a virus-containing liquid using hydrostatic pressure. In the virus inactivation apparatus 1, the virus is destroyed by compressing the liquid containing the virus (liquid to be treated) with a pressure medium pressurized to a high pressure and generating a high hydrostatic pressure in the liquid. By processing the liquid containing liquid (processed liquid) using such high hydrostatic pressure, a liquid (processed liquid) in which the virus is inactivated and infectivity is lost can be obtained. .

  The pressure vessel 10 is provided for holding the liquid to be treated introduced under high pressure and inactivating the virus by the hydrostatic pressure of the liquid. The pressure vessel 10 is a pressure resistant vessel made of stainless steel or the like. The maximum allowable pressure of the pressure vessel 10 is at least 250 MPa or more, preferably 400 MPa or more.

  The pressure vessel 10 can be provided, for example, as a tubular pressure pipe having a long aspect ratio and a high aspect ratio as compared with the pipe diameter. The pressure vessel 10 shown in FIG. 1 has openings at both ends, an opening (inlet 10a) into which the liquid to be treated flows into one end side of the pressure vessel 10, and a treated liquid flows out into the other end side. An opening (exit 10b) is provided.

  The pressure vessel 10 can be provided in an appropriate size based on the size of the hydrostatic pressure generated inside, the total amount of liquid to be treated, the treatment time that can be assigned to virus inactivation treatment, and the like. For example, the pressure pipe can be provided with a pipe diameter (inner diameter) of several mm to several cm and a pipe length of several m to several hundred m. The pressure pipe can be used in an appropriate pipe shape such as a straight line, a spiral, a curve, or a ring.

  The pressure vessel 10 may have openings at both ends arranged at the same height in the vertical direction, or may be arranged at different heights that can generate a hydrostatic pressure difference. However, from the viewpoint of easily filling the liquid to be processed and discharging the liquid to be processed using gravity, it is preferable to arrange the openings so that the opening into which the liquid to be processed flows is at a relatively high position.

  The supply pipe 20 is provided to supply a liquid containing a virus (liquid to be processed) to the pressure vessel 10. The supply pipe 20 connects between the liquid header to be processed for supplying the liquid to be processed and the pressure vessel 10, and communicates with the inlet 10 a of the pressure vessel 10. The liquid to be processed flows through the supply pipe 20 from the header of the liquid to be processed toward the pressure vessel 10 and is filled into the pressure vessel 10 through the inlet 10a.

  The supply pipe valve V <b> 1 is provided in the supply pipe 20, and is provided so that the pipe line of the supply pipe 20 can be opened and closed. The supply pipe valve V1 is a pressure-resistant high-pressure valve or the like that can maintain the internal pressure of the pressure vessel 10 when the pipe line of the filling pipe 20 is closed. The maximum allowable pressure of the supply pipe valve V1 is at least 250 MPa, preferably 400 MPa or more. As the supply pipe valve V1, for example, a high-pressure needle valve, a ball valve, or the like can be used.

  The pressure medium introduction pipe 30 is provided to introduce a pressure medium such as pressurized water into the pressure vessel 10. The pressure medium introduction pipe 30 connects between the pressure medium header that supplies the pressure medium and the pressure vessel 10, and communicates with the outlet 10 b of the pressure vessel 10. The pressure medium introduction pipe 30 is provided with a high pressure pump P1 capable of pressurizing the pressure medium to a pressure of 250 MPa or more. The pressure medium flows through the pressure medium introduction pipe 30 from the pressure medium header toward the pressure vessel 10 and is pressurized to a high pressure by the high-pressure pump P1, and then introduced into the pressure vessel 10 through the outlet 10b. ing.

  As the pressure medium, for example, water such as purified water, distilled water, or pure water, or an aqueous solution can be used. Examples of the aqueous solution include solutions having low biological toxicity such as a buffer solution, an electrolyte aqueous solution, a surfactant aqueous solution, an amino acid aqueous solution, and a sugar aqueous solution. These solutions are preferably sterilized and excluding pyrogens.

  Examples of the buffer include acetic acid, sodium acetate, sodium acetate hydrate, citric acid, citric acid hydrate, sodium citrate, sodium citrate hydrate, phosphoric acid, sodium dihydrogen phosphate, diphosphate Examples include aqueous solutions of sodium hydrogen hydrate, disodium hydrogen phosphate, disodium hydrogen phosphate hydrate, sodium phosphate, and the like. By using a buffer solution, it is possible to suppress denaturation due to pH fluctuations for proteinaceous useful substances. Examples of the aqueous electrolyte solution include aqueous solutions of sodium chloride, lactate, acetate, and the like. By using the aqueous electrolyte solution, the isoelectric point, hydrophobic interaction, and the like of the proteinaceous useful substance can be adjusted, so that formation of protein aggregates is prevented.

  Examples of the surfactant aqueous solution include sorbitan fatty acid esters such as polyoxyethylene sorbitan monolaurate and polyoxyethylene sorbate oleate, and nonionic aqueous solutions such as polyoxyethylene polyoxypropylene glycol. By using the surfactant aqueous solution, it is possible to prevent the proteinaceous useful substance from adsorbing to the pressure vessel 10 or the wall surface of the pipe. Examples of the aqueous amino acid solution include aqueous solutions of glycine, L-arginine, L-histidine, histidine hydrochloride, and the like. By using an amino acid aqueous solution, it is possible to suppress the cleavage of peptide bonds and disulfide bonds, so that deterioration of useful proteinaceous substances is prevented.

  Examples of the sugar aqueous solution include trehalose (glass transition point 107 ° C.), maltose (glass transition point 92 ° C.), sucrose (glass transition point 65 ° C.), D-mannitol (glass transition point 130 ° C.), dextran (glass transition point). 160 ° C) and the like. By using a sugar aqueous solution, a hydrogen bond can be formed between the proteinaceous useful substance and the sugar, so that the useful substance is stabilized and hardly deteriorated. In particular, when the treated liquid is dried, sugars bound to proteinaceous useful substances are in an amorphous state. Therefore, the higher the glass transition temperature of the sugar, the higher the effect of suppressing the molecular motion of the useful substance in virus inactivation treatment performed near room temperature. However, the sugar is preferably a non-reducing sugar. If the sugar is a reducing sugar, it may react with a useful proteinous substance and the surface functional groups may be denatured.

  The pressure medium introduction pipe valve V <b> 2 is provided in the pressure medium introduction pipe 30, and is provided so that the conduit of the pressure medium introduction pipe 30 can be opened and closed. The pressure medium introduction pipe valve V2 is a pressure-resistant high-pressure valve or the like that can maintain the internal pressure of the pressure vessel 10 when the conduit of the pressure medium introduction pipe 30 is closed. The maximum allowable pressure of the pressure medium introduction pipe valve V2 is at least 250 MPa, preferably 400 MPa. As the pressure medium introduction pipe valve V2, for example, a high-pressure needle valve, a ball valve, or the like can be used.

  The discharge pipe 40 is provided for discharging the liquid in which the virus held in the pressure vessel 10 is inactivated (processed solution) from the pressure vessel 10. The discharge pipe 40 connects between the pressure vessel 10 and a fraction tank or the like that receives the treated liquid, and has the same opening (outlet 10b) as the opening (outlet 10b) of the pressure vessel 10 through which the pressure medium introduction pipe 30 communicates. It communicates with the outlet 10b). The treated liquid is discharged from the pressure vessel 10 through the same opening (outlet 10b) as the opening through the pressure medium (outlet 10b), flows through the discharge pipe 40, and is then collected in a fraction tank (not shown) or the like. It has become.

  The discharge pipe valve V3 is provided in the discharge pipe 40, and the pipe line of the discharge pipe 40 is provided to be openable and closable. The discharge pipe valve V3 is a pressure-resistant high-pressure valve that can maintain the internal pressure of the pressure vessel 10 when the pipe line of the discharge pipe 40 is closed. The maximum allowable pressure of the discharge pipe valve V3 is at least 250 MPa, preferably 400 MPa. As the discharge pipe valve V3, for example, a high-pressure needle valve, a ball valve, or the like can be used. In particular, a straight type valve having a straight center line at the inlet and outlet and low fluid resistance is preferable.

  Next, a virus inactivation method performed using the virus inactivation apparatus 1 will be described.

  In the virus inactivation apparatus 1, the pressure medium introduction pipe 30 and the discharge pipe 40 are openings (outlet 10 b) different from openings (inlet 10 a) through which the supply pipe 20 communicates among openings provided in the pressure vessel 10. ) Are connected to each other. Therefore, in the virus inactivation method performed using the virus inactivation apparatus 1, the introduction of the liquid to be processed through the supply pipe 20 and the discharge of the processed liquid through the discharge pipe 40 are mutually performed with respect to the pressure vessel 10. It is performed so as to be a forward liquid flow direction.

  FIG. 2 is a diagram showing a step of filling a liquid to be treated in the virus inactivation method according to an embodiment of the present invention, FIG. 3 is a diagram showing a step of introducing a pressure medium, and FIG. FIG. 5 is a diagram showing a step of activating, and FIG. 5 is a diagram showing a step of discharging the treated liquid.

  In the virus inactivation method according to the present embodiment, first, as shown in FIG. 2, the liquid containing the virus (liquid to be processed) is filled into the pressure vessel 10. When the pressure vessel 10 is filled with the liquid to be treated, the supply pipe valve V1 is opened (shown in white in the figure), and the pressure medium introduction pipe valve V2 and the discharge pipe valve V3 are closed ( (Shown in black in the figure). The liquid to be processed is filled into the pressure vessel 10 through the inlet 10a. And after the to-be-processed liquid is satisfy | filled to the state where the inside of the pressure vessel 10 is substantially full, the supply pipe valve V1 is closed.

  Subsequently, as shown in FIG. 3, a pressure medium such as pressurized water is introduced into the pressure vessel 10 filled with a liquid containing a virus (liquid to be processed). When the pressure medium is introduced into the pressure vessel 10, the closed pressure medium introduction pipe valve V2 is opened. After the pressure medium is pressurized to a high pressure by the high-pressure pump P1, the pressure medium is introduced into the inside of the pressure vessel 10 filled with the liquid to be processed through the outlet 10b. And the to-be-processed liquid with which the pressure vessel 10 is filled is compressed with the pressure by a pressure medium, and the pressure medium introduction pipe valve V2 is closed in the state where high hydrostatic pressure is applied to the liquid hold | maintained at the pressure vessel 10. The

  Subsequently, as shown in FIG. 4, the pressure inside the pressure vessel 10 into which the pressure medium has been introduced is maintained, and the virus contained in the liquid to be treated is hydrostatic pressure of the liquid held in the pressure vessel 10. Inactivate. When the virus contained in the liquid to be treated is inactivated, the opened pressure medium introduction pipe valve V2 is closed. The liquid to be treated is held in the pressure vessel 10 together with the pressure medium for a predetermined time, so that viruses that may be mixed are reliably inactivated.

  In general, an enveloped virus is destroyed at a room temperature at a pressure of 100 MPa or more. This is because when the high pressure is applied to the lipid bilayer membrane constituting the envelope, the equilibrium moves in the direction of decreasing the volume, and the shape cannot be maintained at a pressure of 100 MPa or more. On the other hand, viruses that do not have an envelope are destroyed at a room temperature at a pressure of 250 MPa or more. From the observation of the virus by the present inventor, it has been found that the capsid that forms the outer shell of the virus is destroyed when water is compressed to cause a volume change of about 10% or more. Although water is an incompressible fluid, a volume change of 10% or more can be caused by applying a pressure of about 250 MPa or more at room temperature.

  Therefore, in the virus inactivation process, it is preferable to pressurize the liquid (liquid to be processed) containing the virus filled in the pressure vessel 10 to a pressure of 250 MPa or more and 400 MPa or less in absolute pressure at room temperature. By pressurizing the liquid to be treated to a pressure of 250 MPa or more, the entire virus can be inactivated regardless of the presence or absence of the envelope. On the other hand, when the pressure is 400 MPa or less, it is possible to avoid loss of higher-order structures and the like due to hydrostatic pressure when the liquid to be treated contains a useful proteinaceous substance.

  In the virus inactivation treatment, it is preferable to maintain the liquid (liquid to be treated) containing the virus filled in the pressure vessel 10 at a temperature of 0 ° C. or higher and 30 ° C. or lower. If the liquid to be treated is kept at a temperature of 30 ° C. or lower, the useful substance contained in the liquid to be treated does not have to be exposed to high heat, so that thermal denaturation of the useful substance is suppressed. Further, if the liquid to be treated is kept at a temperature of 0 ° C. or higher, it is not necessary to extremely increase the viscosity of the pressure medium such as water, so that it is difficult for strong shearing force and frictional force to be generated. Degeneration of useful substances due to is suppressed.

  In the virus inactivation treatment, it is preferable to hold the liquid (liquid to be treated) containing the virus filled in the pressure vessel 10 under a pressure of 250 MPa or more for 5 minutes or more. If the liquid to be treated is kept under high pressure for 5 minutes or longer, all viruses that may be mixed in the liquid to be treated can be surely inactivated.

  Subsequently, as shown in FIG. 5, the liquid in which the virus held in the pressure vessel 10 has been inactivated (processed solution) is opened to the same opening (exit 10 b) as the pressure vessel 10 through the pressure medium (exit 10 b). It discharges from the pressure vessel 10 through the outlet pipe valve V3 through the outlet 10b). When the processed liquid held in the pressure vessel 10 is discharged, the closed discharge pipe valve V3 is opened. Before the virus-inactivated treatment liquid and the pressure medium are sufficiently mixed with each other, the virus-inactivated treatment liquid is recovered from the inside of the pressure vessel 10 through the outlet 10b.

  According to the virus inactivation apparatus and the virus inactivation method described above, the liquid held in the pressure vessel is filled by filling the pressure vessel with the liquid containing the useful substance and introducing the pressure medium into the pressure vessel. Can be subjected to high hydrostatic pressure. Unlike the heat treatment, acid treatment, ultraviolet ray treatment, gamma ray treatment, etc., the virus inactivation treatment using hydrostatic pressure suppresses the denaturation of the substance itself, so it mixes in the liquid containing the useful substance. Can be inactivated with little deterioration of useful substances.

  In addition, according to the virus inactivation apparatus and the virus inactivation method described above, after the treatment liquid containing the useful substance is virus inactivated, the treated liquid in which the virus is inactivated is opened through the pressure medium. Is discharged through the same opening. That is, the processed liquid and the pressure medium held at a high pressure in the pressure vessel can be sequentially discharged from the pressure vessel before being sufficiently mixed with each other. In the process in which the liquid held at high pressure in the pressure vessel is discharged while being rapidly depressurized when the valve is opened, strong shear force due to flow turbulence, collision, cavitation, etc., and high heat due to friction are applied There is a high risk. However, according to the virus inactivating apparatus and the virus inactivating method, the pressure medium that does not contain the useful substance is discharged before the treated liquid containing the useful substance and is rapidly depressurized. It passes through the pipe and the discharge pipe valve which exerts high fluid resistance first. And the processed liquid containing a useful substance is discharged | emitted after that, and passes a discharge pipe and a discharge pipe valve at a slow speed, carrying out gentle pressure reduction. Therefore, it is possible to avoid that the useful substance contained in the virus-inactivated liquid is greatly deteriorated due to physical stimulation such as shearing force or high heat due to friction.

  Further, according to the virus inactivation apparatus and the virus inactivation method described above, the introduction of the liquid to be processed through the supply pipe and the discharge of the processed liquid through the discharge pipe are performed in a forward direction with respect to the pressure vessel. It is done in the direction. Therefore, by standing the pressure vessel in the vertical direction, it is possible to perform both filling and discharging by causing the liquid to flow down by gravity. By causing the liquid to flow down by gravity, power for filling and discharging becomes unnecessary, and it is possible to avoid that the useful substance is subjected to strong physical stimulation by an external force applied for filling and discharging.

  Next, a virus inactivation method and a virus inactivation apparatus according to a modification of the present invention will be described with reference to the drawings.

FIG. 6 is a diagram schematically illustrating a virus inactivation apparatus according to a first modification.
As shown in FIG. 6, the virus inactivation apparatus includes a gas pressure feeding pipe 50 that pressure-feeds a gas such as air to the pressure vessel 10, and a liquid in which the virus held in the pressure vessel 10 is deactivated (treated liquid). ) Can be used as a device (the virus inactivation device 2 according to the first modification) that pumps and discharges gas to the pressure vessel 10 and extrudes the treated liquid in the forward direction and in the forward direction.

  Similar to the virus inactivation apparatus 1, the virus inactivation apparatus 2 according to the first modification includes a pressure vessel 10, a supply pipe 20, a pressure medium introduction pipe 30, a discharge pipe 40, a supply pipe valve ( A first valve) V1, a pressure medium introduction pipe valve (second valve) V2, a discharge pipe valve (third valve) V3, and a high-pressure pump P1, and further includes a gas pressure feed pipe 50, a gas And a pressure feed pipe valve (fourth valve) V4.

  The gas pressure feed pipe 50 connects between a gas header that supplies a gas such as air and the pressure vessel 10, and has the same opening (inlet) as the opening (inlet 10 a) of the pressure vessel 10 through which the supply pipe 20 communicates. 10a). A gas such as air flows through the gas pressure feed pipe 50 from the air header toward the pressure vessel 10 and is fed into the pressure vessel 10 through the inlet 10a.

  The gas pressure feed pipe valve V4 is provided in the gas pressure feed pipe 50, and a pipe line of the gas pressure feed pipe 50 is provided to be freely opened and closed. The gas pressure feed valve V4 is a pressure-resistant high pressure valve or the like that can maintain the internal pressure of the pressure vessel 10 when the line of the gas pressure feed pipe 50 is closed together with the line of the supply pipe 20. The maximum allowable pressure of the gas pressure feeding tube valve V4 is at least 250 MPa or more, preferably 400 MPa or more. As the gas pressure feeding tube valve V4, for example, a high pressure needle valve, a ball valve or the like can be used.

  Next, the virus inactivation method performed using the virus inactivation apparatus 2 which concerns on a 1st modification is demonstrated.

  In the virus inactivating device 2, the gas pressure feeding pipe 50 is connected to the same opening (inlet 10a) as the opening (inlet 10a) with which the supply pipe 20 is communicated with each other through the pipes. On the other hand, the pressure medium introduction pipe 30 and the discharge pipe 40 are mutually connected to an opening (outlet 10b) different from the opening (inlet 10a) through which the supply pipe 20 communicates among the openings provided in the pressure vessel 10. The roads merge and communicate with each other. Therefore, in the virus inactivation method performed using the virus inactivation apparatus 2, the introduction of the liquid to be processed through the supply pipe 20 and the discharge of the processed liquid through the discharge pipe 40 are mutually performed with respect to the pressure vessel 10. It is performed so as to be a forward liquid flow direction.

  FIG. 7 is a diagram showing a step of introducing a pressure medium of the virus inactivation method according to the first modification, and FIG. 8 is a diagram showing a step of discharging the treated liquid.

  In the virus inactivation method according to the first modification performed using the virus inactivation apparatus 2, first, similarly to the virus inactivation apparatus 1, a liquid (liquid to be treated) containing a virus is used as a pressure vessel. 10 is filled. When the pressure vessel 10 is filled with the liquid to be treated, the supply pipe valve V1 is opened, and the pressure medium introduction pipe valve V2, the discharge pipe valve V3, and the gas pressure feed pipe valve V4 are closed ( (See FIG. 2). Then, after the liquid to be treated is filled in the pressure vessel 10, the supply pipe valve V1 is closed.

  Subsequently, as shown in FIG. 7, a pressure medium such as pressurized water is introduced into the pressure vessel 10 filled with a liquid containing liquid (liquid to be treated). When the pressure medium is introduced into the pressure vessel 10, the supply pipe valve V1, the discharge pipe valve V3, and the gas pressure feed pipe valve V4 are closed. On the other hand, the closed pressure medium introduction pipe valve V2 is opened. Then, similarly to the virus inactivation apparatus 1, after the pressure medium introduction pipe valve V2 is closed, the virus contained in the liquid to be treated filled in the pressure vessel 10 is inactivated by hydrostatic pressure.

  Then, as shown in FIG. 8, the liquid (processed liquid) in which the virus hold | maintained at the pressure vessel 10 was inactivated is gas-fed into the pressure vessel 10, and it extrudes and discharges with gas. When the treated liquid held in the pressure vessel 10 is discharged, the closed discharge pipe valve V3 and gas pressure feed pipe valve V4 are opened. A gas such as air is pumped through the same opening (inlet 10a) as the opening (inlet 10a) of the pressure vessel 10 through the liquid to be treated containing virus. Then, before the virus-inactivated treatment liquid and the pressure medium are sufficiently mixed with each other, the pressure vessel 10 passes through the same opening (outlet 10b) as the opening (outlet 10b) of the pressure vessel 10 through the pressure medium. Is discharged through a discharge pipe valve V3.

  According to the virus inactivation apparatus and the virus inactivation method described above, the introduction of the liquid to be processed through the supply pipe and the discharge of the processed liquid through the discharge pipe are in the direction of the forward liquid flow with respect to the pressure vessel. The treated liquid in which the virus is inactivated is pushed out in a gas from the pressure vessel and forcibly discharged. Therefore, the treated liquid in which the virus has been inactivated can be recovered with little residual regardless of the orientation of the pressure container with respect to the vertical direction, the tube diameter of the pressure container, and the like. The gas pumped to the pressure pipe is held in the pressure vessel when the liquid is pushed out by gravity while flowing down or when a pressure vessel having an extremely long pipe length compared to the pipe diameter is used. The treated liquid can be discharged at a constant speed with less pulsation. Therefore, it is possible to suppress the occurrence of a strong shearing force or the like in the process of discharging the treated liquid.

FIG. 9 is a diagram schematically showing a virus inactivation apparatus according to a second modification.
As shown in FIG. 9, the virus inactivation apparatus includes a gas pressure feeding pipe 50 that pressure-feeds gas to the pressure vessel 10, and a liquid (treated solution) in which the virus held in the pressure vessel 10 is inactivated, It can also be set as the apparatus (virus inactivation apparatus 3 which concerns on a 2nd modification) which pumps gas to the pressure vessel 10 and pushes out with gas in the direction opposite to the introduction direction of a to-be-processed liquid.

  Similar to the virus inactivation device 2, the virus inactivation device 3 according to the second modified example includes a pressure vessel 10, a supply pipe 20, a pressure medium introduction pipe 30, a discharge pipe 40, a supply pipe valve ( First valve) V1, pressure medium introduction pipe valve (second valve) V2, discharge pipe valve (third valve) V3, high pressure pump P1, gas pressure feed pipe 50, gas pressure feed pipe valve (fourth valve) ) V4.

  The pressure vessel 10 shown in FIG. 9 has openings at both ends, and one end of the pressure vessel 10 has an opening (entrance / exit 10c) through which the liquid to be processed flows and the processed liquid flows out. An opening (air vent 10d) through which air (gas) flows is provided on the end side.

  In the virus inactivation apparatus 3, the supply pipe 20 connects between the liquid header to be processed for supplying the liquid to be processed and the pressure vessel 10, and communicates with the inlet / outlet 10 c of the pressure vessel 10. The pressure medium introduction pipe 30 connects between the pressure medium header that supplies the pressure medium and the pressure vessel 10, and communicates with the inlet / outlet 10 c of the pressure vessel 10. The discharge pipe 40 connects between the pressure vessel 10 and a fraction tank or the like that receives the treated liquid, and is the same as the opening (entrance / exit 10c) of the pressure vessel 10 through which the pressure medium introduction pipe 30 communicates. To the opening (entrance 10c).

  Next, the virus inactivation method performed using the virus inactivation apparatus 3 which concerns on a 2nd modification is demonstrated.

  In the virus inactivating device 3, the pressure medium introduction pipe 30 and the discharge pipe 40 are joined to each other through the same opening (entrance / exit 10c) as the opening (entrance / exit 10c) through which the supply pipe 20 communicates. Communicate. On the other hand, the gas pressure feeding pipe 50 communicates with an opening (vent hole 10d) different from the opening (entrance / exit 10c) with which the supply pipe 20 communicates among the openings provided in the pressure vessel 10. Therefore, in the virus inactivation method performed using the virus inactivation apparatus 3, the introduction of the liquid to be processed through the supply pipe 20 and the discharge of the processed liquid through the discharge pipe 40 are mutually performed with respect to the pressure vessel 10. The liquid flow direction is the opposite direction.

  FIG. 10 is a diagram showing a step of introducing a pressure medium of the virus inactivating method according to the second modification, and FIG. 11 is a diagram showing a step of discharging the treated liquid.

  In the virus inactivation method according to the second modification performed using the virus inactivation apparatus 3, first, as in the case of the virus inactivation apparatus 2, a liquid (liquid to be treated) containing a virus is used as a pressure vessel. 10 is filled. When the pressure vessel 10 is filled with the liquid to be treated, the supply pipe valve V1 is opened, and the pressure medium introduction pipe valve V2, the discharge pipe valve V3, and the gas pressure feed pipe valve V4 are closed ( (See FIG. 2). Then, after the liquid to be treated is filled in the pressure vessel 10, the supply pipe valve V1 is closed.

  Subsequently, as shown in FIG. 10, a pressure medium such as pressurized water is introduced into the pressure vessel 10 filled with a liquid containing a virus (liquid to be treated). When the pressure medium is introduced into the pressure vessel 10, the supply pipe valve V1, the discharge pipe valve V3, and the gas pressure feed pipe valve V4 are closed. On the other hand, the closed pressure medium introduction pipe valve V2 is opened. As in the case of the virus inactivation apparatus 2, after the pressure medium introduction tube valve V2 is closed, the virus contained in the liquid to be treated filled in the pressure vessel 10 is inactivated by hydrostatic pressure.

  Then, as shown in FIG. 11, the liquid (processed liquid) in which the virus hold | maintained at the pressure vessel 10 was inactivated is gas-fed into the pressure vessel 10, and it extrudes and discharges with gas. When the treated liquid held in the pressure vessel 10 is discharged, the closed discharge pipe valve V3 and gas pressure feed pipe valve V4 are opened. A gas such as air is pumped through an opening (vent hole 10d) different from the opening (entrance / exit 10c) of the pressure vessel 10 through a liquid (liquid to be treated) containing virus. Then, before the virus-inactivated treatment liquid and the pressure medium are sufficiently mixed with each other, the pressure is applied through the same opening (inlet / outlet 10c) as the opening of the pressure vessel 10 (inlet / outlet 10c) through the pressure medium. It is discharged from the container 10 through the discharge pipe valve V3.

  According to the virus inactivation apparatus and the virus inactivation method described above, the introduction of the liquid to be processed through the supply pipe and the discharge of the processed liquid through the discharge pipe are in a direction opposite to each other with respect to the pressure vessel. In spite of this, the treated liquid in which the virus is inactivated is forced out by being pushed out of the pressure vessel with gas. Therefore, the treated liquid in which the virus has been inactivated can be recovered with little residual regardless of the orientation of the pressure container with respect to the vertical direction, the tube diameter of the pressure container, and the like. Further, since the introduction of the liquid to be processed and the discharge of the processed liquid are performed through the same opening provided in the pressure vessel, it is not necessary to provide openings of the same size at both ends of the pressure vessel 10, The pressure vessel 10 can be configured to easily maintain the internal pressure. The gas pumped to the pressure pipe discharges the treated liquid held in the pressure vessel at a constant speed with less pulsation when using a pressure vessel whose tube length is extremely long compared to the tube diameter. Can be made. Therefore, it is possible to suppress the occurrence of a strong shearing force or the like in the process of discharging the treated liquid.

FIG. 12 is a diagram schematically showing a virus inactivation apparatus according to a third modification.
As shown in FIG. 12, the virus inactivation apparatus includes a plurality of pressure vessels 10 for inactivating viruses with a hydrostatic pressure of liquid in parallel, and numbers virus inactivation treatment of a large amount of liquid (liquid to be treated). It can also be set as a corresponding apparatus (virus inactivation apparatus 4 according to the third modified example).

Similar to the virus inactivation apparatus 1, the virus inactivation apparatus 4 according to the third modified example includes a pressure vessel 10, a supply pipe 20, a pressure medium introduction pipe 30, a discharge pipe 40, a supply pipe valve ( A first valve) V1, a pressure medium introduction pipe valve (second valve) V2, a discharge pipe valve (third valve) V3, and a high-pressure pump P1 are provided. As the pressure vessel 10, a plurality of pressure vessels (10 ₁ , 10 ₂ ... 10 _n ) are provided in parallel with each other.

Each of the plurality of pressure vessels (10 ₁ , 10 ₂ ... 10 _n ) provided in parallel is connected to a supply pipe 20, a pressure medium introduction pipe 30, and a discharge pipe 40 through a pipe having a branch. It is connected. The supply pipe 20 communicates with the inlet 10a through a pipe having a branch. On the other hand, the pressure medium introduction pipe 30 and the discharge pipe 40 communicate with the outlet 10b through a pipe having a branch. That is, the discharge pipe 40 communicates with the same opening (exit 10b) as the opening (exit 10b) with which the pressure medium introduction pipe 30 communicates.

In the virus inactivation method performed using the virus inactivation apparatus 4, first, the liquid to be processed containing the virus is distributed to each of the plurality of pressure vessels (10 ₁ , 10 ₂ ... 10 _n ). Fill. Subsequently, a pressure medium such as pressurized water is introduced into each of the plurality of pressure vessels (10 ₁ , 10 ₂ ... 10 _n ) filled with the liquid to be treated containing virus. Thereafter, similar to the virus inactivation apparatus 1, the liquid is hydrostatic pressure held in each of the plurality of pressure vessels (10 ₁ , 10 ₂ ... 10 _n ) and is contained in the liquid to be treated. The virus is inactivated, the treated liquid in which the virus is inactivated is discharged through the same opening (outlet 10b) as the opening through the pressure medium (outlet 10b), and the treated liquid in which the virus is inactivated is recovered. .

The number of numbered up pressure vessels (10 ₁ , 10 ₂ ... 10 _n ) provided in the virus inactivating device 4 is preferably 4 or more. For example, when the tube length of the pressure vessel 10 is 100 m and the tube diameter is 1 cm, batch processing of about 127.4 times is required to inactivate 1000 L of the liquid to be processed for virus inactivation. Therefore, if the retention time of the liquid to be processed in one process is set to 5 minutes or more, it takes 10 hours or more to inactivate the 1000 L of liquid to be processed, and it is generally performed in about 3 hours. Compared with the conventional acid treatment, the treatment time is about 3 times or more. Therefore, in order to perform processing in a shorter time than before regardless of the tube length or tube diameter of the pressure vessel 10, it is preferable to include at least four pressure vessels 10.

  According to the virus inactivation apparatus and the virus inactivation method described above, the liquid to be treated containing the virus is distributed to a plurality of pressure vessels and processed at the same time, so that a large amount of liquid is inactivated in a short time. be able to. Therefore, it is not necessary to wait for a long time before subjecting the treatment liquid containing virus to the virus inactivation treatment, and the useful substances contained in the treatment liquid are not deteriorated during storage. Can be suppressed. Furthermore, since a large amount of liquid can be inactivated in a short time, it is not necessary to increase the capacity of the pressure vessel, and it is possible to reliably apply hydrostatic pressure to the virus.

  Next, examples of the virus inactivation method and virus inactivation apparatus of the present invention will be described with reference to the drawings.

FIG. 13 is a diagram illustrating a first example of a virus inactivating apparatus according to an embodiment of the present invention.
As shown in FIG. 13, the above-mentioned virus inactivating apparatus can be used by being placed in the subsequent stage of the affinity column 120 that performs purification using the action of the temperature-responsive polymer. In the subsequent stage of the affinity column 120, the supply pipe 20 is connected so as to connect the outlet of the affinity column 120 and the pressure vessel 10. A liquid (treatment liquid) containing a useful substance and possibly contaminated with a virus is supplied to the affinity column 120 and affinity purified using the useful substance as a target substance for purification. Thereafter, the liquid to be treated, in which the useful substance is purified, is filled in the pressure vessel 10 and subjected to virus inactivation treatment.

  The affinity column 120 is filled with a filler that includes a carrier, a temperature-responsive polymer bonded to the carrier, and a binding substance that is bonded to the temperature-responsive polymer and specifically binds to the target substance. Is done. The temperature-responsive polymer is a polymer having a property of reversibly changing the three-dimensional structure in accordance with a temperature change. When the temperature change occurs, the three-dimensional structure of the temperature-responsive polymer changes, and the affinity of the binding between the target substance and the binding substance also changes greatly. Therefore, by causing a temperature change, the target substance and the binding substance can be freely bound or dissociated.

  Examples of the temperature-responsive polymer include polyamino acids such as polylysine, polyglutamic acid, and polyasparagine, and polyamino acids having side chains introduced therein. As the side chain, any of an amphiphilic group, a hydrophobic group, and a hydrophilic group can be introduced. Examples of the amphiphilic group include valeric acid and butyric acid. Examples of the hydrophobic group include alkylamines such as dodecylamine. Examples of the hydrophilic group include amino alcohols such as 3-aminopropanol. The polyamino acid preferably has both a hydrophobic group and a hydrophilic group as side chains.

  The change in the three-dimensional structure of the temperature-responsive polymer is preferably generated in the range of 0 to 60 ° C, more preferably in the range of 4 to 40 ° C. The temperature-responsive polymer preferably forms a three-dimensional structure that can bind to the target substance on the low temperature side, and forms a three-dimensional structure that cannot bind to the target substance on the high temperature side. With such properties, the target substance does not have to be exposed to high temperatures for a long time during the purification process. In addition, since the three-dimensional structure of the temperature-responsive polymer can be changed by heating a small amount of the eluate rather than the liquid to be treated before purification, the heating cost can be reduced. The temperature at which the three-dimensional structure of the temperature-responsive polymer changes can be adjusted by changing the molecular weight, side chain, and the like. For example, the molecular weight of the polyamino acid is preferably 1 to 20 kDa, more preferably 2 to 18 kDa, and further preferably 3 to 16 kDa.

  Specific examples of the temperature-responsive polymer include polyasparagine containing a monomer unit having a dodecylamine side chain and a monomer unit having an amino alcohol side chain. Such a polyamino acid can cause a change in the three-dimensional structure of the molecule in the range of 0 to 60 ° C., so that, for example, the target substance and the binding substance can be bound at a low temperature of 20 ° C. or lower, or 37 It is possible to dissociate the target substance and the binding substance at a high temperature of about 0C.

  The binding substance may be either a low molecule or a polymer as long as it can specifically bind to a useful substance. For example, peptides, proteins, and the like that can specifically bind to antibodies, enzymes, physiologically active substances, and the like can be used. In particular, protein A and protein G that have binding ability to antibodies are preferably used. The binding force of the binding between the binding substance and the target substance may be such that it can be reversibly adsorbed and desorbed by a change in the three-dimensional structure of the temperature-responsive polymer.

  In the affinity column 120, useful substances contained in the liquid to be treated are purified through a binding step, a washing step, a separation step, and an elution step.

  In the binding step, the liquid to be treated containing the useful substance is passed through the affinity column 120 to bind the useful substance to the binding substance. The temperature in the binding step is a temperature at which the temperature-responsive polymer forms a three-dimensional structure capable of binding with a useful substance that is a target substance. For example, it is 0-60 degreeC, Preferably it is 2-20 degreeC, More preferably, it is 4-10 degreeC. Moreover, pH in a coupling | bonding process shall be 5-9, for example, Preferably it is 6-8.

  In the washing step, impurities are washed and removed without dissociating the useful substance and the binding substance that are bonded to each other. For example, the buffer solution is passed through the affinity column 120 to remove foreign substances adsorbed nonspecifically in the column. The temperature and pH conditions in the washing step can be set to the same level as in the bonding step.

  In the separation step, a temperature change is applied to the temperature-responsive polymer to dissociate useful substances bonded to the binding substance. For example, a buffer solution whose temperature and pH are adjusted to predetermined ranges is passed through the affinity column 120, or the temperature is changed by adjusting the temperature of the affinity column 120. The temperature in the separation step is a temperature at which the temperature-responsive polymer forms a three-dimensional structure that cannot bind to a useful substance that is a target substance. For example, it is 0-60 degreeC, Preferably it is 20-50 degreeC, More preferably, it is 35-40 degreeC. Moreover, pH in a coupling | bonding process shall be 5-9, for example, Preferably it is 6-8.

  In the elution step, the useful substance dissociated from the binding substance is eluted from the affinity column 120. For example, a buffer solution whose temperature and pH are adjusted to a predetermined range is passed through the affinity column 120, and useful substances in the column are eluted and recovered in the buffer solution. The temperature and pH conditions in the elution step can be set to the same level as in the separation step.

  Subsequently, the purified solution purified in the affinity column 120 is filled in the pressure vessel 10 of the virus inactivation apparatus 1. Then, a pressure medium such as water is introduced, and the virus is inactivated using the hydrostatic pressure of the liquid. Thereafter, the treated liquid is discharged from the pressure vessel 10 through the same opening (outlet 10b) as the opening (outlet 10b) of the pressure vessel 10 through the pressure medium.

  As described above, when the virus inactivation apparatus is used in the subsequent stage of the affinity column 120, the purified solution purified by utilizing the action of the temperature-responsive polymer is subjected to virus inactivation treatment with liquid hydrostatic pressure. In the affinity column 120, adsorption / desorption of useful substances is performed in a mild temperature range and a pH near neutral, so that an acid or the like need not be used to elute the useful substances. In addition, since the hydrostatic pressure is used in the virus inactivation treatment, unlike the virus inactivation method by heat treatment, acid treatment, ultraviolet treatment, gamma ray treatment, etc., the modification of the useful substance itself is low. Therefore, when the virus inactivation apparatus 1 is used in the subsequent stage of the affinity column 120, a purified solution in which the mixed virus is inactivated can be obtained without greatly degrading useful substances.

FIG. 14 is a diagram showing a second example of the virus inactivating apparatus according to one embodiment of the present invention.
As shown in FIG. 14, the virus inactivation apparatus 1 can be used by being incorporated in the antibody purification system S. In the antibody purification system S shown in FIG. 14, the virus inactivation apparatus 1 is arranged at the subsequent stage of the affinity column 120 that performs purification using the action of the temperature-responsive polymer and the subsequent stage of the purification column 320 that performs ion-exchange chromatography. Has been.

  As shown in FIG. 14, the antibody purification system S includes an affinity purification unit 100 that performs purification by affinity chromatography, a cation exchange purification unit 200 that performs purification by cation exchange chromatography using a cation exchanger, and an anion. It comprises an anion exchange purification unit 300 that performs purification by anion exchange chromatography using an exchanger, and a membrane separation unit 400 that performs filtration.

  The affinity purification unit 100 is arranged at the latter stage of the culture process for culturing antibody-producing cells. The affinity purification unit 100 includes a culture solution receiving tank 110 that receives a culture solution, an affinity column 120 that performs purification using the action of a temperature-responsive polymer, a fraction tank 130 that receives an elution fraction from the column, a filtration A filter F and a receiving tank 140 for receiving a crude purified solution purified by affinity chromatography are provided in this order.

  In the affinity purification unit 100, the virus inactivation apparatus 1 is arranged immediately after the affinity column 120. A culture medium in which desired antibody-producing cells are cultured is sent from the culture chamber to the affinity purification unit 100 after the cells and coarse impurities are separated and removed. The supernatant of the culture solution is received by the receiving tank 110 and then roughly purified in the affinity column 120 through a binding step, a washing step, a separation step, and an elution step. Contaminants such as host cell-derived protein, DNA, RNA, etc. and medium-derived albumin are discarded through the drain in the washing step. On the other hand, the crudely purified solution obtained by roughly purifying the antibody is collected in the fraction tank 130 in the elution step.

  The cation exchange purification unit 200 is arranged at the subsequent stage of the affinity purification unit 100. The cation exchange purification unit 200 includes a cation exchange column 220 that performs purification by cation exchange chromatography and a fraction tank 230 that receives an elution fraction from the column in this order.

  A crude purified solution obtained by roughly purifying the antibody is sent from the affinity purification unit 100 to the cation exchange purification unit 200. The crude purified solution is passed through an equilibrated cation exchange column 220 and subjected to intermediate purification, and impurities that have not yet been separated, protein A that has fallen from the carrier, antibody aggregates, multimers, Charge change bodies and the like are eliminated. Thereafter, the intermediate purified solution from which the antibody has been purified is collected in the fraction tank 230.

  The anion exchange purification unit 300 is arranged at the subsequent stage of the cation exchange purification unit 200. The anion exchange purification unit 300 includes an anion exchange column 320 that performs purification by anion exchange chromatography, and a fraction tank 330 that receives an eluted fraction from the column in this order.

  The anion exchange purification unit 300 receives an intermediate purified solution from which the antibody has been purified from the cation exchange purification unit 200. Then, the intermediate purified solution is passed through an anion exchange column 320 that has been equilibrated and finally purified, and impurities such as proteins and nucleic acids that have not yet been separated, endotoxins, and the like are excluded. Thereafter, the final purified solution obtained by purifying the antibody is collected in the fraction tank 330.

  The membrane separation unit 400 is disposed at the subsequent stage of the anion exchange purification unit 300. The membrane separation unit 400 includes a purified solution receiving tank 410 that receives the final purified solution, a filtration device 420 that performs ultrafiltration to concentrate the antibody, a two-stage filtration filter F, an ultrafiltration and two-stage removal. Three fraction tanks 430, 440, and 450 for receiving bacteria-treated filtrates are provided in order.

  In the membrane separation unit 400, the virus inactivation device 1 is disposed immediately before the filtration device 420. The final purified solution obtained by purifying the antibody is sent from the anion exchange purification unit 300 to the membrane separation unit 400. Then, after the virus is inactivated by the virus inactivating device 1, the final purified solution is ultrafiltered by the filtering device 420, concentrated, and the remaining contaminants are excluded. Then, it is filtered by the filtration filter F, sent to the filling chamber, and formulated as an antibody drug or the like.

  Thus, when a virus inactivation apparatus is incorporated into the antibody purification system S and used, a purified solution purified by affinity chromatography utilizing the action of a temperature-responsive polymer or a purified solution purified by ion exchange chromatography is treated. It is supplied to the virus inactivation apparatus 1 as a liquid and subjected to virus inactivation processing. In the affinity column 120 using the action of the temperature-responsive polymer, the adsorption / desorption of the antibody can be performed in a mild temperature range and at a pH near neutral. Etc. need not be used. Further, in the cation exchange column 220 and the anion exchange column 320, an extreme pH condition in which the antibody is easily denatured can be avoided by setting the elution condition to a neutral pH. In the virus inactivation treatment, since hydrostatic pressure is used, unlike the virus inactivation method by heat treatment, acid treatment, ultraviolet treatment, gamma ray treatment, etc., the antibody itself can be denatured at a low level. Therefore, according to the antibody purification system S in which the virus inactivation apparatus 1 is incorporated, an antibody purification solution in which the mixed virus is inactivated can be obtained without greatly degrading the antibody.

  As mentioned above, although embodiment, the modification, and the Example of this invention were demonstrated, this invention is not limited to the said form, A various change is possible in the range which does not deviate from the meaning of this invention. For example, the present invention is not necessarily limited to the one having all the configurations included in the above-described embodiments and modifications. Part of the configuration of a certain embodiment or modification may be replaced with the configuration of another embodiment or modification, or part of the configuration of a certain embodiment or modification may be added to another embodiment or modification. It is also possible to omit a part of the configuration of a certain embodiment or modification.

  For example, any of the virus inactivation devices 1, 2, and 3 may be numbered up like the virus inactivation device 4 according to the third modification. Moreover, about the virus inactivation apparatus 3 which concerns on a 2nd modification, it is good also as a system which abbreviate | omits the gas pressure feeding pipe 50 and the gas pressure feeding pipe valve (4th valve) V4, and does not pump gas.

  In the above-described embodiment and modification, a pressure pipe is provided as the pressure vessel 10. However, the pressure vessel 10 is not limited to the pressure pipe type, and may be a tank type or the like. Further, the supply pipe 20, the pressure medium introduction pipe 30, the discharge pipe 40, and the gas pressure feed pipe 50 are connected to an arbitrary opening with respect to the pressure pipe 10, or arbitrarily branched or merged unless otherwise limited. Can do. Further, the supply pipe valve V1, the pressure medium introduction pipe valve V2, the discharge pipe valve V3 and the gas pressure feed pipe valve V4 may be provided on the pipe as long as the flow in each pipe can be opened and shut off. You may prepare on extension of the piping. For example, in the virus inactivation apparatus 4 that has been numbered up, each valve may be provided for each of the plurality of pressure vessels 10.

  Moreover, in the said Example, the virus inactivation apparatus 1 which concerns on embodiment is provided as the pressure vessel 10 as a virus inactivation apparatus. However, the virus inactivating device may be the above-described virus inactivating device 2, 3, 4 or other device. In addition, the virus inactivation device is placed behind the column that performs various purifications such as cation exchange chromatography, anion exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography, and adsorption chromatography instead of affinity chromatography. Also good.

  In the antibody purification system S, the order of the cation exchange purification unit 200 and the anion exchange purification unit 300 may be reversed. In such an antibody purification system S, the virus inactivation apparatus may be provided only in the subsequent stage of the affinity column 120 or only in the subsequent stage of the cation exchange column 220 or the anion exchange column 320.

DESCRIPTION OF SYMBOLS 1 Virus inactivation apparatus 10 Pressure vessel 10a Inlet 10b Outlet 10c Inlet / outlet 10d Vent 20 Supply pipe 30 Pressure medium introduction pipe 40 Discharge pipe 50 Gas pressure feed pipe V1 Supply pipe valve (first valve)
V2 Pressure medium introduction pipe valve (second valve)
V3 discharge pipe valve (third valve)
V4 Gas pressure pipe valve (4th valve)
P1 High pressure pump

Claims (14)

Fill the pressure vessel with the liquid containing the virus,
Introducing a pressurized pressure medium into the pressure vessel filled with the liquid;
Inactivate the virus with the hydrostatic pressure of the liquid held in the pressure vessel,
A virus inactivation method for discharging the inactivated liquid held in the pressure vessel from the pressure vessel through the same opening as the opening of the pressure vessel through the pressure medium. In the virus inactivation method according to claim 1,
A virus inactivation method in which the inactivated liquid held in the pressure vessel is pumped with a gas to the pressure vessel, and is ejected by the gas. The virus inactivation method according to claim 2,
A virus inactivation method in which the gas is pumped through the same opening as the pressure vessel through the liquid containing the virus. The virus inactivation method according to claim 2,
A virus inactivation method wherein the gas is pumped through an opening different from the opening of the pressure vessel through the liquid containing virus. In the virus inactivation method according to claim 1,
A virus inactivation method in which the liquid filled in the pressure vessel is pressurized to a pressure of 250 MPa to 400 MPa. In the virus inactivation method according to claim 1,
Distributing and filling the liquid containing the virus into a plurality of pressure vessels;
Introducing a pressurized pressure medium into each of the pressure vessels filled with the liquid;
Inactivate the virus with the hydrostatic pressure of the liquid held in each of the pressure vessels,
A virus inactivation method for discharging the inactivated liquid held in each of the pressure vessels from each of the pressure vessels through the same opening as the opening of the pressure vessel through the pressure medium. In the virus inactivation method according to claim 1,
A virus inactivation method, wherein the liquid containing a virus is a purified solution obtained by subjecting to an affinity chromatography utilizing the action of a temperature-responsive polymer. In the virus inactivation method according to claim 1,
The liquid containing the virus is a liquid containing an antibody;
After the liquid containing the antibody is subjected to the supernatant of the culture medium in which antibody-producing cells are cultured, the crude purified liquid is recovered, and the crude purified liquid is subjected to cation exchange chromatography to recover the intermediate purified liquid. The virus inactivation method, which is a final purified solution obtained by subjecting the intermediate purified solution to anion exchange chromatography. A pressure vessel for inactivating the virus with liquid hydrostatic pressure,
A supply pipe for supplying a liquid containing virus to the pressure vessel;
A pressure medium introduction pipe for introducing a pressurized pressure medium into the pressure vessel;
A discharge pipe for discharging the inactivated liquid held in the pressure vessel from the pressure vessel;
A first valve capable of opening and closing the supply pipe;
A second valve capable of freely opening and closing the pressure medium introduction pipe;
A third valve that can freely open and close the discharge pipe;
With
The virus inactivation device wherein the discharge pipe communicates with the same opening as the opening of the pressure vessel with which the pressure medium introduction pipe communicates. The virus inactivation apparatus according to claim 9,
A gas pumping tube for pumping gas to the pressure vessel;
A fourth valve capable of freely opening and closing the gas pressure pipe;
A virus inactivation apparatus further comprising: The virus inactivation apparatus according to claim 10,
The virus inactivating apparatus in which the gas pressure feeding pipe communicates with the same opening as the opening of the pressure vessel with which the supply pipe communicates. The virus inactivation apparatus according to claim 10,
The virus inactivating apparatus in which the gas pressure feeding pipe communicates with an opening different from the opening of the pressure vessel with which the supply pipe communicates. The virus inactivation apparatus according to claim 9,
A virus inactivation apparatus in which the pressure medium introduction pipe includes a high-pressure pump capable of pressurizing the pressure medium to a pressure of 250 MPa or more. The virus inactivation apparatus according to claim 9,
A plurality of the pressure vessels are provided in parallel,
Each of the plurality of pressure vessels is connected to the supply pipe, the pressure medium introduction pipe, and the discharge pipe,
The virus inactivating device in which the discharge pipe communicates with the same opening as each of the plurality of pressure vessels with which the pressure medium introduction pipe communicates.

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