Method and apparatus for exchanging dissolved gas in hemoglobin-containing solution

Abstract

This method for exchanging dissolved gas includes a first dissolved gas exchange step, in which the other surface of a first dissolved gas exchange membrane exposes a gas phase of a negative pressure up to a saturated water vapor pressure, and a final dissolved gas exchange step, in which the other surface of a final dissolved gas exchange membrane exposes a gas phase of a positive pressure up to 0.5 MPa of the required gas. This device for exchanging dissolved gas may include at least one dissolved gas exchange membrane module, and the dissolved gas exchange membrane of the dissolved gas exchange membrane module may be a hollow fiber membrane.

Description

Dissolved gas exchange method and device for hemoglobin-containing solution

[0001] The present invention relates to a method for exchanging a dissolved gas in a hemoglobin-containing solution and a dissolved gas exchange device for exchanging a dissolved gas in a hemoglobin-containing solution for carrying out the method, and more specifically to a method for exchanging a dissolved gas in a hemoglobin-containing solution, which includes a step of passing the hemoglobin-containing solution through first and second hollow fiber membrane modules, and a dissolved gas exchange device for exchanging a dissolved gas in a hemoglobin-containing solution for carrying out the method.

[0002] Hemoglobin (Hb) exists in red blood cells and is responsible for oxygen transport. The iron in the heme center is in a divalent state and transports oxygen (O ２ Since hemoglobin can reversibly adsorb oxygen, various oxygen carriers using purified and isolated hemoglobin have been developed. However, in the case of trivalent methemoglobin (metHb), in which the central iron is oxidized, O ２ On the other hand, carbon monoxide (CO) cannot bond with O ２ Hemoglobin binds to the divalent iron center 200 times more strongly than oxyhemoglobin (HbCO), inhibiting oxidation of the iron center. Furthermore, the thermal denaturation temperature of carbonylhemoglobin (HbCO) is 78°C, and the thermal denaturation temperature of oxyhemoglobin (HbO) is 78°C. ２ ) (62°C) and that of other proteins. Utilizing this property, a method for purifying hemoglobin has been established that involves removing the denaturation of water-soluble proteins other than HbCO and inactivating viruses through heat treatment. Furthermore, the denaturation that occurs when hemoglobin is processed into an oxygen carrier (Non-Patent Document 1) can also be suppressed by converting it to HbCO. Furthermore, because HbCO does not have the ability to bind oxygen, converting HbCO to HbO ２ Also, HbO ２ During storage, the iron in the heme center gradually oxidizes to form trivalent methemoglobin (metHb). ２ In the production of an oxygen carrier using hemoglobin, it is necessary to completely remove the gaseous ligand that binds to the heme central iron of hemoglobin. ２ to CO, or CO to O ２ Or replace it with O ２The process of completely removing CO is referred to herein as the gas ligand exchange process. ２ binds to the N-terminus of the globin chain of hemoglobin. ２ , N ２ and Ar dissolve in the hemoglobin-containing solution without binding to hemoglobin. Therefore, in this specification, the process of exchanging gases dissolved in the hemoglobin-containing solution, including the binding to the heme central iron of hemoglobin, the binding to the globin chains of hemoglobin, and the dissolution into the hemoglobin-containing solution, is referred to as the process of exchanging dissolved gases in the hemoglobin-containing solution.

[0003] The manufacturing process of artificial red blood cells using hemoglobin as an oxygen carrier includes the following dissolved gas exchange steps (i) to (iii): (i) O of hemoglobin ２ with CO (CO conversion process); HbO2 + CO → HbCO2 + O2↑

[0004] (ii) CO bound to hemoglobin is dissociated by a photoreaction, and O ２ (deCO2 decomposition step); HbCO2 + O2 → HbO2 + CO↑

[0005] and (iii) O that binds to hemoglobin. ２ HbO2 → deoxyHb + O2↑

[0006] Currently, artificial red blood cells (RBCs) composed of purified concentrated hemoglobin encapsulated in lipid bilayers are being developed as oxygen carriers to replace human RBCs for transfusion. Artificial RBCs include, but are not limited to, hemoglobin vesicles (HbVs) and self-assembling vesicles containing hemoglobin. Hemoglobin vesicles are oxygen carriers in which purified concentrated hemoglobin solutions are encapsulated in liposomes. Self-assembling vesicles are oxygen carriers in which lipid-amphiphile precursors are encapsulated in vesicular structures configured to self-assemble from a solution mixture of phospholipids and cholesterol in the presence of purified concentrated hemoglobin and an allosteric effector. For transfusions using donated blood, a circulating hemoglobin concentration of 7 g / dL is recommended as the threshold for transfusion eligibility for both adults and children (e.g., Non-Patent Document 2). The hemoglobin concentration of blood is 15 g / dL, and the hemoglobin concentration of artificial red blood cells is, for example, 10 g / dL in terms of HbV. To increase the hemoglobin concentration of a subject, artificial red blood cells administered as an alternative to blood transfusion also require a hemoglobin concentration of at least 7 g / dL. However, because bubbles are difficult to eliminate in such a high-concentration hemoglobin solution, the bubble-type dissolved gas exchange method used with low-concentration hemoglobin solutions is not practical.

[0007] In the current production method of artificial red blood cells, a reaction vessel such as a round-bottom flask is used in the dissolved gas exchange step. The wall of the reaction vessel used in the dissolved gas exchange step is made of a material that is substantially impermeable to aqueous solutions and gases. In step (i), a hemoglobin-containing solution (HbO ２ In the step (ii), a reaction vessel containing carboxyhemoglobin (HbCO) is placed in a reaction vessel made of a material that is highly transparent to visible light, such as glass, and the reaction vessel is rotated to remove the liquid film that forms on the inner wall from the reaction vessel (Patent Document 1). ２ The method involves exposing the vessel to visible light while irradiating it with visible light from the outside, thereby gradually removing CO2 (Patent Document 2). ２) and rotate the reaction vessel to remove the liquid film that forms on the inner wall with N ２ In this method, the dissolved gas is gradually deoxygenated by exposing the solution to oxygen. Regarding steps (i) and (iii), a method of exchanging the dissolved gas using a medical oxygenator is also known (Non-Patent Documents 3 and 4). Regarding step (ii), a method of exchanging the dissolved gas using a hollow fiber separation membrane for dialysis is also known (Patent Document 3). Regarding step (iii), a method of chemically deoxygenating the solution by adding sulfites or thiols is also known. ２ Although methods for eliminating the dissolved gases are known (Patent Documents 4 and 5), it is unknown how the addition of drugs affects the safety of the preparation. Therefore, in the production of artificial red blood cells using hemoglobin as an oxygen carrier for use in living organisms, the dissolved gases are generally exchanged physically rather than chemically.

[0008] The exchange reaction of dissolved gases occurs at the interface between the gas phase and the liquid phase, but in the method using the reaction vessel, the area of ​​the interface is insufficient, so the exchange of dissolved gases takes a long time. Methods using medical oxygenators (Non-Patent Documents 3 to 5) have problems such as the hollow fiber membrane and housing of the oxygenator not being durable, there being limitations on the reduced pressure and supply gas pressure (basically the gas outlet of the oxygenator is open, and the gas is released and vented with almost no pressure difference between the inlet and outlet), they are expensive, cannot be cleaned or reused, and are not industrially scaled up. In the method using hollow fiber membranes for hemodialysis (Patent Document 3), HbCO solution and O are mixed through the hollow fiber membrane. ２ A dissolved solution containing CO and O ２ The exchange of dissolved gases occurs between the liquid phases of an aqueous solution containing dissolved oxygen, which binds to the iron in the heme center of hemoglobin, so the efficiency of the exchange of dissolved gases is low.

[0009] Japanese Patent Laid-Open No. 6-16693 Japanese Patent Laid-Open No. 7-179357 Japanese Patent Laid-Open No. 2006-316042 Japanese Patent Laid-Open No. 2006-104069 Japanese Patent Laid-Open No. 2001-72595

[0010] Winslow RM et al., Methods Enzymol 1994; 231:3-16 Carson, J L et al., JAMA 2023; 330(19): 1892-1902 Schmukler R and Chien S, Biorheology 1985;22(1):21-9Fukutomi I et al., J Artif Organs 2002;5:102-7Huang Y et al., Biotechnol Progr 2002;18:101-7

[0011] An object of the present invention is to provide a dissolved gas exchange device for a hemoglobin-containing solution, which allows for switching the type of gas in the gas phase during dissolved gas exchange, switching the gas pressure to a positive pressure of up to 0.5 MPa or a negative pressure of up to saturated water vapor pressure, cleaning and / or sterilization before and after use, and scale-up of the production process, and which allows for efficient dissolved gas exchange at relatively low cost, and a method for dissolving gas exchange for hemoglobin using the dissolved gas exchange device.

[0012] [1] The present invention provides a method for dissolving a dissolved gas in a hemoglobin-containing solution (hereinafter referred to as the "dissolved gas exchange method"), which comprises: (1) an initial dissolved gas exchange step in which the hemoglobin-containing solution before being subjected to the dissolved gas exchange method is exposed to one surface of a first dissolved gas exchange membrane to obtain a solution after the initial dissolved gas exchange, with the other surface of the initial dissolved gas exchange membrane being exposed to a gas phase at a negative pressure up to the saturated water vapor pressure; and (2) a final dissolved gas exchange step in which the hemoglobin-containing solution immediately before the final dissolved gas exchange is exposed to one surface of a final dissolved gas exchange membrane to obtain a hemoglobin-containing solution after the final dissolved gas exchange, with the other surface of the final dissolved gas exchange membrane being exposed to a gas phase at a positive pressure of up to 0.5 MPa of the required gas. The gas dissolved in the hemoglobin-containing solution before the dissolved gas exchange method (hereinafter referred to as "dissolved gas before gas exchange") is exchanged with the gas dissolved in the hemoglobin-containing solution after the exchange method (hereinafter referred to as "required gas").

[0013] [2] In the dissolved gas exchange method of [1], the first dissolved gas exchange step (1) and the last dissolved gas exchange step (2) may be carried out using one dissolved gas exchange membrane module.

[0014] [3] In the dissolved gas exchange method of [1], following the first dissolved gas exchange step (1), (3) a second dissolved gas exchange step may be further included in which the hemoglobin-containing solution after the first dissolved gas exchange is exposed to one surface of a second dissolved gas exchange membrane to obtain a hemoglobin-containing solution after a second dissolved gas exchange, and the other surface of the second dissolved gas exchange membrane is exposed to a gas phase at a negative pressure up to the saturated water vapor pressure or a gas phase at a positive pressure up to 0.5 MPa of the required gas.

[0015] [4] In the dissolved gas exchange method of [1], when the number of all dissolved gas exchange processes is M (M is a natural number of 4 or more), the mth dissolved gas exchange process may further include a process in which the hemoglobin-containing solution after the (m-1)th dissolved gas exchange (m is a natural number satisfying 2≦m≦M-1) is exposed to one surface of the mth dissolved gas exchange membrane to obtain the hemoglobin-containing solution after the mth dissolved gas exchange, and the other surface of the mth dissolved gas exchange membrane is exposed to either the same gas phase as the gas phase to which the other surface of the dissolved gas exchange membrane in the (m-1)th dissolved gas exchange process is exposed, or a gas phase of the required gas at a positive pressure of up to 0.5 MPa.

[0016] [5] In the dissolved gas exchange method described in [1], [3] or [4], the hemoglobin-containing solution after the final dissolved gas exchange may be recovered as the hemoglobin-containing solution after the completion of dissolved gas exchange.

[0017] [6] In the dissolved gas exchange method described in any one of [1] and [3] to [5], the hemoglobin-containing solution after the final dissolved gas exchange may be mixed with the hemoglobin-containing solution before being subjected to the dissolved gas exchange step and subjected to the initial dissolved gas exchange step. The steps from the initial dissolved gas exchange step to the final dissolved gas exchange step may be repeated until the gas concentrations of all the hemoglobin-containing solutions after the dissolved gas exchange are not saturated, or the steps from the initial dissolved gas exchange step to the final dissolved gas exchange step may be repeated until the dissolved gas concentrations of all the hemoglobin-containing solutions after the dissolved gas exchange are saturated, and the hemoglobin-containing solution after the dissolved gas exchange may be recovered as the hemoglobin-containing solution after the completion of the dissolved gas exchange.

[0018] [7] In the dissolved gas exchange method according to any one of [1] to [6], the dissolved gas before gas exchange is CO gas, O ２ Gas, N ２ The required gas may be CO gas, NO gas, O ２ Gas, N ２ Gas, CO ２ Gas, Ar gas, H ２ The gas may be gas, air, or a mixture of these.

[0019] [8] In the final dissolved gas exchange step of the dissolved gas exchange method described in any one of [1] to [7], CO is bound to the heme central iron of hemoglobin in the hemoglobin-containing solution immediately before the final dissolved gas exchange step; (4) the method further includes a photodissociation step of irradiating light onto the hemoglobin-containing solution exposed to one surface of the final dissolved gas exchange membrane to photodissociate the gaseous ligand CO from the heme central iron of hemoglobin in the hemoglobin-containing solution; and in this step, the dissolved gas may be exchanged so that CO is not substantially dissolved in the hemoglobin-containing solution by exposing the other surface of the final dissolved gas exchange membrane to a gas phase with a negative pressure up to the saturated water vapor pressure.

[0020] [9] The present invention provides an exchange device for dissolved gases in a hemoglobin-containing solution (hereinafter referred to as the "dissolved gas exchange device") for carrying out the dissolved gas exchange method described in any one of [1] and [3] to [6]. The dissolved gas exchange device of the present invention comprises at least two dissolved gas exchange membrane modules, including a first dissolved gas exchange membrane module and a last dissolved gas exchange membrane module; a container for storing a hemoglobin-containing solution; and a conduit connecting these modules, wherein all of the dissolved gas exchange membrane modules comprise a liquid phase section, a gas phase section, and a dissolved gas exchange membrane section, and the liquid phase section and the gas phase section are arranged with the dissolved gas exchange membrane section between them, wherein the liquid in the liquid phase section is exposed to one surface of the dissolved gas exchange membrane of the dissolved gas exchange membrane section, and the gas in the gas phase section is exposed to the other surface of the dissolved gas exchange membrane of the dissolved gas exchange membrane section, and the dissolved gas exchange membrane section does not permeate the liquid in the liquid phase section but exchanges the dissolved gas in the liquid with the gas in the gas phase section, the conduit connects a container for storing the hemoglobin-containing solution before the dissolved gas exchange to the liquid supply port of the liquid phase section of the first dissolved gas exchange membrane module; when the total number of dissolved gas exchange processes is 2, the conduit connects the liquid discharge port of the liquid phase section of the first dissolved gas exchange membrane module to the liquid supply port of the liquid phase section of the last dissolved gas exchange membrane module; when the total number of dissolved gas exchange processes is 3, the conduit connects the liquid discharge port of the liquid phase section of the first dissolved gas exchange membrane module to the liquid supply port of the liquid phase section of the second dissolved gas exchange membrane module, and connects the liquid discharge port of the liquid phase section of the second dissolved gas exchange membrane module to the liquid supply port of the liquid phase section of the last dissolved gas exchange membrane module; When the number of all dissolved gas exchange processes is M (M is a natural number of 4 or more), the discharge port of the liquid phase part of the (m-1)th (m is a natural number satisfying 2≦m≦M-1) dissolved gas exchange membrane module is connected to the liquid supply port of the liquid phase part of the mth dissolved gas exchange membrane module, where the first dissolved gas exchange membrane module is the first dissolved gas exchange membrane module and the Mth dissolved gas exchange membrane module is the last dissolved gas exchange membrane module; the hemoglobin-containing solution before being subjected to the dissolved gas exchange process is passed in series from the liquid phase part of the first dissolved gas exchange membrane module to the liquid phase part of the last dissolved gas exchange membrane module;Here, the gas phase of the first dissolved gas exchange membrane module is set to a negative pressure up to the saturated water vapor pressure, and the gas phase of the last dissolved gas exchange membrane module is set to a positive pressure of the required gas with a maximum of 0.5 MPa, thereby exchanging the dissolved gas so that the heme central iron of the hemoglobin-containing solution binds to the required gas as a dissolved gas, or so that the heme central iron does not substantially bind to any dissolved gas.

[0021]

[10] In the dissolved gas exchange device described in [9], when there are a total of three dissolved gas exchange membrane modules, the gas phase of the second dissolved gas exchange membrane module following the first dissolved gas exchange membrane module may be at a negative pressure up to the saturated water vapor pressure, or the required gas may be at a positive pressure up to 0.5 MPa.

[0022]

[11] In the dissolved gas exchange device described in [9], when the total number of dissolved gas exchange membrane modules is N (N is a natural number greater than or equal to 4), the gas phase portions of the dissolved gas exchange membrane modules from the first to the nth (n is a natural number satisfying 2≦n≦N-1) dissolved gas exchange membrane modules are all at a negative pressure up to the saturated water vapor pressure, and the gas phase portions of all dissolved gas exchange membrane modules from the n+1th dissolved gas exchange membrane module to the last dissolved gas exchange membrane module are at a positive pressure of up to 0.5 MPa for the required gas.

[0023]

[12] In the dissolved gas exchange device described in any one of [9] to

[11] , the hemoglobin-containing solution that has passed through the liquid phase portion of the last dissolved gas exchange membrane module may be recovered as the hemoglobin-containing solution after the completion of dissolved gas exchange, or the hemoglobin-containing solution that has passed through the liquid phase portion of the last dissolved gas exchange membrane module may be introduced into a container for storing the hemoglobin-containing solution before being subjected to the dissolved gas exchange process, mixed with the hemoglobin-containing solution before being subjected to the dissolved gas exchange process, and introduced into the liquid phase portion of the first dissolved gas exchange membrane module to repeat the dissolved gas exchange.

[0024]

[13] In the dissolved gas exchange device according to any one of [9] to

[12] , the dissolved gas before gas exchange is CO gas, O ２ Gas, N ２gas, air or a mixture thereof, and the required gas is CO gas, NO gas, O ２ Gas, N ２ Gas, CO ２ Gas, Ar gas, H ２ The gas may be gas, air, or a mixture of these.

[0025]

[14] In the dissolved gas exchange device according to any one of [9] to

[13] , the dissolved gas exchange membrane may be a hollow fiber membrane.

[0026]

[15] The dissolved gas exchange device according to any one of [9] to

[14] further includes a light source, the housing of the last dissolved gas exchange membrane module is transparent, the gas phase of the last dissolved gas exchange membrane module is kept at a negative pressure up to the saturated water vapor pressure, and the light source arranged outside the housing of the last dissolved gas exchange membrane module may irradiate the hemoglobin-containing solution in the liquid phase of the dissolved gas exchange membrane module with light, thereby photodissociating CO from hemoglobin in the hemoglobin-containing solution passing through the liquid phase.

[0027]

[16] The present invention provides a system for exchanging dissolved gases in a hemoglobin-containing solution (hereinafter referred to as a "dissolved gas exchange system"), which includes at least P (P is a natural number of 2 or more) dissolved gas exchange devices according to any one of [9] to

[14] , introducing hemoglobin-containing solution stored in a container for storing hemoglobin-containing solution after completion of dissolved gas exchange in a (p-1)th (p is a natural number satisfying 2≦p≦P) dissolved gas exchange device into a container for storing hemoglobin-containing solution before being subjected to the dissolved gas exchange in the pth dissolved gas exchange device, filling the hemoglobin-containing solution requiring dissolved gas exchange in the container for storing hemoglobin-containing solution before dissolved gas exchange to a required gas in the first dissolved gas exchange device for a hemoglobin-containing solution according to any one of [9] to

[14] , The hemoglobin-containing solution stored in a container for storing hemoglobin-containing solution after dissolved gas exchange in the pth dissolved gas exchange device for hemoglobin-containing solution described in any one of [9] to

[14] is considered to be a hemoglobin-containing solution for which dissolved gas exchange has been completed, and the dissolved gas exchanged in the first dissolved gas exchange membrane module of the first dissolved gas exchange device is different from the dissolved gas exchanged in the last dissolved gas exchange membrane module of the pth dissolved gas exchange device.

[0028]

[17] The present invention provides a dissolved gas exchange system, which includes at least Q (Q is a natural number of 2 or more) dissolved gas exchange devices according to any one of [9] to

[14] and one dissolved gas exchange device according to

[15] , and introduces hemoglobin-containing solution stored in a container for storing hemoglobin-containing solution after completion of dissolved gas exchange in the q-1th (q is a natural number satisfying 2≦q≦Q) dissolved gas exchange device according to any one of [9] to

[14] into a qth container for storing hemoglobin-containing solution before dissolved gas exchange into a required gas in the dissolved gas exchange device according to any one of [9] to

[14] , The hemoglobin-containing solution stored in a container for storing the hemoglobin-containing solution after completion of dissolved gas exchange in the dissolved gas exchange device for hemoglobin-containing solution described in any one of [9] to

[14] in the qth item is introduced into a container for storing the hemoglobin-containing solution before dissolved gas exchange to a required gas in the dissolved gas exchange device for hemoglobin-containing solution described in the above

[15] ; the hemoglobin-containing solution requiring dissolved gas exchange is filled into a container for storing the hemoglobin-containing solution before dissolved gas exchange to a required gas in the dissolved gas exchange device for hemoglobin-containing solution described in the first item [9] to

[14] ; the hemoglobin-containing solution stored in the container for storing the hemoglobin-containing solution after completion of dissolved gas exchange in the dissolved gas exchange device for hemoglobin-containing solution described in the above

[15] is regarded as the hemoglobin-containing solution for which dissolved gas exchange has been completed; The dissolved gas exchanged in the first dissolved gas exchange membrane module of the dissolved gas exchange device for a hemoglobin-containing solution described in any one of the first [9] to

[14] items is different from the dissolved gas exchanged in the last dissolved gas exchange membrane module of the dissolved gas exchange device for a hemoglobin-containing solution described in the above

[15] item.

[0029]

[18] The present invention provides a method for producing a hemoglobin-containing solution. The method for producing a hemoglobin-containing solution of the present invention comprises: ２The method includes a step of obtaining a hemoglobin-containing solution in which the heme central iron of hemoglobin does not bind oxygen by the dissolved gas exchange method according to any one of [1] to [7], which does not contain gas, and the oxygen partial pressure of the hemoglobin-containing solution is 0.1 Torr or less.

[0030]

[19] The present invention provides an apparatus for exchanging dissolved gases in a hemoglobin-containing solution for carrying out the dissolved gas exchange method described in [2]. The dissolved gas exchange apparatus of the present invention comprises one dissolved gas exchange membrane module, at least one container, and a conduit connecting these, wherein the dissolved gas exchange membrane module comprises a liquid phase section, a gas phase section, and a dissolved gas exchange membrane section, the liquid phase section and the gas phase section are arranged across the dissolved gas exchange membrane section, wherein the liquid in the liquid phase section is exposed to one surface of the dissolved gas exchange membrane of the dissolved gas exchange membrane section, and the gas in the gas phase section is exposed to the other surface of the dissolved gas exchange membrane of the dissolved gas exchange membrane section, and the dissolved gas exchange membrane section does not permeate the liquid in the liquid phase section but exchanges the dissolved gas in the liquid with the gas in the gas phase section, (a) In the first dissolved gas exchange step (1), the conduit connects the container to the liquid inlet of the liquid phase part of the dissolved gas exchange membrane module and connects the discharge port of the liquid phase part of the dissolved gas exchange membrane module to the container, thereby reducing the partial pressure of the dissolved gas bound to the heme central iron of the hemoglobin-containing solution to the saturated water vapor pressure by creating a negative pressure in the gas phase part of the dissolved gas exchange membrane module up to the saturated water vapor pressure; (b) In the last dissolved gas exchange step (2), the conduit connects the second container to the liquid inlet of the liquid phase part of the dissolved gas exchange membrane module and connects the discharge port of the liquid phase part of the dissolved gas exchange membrane module to the first container, thereby creating a positive pressure in the gas phase part of the dissolved gas exchange membrane module up to 0.5 MPa of the required gas, thereby replacing the dissolved gas in the hemoglobin-containing solution with the required gas.

[0031] According to the present invention, it is possible to provide a method for dissolving gases in a hemoglobin-containing solution and a dissolved gas exchange device for a hemoglobin-containing solution, which can freely switch the type of gas in the gas phase during dissolved gas exchange and switch the gas pressure to a positive pressure of up to 0.5 MPa or a negative pressure of up to saturated water vapor pressure by changing the flow path of the gas phase part of the dissolved gas exchange membrane module without changing the flow path of the hemoglobin-containing solution.

[0032] Schematic diagram of a saline deoxygenation device described in Example 1. While maintaining airtightness, a tank (referred to as "reservoir") containing 1 L of saline was stirred at approximately 300 rpm, and saline was fed from the tank to the liquid supply port of the liquid phase section of a hollow fiber membrane module (referred to as "HFMM") at a flow rate of 600 mL per minute using a liquid feed pump (referred to as "pump"). The saline discharged from the liquid discharge port of the liquid phase section of the hollow fiber membrane module was returned to the tank. A vacuum source (referred to as "vacuum") was connected to both the air supply port and the exhaust port of the gas phase section of the hollow fiber membrane module, and degassing was performed at a negative pressure of -0.098 MPa (1 atmosphere = 0.1013 MPa, saturated water vapor pressure = 0.003 MPa, reaching 0.003 - 0.10133 = -0.0983 MPa). The tank was fed with 1 L of N per minute. ２ While supplying gas, the gas phase of the tank was released to atmospheric pressure. ２ Probe ("O ２ probe) and irrigate with saline. ２ The change in partial pressure over time was measured. In Fig. 1-A, the liquid flow path is indicated by a thicker line than the gas flow path. ２ A graph showing the change in partial pressure over time. The vertical axis of the graph is the O2 partial pressure of the saline solution in the tank. ２ The horizontal axis represents the elapsed time (unit: minutes) and the horizontal axis represents the partial pressure (unit: Torr). ２ The physiological saline solution was deoxygenated under the same conditions as in Example 1, except that a vacuum pump was connected to the exhaust port and degassed at a negative pressure of -0.090 MPa while introducing oxygen. In Figure 2-A, the liquid flow path is indicated by a thicker line than the gas flow path.２ A graph showing the change in partial pressure over time. The vertical axis of the graph is the O2 partial pressure of the saline solution in the tank. ２ The horizontal axis represents the partial pressure (unit: Torr), and the horizontal axis represents the elapsed time (unit: minutes). Schematic diagram of the deoxygenation device for saline solution described in Example 3. Two hollow fiber membrane modules were prepared, and the gas phase of the first hollow fiber membrane module (center), into which saline solution flows from the tank, was degassed at a negative pressure of -0.098 MPa by connecting a vacuum pump to both the air inlet and the exhaust port. Saline solution from the liquid phase outlet of the first hollow fiber membrane module was introduced into the liquid phase inlet of the last hollow fiber membrane module (left). The gas phase of the last hollow fiber membrane module was degassed at a rate of 1 L N per minute from the air inlet. ２ Gas was supplied and the exhaust port was released to atmospheric pressure. The other conditions were the same as in Example 1, and deoxygenation of the saline solution was carried out. In Figure 3-A, the liquid flow path is indicated by a thicker line than the gas flow path. ２ A graph showing the change in partial pressure over time. The vertical axis of the graph is the O2 partial pressure of the saline solution in the tank. ２ The partial pressure (unit: Torr) is plotted on the horizontal axis, and the elapsed time (unit: minutes) is plotted on the horizontal axis. ２ Partial pressure of O ２ The time-dependent change curves at partial pressures of 0.20 Torr or less (Figs. 1B and 3B, respectively) are shown for O ２ The graph is a plot of partial pressure (unit: Torr) on the vertical axis and elapsed time (unit: minutes) on the horizontal axis. ２ The change in partial pressure over time is shown. The black circles represent the O ２ 4A shows the change in partial pressure over time. Schematic diagram of a deoxygenation device for a human red blood cell dispersion described in Example 4. The human red blood cell dispersion was prepared by diluting concentrated human red blood cells, which were donated for a fee by the certified corporation, the Japanese Red Cross Society, two weeks after blood collection, with physiological saline to a hemoglobin concentration of 11.5 g / dL. Deoxygenation of human red blood cells was carried out under the same conditions as in Example 3, except that the human red blood cell dispersion was used instead of physiological saline. In FIG. 4A, the liquid flow path is indicated by a thicker line than the gas flow path. O2 of the human red blood cell dispersion in Example 4 ２ The graph shows the change in partial pressure over time. The vertical axis of the graph shows the O partial pressure of the human red blood cell dispersion in the tank.２ The horizontal axis represents the partial pressure (unit: Torr), and the horizontal axis represents the elapsed time (unit: minutes). A schematic diagram of a deoxygenation device for an artificial red blood cell (HbV) dispersion liquid described in Example 5. Deoxygenation was carried out under the same conditions as in Example 4, except that an HbV dispersion liquid with a hemoglobin concentration of 10 g / dL was used. In Figure 5-A, the liquid flow path is indicated by a thicker line than the gas flow path. ２ Graph showing the change in partial pressure over time. The vertical axis of the graph is the O partial pressure of the HbV dispersion in the tank. ２The horizontal axis represents the partial pressure (unit: Torr), and the horizontal axis represents the elapsed time (unit: minutes). This is a schematic diagram of a CO2 conversion device for an artificial red blood cell (HbV) dispersion, as described in Example 6. After the experiment of Example 5, the pressure reduction in the gas phase of the first hollow fiber membrane module was stopped and the pressure was returned to normal. The gas phase of the last hollow fiber membrane module was supplied with CO2 gas at less than 1 L per minute through the air inlet, and the exhaust port was released to atmospheric pressure. The CO2 conversion of an HbV dispersion was performed under the same conditions as in Example 3. In Figure 6, the liquid flow path is indicated by a thicker line than the gas flow path. This is a schematic diagram of an apparatus for removing CO2 from a human red blood cell dispersion (CO2-bound) by photoreaction, as described in Example 7. The housing of the second hollow fiber membrane module is made of acrylic resin. Visible light irradiated from the outside reaches the hemoglobin, releasing CO2 by photodissociation. The released CO2 was then removed by reducing the pressure. In Figure 7-A, the liquid flow path is indicated by a thicker line than the gas flow path. Graphs showing the results of spectral analysis of the human red blood cell dispersion in Example 7 over time in the visible-ultraviolet region. The vertical axis of the graph represents absorbance, and the horizontal axis represents wavelength (nm). Figure 7-B shows the superimposed absorption spectra from 370 to 500 nm at 0, 40, 80, 120, 180, 240, 300, and 420 minutes after the start of the photoreaction. The 419 nm peak characteristic of HbCO disappeared as the photoreaction progressed, and instead, a 430 nm peak characteristic of deoxyHb was detected. The downward arrow indicates the disappearance of the 419 nm peak, and the upward arrow indicates the detection of the 430 nm peak. Graph showing the change over time in the CO binding rate (CO binding rate) of the human red blood cell dispersion in Example 7 (Hb concentration 10 g / dL, flow rate 600 mL / min). The vertical axis of the graph represents the CO conversion rate (unit: %), and the horizontal axis represents the elapsed time (unit: minutes). Graph showing the change over time in the CO binding rate (CO conversion rate) of a human red blood cell dispersion (Hb concentration 5 g / dL, flow rate 1500 mL / min) in Example 8. The vertical axis of the graph represents the CO conversion rate (unit: %), and the horizontal axis represents the elapsed time (unit: minutes).

[0033] A first embodiment of the present invention comprises: (1) an initial dissolved gas exchange step in which the hemoglobin-containing solution before being subjected to a dissolved gas exchange method is exposed to one surface of a first dissolved gas exchange membrane to obtain a solution after the initial dissolved gas exchange, and the other surface of the initial dissolved gas exchange membrane is exposed to a gas phase at a negative pressure up to the saturated water vapor pressure; and (2) a final dissolved gas exchange step in which the hemoglobin-containing solution immediately before the final dissolved gas exchange is exposed to one surface of a final dissolved gas exchange membrane to obtain a hemoglobin-containing solution after the final dissolved gas exchange, and the other surface of the final dissolved gas exchange membrane is exposed to a gas phase at a positive pressure of up to 0.5 MPa of the required gas. This is a dissolved gas exchange method in which the gas dissolved in the hemoglobin-containing solution before the dissolved gas exchange method (hereinafter referred to as "dissolved gas before gas exchange") is exchanged for the gas dissolved in the hemoglobin-containing solution after the exchange method (hereinafter referred to as "required gas").

[0034] In the present invention, the term "hemoglobin-containing solution" refers to a mixture of hemoglobin and an aqueous solvent having the pH, ionic strength, etc. necessary for hemoglobin to function. The hemoglobin may be in the form of biologically derived red blood cells, a red blood cell lysate, a tetramer of isolated or purified hemoglobin derived from red blood cells, a chemically modified form in which a water-soluble polymer is covalently bound to the hemoglobin, or artificial red blood cells containing hemoglobin vesicles or self-assembled vesicles. The hemoglobin in the hemoglobin-containing solution of the present invention is preferably derived from the same species as the subject to which the hemoglobin is administered, including, but not limited to, livestock such as humans, cows, pigs, horses, sheep, goats, guinea pigs, camels, and rabbits, pets such as dogs and cats, and other mammals. However, depending on the purpose of administration, the hemoglobin does not necessarily have to be derived from the same species as the subject.

[0035] The hemoglobin-containing solution of the present invention may have any viscosity, provided that it can be flowed using a liquid pump so as to be exposed to one surface of the dissolved gas exchange membrane. The hemoglobin concentration in the hemoglobin-containing solution of the present invention is determined by the O binding to the heme center iron of the hemoglobin. ２The concentration of the dissolved gas may be any concentration, provided that the dissolved gases such as CO can be desorbed from the central iron by the dissolved gas exchange membrane of the present invention or exchanged with other dissolved gases.

[0036] When the hemoglobin-containing solution of the present invention is a purified hemoglobin solution obtained by hemolysis of red blood cells, a high-concentration hemoglobin solution of about 40-45 g / dL can be obtained by ultrafiltration or the like. The viscosity at 40 g / dL is measured by a rotational viscometer at a shear rate of 113 s －１ It has been reported that the hemoglobin concentration is 49.7 cP (Kure & Sakai, ACS Biomaterials Sci. & Eng. 2021, 7, 2835-2844). Therefore, when the hemoglobin-containing solution of the present invention is purified hemoglobin, the hemoglobin concentration in the hemoglobin-containing solution of the present invention may be 45 g / dL or less. Furthermore, since a hemoglobin solution with a concentration of 45 g / dL has sufficient fluidity, a hemoglobin solution with a concentration of 50 g / dL or 55 g / dL is within the scope of the present invention or an equivalent range of the present invention as long as it can be flowed using a liquid pump so as to be exposed to one surface of the dissolved gas exchange membrane. Therefore, when the hemoglobin-containing solution of the present invention is a purified hemoglobin solution, the hemoglobin concentration in the hemoglobin-containing solution of the present invention may be at least about 3 g / dL, at least about 5 g / dL, at least about 7 g / dL, at least about 8 g / dL, at least about 9 g / dL, at least 10 g / dL, at least 15 g / dL, at least 20 g / dL, at least 22 g / dL, at least 25 g / dL, at least 30 g / dL, or at least 40 g / dL, and may be 55 g / dL or less, 50 g / dL or less, or 45 g / dL or less.

[0037] It is known that in human blood (red blood cell dispersion) obtained by blood donation, when the hemoglobin concentration is about 15 g / dL, the hematocrit value is about 50%. The hematocrit value is the percentage of the volume occupied by red blood cells in the total volume of the red blood cell dispersion. Therefore, if the hemoglobin concentration in the red blood cell dispersion is about 30 g / dL, the percentage of the volume occupied by red blood cells in the dispersion will be nearly 100%. In other words, if the hemoglobin concentration in a human red blood cell dispersion is about 30 g / dL, the dispersion will have almost no fluidity. Therefore, if the hemoglobin-containing solution of the present invention is human red blood cells, the concentration must be less than about 30 g / dL. Similarly, when the hemoglobin-containing solution of the present invention is a hemoglobin vesicle as described below, when the hemoglobin concentration is about 10 g / dL, the percentage of the volume occupied by hemoglobin vesicles in the hemoglobin vesicle dispersion is about 40% (Sakai et al., Clin Chem Lab Med. 2003; 41(2): 222-231). Therefore, when the hemoglobin concentration in the hemoglobin vesicle dispersion is about 25 g / dL, the dispersion becomes almost non-fluid. Therefore, when the hemoglobin-containing solution of the present invention is a hemoglobin vesicle, the concentration must be less than about 25 g / dL. Even when the hemoglobin-containing solution of the present invention is a self-assembling vesicle, the hemoglobin concentration at which the self-assembling vesicle dispersion becomes almost non-fluid can be determined from the percentage of the volume occupied by the self-assembling vesicles in the dispersion of the self-assembling vesicles. Therefore, when the hemoglobin-containing solution of the present invention is an artificial red blood cell, the hemoglobin concentration in the hemoglobin-containing solution of the present invention may be 10 to 50 g / dL (1.55 mM to 7.75 mM), which is the concentration of hemoglobin encapsulated in the liposomal hemoglobin, self-assembling vesicles, or artificial red blood cells, or may be 7 g / dL to 50 g / dL (1.085 mM to 7.75 mM), which is the hemoglobin concentration in the circulating blood of a subject, which is the recommended threshold for transfusion eligibility.

[0038] The hemoglobin-containing solution of the present invention contains salts, enzymes, etc. necessary for gas molecules to bind to the heme central iron or globin chains of hemoglobin and maintain the function of the subunit tetramer structure. When the hemoglobin-containing solution of the present invention encapsulates hemoglobin as red blood cells or artificial red blood cells, the hemoglobin-containing solution of the present invention contains salts, etc. necessary for stably maintaining the red blood cells or artificial red blood cells. Furthermore, when the hemoglobin-containing solution of the present invention encapsulates hemoglobin as artificial red blood cells, the hemoglobin-containing solution of the present invention may contain salts, etc., polymers, etc. in order to adjust the crystalloid osmotic pressure and colloid osmotic pressure of the artificial red blood cell dispersion to physiological conditions. The salts, etc., polymers, etc. may include physiological saline, aqueous solutions of plasma substitutes such as human serum-derived albumin, recombinant albumin, hydroxyethyl starch, dextran, and modified gelatin, electrolytes, carbohydrates, amino acids, etc. The hemoglobin-containing solution of the present invention is preferably adjusted to physiologically acceptable crystalloid osmotic pressure (200 mOsm / L to 2500 mOsm / L, colloid osmotic pressure (0 to 80 mmHg), and pH (4 to 9). The hemoglobin-containing solution of the present invention further comprises adjusting the O ２ As an allosteric factor for adjusting affinity, pyridoxal 5'-phosphate (PLP) may be added at a molar ratio to hemoglobin of 0.5 to 3. Furthermore, to delay or inhibit deterioration due to autoxidation and oxidative stress, NADH and / or NADPH may be added at a molar ratio to hemoglobin of 0.5 to 10 (International Publication No. WO2017 / 150637).

[0039] When the hemoglobin-containing solution of the present invention is a hemoglobin vesicle, the capsule of the vesicle is composed of a phosphatidylcholine-type phospholipid, 1,2-dipalmitoyl-sn-glycero-3-phosphotidylcholine (DPPC), cholesterol, a negatively charged lipid, 1,5-O-dihexadecyl-N-succinyl-glutamate (DHSG), and a lipid bound with polyethylene glycol, 1,2-distearoyl-sn-glycero-3-phosphotidylethanolamine-N-poly(oxyethylene) 5000 (molecular weight of polyethylene glycol chain: 5000, DSPE-PEG). ５０００ ), or may be composed of phosphatidylcholine-type phospholipids having different fatty acid chain lengths, or lipids to which water-soluble polymers (polyoxazoline, polysarcosine, etc.) instead of polyethylene glycol are bound.

[0040] When the hemoglobin-containing solution of the present invention is a self-assembled vesicle, the capsule of the vesicle can be composed of a compound containing a lipid such as a lipid-amphiphile precursor compound, e.g., 1-palmitoyl-2-(2S-amino-5-guanidinopentanoyl)-sn-glycero-3-phosphocholine, and an allosteric effector such as 2,3-DPG, RSR-13, inositol phosphate, inositol hexaphosphate (IP6), phytic acid, and guanosine triphosphate, as disclosed in International Publication No. WO 2021217010. The self-assembled vesicle can also contain the lipids listed as components of the hemoglobin vesicle, provided that the lipids do not interfere with the self-assembly action of the lipid-amphiphile precursor compound and the allosteric effector.

[0041] The gas ligand that binds to the heme center iron of hemoglobin of the present invention is a molecule that coordinates with the heme center iron of hemoglobin, and is O ２ The gaseous ligand that binds to the globin chain of hemoglobin of the present invention refers to a molecule derived from a gas dissolved in a hemoglobin-containing solution selected from the group consisting of CO, CO, and NO. ２The gases that can be dissolved in the hemoglobin-containing solution are O ２ gas, CO gas, NO gas and CO ２ In addition to gas, N ２ Gas, Ar gas, H ２ However, N ２ , Ar and H ２ None of these gases form coordinate bonds with the iron at the heme center of hemoglobin or with the globin chains of hemoglobin. Therefore, in the present invention, the term "dissolved gas in a hemoglobin-containing solution" is used to encompass all of the gases that form coordinate bonds with the iron at the heme center of hemoglobin, the gases that bind to the globin chains of hemoglobin, and the gases that do not directly bind to hemoglobin itself but are dissolved in the hemoglobin-containing solution.

[0042] The dissolved gas exchange membrane of the present invention is ２ , O ２ , CO, CO ２ , NO, H ２ The dissolved gas exchange membrane of the present invention diffuses and permeates as molecules, but cannot permeate as liquid or bubbles. Therefore, even when the pressure is reduced to the saturated water vapor pressure, bubbles do not form on the liquid phase side, and bumping does not occur. ２ , O ２ , CO, CO ２ , NO, H ２ The gas exchange membrane may be any gas exchange membrane having pores the size of water vapor molecules, provided that it is durable against pressurization up to 0.5 MPa and decompression down to saturated water vapor pressure, and / or is stable against aqueous sodium hydroxide solutions, surfactant solutions, and the like used for chemical cleaning of the hollow fiber membrane. The pressure limit of the dissolved gas exchange membrane of the present invention is not particularly limited, provided that it is within the durability range of the gas exchange membrane. The hollow fiber membrane modules used in the examples of the present invention (SEPAREL EF-002A and SEPAREL EF-002A-CL (custom-made) manufactured by DIC Corporation (Tokyo)) have a pressure resistance of 0.3 MPa. Also, the pressure resistance of 20M1500A manufactured by Mitsubishi Chemical Corporation is 0.3 MPa.

[0043] The positive pressure of the gas phase of the dissolved gas exchange membrane of the present invention means that the pressure on the gas phase side of the dissolved gas exchange membrane is higher than the pressure on the liquid phase side, and when the liquid phase side is not under pressure above atmospheric pressure, it means that the pressure is at least about 0.101 MPa. The negative pressure of the gas phase of the dissolved gas exchange membrane of the present invention means that the pressure on the gas phase side of the dissolved gas exchange membrane is lower than the pressure on the liquid phase side, and when the liquid phase side is not under pressure above atmospheric pressure, it means that the negative pressure is -0.098 MPa (1 atmosphere = 0.1013 MPa, saturated water vapor pressure = 0.003 MPa, so 0.003 - 0.10133 = -0.0983 MPa). This makes it possible to avoid bubbles being mixed into the liquid in the liquid phase due to bumping.

[0044] Materials for the dissolved gas exchange membrane of the present invention include, but are not limited to, substances with high gas permeability coefficients, such as silicone (dimethylsiloxane), and crystal-oriented polymers that can form micropores by stretching. The dissolved gas exchange membrane of the present invention can have a homogeneous membrane, a composite membrane, or an asymmetric membrane. Composite membranes include those consisting of a non-porous, ultra-thin layer with high gas permeability and a porous layer that mechanically supports it. Asymmetric membranes include those in which a dense layer and a porous layer are formed using the same membrane material. The dissolved gas exchange membrane of the present invention can have a planar shape or a hollow fiber shape. Hollow fiber membranes have advantages such as a larger membrane area per unit volume and superior productivity compared to flat membranes. The hollow fiber membrane as the dissolved gas exchange membrane of the present invention includes a hollow fiber membrane at least partially manufactured from a highly hydrophobic material. The highly hydrophobic material includes, but is not limited to, polyolefin resins such as poly(4-methylpentene-1), polyethylene, polypropylene, and poly(3-methylbutene-1), as well as fluorine-containing polymers such as polyvinylidene fluoride and polytetrafluoroethylene, provided that the liquid of the hemoglobin-containing solution does not permeate the hollow fiber membrane and the dissolved gas that binds to the heme central iron of hemoglobin in the hemoglobin-containing solution permeates the hollow fiber membrane as a gas. The structure of the membrane is not particularly limited as long as it comprises at least a skin layer (dense layer) and a layer having pores (porous layer), but may be a heterogeneous membrane comprising a skin layer (dense layer) and a support layer having pores (porous layer) laminated thereto, or may be a heterogeneous membrane comprising a skin layer (dense layer) on the outside and a support layer having pores (porous layer) on the inside. The pore size of the pores is not particularly limited, but may be in the range of more than 0 nm or 0.1 nm or more, or may be in the range of 100 nm or less, or 50 nm or less. When the dissolved gas exchange membrane of the present invention is a hollow fiber membrane, one surface of the dissolved gas exchange membrane is the inner surface of the hollow fiber membrane, and the other surface of the dissolved gas exchange membrane is the outer surface of the hollow fiber membrane, or one surface of the dissolved gas exchange membrane is the outer surface of the hollow fiber membrane, and the other surface of the dissolved gas exchange membrane is the inner surface of the hollow fiber membrane.When a hollow fiber membrane is used as a dissolved gas exchange membrane for a hemoglobin-containing solution, the hemoglobin-containing solution can be perfused through the inside of the hollow fiber membrane, but perfusion through the outside of the hollow fiber membrane is preferred because this increases the gas diffusion efficiency.

[0045] In a first embodiment of the present invention, in an initial dissolved gas exchange step, a hemoglobin-containing solution is exposed to a gas phase with a negative pressure up to the saturated water vapor pressure, and gases dissolved in the hemoglobin-containing solution before the exchange step, i.e., the hemoglobin-containing solution before being subjected to a dissolved gas exchange method (hereinafter referred to as "pre-gas exchange dissolved gases") are removed. The mass of the pre-gas exchange dissolved gases per unit volume of the hemoglobin-containing solution (hereinafter referred to as "pre-gas exchange dissolved gas concentration") is significantly reduced in the initial dissolved gas exchange step, but the pre-gas exchange dissolved gases are still dissolved in the hemoglobin-containing solution at a concentration of at least the pre-gas exchange dissolved gas corresponding to the partial pressure of the saturated water vapor pressure. However, by exposing the hemoglobin-containing solution to the gas phase of the required gas at positive pressure in the final dissolved gas exchange step, the dissolved gas before gas exchange is diluted by the required gas, and the dissolved gas concentration of the dissolved gas before gas exchange dissolved in the hemoglobin-containing solution after the final dissolved gas exchange step is further reduced.

[0046] In a second embodiment of the present invention, in the dissolved gas exchange method of the first embodiment of the present invention, the first dissolved gas exchange step (1) and the final dissolved gas exchange step (2) are carried out using one dissolved gas exchange membrane module.

[0047] In a second embodiment of the present invention, the dissolved gas exchange steps (1) and (2) are carried out using one dissolved gas exchange membrane module. Here, it is preferable to improve the efficiency of the dissolved gas exchange to the required gas by passing a required gas through a container storing the hemoglobin-containing solution after the dissolved gas exchange in the dissolved gas exchange steps (1) and (2) and stirring the hemoglobin-containing solution as necessary.

[0048] A third embodiment of the present invention is the dissolved gas exchange method of the first embodiment, further comprising, following the initial dissolved gas exchange step (1), (3) a second dissolved gas exchange step in which the hemoglobin-containing solution after the initial dissolved gas exchange is exposed to one surface of a second dissolved gas exchange membrane to obtain a hemoglobin-containing solution after the second dissolved gas exchange, and the other surface of the second dissolved gas exchange membrane is exposed to a gas phase at a negative pressure up to the saturated water vapor pressure or a gas phase at a positive pressure up to 0.5 MPa of the required gas.

[0049] In the first embodiment, one surface of the dissolved gas exchange membrane used in the first and last dissolved gas exchange steps is exposed to the liquid phase of the hemoglobin-containing solution, while the other surface of the dissolved gas exchange membrane used in the first dissolved gas exchange step is exposed to a gas phase at a negative pressure up to the saturated water vapor pressure, and the other surface of the last dissolved gas exchange membrane is exposed to a gas phase at a positive pressure up to 0.5 MPa of the required gas. In contrast, in the third embodiment, a second dissolved gas exchange step is included after the first dissolved gas exchange step and before the last dissolved gas exchange step, and one surface of the second dissolved gas exchange membrane is exposed to the liquid phase of the hemoglobin-containing solution, but the other surface of the second dissolved gas exchange membrane may be exposed to a gas phase at a negative pressure up to the saturated water vapor pressure, as in the first dissolved gas exchange step, or may be exposed to a gas phase at a positive pressure up to 0.5 MPa of the required gas, as in the last dissolved gas exchange step.

[0050] A fourth embodiment of the present invention is a general embodiment in which the number of all dissolved gas exchange steps is M (M is a natural number of 4 or more) in the dissolved gas exchange method of the first embodiment of the present invention. That is, the mth dissolved gas exchange step further includes a step of exposing the hemoglobin-containing solution after the (m-1)th dissolved gas exchange (m is a natural number satisfying 2≦m≦M) to one surface of the mth dissolved gas exchange membrane to obtain the hemoglobin-containing solution after the mth dissolved gas exchange, and exposing the other surface of the mth dissolved gas exchange membrane to either the same gas phase as the gas phase to which the other surface of the dissolved gas exchange membrane in the (m-1)th dissolved gas exchange step is exposed, or a gas phase of the required gas at a positive pressure of up to 0.5 MPa. For a natural number m ranging from 2 to M, "the other surface of the mth dissolved gas exchange membrane is exposed to either the same gas phase as the gas phase to which the other surface of the dissolved gas exchange membrane in the (m-1)th dissolved gas exchange step is exposed, or a gas phase of the required gas at a positive pressure of up to 0.5 MPa" means that, when m' is any natural number in the range of 2≦m'≦M, from the first dissolved gas exchange step to the (m'-1)th dissolved gas exchange step, the gas phase is at a negative pressure of up to the saturated water vapor pressure, and from the (m')th dissolved gas exchange step to the last dissolved gas exchange step, the gas phase is at a positive pressure of up to 0.5 MPa of the required gas. In other words, when the total number of dissolved gas exchange processes is four or more, the gas phase will be either a "gas phase under negative pressure up to saturated water vapor pressure" as in the first dissolved gas exchange process, or a "gas phase under positive pressure up to 0.5 MPa of the required gas" as in the last dissolved gas exchange process; once it changes from a "gas phase under negative pressure up to saturated water vapor pressure" to a "gas phase under positive pressure up to 0.5 MPa of the required gas" midway, it will remain at a "positive pressure up to 0.5 MPa of the required gas" until the last dissolved gas exchange process, and will never return to a "gas phase under negative pressure up to saturated water vapor pressure" again.

[0051] In a fifth embodiment of the present invention, in the dissolved gas exchange method of the first, third or fourth embodiment of the present invention, the hemoglobin-containing solution after the final dissolved gas exchange is recovered as the hemoglobin-containing solution after the completion of dissolved gas exchange.

[0052] A sixth embodiment of the present invention is a dissolved gas exchange method according to any one of the first and third to fifth embodiments of the present invention, wherein the hemoglobin-containing solution after the final dissolved gas exchange is mixed with the hemoglobin-containing solution before being subjected to the dissolved gas exchange step and subjected to the first dissolved gas exchange step, and the steps from the first dissolved gas exchange step to the last dissolved gas exchange step are repeated until the gas concentrations of all the hemoglobin-containing solutions after the dissolved gas exchange reach saturation, and the hemoglobin-containing solution is recovered as the hemoglobin-containing solution after the completion of the dissolved gas exchange.

[0053] In the fifth embodiment, the dissolved gas exchange is completed when one cycle of the dissolved gas exchange process is completed, and the hemoglobin-containing solution after the final dissolved gas exchange is recovered as the hemoglobin-containing solution after the dissolved gas exchange is completed. In contrast, in the sixth embodiment, the dissolved gas exchange is completed not based on the number of times the dissolved gas exchange process is repeated, but when the gas concentration of the desired gas in all hemoglobin-containing solutions after the dissolved gas exchange reaches a saturated state, and the hemoglobin-containing solution at that time is recovered as the hemoglobin-containing solution after the dissolved gas exchange is completed. Here, "the gas concentration in the hemoglobin-containing solution after the dissolved gas exchange reaches a saturated state" means that the change over time in the mass of the desired gas per unit volume of the hemoglobin-containing solution after the dissolved gas exchange of the hemoglobin-containing solution (hereinafter referred to as "dissolved gas concentration of the desired gas") reaches a plateau. The change over time in the dissolved gas concentration of the required gas in all hemoglobin-containing solutions has reached a plateau state, for example, by measuring the dissolved gas concentration of the required gas in the hemoglobin-containing solutions before the first dissolved gas exchange step or after the last dissolved gas exchange step each time the dissolved gas exchange step is repeated, and when the dissolved gas concentration of the required gas shows almost no change even as the number of times the dissolved gas exchange step is repeated increases, it is determined that the change over time in the dissolved gas concentration of the required gas in all hemoglobin-containing solutions after dissolved gas exchange has reached a plateau state. Here, "when the dissolved gas concentration of the required gas hardly changes even when the number of times the dissolved gas exchange process is repeated increases" means, for example, when the difference between the dissolved gas concentration of the required gas at the time when it is determined whether a plateau state has been reached and the dissolved gas concentration of the required gas after the dissolved gas exchange process immediately before that time is within 10% of the dissolved gas concentration of the required gas at that time, and when the difference between the dissolved gas concentration of the required gas after the dissolved gas exchange process immediately before that time and the dissolved gas concentration of the required gas after the dissolved gas exchange process two processes before that time is within 10% of the dissolved gas concentration of the required gas after the dissolved gas exchange process immediately before that time.

[0054] In a seventh embodiment of the present invention, in the dissolved gas exchange method according to any one of the first to sixth embodiments of the present invention, the dissolved gas before gas exchange is CO gas, O ２ Gas, N２ gas, air or a mixture thereof, and the required gas is CO gas, NO gas, O ２ Gas, N ２ Gas, CO ２ Gas, Ar gas, H ２ The gas may be CO gas, NO gas, O gas, or a mixture thereof. ２ Gas and air (component O ２ The gases (CO, NO, O) are dissolved gases that form coordinate bonds with the iron center of the heme. ２ When the heme is exposed to gas, air, or a mixture of these gases, it can be exchanged for a gas molecule that coordinates with the iron at the heme center. ２ gas, Ar gas and H ２ The gas is a dissolved gas that does not form a coordinate bond with the heme center iron. ２ Gases bind to the N-terminus of the globin chain of hemoglobin. ２ Gas, N ２ Gas, air or a mixture thereof is dissolved gas before gas exchange, and N ２ Gas, Ar gas, H ２ Gas, CO ２ When a desired gas or a mixture of these gases is used as the gas phase to which the other surface of the dissolved gas exchange membrane is exposed in the gas-ligand exchange step, the partial pressure of the dissolved gas in the gas phase before gas exchange is reduced, so that the dissolved gas before gas exchange dissociates from the heme iron center, leaving the heme iron unbound to any gas ligands. The dissolved gas exchange method of the present invention does not include cases where the dissolved gas before gas exchange and the desired gas are the same gas, except for cases where they differ due to the presence or absence of an isotope.

[0055] An eighth embodiment of the present invention is the dissolved gas exchange method according to any one of the first to seventh embodiments of the present invention, wherein when the heme central iron of hemoglobin in the hemoglobin-containing solution immediately before the final dissolved gas exchange is bound to CO as a gas ligand, the final dissolved gas exchange step comprises a step of photodissociating CO as a gas ligand from the hemoglobin-containing solution immediately before the final dissolved gas exchange.

[0056] In the present invention, a state in which a hemoglobin-containing solution is not substantially bound to any dissolved gas means that the partial pressure of the dissolved gas in hemoglobin bound to the dissolved gas is 1 Torr or less, 0.5 Torr or less, 0.2 Torr or less, 0.1 Torr or less, 0.05 Torr or less, 0.02 Torr or less, or 0.01 Torr or less.

[0057] Since CO2-bound hemoglobin has absorption across a wide range of visible light, any wavelength in the visible light range can be used for photodissociation. However, it is desirable to block ultraviolet and infrared rays to prevent denaturation and decomposition of the dissolved gas exchange membrane and hemoglobin due to irradiation with light for photodissociation. Therefore, sodium lamps and LED lighting are preferred as light sources. However, since these lighting devices themselves generate a considerable amount of heat, it is desirable to cool them as needed to prevent denaturation and decomposition of the dissolved gas exchange membrane and hemoglobin due to heat transfer.

[0058] A ninth embodiment of the present invention is an exchange device for dissolved gases in a hemoglobin-containing solution (hereinafter referred to as a "dissolved gas exchange device") for carrying out the dissolved gas exchange method of any one of the first and third to sixth embodiments. The dissolved gas exchange device of the ninth embodiment includes at least two dissolved gas exchange membrane modules including a first dissolved gas exchange membrane module and a last dissolved gas exchange membrane module, a container for storing the hemoglobin-containing solution, and a conduit connecting them.

[0059] In a ninth embodiment of the present invention, all of the dissolved gas exchange membrane modules include a liquid phase section, a gas phase section, and a dissolved gas exchange membrane section, and the liquid phase section and the gas phase section are arranged separated by the dissolved gas exchange membrane section, wherein the liquid in the liquid phase section is exposed to one surface of the dissolved gas exchange membrane of the dissolved gas exchange membrane section, and the gas in the gas phase section is exposed to the other surface of the dissolved gas exchange membrane of the dissolved gas exchange membrane section, and the dissolved gas exchange membrane section does not allow the liquid in the liquid phase section to pass through but exchanges the dissolved gas in the liquid with the gas in the gas phase section.

[0060] The conduit connects a container for storing the hemoglobin-containing solution before the dissolved gas exchange to the liquid supply port of the liquid phase part of the first dissolved gas exchange membrane module, and when the total number of dissolved gas exchange processes is 2, connects the liquid discharge port of the liquid phase part of the first dissolved gas exchange membrane module to the liquid supply port of the liquid phase part of the last dissolved gas exchange membrane module, and when the total number of dissolved gas exchange processes is 3, connects the liquid discharge port of the liquid phase part of the first dissolved gas exchange membrane module to the liquid supply port of the liquid phase part of the second dissolved gas exchange membrane module, and connects the liquid discharge port of the liquid phase part of the second dissolved gas exchange membrane module to the liquid supply port of the liquid phase part of the last dissolved gas exchange membrane module. are connected, and when the number of all dissolved gas exchange processes is M (M is a natural number of 4 or more), the discharge port of the liquid phase part of the (m-1)th (m is a natural number satisfying 2≦m≦M-1) dissolved gas exchange membrane module is connected to the liquid supply port of the liquid phase part of the mth dissolved gas exchange membrane module, where the first dissolved gas exchange membrane module is the first dissolved gas exchange membrane module and the Mth dissolved gas exchange membrane module is the last dissolved gas exchange membrane module, and the hemoglobin-containing solution before being subjected to the dissolved gas exchange process is passed in series from the liquid phase part of the first dissolved gas exchange membrane module to the liquid phase part of the last dissolved gas exchange membrane module.

[0061] Here, the gas phase of the first dissolved gas exchange membrane module is set to a negative pressure up to the saturated water vapor pressure, and the gas phase of the last dissolved gas exchange membrane module is set to a positive pressure of the required gas with a maximum of 0.5 MPa, thereby exchanging the required gas so that the hemoglobin-containing solution dissolves the required gas as a dissolved gas, or exchanging the dissolved gas so that the hemoglobin-containing solution does not substantially bind to any gas.

[0062] In a ninth embodiment of the present invention, the dissolved gas exchange membrane portion of the dissolved gas module includes the dissolved gas exchange membrane described in the first embodiment of the present invention.

[0063] In a ninth embodiment of the present invention, the liquid phase and gas phase of the dissolved gas exchange membrane module are separated by the dissolved gas exchange membrane, i.e., the liquid phase is in contact with one surface of the dissolved gas exchange membrane and the gas phase is in contact with the other surface of the dissolved gas exchange membrane. Here, the dissolved gas exchange membrane may have the shape of a tube or a surface.

[0064] The gas phase portion and dissolved gas exchange membrane portion of the dissolved gas exchange membrane module are made of materials that are durable against pressurization up to 0.5 MPa and decompression down to saturated water vapor pressure. The liquid phase portion and dissolved gas exchange membrane portion of the dissolved gas exchange membrane module are made of materials that are stable against sterilization procedures, such as sodium hydroxide aqueous solution and surfactant aqueous solution used for chemical cleaning of the hollow fiber membrane. The liquid phase portion, gas phase portion, and dissolved gas exchange membrane portion of the dissolved gas exchange membrane module are made of materials that are stable against sterilization procedures, etc. In the present invention, the hemoglobin-containing solution in the liquid phase portion does not permeate the dissolved gas exchange membrane, but gas dissolved in the hemoglobin-containing solution in the liquid phase portion can be exchanged with gas in the gas phase portion. When the gas dissolved in the hemoglobin-containing solution in the liquid phase portion is exchanged, the dissolved gas that binds to the heme central iron of hemoglobin in the solution is also exchanged.

[0065] As described in the first embodiment of the present invention, the material of the dissolved gas exchange membrane in the dissolved gas exchange membrane section of the dissolved gas exchange membrane module includes, but is not limited to, a substance with a high gas permeability coefficient, such as silicone (dimethylsiloxane), or a crystal-oriented polymer that can form micropores by stretching. The hollow fiber membrane as the dissolved gas exchange membrane of the present invention includes a hollow fiber membrane at least partially manufactured from a highly hydrophobic material. The highly hydrophobic material includes, but is not limited to, polyolefin resins such as poly(4-methylpentene-1), polyethylene, polypropylene, and poly(3-methylbutene-1), as well as fluorine-based polymers such as polyvinylidene fluoride and polytetrafluoroethylene, provided that the liquid of the hemoglobin-containing solution does not permeate the hollow fiber membrane and the dissolved gas that binds to the heme central iron of hemoglobin in the hemoglobin-containing solution permeates the hollow fiber membrane as a gas.

[0066] Of the dissolved gas exchange membrane portion of the dissolved gas exchange membrane module, the portion other than the dissolved gas exchange membrane, and the liquid phase portion and gas phase portion of the dissolved gas exchange membrane module are formed from materials such as polypropylene, rigid polyvinyl chloride, Teflon (registered trademark), acrylic, polycarbonate, stainless steel, etc.

[0067] The housing of the dissolved gas exchange membrane module, i.e., the casing that entirely covers the liquid phase section, the gas phase section, and the dissolved gas exchange membrane section, is formed from materials such as polypropylene, rigid polyvinyl chloride, Teflon (registered trademark), acrylic, polycarbonate, stainless steel, etc. Since the dissolved gas exchange membrane and hemoglobin need to be protected from infrared and ultraviolet rays, it is desirable that the housing of the dissolved gas exchange membrane module be light-blocking, except for the embodiment in which the photodissociation reaction described below is performed.

[0068] The pressure limit of the dissolved gas exchange membrane of the present invention is not particularly limited, provided that it is within the durability range of the gas exchange membrane. The hollow fiber membrane modules (SEPAREL EF-002A and SEPAREL EF-002A-CL (custom-made) manufactured by DIC Corporation (Tokyo)) used in the examples of the present invention have a pressure resistance of 0.3 M. The 20M1500A manufactured by Mitsubishi Chemical Corporation also has a pressure resistance of 0.3 M.

[0069] The dissolved gas exchange apparatus of the ninth embodiment of the present invention may include at least two containers, including a container for storing hemoglobin-containing solution before dissolved gas exchange and a container for storing hemoglobin-containing solution after dissolved gas exchange is completed. Alternatively, the dissolved gas exchange apparatus of the ninth embodiment of the present invention may include only one container for storing hemoglobin-containing solution. In the dissolved gas exchange apparatus of the ninth embodiment of the present invention having one container, the hemoglobin-containing solution after dissolved gas exchange is completed is returned to the container for storing hemoglobin-containing solution before dissolved gas exchange. The container for storing the hemoglobin-containing solution may be made of any material, provided that it has low (or no) gas permeability to the gas used for dissolved gas exchange. Preferred materials for the container include metals such as glass and stainless steel, and it is essential that the container be capable of alkaline cleaning or high-pressure steam sterilization. The container for storing the hemoglobin-containing solution is airtight and liquid-tight, and includes a stirrer for stirring the hemoglobin-containing solution, a sensor for detecting gas dissolved in the hemoglobin-containing solution and / or a gas present in the space above the liquid surface of the hemoglobin-containing solution, e.g., O ２ The probe (O ２The hemoglobin-containing solution may be provided with a probe, a conduit for guiding the hemoglobin-containing solution stored in the container to the inlet of the dissolved gas exchange membrane module, a conduit for guiding the hemoglobin-containing solution from the outlet of the dissolved gas exchange membrane module to the container, a conduit for blowing a required gas into the space above the liquid level of the hemoglobin-containing solution in the container, and a conduit for discharging the required gas from the space above the liquid level of the hemoglobin-containing solution in the container. The stirrer for stirring the hemoglobin-containing solution may be an agitator installed at the bottom of the container, and may be stirred at a stirring speed (e.g., about 300 rpm) that does not cause foaming of the stored hemoglobin-containing solution. The gas sensor may transmit data via a wired or wireless connection to a computer for control. The conduit for blowing the required gas into the space above the liquid level of the hemoglobin-containing solution in the container may be provided with a flow regulator so that the inflow pressure or inflow volume per unit time of the required gas blown into the container is set to a constant value, for example, 1 L per minute. The conduit for discharging the required gas from the space above the liquid level of the hemoglobin-containing solution in the container may be open to atmospheric pressure, or a pressure regulator may be used to maintain a constant gas pressure in the space above the liquid level of the hemoglobin-containing solution in the container.

[0070] The dissolved gas exchange device of the present invention is manufactured so that the entire device can be sterilized. Sterilization treatments include, but are not limited to, high-pressure steam sterilization using an autoclave, gas sterilization using ethylene oxide gas, and / or chemical sterilization using a disinfectant solution. The dissolved gas exchange device of the present invention may be provided with a filter at the intake port that receives gas used for dissolved gas exchange into the tank or hollow fiber membrane module, for capturing microorganisms and other particles suspended in the gas. Furthermore, the dissolved gas exchange device of the present invention may be provided with a filter at the exhaust port of the tank or hollow fiber membrane module, which is open to atmospheric pressure, for capturing microorganisms and other particles suspended in the outside air that flows into the exhaust port due to changes in air pressure. Alternatively, the dissolved gas exchange device of the present invention and the entire supply source of gas used for dissolved gas exchange may be installed in a so-called clean room, where microorganisms and other particles have been removed.

[0071] The conduit used in the dissolved gas exchange device of the ninth embodiment of the present invention is a conduit for CO gas, NO gas, O gas, etc. ２ Gas, N ２ Gas, CO ２ Gas, Ar gas, H ２ The conduit is made of a material that has low permeability to gas, air, or a mixture thereof. A preferred material for the conduit is metal such as stainless steel, but if flexibility is required for the piping, materials include, but are not limited to, butyl rubber, high nitrile rubber, urethane rubber, polysulfide rubber, and epichlorohydrin rubber. A preferred example of a commercially available conduit is PharMed® BPT, a polypropylene-based thermoplastic elastomer manufactured by Saint-Gobain Performance Plastics.

[0072] The dissolved gas exchange device of the ninth embodiment of the present invention may use a pump to deliver the hemoglobin-containing solution. Because the hemoglobin-containing solution of the present invention is used for human treatment, a rotor pump that pushes the flexible conduit from the outside to push out the hemoglobin-containing solution in the conduit is preferred from the viewpoint of safety to living bodies. A preferred rotor pump is, for example, any delivery pump (e.g., Easy-Load (registered trademark), Masterflex (registered trademark), Avantor, Inc.) that can flow the hemoglobin-containing solution at a flow rate of about 600 mL per minute.

[0073] In the ninth embodiment of the dissolved gas exchange apparatus of the present invention, when the gas phase of the dissolved gas exchange membrane module is pressurized positively, a required gas at a constant pressure may be supplied to the gas phase using a high-pressure cylinder and a pressure regulator. In the ninth embodiment of the dissolved gas exchange apparatus of the present invention, when the gas phase of the dissolved gas exchange membrane module is pressurized negatively, a vacuum pump may be used. Vacuum pumps based on various principles are known to be suitable for the dissolved gas exchange apparatus of the present invention. Mechanical vacuum pumps that do not use oil or liquid, such as diaphragm-type dry vacuum pumps, are particularly suitable for applications requiring a clean atmosphere free from contamination by oil vapors, as they are capable of clean exhaust and achieving a high degree of vacuum. In the present invention, it is necessary to be able to reduce the pressure down to water vapor pressure, and to be able to remove water from the pump in the event of condensation of water vapor. When the gas phase of the dissolved gas exchange membrane module is pressurized negatively, the degree of negative pressure can be set using a pressure regulator, such as a needle valve unit (AS ONE Corporation).

[0074] The dissolved gas exchange device of the ninth embodiment of the present invention is a device for storing dissolved O in the hemoglobin-containing solution in at least one of a container for storing the hemoglobin-containing solution before being subjected to dissolved gas exchange and a container for storing the hemoglobin-containing solution after the completion of dissolved gas exchange. ２ O for measuring concentration ２ A probe may be placed on the interior wall of the vessel.

[0075] A dissolved gas exchange apparatus according to a tenth embodiment of the present invention is the dissolved gas exchange apparatus according to the ninth embodiment, wherein the dissolved gas exchange membrane modules are three in total, and the gas phase of the second dissolved gas exchange membrane module following the first dissolved gas exchange membrane module is either at a negative pressure up to the saturated water vapor pressure or at a positive pressure up to 0.5 MPa of the required gas. While the ninth embodiment requires at least two dissolved gas exchange membrane modules in total, the tenth embodiment requires three. The dissolved gas exchange apparatus according to the tenth embodiment corresponds to the dissolved gas exchange method according to the third embodiment.

[0076] The dissolved gas exchange apparatus of an eleventh embodiment of the present invention is the dissolved gas exchange apparatus of the ninth embodiment, wherein the total number of dissolved gas exchange membrane modules is N (N is a natural number of 4 or more), and the gas phase portions of the dissolved gas exchange membrane modules from the first to the nth (n is a natural number satisfying the range of 2≦n≦N−1) dissolved gas exchange membrane modules are all at a negative pressure up to the saturated water vapor pressure, and the gas phase portions of all dissolved gas exchange membrane modules from the n+1th dissolved gas exchange membrane module to the last dissolved gas exchange membrane module are at a positive pressure of up to 0.5 MPa for the required gas. The dissolved gas exchange apparatus of the eleventh embodiment corresponds to the dissolved gas exchange method of the fourth embodiment.

[0077] A twelfth embodiment of the dissolved gas exchange apparatus of the present invention is the dissolved gas exchange apparatus of any one of the ninth to eleventh embodiments, in which the hemoglobin-containing solution that has passed through the liquid phase portion of the last dissolved gas exchange membrane module is recovered as the hemoglobin-containing solution after completion of dissolved gas exchange, or the hemoglobin-containing solution that has passed through the liquid phase portion of the last dissolved gas exchange membrane module is introduced into a container for storing the hemoglobin-containing solution before being subjected to the dissolved gas exchange step, mixed with the hemoglobin-containing solution before being subjected to the dissolved gas exchange step, and introduced into the liquid phase portion of the first dissolved gas exchange membrane module, thereby repeating the dissolved gas exchange. The dissolved gas exchange apparatus of the twelfth embodiment corresponds to the dissolved gas exchange method of the fifth or sixth embodiment.

[0078] A dissolved gas exchange apparatus according to a thirteenth embodiment of the present invention is the dissolved gas exchange apparatus according to any one of the ninth to twelfth embodiments, wherein the dissolved gas before gas exchange is CO gas, O ２ Gas, N ２ gas, air or a mixture thereof, and the required gas is CO gas, NO gas, O ２ Gas, N ２ Gas, CO ２ Gas, Ar gas, H ２ The dissolved gas exchange device of the thirteenth embodiment corresponds to the dissolved gas exchange method of the seventh embodiment. The dissolved gas exchange device of the present invention does not include cases where the dissolved gas before gas exchange and the desired gas are the same gas, except for cases where there is no difference due to the presence or absence of an isotope.

[0079] The gas used in the dissolved gas exchange method and dissolved gas exchange apparatus according to the embodiment of the present invention is a CO / N ratio of 1-100% in the step of binding CO as a dissolved gas to the heme center iron of hemoglobin. ２ Then, CO is dissociated from the dissolved gas in the iron at the heme center of hemoglobin to form O ２ In the step of binding, 10-100% O ２ / N ２ Mixed gas, O ２ A mixed gas of O / Ar or air may be used. ２ In the step of removing N with a purity of 99.95% or more ２ gas, Ar gas, and H ２ H mixed with 1-5% ２ / N ２ Mixed gas, H ２ / Ar mixed gas. ２ does not bind to hemoglobin, but is physically dissolved in the dispersion of the artificial red blood cell preparation. ２ When a mixed gas containing the above is dissolved in a hemoglobin-containing solution, for example, in the case where the hemoglobin-containing solution is artificial red blood cells, active oxygen in the body can be eliminated and ischemia-reperfusion injury can be reduced when the artificial red blood cells are administered to a living body.

[0080] A fourteenth embodiment of the dissolved gas exchange device of the present invention is the dissolved gas exchange device of any one of the ninth to thirteenth embodiments, wherein the dissolved gas exchange membrane is a hollow fiber membrane. When the dissolved gas exchange membrane is a hollow fiber membrane, the lumen may be the gas phase and the exterior may be the liquid phase, or the lumen may be the liquid phase and the exterior may be the gas phase. When the dissolved gas exchange membrane is a hollow fiber membrane and the lumen is the liquid phase and the exterior is the gas phase, the liquid phase branches in parallel from the liquid inlet of the dissolved gas exchange membrane module, passes through the lumen of the hollow fiber membrane, and then joins and extends to the liquid outlet of the dissolved gas exchange membrane module. The gas phase extends from the air inlet of the dissolved gas exchange membrane module, passes outside the dissolved gas exchange membrane, and extends to the exhaust port of the dissolved gas exchange membrane module. When the dissolved gas exchange membrane is a hollow fiber membrane with its inner cavity as a gas phase and its exterior as a liquid phase, the gas phase branches in parallel from the air inlet of the dissolved gas exchange membrane module, passes through the inner cavity of the hollow fiber membrane, and then joins together and extends to the exhaust port of the dissolved gas exchange membrane module. The liquid phase extends from the liquid inlet of the dissolved gas exchange membrane module, passes outside the hollow fiber membrane, and extends to the discharge port of the dissolved gas exchange membrane module.

[0081] A fifteenth embodiment of the present invention is the dissolved gas exchange device of any one of the ninth to fourteenth embodiments, further comprising a light source, the final dissolved gas exchange membrane module having a transparent housing, the gas phase of the final dissolved gas exchange membrane module being kept at a negative pressure up to the saturated water vapor pressure, and the light source disposed outside the housing of the dissolved gas exchange membrane module irradiates the hemoglobin-containing solution in the liquid phase of the dissolved gas exchange membrane module with light, thereby photodissociating the dissolved gas CO from the heme central iron of hemoglobin in the hemoglobin-containing solution passing through the liquid phase. The housing is preferably made of a highly light-transmitting material such as acrylic resin, polycarbonate, or glass.

[0082] In a dissolved gas exchange device according to a fifteenth embodiment of the present invention, CO photodissociated from the iron heme center of hemoglobin in a hemoglobin-containing solution is discharged via a dissolved gas exchange membrane module. Because CO is toxic, the dissolved gas exchange device is preferably installed in a draft chamber. Gases potentially containing CO from the exhaust ports of the draft chamber and the dissolved gas exchange membrane module are forcibly exhausted through an exhaust duct by an exhaust fan. If necessary, CO may be removed by an exhaust gas treatment device, and the CO-free exhaust may then be released into the atmosphere.

[0083] Hemoglobin bound to CO2 has absorption across a wide range of visible light, and any wavelength in the visible light range can be used for photodissociation. Possible light sources include tungsten lamps, halogen lamps, sodium lamps, and LED lighting. However, sodium lamps and LED lighting are preferred because they block ultraviolet and infrared rays to prevent denaturation and decomposition of the dissolved gas exchange membrane module and hemoglobin. Furthermore, since these lighting devices themselves generate considerable heat, it is desirable to cool them as needed to prevent denaturation and decomposition of the dissolved gas exchange membrane module and hemoglobin.

[0084] A sixteenth embodiment of the present invention is a system for exchanging dissolved gases in a hemoglobin-containing solution (hereinafter referred to as a "dissolved gas exchange system"). A dissolved gas exchange system according to a sixteenth embodiment of the present invention includes at least P groups (P is a natural number of 2 or more) of the dissolved gas exchange devices according to any one of the ninth to fourteenth embodiments, and introduces hemoglobin-containing solution stored in a container for storing hemoglobin-containing solution after completion of dissolved gas exchange in the (p-1)th (p is a natural number satisfying 2≦p≦P-1) dissolved gas exchange device among the dissolved gas exchange devices according to any one of the ninth to fourteenth embodiments of the present invention into a container for storing hemoglobin-containing solution before being subjected to the dissolved gas exchange in the pth dissolved gas exchange device, and fills the hemoglobin-containing solution requiring dissolved gas exchange in a container for storing hemoglobin-containing solution before dissolved gas exchange to a required gas in the first dissolved gas exchange device among the dissolved gas exchange devices according to any one of the ninth to fourteenth embodiments of the present invention, In any one of the 9th to 14th embodiments of the dissolved gas exchange device of the present invention, the hemoglobin-containing solution stored in the container for storing the hemoglobin-containing solution after the completion of dissolved gas exchange in the pth dissolved gas exchange device is the hemoglobin-containing solution for which dissolved gas exchange has been completed, and the dissolved gas exchanged in the first dissolved gas exchange membrane module of the first dissolved gas exchange device is different from the dissolved gas exchanged in the last dissolved gas exchange membrane module of the pth dissolved gas exchange device.

[0085] The dissolved gas exchange system of the 17th embodiment of the present invention comprises at least Q (Q is a natural number of 2 or more) dissolved gas exchange devices of any one of the 9th to 14th embodiments and one dissolved gas exchange device of the 15th embodiment. The hemoglobin-containing solution stored in a container for storing hemoglobin-containing solution after completion of dissolved gas exchange in the q-1th (q is a natural number satisfying 2≦q≦Q-1) dissolved gas exchange device of any one of the 9th to 14th embodiments of the present invention is introduced into a container for storing hemoglobin-containing solution before dissolved gas exchange to the required gas in the qth dissolved gas exchange device of any one of the 9th to 14th embodiments of the present invention; The hemoglobin-containing solution stored in a container for storing hemoglobin-containing solution after completion of dissolved gas exchange in the qth dissolved gas exchange device of any one of the 9th to 14th embodiments of the present invention is introduced into a container for storing hemoglobin-containing solution before dissolved gas exchange to the required gas in the dissolved gas exchange device of the 15th embodiment; In the dissolved gas exchange device of any one of the 9th to 14th embodiments of the present invention, a container for storing hemoglobin-containing solution before dissolved gas exchange to a required gas in the first dissolved gas exchange device is filled with hemoglobin-containing solution requiring dissolved gas exchange; the hemoglobin-containing solution stored in a container for storing hemoglobin-containing solution after dissolved gas exchange in the dissolved gas exchange device of the 15th embodiment is used as the hemoglobin-containing solution for which dissolved gas exchange has been completed; and in the dissolved gas exchange device of any one of the 9th to 14th embodiments of the present invention, the dissolved gas exchanged in the first dissolved gas exchange membrane module of the first dissolved gas exchange device is different from the dissolved gas exchanged in the last dissolved gas exchange membrane module of the dissolved gas exchange device of the 15th embodiment.

[0086] The dissolved gas exchange system of the 16th embodiment of the present invention is different in that it is composed only of a dissolved gas exchange device of any one of the 9th to 14th embodiments, whereas the dissolved gas exchange system of the 17th embodiment of the present invention is composed of a dissolved gas exchange device of any one of the 9th to 14th embodiments and a dissolved gas exchange device of the 15th embodiment.

[0087] The method for producing a hemoglobin-containing solution according to the eighteenth embodiment of the present invention comprises the steps of: ２ The present invention also includes a step of obtaining a hemoglobin-containing solution in which the iron at the heme center of hemoglobin does not bind oxygen by the dissolved gas exchange method of any one of the first to seventh embodiments of the present invention, which does not contain any gas, and the oxygen partial pressure of the hemoglobin-containing solution is 0.1 Torr or less. The hemoglobin-containing solution in which the iron at the heme center of hemoglobin does not bind oxygen, which is produced by the eighteenth embodiment of the present invention, is HbO in which the iron at the heme center of hemoglobin binds oxygen. ２ Unlike erythrocytes, the iron at the heme center does not oxidize to form trivalent methemoglobin (metHb) during storage.

[0088] A nineteenth embodiment of the present invention relates to an apparatus for exchanging dissolved gases in a hemoglobin-containing solution, which is an apparatus for carrying out the dissolved gas exchange method of the second embodiment of the present invention, and which comprises one dissolved gas exchange membrane module, at least one container, and a conduit connecting them. The dissolved gas exchange membrane module comprises a liquid phase section, a gas phase section, and a dissolved gas exchange membrane section, and the liquid phase section and the gas phase section are arranged across the dissolved gas exchange membrane section, wherein the liquid in the liquid phase section is exposed to one surface of the dissolved gas exchange membrane of the dissolved gas exchange membrane section, and the gas in the gas phase section is exposed to the other surface of the dissolved gas exchange membrane of the dissolved gas exchange membrane section, and the dissolved gas exchange membrane section does not permeate the liquid in the liquid phase section but exchanges the dissolved gas in the liquid with the gas in the gas phase section. (a) When performing the first dissolved gas exchange step (1) in the dissolved gas exchange method of the second embodiment of the present invention, the conduit connects the container to the liquid supply port of the liquid phase part of the dissolved gas exchange membrane module, and connects the liquid discharge port of the liquid phase part of the dissolved gas exchange membrane module to the container, and thereby reduces the partial pressure of the dissolved gas in the hemoglobin-containing solution by creating a negative pressure in the gas phase part of the dissolved gas exchange membrane module up to the saturated water vapor pressure. (b) When carrying out the final dissolved gas exchange step (2) in the dissolved gas exchange method of the second embodiment of the present invention, the conduit connects the second container to the liquid supply port of the dissolved gas exchange membrane module, and connects the liquid discharge port of the dissolved gas exchange membrane module to the first container, and the gas phase of the dissolved gas exchange membrane module is made a positive gas phase of up to 0.5 MPa of the required gas, thereby replacing the dissolved gas in the hemoglobin-containing solution with the required gas. According to the dissolved gas exchange device for hemoglobin-containing solution of the nineteenth embodiment of the present invention, the dissolved gas exchange method of the second embodiment of the present invention can be carried out with just one dissolved gas exchange membrane module.

[0089] In this specification, the conjunction "about" used to modify a numerical value means a numerical range of 90% or more and 110% or less of the numerical value. For example, ２ The partial pressure of "about 0.10 Torr" means that the O ２The partial pressure is a value within a range including the lower and upper limits of 0.09 Torr and 0.11 Torr, respectively.

[0090] All documents mentioned herein are incorporated by reference in their entirety.

[0091] The following examples of the present invention are for illustrative purposes only and do not limit the technical scope of the present invention. The technical scope of the present invention is limited only by the claims. Modifications of the present invention, such as additions, deletions, and substitutions of constituent elements of the present invention, can be made without departing from the spirit of the present invention.

[0092] Example 1 In this example, deoxygenation of saline was carried out using a hollow fiber membrane module (HFMM). As shown in FIG. 1-A, approximately 1 L of saline and a stirrer were placed in a tank (reservoir) while maintaining airtightness, and the mixture was stirred with a stirrer. A glass container (manufactured by PYREX (registered trademark)) was used as the airtight tank, and Otsuka Saline Injection (manufactured by Otsuka Pharmaceutical Factory, Inc., Naruto City, Tokushima Prefecture) was used as the saline. Stirring was carried out at approximately 300 rpm with a powerful magnetic stirrer (RCX-1100, manufactured by Tokyo Rikakikai Co., Ltd., Tokyo). A SEPAREL EF-002A (liquid contact area approximately 1.8 m) was used as the hollow fiber membrane module. ２One unit of a PTFE-based pump (manufactured by DIC Corporation, Tokyo) was used. An Easy-Load (registered trademark), Masterflex (registered trademark), manufactured by Avantor, Inc., model number 07557-02, was used as the liquid feed pump. Physiological saline was fed from the glass container to the liquid feed port of the liquid phase section of the hollow fiber membrane module at a flow rate of approximately 600 mL per minute by the liquid feed pump. The physiological saline discharged from the liquid drain port of the liquid phase section of the hollow fiber membrane module was returned to the glass container. The tube used for liquid feed was PharMed (registered trademark) tubing (#L / S) manufactured by Saint-Gobain Life Sciences. 36) was used. A vacuum source was connected to both the air inlet and exhaust port of the gas phase part of the hollow fiber membrane module. As a vacuum source, a diaphragm type vacuum pump (Tokyo Rikakikai Co., Ltd., EVP-1100 model) was connected via a pressure-resistant tube (silicone exhaust tube, inner diameter 5 mm, outer diameter 19 mm, AS ONE Corporation), and degassing was performed at a negative pressure of approximately -0.098 MPa. The level of negative pressure was adjusted using a needle valve unit (AS ONE Corporation). The glass container was supplied with N at a rate of 1 L per minute. ２ While supplying gas, the gas phase of the tank was released to atmospheric pressure. ２ A probe (Oxygen Sensor Spot SP-PSt6-YAU-D5-YOP, manufactured by PreSens Precision Sensing GmbH, Germany) was installed to measure O in the liquid and gas phases. ２ The O partial pressure was measured from the outside of the container using a Fibox 3 (manufactured by PreSens Precision Sensing GmbH). ２ The probe was irradiated with laser light, and the fluorescence lifetime was used to determine the O ２ The change in partial pressure over time was measured. In Figure 1-A, the liquid flow path is shown with a thicker line than the gas flow path.

[0093] FIG. 1-B shows the O of physiological saline in Example 1. ２ 1 is a graph showing the change in partial pressure over time. The vertical axis of the graph is the O partial pressure of the saline solution in the glass container. ２ The partial pressure (unit: Torr) is on the horizontal axis, and the elapsed time (unit: minutes) is on the horizontal axis. As shown in the graph, the O ２The partial pressure dropped to 1.00 Torr after 10 minutes, but after 20 minutes, ２ The rate of decrease in partial pressure slowed down, and it did not reach 0.10 Torr even after 60 minutes. ２ The partial pressure reached 0.03 Torr after 3 minutes, and dropped to less than 0.01 Torr after 5 minutes. In principle, degassing using a negative pressure lower than the saturated water vapor pressure (24 Torr, 32 hPa) is thought to be inefficient.

[0094] Example 2 In this example, as shown in FIG. 2-A, N 2 was introduced from the gas inlet of the gas phase of a hollow fiber membrane module (SEPAREL EF-002A). ２ The physiological saline solution was deoxygenated under the same conditions as in Example 1, except that a vacuum pump was connected to the exhaust port to degas the solution at a negative pressure of approximately -0.090 MPa while introducing oxygen. In Figure 2-A, the liquid flow path is indicated by a thicker line than the gas flow path.

[0095] FIG. 2-B shows the O ２ 1 is a graph showing the change in partial pressure over time. As shown in the graph, the O 2 in the liquid phase of the glass vessel ２ The partial pressure dropped to 1.00 Torr after 5 minutes, but after 10 minutes, ２ The rate of decrease in partial pressure slowed down, and even after 60 minutes, it was still 0.13 Torr.

[0096] Example 3 In this example, as shown in Figure 3-A, two hollow fiber membrane modules (SEPAREL EF-002A) were prepared, and the gas phase of the first hollow fiber membrane module (center), into which saline solution flowed from the glass container, was degassed at a negative pressure of approximately -0.098 MPa by connecting a vacuum pump to both the air inlet and the exhaust port. Saline solution from the liquid phase outlet of the first hollow fiber membrane module was introduced into the liquid phase inlet of the last hollow fiber membrane module (left). The gas phase of the last hollow fiber membrane module was degassed at a rate of approximately 1 L N per minute from the air inlet. ２ Gas was supplied and the exhaust port was released to atmospheric pressure. The physiological saline solution was deoxygenated under the same conditions as in Example 1. In Figure 3-A, the liquid flow path is indicated by a thicker line than the gas flow path.

[0097] FIG. 3-B shows the O２ 1 is a graph showing the change in partial pressure over time. The vertical axis of the graph is the O partial pressure of the saline solution in the glass container. ２ The graph shows partial pressure (unit: Torr) and the horizontal axis shows elapsed time (unit: minutes). As shown in the graph, the O ２ The partial pressure dropped to 0.1 Torr after 34 minutes, and to 0.07 Torr after 90 minutes. ２ The partial pressure reached less than 0.01 Torr after 3 minutes. From the results of Example 3, it was found that saline was passed through two hollow fiber membrane modules in sequence, and the gas phase of the first hollow fiber membrane module was degassed by applying negative pressure up to the saturated water vapor pressure, and the gas phase of the last hollow fiber membrane module was degassed by applying N ２ Gas insufflation was the most effective at deoxygenating saline.

[0098] FIG. 3-C shows the O2 concentration of physiological saline in Examples 1 and 3. ２ Partial pressure of O ２ The time-dependent change curves at partial pressures of 0.20 Torr or less (Figs. 1B and 3B, respectively) are shown for O ２ 1 is a graph plotting partial pressure (unit: Torr) on the vertical axis and elapsed time (unit: minutes) on the horizontal axis. ２ The change in partial pressure over time is shown. The black circles represent the O ２ 3-C clearly shows that the deoxygenation efficiency of saline solution is higher under the conditions of Example 3 than under the conditions of Example 1.

[0099] Example 4 In this example, as shown in FIG. 4-A, deoxygenation of a human red blood cell dispersion was carried out under the same conditions as in Example 3. The human red blood cell dispersion was prepared by diluting human concentrated red blood cells, donated for a fee by the Japanese Red Cross Society two weeks after collection, with physiological saline to a hemoglobin concentration of approximately 11.5 g / dL. This is slightly higher than the concentration of approximately 10 g / dL of the inventors' artificial red blood cells (HbV, see International Publication No. WO 2017 / 150637 and others). The human red blood cell dispersion was deoxygenated under the same conditions as in Example 3, except that the human red blood cell dispersion was used instead of physiological saline. In FIG. 4-A, the liquid flow path is indicated by a thicker line than the gas flow path.

[0100] FIG. 4-B shows the O of the human red blood cell dispersion in Example 4. ２ 1 is a graph showing the change in partial pressure over time. The vertical axis of the graph is the O partial pressure of the human red blood cell dispersion in the glass container. ２ The partial pressure (unit: Torr) is on the horizontal axis, and the elapsed time (unit: minutes) is on the horizontal axis. As shown in the graph, the O ２ The partial pressure dropped to 0.09 Torr after 35 minutes, and to 0.01 Torr after 65 minutes. ２ The time course curve of partial pressure is the O ２ Unlike the time-dependent change curve of partial pressure, the curve was S-shaped. ２ This is because an allosteric effect is observed in the change in the binding state with the liquid phase O ２ The partial pressure initially dropped to 0.01 Torr, and then showed a tendency to rise. This was because water had accumulated in the vacuum pump that degassed the gas phase of the first hollow fiber membrane module, causing the negative pressure to rise to -0.08 MPa. The human red blood cell dispersion before deoxygenation was bright red, but after deoxygenation, the human red blood cell dispersion turned dark reddish purple. The O in the gas phase of the glass container ２ The partial pressure reached less than 0.01 Torr after 10 minutes. This result suggests that deoxygenation can be achieved not only in human red blood cells but also in various hemoglobin-containing solutions.

[0101] In this example, an HbV dispersion was deoxygenated as shown in Figure 5-A. Deoxygenation was carried out under the same conditions as in Example 4, except that an HbV dispersion with a hemoglobin concentration of 10 g / dL was used. In Figure 5-A, the liquid flow path is indicated by a thicker line than the gas flow path.

[0102] FIG. 5-B shows the O of the HbV dispersion in Example 5. ２ 1 is a graph showing the change in partial pressure over time. As shown in the graph, the O 2 partial pressure in the liquid phase of the airtight glass container ２ The partial pressure fell below 0.1 Torr after 59 minutes and reached less than 0.01 Torr after 79 minutes. ２The partial pressure reached less than 0.01 Torr after 21 minutes. The HbV dispersion before deoxygenation was bright red, but after deoxygenation, the HbV dispersion turned dark reddish purple.

[0103] Example 6 In this example, as shown in Figure 6, the HbV dispersion deoxygenated in Example 5 was converted to CO. Immediately after the experiment in Example 5, the decompression of the gas phase of the first hollow fiber membrane module was stopped and returned to normal pressure, and CO gas was supplied to the gas phase of the last hollow fiber membrane module through the gas inlet at a rate of less than about 1 L per minute, and the exhaust port was released to atmospheric pressure. The HbV dispersion was converted to CO under the same conditions as in Example 3, except for the above. In Figure 6, the liquid flow path is indicated by a thicker line than the gas flow path.

[0104] Ten minutes after the start of CO2 conversion of the HbV dispersion, a clear change in color tone was observed, and the HbV dispersion turned bright red. At 10, 20, and 30 minutes after the start of CO2 conversion, a small amount of the HbV dispersion was sampled from the glass container, diluted with physiological saline, and subjected to visible-ultraviolet spectral analysis (V-650 equipped with a 60 mm integrating sphere, manufactured by JASCO Corporation). A peak at approximately 419 nm in the Soret band characteristic of HbCO was detected in the HbV dispersion sample after 10 minutes. The spectral analysis showed almost no change in the shape of the peak at approximately 419 nm and the surrounding spectrum for the HbV dispersion samples after 20 and 30 minutes. Therefore, it was determined that the CO2 conversion of the HbV dispersion was completed 10 minutes after the start of CO2 conversion. Furthermore, the O2 concentration of the HbV dispersion was 0.01%. ２ The partial pressure rose slightly to 0.3 Torr during the CO conversion of the HbV dispersion. This was because the gas phase of the first hollow fiber membrane module was left without being degassed, and O ２ It was thought that this was caused by the infiltration of HbV into the HbV dispersion.

[0105] In this example, as shown in FIG. 7-A, a model of a CO-bound HbV dispersion was prepared by dispersing CO-bound human red blood cells in saline (hemoglobin concentration: approximately 9.9 g / dL) in about 1 L of the solution, which was placed in a glass container. The CO bound to the hemoglobin was dissociated by a photoreaction, and the CO was released. ２The gas phase of the first hollow fiber membrane module (SEPAREL EF-002A) was O ２ Gas was supplied at a rate of approximately 1 L / min, and the exhaust port was opened to atmospheric pressure. The second hollow fiber membrane module had an acrylic resin housing and was identical in dimensions (a custom-made SEPAREL EF-002A-CL manufactured by DIC Corporation (Tokyo)). Treatment was performed at a flow rate of approximately 600 mL / min. A sodium lamp (high-pressure sodium lamp, EYE Sunlux Ace 360 ​​W; Iwasaki Electric Co., Ltd.) was used to illuminate the module so that light could reach the interior through the transparent housing. To prevent the temperature of the hollow fiber module from rising due to heat transfer from the light irradiation device, the hollow fiber module was immersed in a stainless steel container filled with cold water (approximately 15-20°C). The light from the sodium lamp was reflected by the stainless steel container and irradiated onto the back side of the hollow fiber module. The module was degassed by applying a negative pressure of approximately -0.098 MPa using a vacuum pump. In Figure 7-A, the liquid flow path is indicated by a thicker line than the gas flow path.

[0106] After the start of the decarbonation procedure, small amounts of the red blood cell dispersion were collected from the glass container over time, and spectral analysis in the visible-ultraviolet region was performed using the following procedure. First, approximately 10 μL of the collected sample was placed in a polystyrene cuvette (1 cm path length) containing 3 mL of phosphate-buffered saline, and a small amount of sodium dithionite powder was added. The cuvette was sealed with parafilm and mixed by inversion. The spectrum was measured using a UV-visible spectrophotometer (V-650, equipped with a 60 mm integrating sphere, manufactured by JASCO Corporation). As shown in Figure 7-B, the peak at approximately 419 nm in the Soret band characteristic of HbCO began to disappear, and instead, a peak at approximately 430 nm characteristic of deoxyHb began to be detected. As shown in Figure 7-C, the HbCO ratio decreased to 40% after 100 minutes, to 24% after 210 minutes, and to 6.5% after 420 minutes. From these results, it was considered that the decarbonization procedure could be carried out in the same manner for CO-bound HbV dispersions.

[0107] In this example, the same apparatus as in Example 7 was used, but the hemoglobin concentration of a solution of CO2-bound human red blood cells dispersed in physiological saline was reduced to half, approximately 5.0 g / dL, and the flow rate was increased to approximately 1500 mL per minute. The HbCO2 ratio was similarly monitored over time. As shown in Figure 8, the HbCO2 ratio decreased to 41% after 45 minutes, 23% after 120 minutes, 5.1% after 270 minutes, and 3.0% after 300 minutes.

[0108] Example 9 In this example, the same apparatus as in Example 4 was used to deoxygenate a human red blood cell dispersion (hemoglobin concentration: approximately 10.0 g / dL, 1 L). ２ Instead of gas, H ２ / N ２ Mixture of gases (H ２ A concentration of approximately 5% was used. ２ The partial pressure decreased to 0 Torr in the same manner as in Example 4. ２ The gas does not bind to hemoglobin, but H ２ Gas (H ２ The solubility of ) in water (20°C) is approximately 0.0016 g / 1000 mL at 1 atmosphere, so approximately 0.0016 / 2 x 0.05 = 4 x 10 －５ It was considered to be physically dissolved in the solution at a concentration of 1 mol / L.

[0109] It should be noted that the present invention allows various embodiments and modifications without departing from the broad spirit and scope of the present invention. Furthermore, the above-described embodiments are intended to explain this disclosure and do not limit the scope of this disclosure. That is, the scope of this disclosure is defined by the claims, not the embodiments. Various modifications made within the scope of the claims and within the meaning of the disclosure equivalent thereto are considered to be within the scope of this disclosure.

[0110] This application is based on Japanese Patent Application No. 2023-141848, filed on August 31, 2023. The entire specification, claims, and drawings of Japanese Patent Application No. 2023-141848 are incorporated herein by reference.

[0111] The dissolved gas exchange method and dissolved gas exchange device of the present invention enable switching of the type of gas in the gas phase during dissolved gas exchange, switching of the gas pressure to a positive pressure of up to 0.5 MPa or a negative pressure of up to saturated water vapor pressure, switching of the gas pressure to a positive or negative pressure while promoting a photoreaction by light irradiation, cleaning and / or sterilization before and after use, and scale-up of the manufacturing process.Therefore, dissolved gas exchange, which must be performed multiple times in the Hb purification process and the manufacturing process of artificial red blood cells using Hb, can be performed much more quickly and efficiently than conventional methods, and a stable supply of high-quality artificial red blood cells can be achieved.

Claims

1. A method for exchanging dissolved gas in a hemoglobin-containing solution (hereinafter referred to as the "dissolved gas exchange method"), comprising: (1) an initial dissolved gas exchange step in which the hemoglobin-containing solution before being subjected to the dissolved gas exchange method is exposed to one surface of an initial dissolved gas exchange membrane to obtain a solution after the initial dissolved gas exchange, the other surface of the initial dissolved gas exchange membrane being exposed to a gas phase at a negative pressure up to the saturated water vapor pressure; and (2) a final dissolved gas exchange step in which the hemoglobin-containing solution immediately before the final dissolved gas exchange is exposed to one surface of a final dissolved gas exchange membrane to obtain a hemoglobin-containing solution after the final dissolved gas exchange, the other surface of the final dissolved gas exchange membrane being exposed to a gas phase at a positive pressure of up to 0.5 MPa of a required gas, A dissolved gas exchange method in which a gas dissolved in a hemoglobin-containing solution before the solution is subjected to the dissolved gas exchange method (hereinafter referred to as "dissolved gas before gas exchange") is exchanged for a gas dissolved in the hemoglobin-containing solution after the exchange method is performed (hereinafter referred to as "required gas").

2. The dissolved gas exchange method according to claim 1, wherein the first dissolved gas exchange step (1) and the last dissolved gas exchange step (2) are carried out using one dissolved gas exchange membrane module.

3. The dissolved gas exchange method according to claim 1, further comprising the step of: (3) following the first dissolved gas exchange step (1), exposing the hemoglobin-containing solution after the first dissolved gas exchange to one surface of a second dissolved gas exchange membrane to obtain a hemoglobin-containing solution after a second dissolved gas exchange, wherein the other surface of the second dissolved gas exchange membrane is exposed to a gas phase at a negative pressure up to the saturated water vapor pressure or a gas phase at a positive pressure up to 0.5 MPa of the required gas.

4. The dissolved gas exchange method according to claim 1, further comprising a step of exposing the hemoglobin-containing solution after the m-1th dissolved gas exchange (m-1) to one surface of the mth dissolved gas exchange membrane to obtain the hemoglobin-containing solution after the mth dissolved gas exchange, where M (M is a natural number equal to or greater than 4) is the number of all dissolved gas exchange steps, and the other surface of the mth dissolved gas exchange membrane is exposed to either the same gas phase as the gas phase to which the other surface of the dissolved gas exchange membrane in the m-1th dissolved gas exchange step is exposed, or a gas phase of the required gas at a positive pressure of up to 0.5 MPa.

5. A method for exchanging a dissolved gas as described in claim 1, 3 or 4, in which the hemoglobin-containing solution after the final dissolved gas exchange is recovered as the hemoglobin-containing solution after the completion of the dissolved gas exchange.

6. A method for dissolved gas exchange according to any one of claims 1 and 3 to 5, in which the hemoglobin-containing solution after the final dissolved gas exchange is mixed with the hemoglobin-containing solution before being subjected to the dissolved gas exchange process and subjected to the initial dissolved gas exchange process, and the process is repeated from the initial dissolved gas exchange process to the final dissolved gas exchange process, and when the gas concentrations after dissolved gas exchange of all hemoglobin-containing solutions do not reach saturation, the process is further repeated from the initial dissolved gas exchange process to the final dissolved gas exchange process, and when the gas concentrations after dissolved gas exchange of all hemoglobin-containing solutions reach saturation, the hemoglobin-containing solution is recovered as the hemoglobin-containing solution after completion of dissolved gas exchange.

7. The dissolved gas before gas exchange is CO gas, O ２ gas, air or a mixture thereof, and the required gas is CO gas, NO gas, O ２ Gas, N ２ Gas, CO ２ Gas, Ar gas, H ２ 7. The method for dissolving gas exchange according to claim 1, wherein the dissolved gas is a gas, air or a mixture thereof.

8. A dissolved gas exchange method according to any one of claims 1 to 7, wherein in the final dissolved gas exchange step, the heme central iron of hemoglobin in the hemoglobin-containing solution immediately before the final dissolved gas exchange step has CO bound to it as a dissolved gas, and (4) further comprises a photodissociation step of irradiating light onto the hemoglobin-containing solution exposed to one surface of the final dissolved gas exchange membrane to photodissociate CO from the hemoglobin in the hemoglobin-containing solution, in which the other surface of the final dissolved gas exchange membrane is exposed to a negative pressure up to the saturated water vapor pressure, thereby exchanging the dissolved gas such that the heme central iron of the hemoglobin contained in the hemoglobin-containing solution is not substantially bound to CO.

9. A device for exchanging dissolved gas in a hemoglobin-containing solution for carrying out the dissolved gas exchange method according to any one of claims 1 and 3 to 6 (hereinafter referred to as the "dissolved gas exchange device"), comprising at least two dissolved gas exchange membrane modules including a first dissolved gas exchange membrane module and a last dissolved gas exchange membrane module, a container for storing the hemoglobin-containing solution, and a conduit connecting them; All of the dissolved gas exchange membrane modules include a liquid phase section, a gas phase section, and a dissolved gas exchange membrane section, and the liquid phase section and the gas phase section are arranged with the dissolved gas exchange membrane section in between, where the liquid in the liquid phase section is exposed to one surface of the dissolved gas exchange membrane of the dissolved gas exchange membrane section, and the gas in the gas phase section is exposed to the other surface of the dissolved gas exchange membrane of the dissolved gas exchange membrane section, and the dissolved gas exchange membrane section does not permeate the liquid in the liquid phase section, but exchanges the dissolved gas in the liquid with the gas in the gas phase section; the conduit connects a container for storing the hemoglobin-containing solution before the dissolved gas exchange and a liquid supply port of the liquid phase section of the first dissolved gas exchange membrane module; when the total number of dissolved gas exchange processes is 2, a liquid discharge port of the liquid phase section of the first dissolved gas exchange membrane module is connected to a liquid supply port of the liquid phase section of the last dissolved gas exchange membrane module; When the total number of dissolved gas exchange processes is 3, the drain port of the liquid phase part of the first dissolved gas exchange membrane module is connected to the liquid supply port of the liquid phase part of the second dissolved gas exchange membrane module, and the drain port of the liquid phase part of the second dissolved gas exchange membrane module is connected to the liquid supply port of the liquid phase part of the last dissolved gas exchange membrane module; when the total number of dissolved gas exchange processes is M (M is a natural number of 4 or more), the drain port of the liquid phase part of the m-1th (m is a natural number of 2≦m≦M-1) dissolved gas exchange membrane module is connected to the liquid supply port of the liquid phase part of the mth dissolved gas exchange membrane module, where the first dissolved gas exchange membrane module is the first dissolved gas exchange membrane module and the Mth dissolved gas exchange membrane module is the last dissolved gas exchange membrane module; The hemoglobin-containing solution before being subjected to the dissolved gas exchange step is passed in series from the liquid phase part of the first dissolved gas exchange membrane module to the liquid phase part of the last dissolved gas exchange membrane module;In this dissolved gas exchange device, the gas phase of the first dissolved gas exchange membrane module is kept at a negative pressure up to the saturated water vapor pressure, and the gas phase of the last dissolved gas exchange membrane module is kept at a positive pressure of the required gas with a maximum of 0.5 MPa, thereby exchanging dissolved gas such that the heme central iron of the hemoglobin-containing solution binds to the required gas as a dissolved gas, or such that the heme central iron does not bind to any dissolved gas.

10. A dissolved gas exchange device as described in claim 9, wherein when the dissolved gas exchange membrane modules are three in total, the gas phase of the second dissolved gas exchange membrane module following the first dissolved gas exchange membrane module is at a negative pressure up to the saturated water vapor pressure or a positive pressure up to 0.5 MPa of the required gas.

11. A dissolved gas exchange device as described in claim 9, wherein when the total number of dissolved gas exchange membrane modules is N (N is a natural number greater than or equal to 4), the gas phase portions of the dissolved gas exchange membrane modules from the first dissolved gas exchange membrane module to the nth dissolved gas exchange membrane module (n is a natural number satisfying 2≦n≦N-1) are all at a negative pressure up to the saturated water vapor pressure, and the gas phase portions of all dissolved gas exchange membrane modules from the n+1th dissolved gas exchange membrane module to the last dissolved gas exchange membrane module are at a positive pressure of up to 0.5 MPa for the required gas.

12. A dissolved gas exchange device as described in any one of claims 9 to 11, wherein the hemoglobin-containing solution that has passed through the liquid phase portion of the last dissolved gas exchange membrane module is recovered as the hemoglobin-containing solution after the completion of dissolved gas exchange, or the hemoglobin-containing solution that has passed through the liquid phase portion of the last dissolved gas exchange membrane module is introduced into a container for storing the hemoglobin-containing solution before being subjected to the dissolved gas exchange process, mixed with the hemoglobin-containing solution before being subjected to the dissolved gas exchange process, and introduced into the liquid phase portion of the first dissolved gas exchange membrane module, thereby repeating the dissolved gas exchange.

13. The dissolved gas before gas exchange is CO gas, O ２ gas, air or a mixture thereof, and the required gas is CO gas, NO gas, O ２ Gas, N ２ Gas, CO ２ Gas, Ar gas, H ２ 13. The dissolved gas exchange device according to any one of claims 9 to 12, wherein the dissolved gas exchange material is a gas, air or a mixture thereof.

14. A dissolved gas exchange device according to any one of claims 9 to 13, wherein the dissolved gas exchange membrane is a hollow fiber membrane.

15. A dissolved gas exchange device as described in any one of claims 9 to 14, further comprising a light source, the housing of the last dissolved gas exchange membrane module being transparent, the gas phase portion of the last dissolved gas exchange membrane module being kept at a negative pressure up to the saturated water vapor pressure, and light being irradiated from a light source arranged outside the housing of the last dissolved gas exchange membrane module onto the hemoglobin-containing solution in the liquid phase portion of the dissolved gas exchange membrane module, thereby photodissociating the dissolved gas CO from the heme central iron of hemoglobin in the hemoglobin-containing solution passing through the liquid phase portion.

16. A system for exchanging dissolved gas in a hemoglobin-containing solution (hereinafter referred to as a "dissolved gas exchange system"), comprising at least P groups (P is a natural number of 2 or more) of the dissolved gas exchange devices according to any one of claims 9 to 14, leading the hemoglobin-containing solution stored in a container for storing the hemoglobin-containing solution after completion of dissolved gas exchange in the (p-1)th (p is a natural number satisfying 2≦p≦P) dissolved gas exchange device to a container for storing the hemoglobin-containing solution before being subjected to the dissolved gas exchange in the pth dissolved gas exchange device, filling the hemoglobin-containing solution requiring dissolved gas exchange in a container for storing the hemoglobin-containing solution before the dissolved gas exchange into a required gas in the first dissolved gas exchange device according to any one of claims 9 to 14, A dissolved gas exchange system, wherein the hemoglobin-containing solution stored in a container for storing the hemoglobin-containing solution after completion of dissolved gas exchange in the p-th dissolved gas exchange device described in any one of claims 9 to 14 is a hemoglobin-containing solution for which dissolved gas exchange has been completed, and the dissolved gas exchanged in the first dissolved gas exchange membrane module of the first dissolved gas exchange device is different from the dissolved gas exchanged in the last dissolved gas exchange membrane module of the p-th dissolved gas exchange device.

17. A dissolved gas exchange system comprising at least Q dissolved gas exchange devices according to any one of claims 9 to 14 (Q being a natural number of 2 or more) and one dissolved gas exchange device according to claim 15, comprising: directing a hemoglobin-containing solution stored in a container for storing a hemoglobin-containing solution after completion of dissolved gas exchange in the dissolved gas exchange device according to any one of claims 9 to 14 (q-1st (q being a natural number satisfying 2≦q≦Q)) to a container for storing a hemoglobin-containing solution before dissolved gas exchange into a required gas in the dissolved gas exchange device according to any one of claims 9 to 14 (q-1st (q being a natural number satisfying 2≦q≦Q)), and directing a hemoglobin-containing solution stored in a container for storing a hemoglobin-containing solution after completion of dissolved gas exchange in the dissolved gas exchange device according to any one of claims 9 to 14 (q-1st (q being a natural number satisfying 2≦q≦Q)), to a container for storing a hemoglobin-containing solution before dissolved gas exchange into a required gas in the dissolved gas exchange device according to any one of claims 9 to 14 (q-1st (q being a natural number satisfying 2≦q≦Q)), A dissolved gas exchange system comprising: a container for storing the hemoglobin-containing solution before dissolved gas exchange to a required gas in the dissolved gas exchange device described in any one of the first claims 9 to 14; a hemoglobin-containing solution requiring dissolved gas exchange; a hemoglobin-containing solution stored in a container for storing the hemoglobin-containing solution after completion of dissolved gas exchange in the dissolved gas exchange device described in claim 15; and a hemoglobin-containing solution for which dissolved gas exchange has been completed; and the dissolved gas exchanged in the first dissolved gas exchange membrane module of the dissolved gas exchange device described in any one of the first claims 9 to 14 is different from the dissolved gas exchanged in the last dissolved gas exchange membrane module of the dissolved gas exchange device described in claim 15.

18. The required gas is O ２ 8. A method for producing a hemoglobin-containing solution, comprising the step of obtaining a hemoglobin-containing solution in which the heme central iron of hemoglobin does not bind oxygen by the dissolved gas exchange method according to any one of claims 1 to 7, wherein the hemoglobin-containing solution has an oxygen partial pressure of 0.1 Torr or less.

19. An exchange device for a dissolved gas in a hemoglobin-containing solution for carrying out the dissolved gas exchange method according to claim 2, comprising one dissolved gas exchange membrane module, at least one container, and a conduit connecting these, said dissolved gas exchange membrane module comprising a liquid phase section, a gas phase section, and a dissolved gas exchange membrane section, said liquid phase section and gas phase section being arranged with said dissolved gas exchange membrane section in between, wherein the liquid in said liquid phase section is exposed to one surface of the dissolved gas exchange membrane of said dissolved gas exchange membrane section, and the gas in said gas phase section is exposed to the other surface of the dissolved gas exchange membrane of said dissolved gas exchange membrane section, said dissolved gas exchange membrane section does not permeate the liquid in said liquid phase section, but exchanges the dissolved gas in said liquid with the gas in said gas phase section, (a) in the first dissolved gas exchange step (1), the conduit connects a first container to the liquid supply port of the liquid phase part of the dissolved gas exchange membrane module, and connects the discharge port of the liquid phase part of the dissolved gas exchange membrane module to a second container, and by making the gas phase part of the dissolved gas exchange membrane module negative pressure up to the saturated water vapor pressure, the partial pressure of the dissolved gas bound to the heme central iron of the hemoglobin-containing solution is reduced to the saturated water vapor pressure; (b) in the last dissolved gas exchange step (2), the conduit connects the second container to the liquid supply port of the liquid phase part of the dissolved gas exchange membrane module, and connects the discharge port of the liquid phase part of the dissolved gas exchange membrane module to the first container; The gas phase part of the dissolved gas exchange membrane module is made a gas phase of a required gas at a positive pressure of up to 0.5 MPa, thereby replacing the dissolved gas in the hemoglobin-containing solution with the required gas.

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