Plasma preparation having characteristics of pathogen reduction and low complement activity

Abstract

Provided are: a plasma preparation that has a reduced complement value and in which mixed viruses have been inactivated while maintaining the biological activity of plasma; and a method for producing the plasma preparation. The method includes: (1) a step for subjecting plasma to a solvent / detergent treatment (S / D treatment); and (2) a step for irradiating the S / D treated plasma with ultraviolet rays having a central wavelength of 200-260 nm from an LED light source for 10 minutes or longer such that the value of ΔA352, which corresponds to the cumulative irradiation amount and is converted by a 1% aqueous potassium iodide solution, is 0.2-0.7. The plasma preparation has a complement value (CH50) of less than 25 CH50 / mL.

Description

Plasma preparations with pathogen-reducing and low complement activity properties

[0001] The present invention relates to a plasma preparation in which viruses have been inactivated and which has a low complement titer, and a method for producing the same.

[0002] Plasma preparations are essential medical preparations, and although individual bags of plasma preparations are subjected to nucleic acid tests and antigen tests to eliminate any contamination with viruses and other pathogens, contamination with viruses and bacteria that could cause infectious diseases cannot be completely ruled out. For this reason, efforts have been made to improve pathogen safety by introducing pathogen inactivation treatments into plasma.

[0003] Examples of viral inactivation treatments applied to plasma include solvent / surfactant treatment (S / D treatment) (Patent Document 1). S / D treatment is an effective inactivation technique for enveloped viruses, and S / D-treated plasma, which is a plasma product that has been subjected to S / D treatment, is widely used around the world. However, while S / D treatment in principle effectively inactivates viruses with lipid membranes, it does not inactivate non-enveloped viruses with non-lipid outer layers. Therefore, cases of infection with hepatitis E virus (HEV), which is classified as a non-enveloped virus, have been reported in patients who used S / D-treated plasma (Non-Patent Document 1).

[0004] As a technique for inactivating non-enveloped viruses, an inactivation method that combines a photosensitizer (such as riboflavin) with ultraviolet irradiation is widely used (Patent Document 2). Furthermore, a method for inactivating viruses while maintaining high activity of blood coagulation factors involves irradiating them with ultraviolet light at a wavelength of 253.7 nm using a mercury lamp as a light source (Patent Document 3) (Non-Patent Document 3). However, these inactivation techniques involve adding additives to individual plasma bags, making it difficult to produce inactivated plasma products on a large scale. Therefore, there has been a demand for an inactivation technique for non-enveloped viruses that can be industrialized.

[0005] Furthermore, from the viewpoint of enhancing the viral safety of plasma products, it is appropriate to combine S / D treatment, which is an industrially widespread method for inactivating enveloped viruses, with an inactivation technique for non-enveloped viruses. It is desirable to have a plasma product that has undergone comprehensive inactivation treatment for both viruses and that maintains high activity of blood coagulation factors (Non-Patent Document 2).

[0006] Furthermore, in pathological conditions requiring plasma exchange, such as organ transplantation, the high complement activity can easily cause thrombotic microangiopathy (TMA), which can lead to tissue damage and death. For plasma exchange in such pathological conditions with high complement activity, plasma preparations with low complement activity are desired (Non-Patent Documents 4 and 5).

[0007] Patent No. 2708632 Patent No. 4464939 WO97 / 33629

[0008] Emerging Infectious Diseases. 2020 Dec; 26(12): 2881-2886. Risk for Hepatitis E Virus Transmission by Solvent / Detergent-Treated Plasma.Vox Sanguinis (2011) 100, 169-178. A comparison of methods of pathogen inactivation of FFP.Blood, Vol 86, No. 11(December 1), 1995: pp 4331-4336. Virucidal Short Wavelength Ultraviolet Light Treatment of Plasma and Factor VIII Concentrate: Protection of Proteins by Antioxidants.Blood(2020) vol.35, Num13, 1049-1057. Complement blockade for TA-TMA: lessons learned from a large pediatric cohort with eculizumab.Clinical and treated Developmental Immunology(2012),12,Initiation and Regulation of Complement during Hemolytic Transfusion Reactions

[0009] In order to solve the above problems, the present invention provides a plasma preparation in which viruses are inactivated while maintaining biological activity and complement activity is reduced by introducing into the plasma preparation an inactivation process based on different principles: enveloped virus inactivation technology using S / D treatment and non-enveloped virus inactivation technology using ultraviolet LED irradiation. The plasma preparation can also be used as a reagent, such as an additive in cell preparations or culture media in regenerative medicine.

[0010] The present inventors have discovered that plasma (also referred to as S / D-UV Plasma) obtained by irradiating S / D-treated plasma (also referred to as S / D Plasma) with ultraviolet LED radiation of a specific wavelength at a low illuminance and a constant cumulative irradiation dose for a certain period of time (also referred to as S / D-UV Plasma) exhibits a higher virus inactivation effect than fresh frozen plasma (also referred to as FFP) irradiated with ultraviolet LED radiation, and furthermore, complement activity is reduced while maintaining biological activity, thereby completing the present invention.

[0011] That is, the present invention is as follows. [1] A method for producing a plasma preparation, comprising the following steps: (1) a step of subjecting plasma to solvent / surfactant treatment (S / D treatment), and (2) a step of irradiating the S / D-treated plasma with ultraviolet light having a center wavelength of 200 to 260 nm from an LED light source for 10 minutes or more, so that the ΔA352, calculated using a 1% potassium iodide aqueous solution equivalent to the cumulative exposure dose, is 0.2 to 0.7. [1-1] The production method according to [1], wherein (1) is a step of performing S / D treatment using 0.5 to 2 parts by weight of a solvent and 0.5 to 2 parts by weight of a surfactant per 100 parts by weight of plasma. [1-2] The production method according to [1] or [1-1], wherein (1) the solvent is a dialkyl or trialkyl phosphate having an alkyl group having 1 to 10 carbon atoms, and the surfactant is an oxyethylated alkylphenol, non-phenolic polyoxyethylene ether, dimethylamine oxide, or a mixture thereof. [2] The production method according to any of [1] to [1-2], further comprising an oil extraction step. [3] The manufacturing method according to [2], further comprising a purification step by solid-phase extraction. [3-1] The manufacturing method according to [3], wherein the purification step by solid-phase extraction is a purification step by reverse-phase chromatography. [4] The manufacturing method according to [3] or [3-1], further comprising a vacuum freeze-drying step. [4-1] The manufacturing method according to [4], wherein the vacuum freeze-drying step comprises: ejecting plasma from a nozzle provided at the upper end of a vacuum freeze-drying tower, the nozzle having a diameter of 50 to 350 μm for ejecting plasma, into a vacuum freeze-drying tower having an internal pressure of 1.0 to 20 Pa under vacuum, at a spray pressure of 0.01 to 0.3 MPa, thereby forming fine raw material liquid particles, and allowing the particles to self-freeze to form fine frozen particles; and capturing the fine frozen particles on a capturing surface within the vacuum freeze-drying tower, heating them, and drying them by sublimation. [5] A plasma preparation obtained by the manufacturing method according to any one of [1] to [3-1]. [6] A dried plasma preparation obtained by the manufacturing method according to [4] or [4-1]. [7] The plasma preparation described in [5], wherein the complement titer (CH50) is less than 25CH50 / mL.[8] The plasma preparation according to [7], which contains one or more selected from the group consisting of 150 mg / dL to 400 mg / dL of factor I (fibrinogen), 0.5 IU / mL to 1.5 IU / mL of factor VIII, 0.5 IU / mL to 1.3 IU / mL of factor V, 0.2 IU / mL to 1.2 IU / mL of α2-antiplasmin, 0.7 IU / mL to 1.5 IU / mL of protein C, 0.5 IU / mL to 1.3 IU / mL of factor II, and 0.6 IU / mL to 1.7 IU / mL of vWF (von Willebrand factor). [9] The plasma preparation according to [7] or [8], which is a culture reagent. [9-1] The plasma preparation according to [7] or [8], which is a pharmaceutical product.

[10] A method for reducing the complement value (CH50) of a plasma product, comprising the following steps: (1) treating the plasma with a solvent / detergent (S / D) treatment; and (2) irradiating the S / D-treated plasma with ultraviolet light having a central wavelength of 200 to 260 nm using an LED light source for 10 minutes or more, so that the ΔA352, calculated using a 1% potassium iodide aqueous solution equivalent to the cumulative irradiation dose, is 0.2 to 0.7.

[11] A method for inactivating viruses in a plasma product, comprising the following steps: (1) treating the plasma with a solvent / detergent (S / D) treatment; and (2) irradiating the S / D-treated plasma with ultraviolet light having a central wavelength of 200 to 260 nm using an LED light source for 10 minutes or more, so that the ΔA352, calculated using a 1% potassium iodide aqueous solution equivalent to the cumulative irradiation dose, is 0.2 to 0.7.

[12] A plasma product obtained by the production method described in Figure 1.

[13] A dried plasma product obtained by the production method described in Figures 1 and 2.

[0012] According to the present invention, it is possible to provide an effective and safe plasma preparation that achieves both virus inactivation and maintenance of high activity of blood coagulation factors. According to the present invention, it is possible to provide a plasma preparation that has a low complement titer and is capable of suppressing hemolysis. According to the present invention, it is possible to provide a virus-inactivated plasma that complies with the reference values ​​for blood coagulation factor activity set forth in the European Pharmacopoeia (EP).

[0013] The manufacturing flow of S / D-UV Plasma and S / D Plasma is shown. The flow of preparation of dried plasma (powder formulation) and reconstituted plasma for S / D-UV Plasma and S / D Plasma is shown. The evaluation items of blood elasticity viscosity test (ROTEM (registered trademark)) measurement and their explanations are shown (Reference: Nobuyuki Katori, Journal of the Japanese Society of Clinical Medicine 2013; 33, 263-271). The evaluation items of thrombogram for measuring thrombin generation amount and their explanations are shown.

[0014] The present invention relates to a method for producing a plasma preparation (also referred to as the production method of the present invention), which includes the steps of: (1) subjecting plasma to a solvent / surfactant treatment (S / D treatment); and (2) irradiating the S / D-treated plasma with ultraviolet light having a central wavelength of 200 to 260 nm from an LED light source for 10 minutes or more so that ΔA352, calculated using a 1% potassium iodide aqueous solution equivalent to the cumulative irradiation dose, is 0.2 to 0.7.

[0015] In the production method of the present invention, raw materials include blood, blood-derived components such as plasma and serum obtained by centrifugation of whole blood, but plasma is usually used. The plasma used in the production method of the present invention may be, for example, plasma prepared just before use, thawed fresh frozen plasma, or lyophilized plasma redissolved in a desired solution. Fresh frozen plasma (also referred to as FFP) is plasma obtained from donated blood and promptly frozen to -40°C or below within six hours of collection, and is commercially available from the Japanese Red Cross Blood Center.

[0016] (1) Solvent / Detergent Treatment (S / D Treatment) Solvent / detergent treatment is a virus inactivation method that uses a biocompatible solvent and detergent, and is also called the solvent-detergent method (hereinafter also referred to as S / D treatment). In S / D treatment, the surfactant breaks the sheath of enveloped viruses and dissolves them in a solvent, and the combined use of a solvent synergistically inactivates viruses having a lipid envelope.

[0017] In the present invention, virus inactivation treatment is performed by contacting plasma with a mixture of a solvent and a surfactant (also referred to as an SD treatment solution). The SD treatment solution is not particularly limited, as long as it is a solvent-surfactant combination known in the technical field of S / D treatment that can chemically inactivate enveloped viruses. Specific examples of the solvent include organic solvents, particularly dialkyl or trialkyl phosphates having an alkyl group containing 1 to 10 carbon atoms, such as tri(n-butyl)phosphate (hereinafter also referred to as TNBP), tri(t-butyl)phosphate, tri(n-hexyl)phosphate, tri(2-ethylhexyl)phosphate, tri(n-decyl)phosphate, and ethyl-di(n-butyl)phosphate. Among these, TNBP is preferred from the viewpoints of virus inactivation effect and ensuring safety.

[0018] Specific examples of surfactants include polyoxyethylene derivatives of fatty acids, polyoxyethylene sorbitan fatty acid esters, oxyethylated alkylphenols, polyoxyethylene alcohols, polyoxyethylene oils, polyoxyethyleneoxypropylene fatty acids, etc. More specific examples include polyoxyethylene derivatives of fatty acids (trade names: Tween 80, Tween 20, etc.), partial esters of sorbitol anhydride (trade name: Polysorbate 80, etc.), oxyethylated alkylphenols such as polyoxyethylene octylphenyl ether (trade name: Triton X-100, etc.), sodium cholate, sodium deoxycholate, sulfobetaines such as N-dodecyl-N,N-dimethyl-2-ammonio-1-ethanesulfonate, nonionic surfactants such as octyl-β,D-glucopyranoside, non-phenolic polyoxyethylene ethers such as Brij C10, and dimethylamine oxides such as LDAO. Among these, from the viewpoint of virus inactivation effect and ensuring safety, oxyethylated alkylphenols, non-phenolic polyoxyethylene ethers, dimethylamine oxide, and the like are preferred, and oxyethylated alkylphenols such as Triton X-100, non-phenolic polyoxyethylene ethers such as Brij C10, and dimethylamine oxides such as LDAO are more preferred, but any equivalent surfactant that can achieve the effects of the present invention can also be preferably used.

[0019] The S / D treatment solution may contain two or more solvents and / or surfactants independently. The S / D treatment solution may also contain other additives, such as a reducing agent, to enhance its effects, as needed. The S / D treatment solution is preferably a mixture of equal volumes of the above two surfactants.

[0020] In the present invention, to ensure reliable virus inactivation, the amounts of the S / D treatment solution used are typically 0.5 to 2 parts by weight of surfactant and 0.5 to 2 parts by weight of solvent, preferably 0.9 to 1.1 parts by weight of surfactant and 0.9 to 1.1 parts by weight of solvent, per 100 parts by weight of plasma. Specifically, the solvent and surfactant are mixed in equal amounts to prepare the S / D treatment solution, which is then added to the plasma at a concentration of 2% (v / v) relative to the plasma volume. That is, the S / D treatment solution is prepared so that the final concentrations after treatment are 1% (v / v) of solvent and 1% (v / v) of surfactant. The S / D treatment is typically carried out at 25 to 35°C, preferably 29 to 31°C. The S / D treatment time is typically 30 to 240 minutes, preferably 60 to 90 minutes, and more preferably 60 to 70 minutes.

[0021] In the manufacturing method of the present invention, other steps are not particularly limited as long as they include the above-mentioned S / D treatment and the step of irradiating specific LED ultraviolet light described below, but may include a purification step for removing the solvent and surfactant after the S / D treatment.

[0022] In the present invention, the purification process may include an oil extraction step to remove the solvent. The oil extraction step can be carried out using conventional methods. In particular, to efficiently irradiate the plasma with ultraviolet light from LEDs (described below), the oil used for extraction can be soybean oil, castor oil, or the like. Castor oil is preferred from the viewpoint of increasing the clarity of the plasma after oil extraction. The temperature for oil extraction is typically 20 to 25°C, preferably 21 to 23°C. The mixture containing plasma treated with the S / D treatment solution and the oil are stirred and mixed for typically 0.5 to 3 hours, preferably 1 to 2 hours, to allow the surfactant to migrate to the upper oil layer, and the mixture is allowed to stand for oil-water separation. The temperature for standing is typically 20 to 25°C, preferably 21 to 23°C, and the time for standing is typically 0.5 to 1.5 hours, preferably 0.5 to 1 hour, but can be appropriately set depending on various conditions. After standing, the aqueous layer is recovered using conventional methods. The liquid recovered after oil extraction is filtered to adsorb the residual oil using filters made of a material that easily adsorbs oil, for example, two types of depth filters: a filter with a pore size of 1 to 9 μm, and a filter with a pore size of 1 μm or less. Specifically, filtration is performed using a first filter, a depth filter for clarification filtration with an average pore size of 1 to 9 μm, and a second filter, a depth filter with a filtration accuracy of 0.2 to 1 μm.

[0023] In the present invention, solid-phase extraction or a pre-column may be used as a purification step to remove surfactants. The solid-phase extraction step can be carried out by a conventional method. For example, as the type of packing material used in solid-phase extraction, a reversed-phase system is usually used because the surfactants remaining in plasma are hydrophobic substances and have a high adsorption capacity for hydrophobic substances. In particular, reversed-phase chromatography using an octadecyl group-bonded (C18) resin is preferably used.

[0024] In the present invention, the purification step by reverse phase chromatography is preferably carried out after the oil extraction step, from the viewpoint of separating impurities derived from oil components together with the surfactant.

[0025] (2) Ultraviolet Irradiation Step The method for producing a plasma preparation of the present invention further comprises an ultraviolet irradiation step following the S / D treatment and purification steps, which removes non-enveloped viruses and allows the production of a virally inactivated plasma preparation.

[0026] In the production method of the present invention, "inactivating viruses in a plasma product" means reducing the activity of viruses present in the plasma product to a level that does not cause health hazards and rendering infectious viruses present in the plasma product non-infectious. Specifically, this means achieving a log reduction value (LRV) for viruses of a predetermined value or greater; since an LRV of 1 or less is generally considered to be ineffective in inactivating viruses, for example, greater than 1.00, preferably 2.00 or greater, more preferably 3.00 or greater, and even more preferably 4.00 or greater.

[0027] As used herein, LRV specifically refers to the viral log reduction value, and is the TCID ５０ The LRV is the logarithmic reduction rate, which is the value obtained by subtracting the index value of the infectivity titer after irradiation from the index value of the infectivity titer calculated by the method. LRV can also be calculated by the following formula: LRV = Log [(V １ ×T １ ))] / [(V ２ ×T ２ ))] V １ : Volume of sample before virus inactivation treatment process T １ V: virus amount (titer) before virus inactivation treatment process ２ : Volume of sample after virus inactivation treatment process T ２ : Virus amount (titer) after virus inactivation treatment process

[0028] The amount of virus in a plasma product can be measured by the infectivity titer or the amount of viral nucleic acid. The infectivity titer can be obtained by inoculating the virus into indicator cells, culturing them for a certain period of time, and measuring the denaturation of the cells after culturing, as well as the viral antigen and nucleic acid (TCID ５０The quantitative polymerase chain reaction (Q-PCR) method for measuring the amount of nucleic acid can be carried out by a method known per se.

[0029] Viruses that may be contained in plasma may be enveloped or non-enveloped, and are not particularly limited, and examples thereof include porcine parvovirus (PPV), bovine parvovirus (BPV), canine parvovirus (CPV), human parvovirus B19, murine encephalomyocarditis virus (EMC), minute virus of mice (MVM), poliovirus, porcine circovirus (PCV), hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis E virus (HEV), human immunodeficiency virus 1 (HIV-1), human T-cell lymphotropic virus type 1 (HTLV-1), bovine viral diarrhea virus (BVDV), adenovirus, and vesivirus 2117. In particular, the present invention is preferably applied to non-enveloped viruses that are generally difficult to inactivate, such as PPV and EMC, from the viewpoint of providing an inactivation method that is effective even against non-enveloped viruses that are resistant to S / D treatment.

[0030] In the manufacturing method of the present invention, the cumulative irradiation amount is also called the cumulative illuminance or the cumulative light amount, and is the cumulative irradiation amount (mJ / cm ２ ) = UV illuminance (mW / cm ２ ) x exposure time (seconds), and is a light intensity that serves as a guide for estimating the inactivation of viruses and the reduction in biological activity. UV irradiance refers to the intensity of UV light emitted from a light source, indicating how much UV light is irradiated onto a certain area. It is measured using an illuminance meter equipped with a hemispherical element (light-receiving element) that receives light of a specific wavelength, assuming a constant irradiance distribution. However, standardization is generally difficult due to differences in light-receiving elements, measurement conditions, and UV characteristics depending on the light source.

[0031] In the present invention, the change in absorbance (ΔA352) at 352 nm of a 1% potassium iodide aqueous solution (1% KI), which is a chemical actinometer, when irradiated for 30 seconds under constant illuminance is measured. ３０　ｓｅｃ) is taken as the ultraviolet irradiance equivalent, and the change in absorbance at 352 nm of 1% KI (ΔA352) over the set irradiation time is taken as the cumulative irradiation equivalent. These values ​​are converted and expressed as numerical values, and are used as a guide for estimating viral inactivation and reduction in biological activity.

[0032] Specifically, the cumulative irradiation dose equivalent ΔA352 is measured as follows: [1] Prepare a 1% potassium iodide aqueous solution. [2] Place the aqueous solution in a container under the same conditions as when actually measuring the sample (same container, same thickness), and irradiate it with a constant illuminance for 30 seconds using an ultraviolet irradiator. [3] Measure the absorbance of the aqueous solution at 352 nm before and after irradiation using a spectrophotometer. [4] Measure the absolute value of the change before and after irradiation as ΔA352. ３０　ｓｅｃ This is determined as the amount equivalent to the UV irradiance. [5] A new 1% potassium iodide aqueous solution is placed in the same container as the previous solution at the same thickness, and UV light is irradiated for the desired time at a constant irradiance. [6] The absorbance at 352 nm before and after irradiation is measured using a spectrophotometer. [7] The absolute value of the change before and after irradiation is determined as ΔA352, which is the amount equivalent to the cumulative irradiation dose.

[0033] Chemical actinometers (solutions for chemical actinometry) use iodides of alkali metals, alkaline earth metals, or ammonia. Iodides are converted to iodine by ultraviolet irradiation, and the color reaction can be measured spectrophotometrically at 352 nm. In the present invention, alkali metal iodides are preferred, and potassium iodide or sodium iodide is more preferred.

[0034] In the manufacturing method of the present invention, a method for adjusting the cumulative irradiation dose equivalent ΔA352 to a desired range so as to achieve a desired effect is to ３０　ｓｅｃ For example, when ΔA352 is increased, ΔA352 ３０　ｓｅｃ Alternatively, the irradiation time or both can be increased, and vice versa. On the other hand, ultraviolet irradiation can be performed without changing the cumulative irradiation amount by adjusting the ultraviolet irradiance and irradiation time.

[0035] The ultraviolet irradiance can be adjusted by adjusting the type of light source, the number of light source elements, the output, and the distance to the measurement sample. For example, the ultraviolet irradiance of an LED light source device can be reduced by reducing the power output of the device and increasing the distance to the measurement sample. In the manufacturing method of the present invention, for example, the ultraviolet irradiance equivalent ΔA352 ３０　ｓｅｃ Although it varies depending on the irradiation time, it is usually 0.010 to 1, preferably 0.030 to 0.148, and more preferably 0.037 to 0.050.

[0036] The production method of the present invention includes a step of irradiating plasma with ultraviolet light for 10 minutes or longer so that the ΔA352, calculated using a 1% potassium iodide aqueous solution, corresponding to the cumulative irradiation dose is 0.2 to 0.7. The cumulative irradiation dose for a test subject may vary depending on factors such as the type of light source, the number of light source elements, output, the distance between the test subject and the light source, the irradiation time, and the amount of test subject. However, by using the above-described measurement method, the cumulative irradiation dose can be standardized by ΔA352 regardless of the variation of each factor and the extent of that variation. In the present invention, the ΔA352, which corresponds to the cumulative irradiation dose, is typically 0.2 or greater, preferably 0.4 or greater, from the viewpoint of virus inactivation. Furthermore, from the viewpoint of maintaining biological activity, it is typically 0.7 or less, preferably 0.6 or less, and more preferably 0.5 or less. For example, the ΔA352 required for virus inactivation and maintaining blood coagulation activity is typically 0.2 to 0.7, preferably 0.2 to 0.6, and more preferably 0.4 to 0.5.

[0037] In the production method of the present invention, the time for irradiating plasma with ultraviolet light is usually 10 minutes to 1 hour, preferably 15 minutes to 20 minutes, from the viewpoint of maintaining the activity of protein factors and inactivating viruses at the same time. Although the time for ultraviolet light irradiation may vary depending on the number of light source elements, the irradiation time relative to the plasma volume can be determined by the method for calculating the cumulative irradiation amount using ΔA352 converted using a 1% potassium iodide aqueous solution. Therefore, the irradiation time can be set based on ΔA352 regardless of the plasma volume. For example, when the ultraviolet irradiance is 800 μW / cm, ２ (Measured using a Topcon UV illuminance meter, model numbers UVR-300 and UD-250) for approximately 15 minutes, the cumulative exposure was 720 mJ / cm ２corresponds to a ΔA352 of approximately 0.442.

[0038] Ultraviolet light includes UVC (200-280 nm), UVB (280-320 nm), and UVA (320-400 nm), but in this specification, ultraviolet light generally refers to UVC. From the viewpoint of maintaining the activity of protein factors and inactivating viruses, the central wavelength of ultraviolet light is generally 265 nm or less, preferably less than 260 nm, and more preferably 258 nm or less, with the lower limit being 200 nm or more, 245 nm or more, and more preferably 252 nm or more. Specifically, 70% or more of the entire wavelength spectrum is in the 245-265 nm range, and the central wavelength of ultraviolet light is in the 252-258 nm range.

[0039] In the production method of the present invention, the ultraviolet irradiation source can be an LED light source or a low-pressure mercury lamp light source, but an LED light source is preferred from the viewpoints of maintaining the activity of protein factors, inactivating viruses, and reducing environmental impact. An ultraviolet LED irradiator having an LED light source is configured with UVLED elements, the total amount of which represents the ultraviolet irradiance of the irradiator. The distance from the light source to the plasma to be irradiated varies depending on the spacing between the UVLED elements and the area of ​​the target, but a certain distance is preferred from the viewpoints of uniform irradiation and preventing contact with the target, for example, 0.5 to 10 cm, with 3 to 5 cm being preferred.

[0040] In the production method of the present invention, the temperature of the plasma is, for example, in the range of 1°C to 40°C, preferably 15°C to 35°C, more preferably 20°C to 35°C throughout all steps of the production method of the present invention.

[0041] In the production method of the present invention, the pH of the plasma is not particularly limited, but is, for example, pH 6 to pH 9.

[0042] In the production method of the present invention, the method for irradiating plasma with ultraviolet light is not particularly limited as long as the effects of the present invention are not impaired, but it is preferable that the plasma be irradiated uniformly with ultraviolet light. For example, the plasma may be irradiated while being shaken or stirred with a stirrer.

[0043] For example, the plasma to be irradiated may be placed in a container as is or in an ultraviolet-transmitting bag or the like and then irradiated. From the viewpoint of uniformly and deeply irradiating the plasma with ultraviolet light, the thickness is usually 100 mm or less, preferably 20 mm or less, and more preferably 5 mm or less. There are no particular restrictions on the lower limit of the thickness, but in consideration of efficiency, it is usually 1 mm or more, preferably 3 mm or more. The area of ​​the plasma to be irradiated also varies depending on the area of ​​the light source of the irradiation part of the irradiator, but the irradiation area can be set by ΔA352 regardless of the plasma volume. For example, a light source area that can be uniformly irradiated is 3600 mm ２ In this case, the area of ​​the plasma to be irradiated is 1 to 148 cm ２ , preferably 9 to 148 cm ２ is.

[0044] The production method of the present invention reduces the number of insoluble particles present in the plasma obtained. It has been reported that, in cell preparations administered intravenously, an increase in the number of insoluble particles 10 μm or larger increases the risk of cell damage (Ref. DOMINIC CLARKE, et al. Managing particulates in cell therapy: Guidance for best practice. Cytotherapy, 2016;18:1063-1076). Furthermore, the Japanese Pharmacopoeia (JP) General Test Method 6.07, "Insoluble Particle Test Method for Injectables, Method 1 (Light-Obscured Particle Counting Method)," specifies the specified number of insoluble particles as follows: for injections of 100 mL or more, there should be no more than 25 particles of 10 μm or larger and no more than 3 particles of 25 μm or larger per mL; and for injections of less than 100 mL, there should be no more than 6,000 particles of 10 μm or larger and no more than 600 particles of 25 μm or larger per container. Although the Japanese Pharmacopoeia does not specify a range for the number of insoluble particles in plasma preparations, it is preferable that the number of insoluble particles is small from the viewpoint of intravenous preparations. In the present invention, the term "insoluble particles" refers to exogenous, insoluble particles that are easily mobile and are not gas bubbles and that are unintentionally mixed into plasma.

[0045] In the present invention, the number of insoluble particles can be measured using "Insoluble Particle Test Method for Injections, Method 1 (Light Obscuration Particle Counting Method)" described in General Test Method 6.07 of the Japanese Pharmacopoeia (JP). (Measurement Conditions) Apparatus: Liquid particle counter HIAC System 9703+ (Beckman Courter) Sensor model: HRLD150 Sampler settings: Syringe size 1 mL, probe size 2.75, 1 / 16 O.D. parameters: Sample volume 0.2 mL Number of measurements: 3 Dilution ratio: 1 Channel size (μm): 2, 3, 5, 10, 15, 20, 25, 30, 40, 50, 99

[0046] The plasma after the ultraviolet irradiation step may further include a sterilization filtration step. Specifically, a sterilization filter (sterilization membrane) is used for the sterilization filtration. For example, the sterilization filter may be made of a material such as regenerated cellulose, polyethersulfone, or PVDF, and may be performed using a combination of sterilization membranes with pore sizes of 0.45 μm and 0.22 μm.

[0047] Furthermore, the production method of the present invention may include steps such as shaking or stirring the plasma, concentration treatment and concentration / buffer exchange treatment, as necessary.

[0048] The plasma obtained by the production method of the present invention may be formulated as a plasma preparation with its liquid composition as is, or may be formulated as a plasma preparation after buffer exchange with a solvent of a different composition.

[0049] The plasma obtained by the production method of the present invention may be supplemented with other components to prepare a plasma preparation, as long as the effects of the present invention are not impaired. Examples of such other components include amino acids, inorganic salts, buffer components, organic solvents, surfactants, sugars, and vitamins.

[0050] Furthermore, the plasma obtained by the production method of the present invention may be powdered in a drying process. Specifically, the plasma is sprayed into a vacuum freeze-drying tower under vacuum (spray pressure: 0.01 to 0.3 MPa) from a nozzle (preferably a dispenser nozzle) with a 50 to 350 μm (preferably 100 to 250 μm) orifice diameter for spraying the plasma through the nozzle (preferably a dispenser nozzle) located at the top of the vacuum freeze-drying tower. The resulting fine raw material liquid particles are then self-freezed to form fine frozen particles. The fine frozen particles are then captured on a capture surface within the vacuum freeze-drying tower (pressure within the vacuum freeze-drying tower: 1.0 to 20 Pa), heated, and sublimated to dry. This powdering process is also referred to herein as a spray-type vacuum freeze-drying process. The dried plasma obtained by this freeze-drying process has excellent particle homogeneity, maintains the biological activity of each plasma component, and exhibits high solubility upon plasma reconstitution, reducing the risk of side effects.

[0051] Another embodiment of the present invention is a plasma preparation obtained by the production method of the present invention (also referred to as the plasma preparation of the present invention). The plasma preparation of the present invention may be the above-described treated plasma itself or a prepared plasma preparation containing, for example, other components, in which viruses have been inactivated, various blood coagulation factors normally required to prevent bleeding from blood are highly active, and the complement value (CH50) has also been reduced.

[0052] "Viruses are inactivated" means that they are rendered non-infectious. Specifically, this means that the log reduction value (LRV) for the virus is greater than 1.00, preferably 2.00 or greater, more preferably 3.00 or greater, and even more preferably 4.00 or greater.

[0053] Blood coagulation factors include Factor I (Fibrinogen), Factor II (Prothrombin), Factor III (Tissue Factor), Factor V (Proaccelerin), Factor VII (Proconvertin), Factor VIII (Antihemophilic Factor), Factor IX (Christmas Factor), Factor X (Stuart Factor), Factor XI (Plasma Thromboplastin Precursor), Factor XII (Hageman Factor), and Factor XIII (Factor XIII). XIII) Fibrin-stabilizing factor, von Willebrand factor (vWF), ADAMTS13, antithrombin III, protein C, protein S, plasminogen, α2-antiplasmin, etc. The activity of each blood coagulation factor in the plasma preparation of the present invention maintains the activity normally required for plasma preparations, and is within the range of the EP standard value, for example.

[0054] Specifically, the activity ranges of each blood coagulation factor in the plasma preparation of the present invention are as follows: 1) Factor I (fibrinogen): 150 mg / dL to 400 mg / dL 2) Factor VIII: 0.5 IU / mL to 1.5 IU / mL 3) Factor V: 0.5 IU / mL to 1.3 IU / mL 4) α2-antiplasmin: 0.2 IU / mL to 1.2 IU / mL 5) Protein C: 0.7 IU / mL to 1.5 IU / mL 6) Factor VII: 0.5 IU / mL to 1.4 IU / mL 7) Factor II: 0.5 IU / mL to 1.3 IU / mL 8) Factor IX: 0.5 IU / mL to 1.3 IU / mL 9) Factor X: 0.7 IU / mL or more 10) Factor XII: 0.5 IU / mL or more and 1.5 IU / mL or less 11) Factor XIII: 0.7 IU / mL or more and 1.4 IU / mL or less 12) Plasminogen: 0.7 IU / mL or more 13) Antithrombin III: 0.7 IU / mL or more and 1.2 IU / mL or less 14) vWF: 0.6 IU / mL or more and 1.7 IU / mL or less 15) ADAMTS13: 0.5 IU / mL or more and 1.5 IU / mL or less

[0055] Here, the fibrinogen values ​​were based on the standard values ​​of the SD-treated plasma product "Octaplas LG (registered trademark)." The above 2) to 5) and 7) are within the range of the EP standard values. The lower limit of hemostatic levels of blood coagulation factors in the body for therapeutic administration in coagulation factor deficiency or prophylactic administration during invasive procedures is 75 mg / dL of factor I (fibrinogen), 0.4 IU / mL of factor II, 0.15 IU / mL of factor V, 0.05 IU / mL of factor VII, 0.1 IU / mL of factor VIII, 0.1 IU / mL of factor IX, 0.1 IU / mL of factor X, 0.1 IU / mL of factor XI, 0.15 IU / mL of factor XIII, and 0.25 IU / mL of von Willebrand factor (Ref: Replacement therapy platelet concentrates, fresh frozen plasma, fibrinogen therapy. Journal of Thrombosis and Hemostasis 17(3); 308-313, 2006). Although the above-mentioned activities of plasma preparations vary somewhat depending on the production lot, von Willebrand factor (vWF), the main factor involved in primary thrombus formation, and factor I (fibrinogen), the main factor involved in secondary thrombus formation, are particularly important factors in the coagulation cascade. vWF binds to platelets in the blood via the GPIb-α / IX receptor on the surface of platelets, forming a primary thrombus (also known as a white thrombus) and causing platelet adhesion. Furthermore, fibrinogen binds to this primary thrombus via the platelet membrane glycoprotein GPIIb / IIIa, causing platelet aggregation. After platelet aggregation occurs, the coagulation cascade is accelerated, producing thrombin that converts fibrinogen to fibrin. The formed fibrin is then crosslinked by Factor XIII to form a strong secondary thrombus (also known as a brown thrombus), resulting in hemostasis. Therefore, it is desirable for the lower limit of fibrinogen and vWF in plasma preparations to be 75 mg / dL and 0.25 IU / mL, respectively. As described above, the plasma preparation of the present invention is highly useful because the activity of various blood coagulation factors is maintained at a level higher than that required for hemostasis. The specific method and conditions for measuring the activity of each blood coagulation factor can be conventional, and for example, an automated coagulation analyzer can be used.

[0056] Complement is a plasma protein produced primarily in the liver that undergoes chain activation in response to bacterial invasion or other triggers, and comprises nine components C1 to C9 and various reaction factors. The complement titer CH50 (CH50 / mL), which indicates the sum of complement activities, represents the amount of complement activity required for 50% hemolysis of a given number of red blood cells. When measured by a complement titer calculation method (Mayer relative turbidimetric method) based on the change in absorbance due to hemolysis using sensitized sheep red blood cells, the complement titer of the plasma preparation of the present invention is lower than the normal range of 25.0 to 48.0 (CH50 / mL), and is usually less than 25 CH50 / mL, preferably 22 CH50 / mL or less, and more preferably 20 CH50 / mL or less.

[0057] From the viewpoint of viral inactivation efficiency by uniformly and deeply irradiating plasma with ultraviolet light, the plasma preparation of the present invention preferably has a low number of insoluble particles in plasma (cumulative number of particles per mL). Typically, the number of insoluble particles of 10 μm or larger is 3,000 or less, and the number of insoluble particles of 25 μm or larger is 200 or less, preferably the number of insoluble particles of 10 μm or larger is 1,000 or less, and the number of insoluble particles of 25 μm or larger is 100 or less, and more preferably the number of insoluble particles of 10 μm or larger is 500 or less, and the number of insoluble particles of 25 μm or larger is 50 or less. Here, the number of insoluble particles is evaluated by "Insoluble Particle Test Method for Injections, Method 1 (Light-Shielding Particle Counting Method)" described in General Test Method 6.07 of the Japanese Pharmacopoeia (JP). As is well known to those skilled in the art, evaluation of the number of insoluble particles in a plasma preparation may require a comprehensive evaluation of the evaluation results of plasma obtained from multiple production lots. The plasma preparation of the present invention is used in cases of bleeding or bleeding tendency due to deficiency of multiple blood coagulation factors, and depending on the purpose of use, it can be used directly for transfusion or stored as frozen fresh plasma, and can be further used as raw material plasma for transfusion or fractionated preparations. Furthermore, because the risk of viral contamination is reduced, it can also be used as a reagent in regenerative medicine and cell culture.

[0058] The plasma preparation of the present invention may also be dried and used as a dried plasma preparation. Compared to FFP, which requires thawing before use and takes time to administer, making it difficult to use for emergency transfusions, dried plasma preparations can be administered immediately, are convenient to store and carry, and are advantageous in terms of transportation. The dried plasma preparation may be dried by a conventional method or through the drying process described above. For example, dried plasma may be obtained by powder processing using a vacuum freeze-drying device. The physical properties of the dried plasma preparation are maintained, such as maintaining high activity of coagulation factors and reducing the complement value (CH50).

[0059] Another aspect of the present invention is a method for reducing the complement titer (CH50) of a plasma product, comprising: (1) subjecting plasma to a solvent / surfactant treatment (S / D treatment); and (2) irradiating the S / D-treated plasma with ultraviolet light having a central wavelength of 200 to 260 nm from an LED light source for 10 minutes or longer so that the ΔA352, calculated using a 1% potassium iodide solution equivalent to the cumulative exposure dose, is 0.2 to 0.7. By employing the above steps (1) and (2), the complement titer of the plasma product can be typically less than 25 CH50 / mL, preferably 15 CH50 / mL to less than 25 CH50 / mL, more preferably 15 CH50 / mL to 22 CH50 / mL, and even more preferably 15 CH50 / mL to 20 CH50 / mL. Other definitions and preferred ranges are as described above.

[0060] Another aspect of the present invention is a method for inactivating viruses in a plasma product, comprising the steps (1) and (2) above. By employing the steps (1) and (2) above, the log reduction value (LRV) of the virus in the plasma product can be made to exceed 1.00, preferably 2.00 or more, more preferably 3.00 or more, and even more preferably 4.00 or more. Other definitions and preferred ranges are as described above.

[0061] The present invention will be described below with reference to examples, although the present invention is not limited to these examples.

[0062] Example 1: Study of Virus Inactivation in S / D-Treated Plasma Using UV LED with a Center Wavelength of 255 nm [Test Overview] (1) Sample Preparation Fresh frozen plasma (FFP) was obtained from the Japanese Red Cross Society. Fresh frozen plasma was prepared by mixing 200 mL of human blood with 28 mL of blood preservation solution (CPD solution), separating and removing most of the leukocytes, and freezing the resulting fresh plasma. CPD solution was prepared by dissolving 26.3 g of sodium citrate hydrate, 3.27 g of citric acid hydrate, 23.2 g of glucose, and 2.51 g of sodium dihydrogen phosphate in water for injection to a total volume of 1000 mL. S / D-treated plasma was prepared as follows. The manufacturing flow charts are shown in Figures 1 and 2.

[0063] Fresh frozen plasma (FFP) was thawed and pooled to produce the FFP pool. The FFP pool was filtered to remove cell debris. To this solution, the solvent / detergents TNBP (final concentration 1%) and Triton X-100 (final concentration 1%) were added and stirred at 30°C for 1 hour for S / D treatment. The S / D solution was subjected to oil extraction with soybean oil or castor oil (5% v / v), transferring TNBP to the upper oil layer, and the lower aqueous layer was collected and filtered. Triton X-100 was adsorbed onto an octadecyl-group-bonded (C18) ODS column, which is commonly used in reversed-phase chromatography. The filtered solution was then passed through the ODS column to remove Triton X-100. The column effluent was collected and designated S / D-treated plasma (SDP). For comparison, plasma not subjected to ultraviolet irradiation was collected from the column effluent and filtered through a sterilizing filter, and this may also be referred to as S / D-treated plasma.

[0064] (2) UV Irradiation UV LED with a central wavelength of 255 nm was irradiated on S / D Plasma and fresh frozen plasma (FFP) as a control for 10 to 30 minutes, respectively, to evaluate the virus inactivation effect and the degree of damage to coagulation factors. S / D Plasma and FFP were added to a petri dish to a thickness of 3 mm, and UV light was irradiated from the UV LED while shaking. UV irradiance was measured using a cuvette cell with a 10 mm optical path length, and the absorbance at 352 nm of 1% KI, a chemical actinometer, after 30 seconds of irradiation at a constant irradiance was measured using a Thermo Fisher Scientific NanoDrop 2000c spectrophotometer. The change in absorbance (ΔA352 ３０　ｓｅｃ The cumulative irradiation dose was evaluated based on the change in absorbance at 352 nm (ΔA352) of 1% KI relative to the set irradiation time. The irradiation time and ΔA352 are shown in Table 1.

[0065] (3) Various Evaluations (3-1) Evaluation of Virus Inactivation Effect The virus inactivation effect was evaluated by adding porcine parvovirus (PPV) to FFP and evaluating the change in infectivity due to ultraviolet irradiation. The infectivity was evaluated using the infectivity titer (TCID ５０ The LRV was calculated by the method, and the LRV was evaluated from the change in infectivity before and after ultraviolet irradiation. The results are shown in Table 1.

[0066] (3-2) Evaluation of Damage to Coagulation Factors The degree of damage to coagulation factors was evaluated by measuring the coagulation activity of fibrinogen (Fib) and blood coagulation factor VIII (Factor VIII) in FFP or S / D plasma before and after UV irradiation, and evaluating the inactivation rate after irradiation, with the activity before UV irradiation set at 100%. The results are shown in Table 1.

[0067]

[0068] [Summary of Results] As shown in Table 1, it was revealed that S / D treatment enhances viral inactivation in plasma caused by UV irradiation. As shown in Table 1, it was revealed that S / D treatment does not affect the damage to coagulation activity in plasma caused by UV irradiation.

[0069] Example 2: Quality evaluation of S / D-UV Plasma preparations subjected to S / D treatment and LED ultraviolet irradiation [Test overview] (1) Sample preparation S / D Plasma was prepared in the same manner as in Example 1. After the S / D Plasma was produced, it was irradiated with an ultraviolet LED having a central wavelength of 255 nm, and sterilized and filtered plasma (also referred to as S / D-UV Plasma) was prepared as follows. As in Example 1, the S / D Plasma was added to a petri dish to a thickness of 3 mm, and irradiated with ultraviolet light from the ultraviolet LED for 15 minutes while shaking (the irradiation conditions were determined in advance to give a ΔA352 in the range of 0.4 to 0.5).

[0070] (2) Measurement of Various Evaluation Items (2-1) Protein Content of Plasma Preparations Protein concentration was determined using the conventional BCA method. Albumin, the main component of plasma, was used as a standard. (2-2) Evaluation of Coagulation Factor Activity Coagulation factor activity was evaluated using a conventional method to assess whether it met the EP standard value for S / D Plasma preparations. Specifically, the coagulation activity of each blood coagulation factor in plasma was measured using a Sysmex automated coagulation analyzer CS2400 and dedicated reagents.

[0071] (Evaluation items) Activated partial thromboplastin time (APTT), prothrombin time (PT), fibrinogen activity (mg / dL), Factor VIII activity (IU / mL), Factor V activity (IU / mL), Factor VII activity (IU / mL), Factor II activity (IU / mL), Factor IX activity (IU / mL), Factor X activity (IU / mL), Factor XII activity (IU / mL) (Measurement method: clotting time method) Measurements were performed by conventional methods depending on each evaluation item.

[0072] (Evaluation items) Protein C activity (IU / mL), α2-antiplasmin activity (IU / mL), Factor XIII activity (IU / mL), Antithrombin III activity (IU / mL) (Measurement method: synthetic substrate method) Measurement was carried out by a conventional method depending on each evaluation item.

[0073] (Evaluation Items) vWF Ristocetin Activity (vWF:Rco) (IU / mL) (Measurement Method: Platelet Aggregation Method) The coagulation activity of blood coagulation factors in the plasma solution was measured using an automated coagulation measuring device CS2400 manufactured by Sysmex Corporation and dedicated reagents for the device. (Device) Automatic coagulation analyzer CS2400 (Sysmex) (Standard) Standard human plasma for blood coagulation tests (SIEMENS / Sysmex) (Dilution buffer) Owren-Veronal buffer containing 1% BSA (Reagents and materials) Owren-Veronal buffer (Sysmex) Standard human plasma for blood coagulation tests (SIEMENS / Sysmex) 0.02 mol / L calcium chloride solution (Sysmex) Thrombocheck APTT (containing rabbit brain-derived cephalin and ellagic acid) (Sysmex) Thrombocheck PT (freeze-dried product containing rabbit brain-derived thromboplastin and calcium lactate) (Sysmex) Thrombocheck Fib (thrombin reagent) (Sysmex) Thrombocheck Factor VIII (Factor VIII deficient plasma) (Sysmex) ThromboCheck Factor V (Factor V deficient plasma) (Sysmex) ThromboCheck Factor VII (Factor VII deficient plasma) (Sysmex) ThromboCheck Factor II (Factor II deficient plasma) (Sysmex) ThromboCheck Factor IX (Factor IX deficient plasma) (Sysmex) ThromboCheck Factor X (Factor X deficient plasma) (Sysmex) ThromboCheck Factor XII (Factor XII deficient plasma) (Sysmex) Verichrome Protein C (Sysmex) Verichrome α ２ - Antiplasmin (Sysmex) - Berichrome Factor XIII (Sysmex) - Berichrome Plasminogen (Sysmex) - Berichrome Antithrombin III (Sysmex) - BC von Willebrand Reagent (Sysmex)

[0074] (2-3) Evaluation of complement value (CH50) Complement value was measured using a method for calculating complement value based on the change in absorbance due to hemolysis using sensitized sheep red blood cells. (Measurement principle) When sensitized sheep red blood cells are reacted with a sample, the sensitized sheep red blood cells are hemolyzed by the complement in the sample. When this hemolysis reaction is interpreted as a change in absorbance, the amount of change is proportional to the complement value in the sample, so a calibration curve is created using a standard complement with a known concentration of complement value, and the complement value in the sample is determined. (Reagent) Auto CH50-L "Seiken" II

[0075] (2-4) Evaluation of C3 and C4 antigen quantity (Device) Immunochemical automatic analyzer Atellica NEPH630 (Siemens Healthcare) (Reagent) N-antiserum C3c, N-antiserum C4 (Siemens Healthcare) (Standard) N Protein Standard SL (Siemens Healthcare)

[0076] (2-5) Evaluation of thrombin generation amount The thrombin generation amount, which is the factor that has the greatest effect on blood coagulation ability, was measured using a calibrated automated thrombogram (CAT) system. (Apparatus) Calibrated automated thrombogram (CAT) system (FingalLink) (Measurement overview) The thrombin generation amount is measured by adding tissue factor (TF), calcium chloride, and phospholipids to citrated plasma, and measuring the generated thrombin using a synthetic substrate method. The measurement reagents used were tissue factor reagent PPP Low (TF concentration 1 pmol / L), calcium chloride, and phospholipids. These reagents were added to a plasma sample, and fluorescence intensity was measured over time. From the thrombogram, Lag Time (time (min) until thrombin generation begins), ETP (total thrombin amount (nmol / L×min)), Peak Thrombin (maximum thrombin generation amount (nmol / L)), and Time to Peak (ttPeak) (time (min) until maximum thrombin generation is reached) were determined. (Reagents and Materials) Thrombin Calibrator (Thrombinoscope) PPP-Reagent Low (Thrombinoscope) (Contains TF concentration of 1 pmol / L, calcium chloride, and phospholipids) FluCa-Kit (Thrombinoscope) (FluCa-Reagent is prepared by adding 40 μL of Fluo-Substrate to one vial of Fluo-Buffer.) (Measurement Method) 20 μL of PPP-Reagent Low or Thrombin Calibrator and 80 μL of the measurement sample were placed in a 96-well microplate. The plate was then set in a plate reader and incubated at 37°C for 10 minutes. 20 μL of FluCa Reagent was added to each well, and the fluorescence intensity was measured for 60 minutes to obtain a thrombogram.

[0077] (2-6) Evaluation of insoluble particles Insoluble particles were measured by the method of "Test method for insoluble particles of injections, Method 1 (light-obscured particle counting method)" described in General Test Method 6.07 of the Japanese Pharmacopoeia (JP). (Measurement method) Apparatus: Liquid particle counter HIAC System 9703+ (Beckman Courter) Sensor model: HRLD150 Sampler settings: Syringe size 1 mL, probe size 2.75, 1 / 16 O.D. Parameters: Sample volume 0.2 mL Number of measurements: 3, Dilution ratio: 1 Channel size (μm): 2, 3, 5, 10, 15, 20, 25, 30, 40, 50, 99

[0078] (2-7) Evaluation by DSC (Differential Scanning Calorimetry) Evaluation by DSC (Differential Scanning Calorimetry) was measured and evaluated by a differential scanning calorimetry analysis method. (Measurement method) (Apparatus) MicroCal PEAQ-DSC Automated (Malvern) (Sample solution) Sample diluted 30 times with PBS (Blank solution) 20 mmol / L glycine (pH 2.4) diluted 30 times with PBS (DSC measurement conditions) Specify MW: 67,000 (Albumin), Scan Settings: Tstart: 25°C, Tend: 100°C, Scan Rate: 90 (°C / hr), Feedback: None, Pre-Scan Thermostat: 5 min, Post-Scan Thermostat: 0 min, Analysis Setting: Activate Assessment (Analysis conditions) Baseline Parameters: Spline, Fitting Module: Non-Two-State

[0079] (2-8) In Vitro / In Vivo Pharmacological Tests (Test Contents) Evaluation of Hemostatic Effect in a Rat Model of Severe Blood Coagulation Disorders (Evaluation Model) Animals: Crl:CD (SD) rats (male, 7 weeks old) Drugs: S / D-UV Plasma (specimen), S / D Plasma (control drug), FFP (control drug), Sarinhes (HES) (negative control), normal rats (untreated) Evaluation Items: ROTEM evaluation (evaluation of rat blood coagulation ability after administration of the formulation) and mortality rate (Experimental Procedures) Rats were anesthetized with sevoflurane inhalation anesthesia, and 27 mL / kg of blood was withdrawn from a catheter-inserted femoral vein at a rate of 1 mL / min. HES was administered through the catheter at a dose of 20 mL / kg and an administration rate of 1 mL / min. The test substance was administered through the catheter at a dose of 30 mL / kg and an administration rate of 0.3 mL / min. Thirty minutes after fluid replacement, the tail was cut 30 mm from the tip to induce bleeding, and the animals were then left to stand for a maximum of 30 minutes until bleeding stopped. The mortality rate after bleeding was then measured. Surviving animals were subjected to citrate blood sampling, and the blood was used to evaluate coagulation ability using ROTEM analysis, as shown in Figure 3. The results are described below.

[0080] ROTEM analysis method (coagulation ability evaluation) (Parameters) CT (Clotting time (sec or min)) The time from the start of measurement to the formation of the initial clot (fibrin) (until the amplitude reaches 2 mm). Equivalent to APTT or PT. CFT (Clot formation time (sec or min)) The time from CT until the amplitude reaches 20 mm. The smaller the value, the faster the fibrin network is formed. MCF (Maximum clot firmness (mm)) The maximum amplitude during measurement. The higher the value, the stronger the clot. (Measurement conditions) (Animal species) Sprague-Dawley rat, male, 7 weeks old (Charles River Japan) (Blood used) Citrated rat blood (blood: 3.8% sodium citrate solution mixed at a ratio of 9:1) (Apparatus and reagents) ROTEM delta (Tem Innovations GmbH), Extem (star-tem, rex-tem) (Measurement time) 30 minutes (Measurement sample) 200 μL of citrated rat blood, 200 μL of physiological saline (blood dilution ratio: 2 times) (Number of measurements) 1

[0081] (3) Results (3-1) Protein Content of Plasma Preparations As shown in Table 2, the protein contents of the FFP pool, S / D Plasma, and S / D-UV Plasma were measured by the BCA method, and the results satisfied the EP standard value of 45 mg / mL or more.

[0082]

[0083] (3-2) Evaluation Results of Coagulation Factor Activity As shown in Table 3, the coagulation factor activity of S / D-UV Plasma was lower than that of FFP, but met the EP standard value of S / D Plasma. Furthermore, as shown in Table 4, the coagulation factor activity of blood coagulation factors without EP standard values ​​was within the concentration range of normal plasma.

[0084]

[0085]

[0086] (3-3) Complement Value (CH50) Results As shown in Table 5, the antigenic content of the third component of complement (C3) and the fourth component of complement (C4) in S / D-UV Plasma and the complement value (CH50) of plasma were measured. The C3 and C4 contents remained unchanged, while the CH50 was lower than that of S / D Plasma and FFP. UVC-LED irradiation was found to be effective in lowering the CH50. Clinically, complement-related diseases can generally be diagnosed by measuring the complement value (CH50) and the C3 and C4 contents. When used as a plasma product, it is desirable for C3 and C4 to be within the normal range. For example, C4 decreases when the classical pathway is activated. Furthermore, decreased C3 and normal C4 are thought to be due to activation of the alternative pathway. In patients with liver cirrhosis, systemic lupus erythematosus, and malignant rheumatoid arthritis, C3, C4, and CH50 are all lower than normal. In addition, in C3 deficiency and acute glomerulonephritis, C4 levels are normal, but C3 and CH50 levels are lower than normal, and in C4 deficiency and C1 inhibitor deficiency, C3 levels are normal, but C4 and CH50 levels are lower than normal. In inflammatory diseases such as rheumatoid arthritis, malignant tumors, vasculitis syndrome, and infections, C3, C4, and CH50 levels are all elevated above normal (Reference: Clinical Laboratory Guide, 2020 revised edition).

[0087]

[0088] (3-4) Results of Thrombin Generation Amount As shown in Table 6 and Figure 4, the thrombin generation amount of the plasma preparations was measured, and the total thrombin generation amount and maximum thrombin concentration (Peak Thrombin) of S / D-UV Plasma were higher than those of FFP. Generally, when the total thrombin generation amount in plasma is high, the frequency of fibrinogen being converted to fibrin increases, increasing the clot formation ability and facilitating hemostasis. On the other hand, plasma with a high total thrombin generation amount but a low maximum thrombin concentration is susceptible to fibrinolysis and has a weakened hemostatic function, so a high maximum thrombin concentration is also important.

[0089]

[0090] (3-5) Results of insoluble particles As shown in Table 7, S / D-UV Plasma had fewer insoluble particles and higher clarity than FFP. Since insoluble particles can cause thrombus formation, a lower number is desirable.

[0091]

[0092] (3-6) Results of DSC (Differential Scanning Calorimetry) As shown in Table 8, the results of DSC showed no difference in the Tm values ​​of albumin and IgG between S / D-UV Plasma and FPP and S / D Plasma, and no phenomenon of albumin or IgG forming aggregates due to LED irradiation was observed.

[0093]

[0094] (3-7) Mortality and Blood Coagulation Assessment by ROTEM Analysis Plasma preparations were administered intravenously to rats with a severe dilutional coagulopathy model, and mortality and blood coagulation were assessed by ROTEM analysis. As shown in Table 9, the mortality rate increased in the FFP and S / D Plasma groups compared to the HES group (negative control), whereas no change in mortality rate was observed with S / D-UV Plasma, demonstrating that mortality was suppressed. As shown in Table 10, ROTEM measurements showed that S / D-UV Plasma did not increase CT (clotting time), but FFP and S / D Plasma increased CT. Furthermore, MCF (maximum clot strength) was comparable for S / D-UV Plasma and FFP.

[0095]

[0096]

[0097] Example 3: Quality evaluation of dried plasma obtained by powder treatment of S / D-UV plasma [Test overview] (1) Sample preparation S / D plasma and S / D-UV plasma were prepared in the same manner as in Example 1. The S / D plasma and S / D-UV plasma were powder-treated using a vacuum freeze-drying device manufactured by ULVAC, Inc. to obtain dried plasma. The dried plasma was dissolved at room temperature using a dedicated dissolving solution (20 mmol / L glycine, pH 2.4). Specifically, 3.0 mL of the dissolving solution was added to 0.3 g of dried plasma to achieve a weight ratio of 10 w / v%, and the reconstituted plasma was used as a sample. S / D-UV Plasma (specimen), S / D-UV Plasma Powder (specimen), S / D Plasma (control agent), S / D Plasma Powder (control agent), FFP (control agent)

[0098] (2) Measurement of Various Evaluation Items (2-1) Measurement of Protein Content Measurement and evaluation were carried out in the same manner as in Example 2.

[0099] (2-2) Evaluation of Complement Value (CH50) Measurement and evaluation were carried out in the same manner as in Example 2.

[0100] (2-3) Measurement of antibody content (Device) Immunochemical automatic analyzer Atellica NEPH630 (Siemens Healthcare) (Reagents) N-antiserum IgG, N-antiserum IgA, N-antiserum IgM (Siemens Healthcare) (Standard) N Protein Standard SL (Siemens Healthcare)

[0101] (2-4) Evaluation of Coagulation Factor Activity Coagulation factor activity was measured and evaluated in the same manner as in Example 2. The evaluation criteria for each coagulation factor activity were the European Pharmacopoeia standard value for S / D Plasma preparations (EP standard value) and the standard value for the commercially available S / D Plasma preparation "OctaplasLG (registered trademark)."

[0102] (2-5) Protein S activity (Measurement method: clotting time method) (Measurement principle) The activity of free protein S in plasma was measured using the prothrombin time method. Protein S activity was measured by measuring the degree of prolongation of prothrombin time using tissue factor, synthetic phospholipid, calcium chloride, and activated protein C as reagents. Protein S activity correlates with the prolongation of clotting time when protein S-deficient plasma is added to a diluted sample, so the change over time was measured to determine protein S activity (%). (Measurement equipment) Blood coagulation automatic analyzer ACL-TOP (Instrumentation Laboratory Co., Ltd.) (Measurement reagents) Hemos IL PS Clot (Instrumentation Laboratory Co., Ltd.) Hemos IL Factor Diluent (Instrumentation Laboratory Co., Ltd.)

[0103] (2-6) ADAMTS13 Activity (Measurement Method: ELISA) (Measurement Principle) This is a solid-phase sandwich ELISA using a monoclonal antibody that specifically recognizes the amino acid Tyr1605 at the end of VWF-A2, which is generated by cleavage by ADAMTS13 present in plasma. (Measurement Reagent) ADAMTS13-act ELISA Kit

[0104] (2-7) ROTEM Analysis Using Human Blood The coagulation ability of S / D-UV Plasma and S / D-UV Plasma Powder in human blood was evaluated.

[0105] ROTEM analysis (coagulation evaluation) (Apparatus and reagents) ROTEM delta (Tem Innovations GmbH), Extem (star-tem, rex-tem) (Human blood) 10 mL of human blood collected with 3.2% citrate is used. Measurement is performed 30 minutes or more after blood collection. (Measurement conditions) star-tem (CaCl ２20 μL of a buffer solution containing 20 μL of Rextem was placed in a measuring cup, and the sample was warmed at 37°C for 3 minutes. 300 μL of the sample was added to the measuring cup, quickly set in the instrument, and measured for 30 minutes. Physiological saline, the sample, or the control drug was added to human blood in a 1.5-fold volume (so that the human blood was 40%), and the measurement was performed using a ROTEM. Human blood from two individuals (Person A and Person B) was used. (Parameters) ・CT (Clotting time (sec or min)) The time from the start of measurement to the initial clot formation (until the amplitude reaches 2 mm). Equivalent to APTT and PT. ・Clot formation time (sec or min)) The time from CT until the amplitude reaches 20 mm. The smaller the value, the faster the fibrin network is formed. A25 (Amplitude after 25 minutes of CT: clot hardness (mm)) The amplitude of the measured specimen 25 minutes after CT. The higher the value, the stronger the clot.

[0106] (3) Results (3-1) Protein Content of Plasma Products As shown in Table 11, the protein content of each product was measured by the BCA method. All products had a protein content of 45 mg / mL or higher, the EP standard value. S / D Plasma and S / D-UV Plasma had concentrations similar to FFP, but S / D Plasma Powder was dissolved to a weight ratio of 10 w / v% when reconstituted with a reconstitution solution (20 mmol / L glycine, pH 2.4). Therefore, the protein content of the reconstituted plasma was higher than that of ordinary FFP, with a concentration effect of about 1.1 to 1.2 times.

[0107]

[0108] (3-2) Complement titer (CH50) results As shown in Table 12, the complement titer (CH50) of S / D-UV Plasma and S / D-UV Plasma Powder was lower than that of S / D Plasma and FFP, and the effect of UVC-LED irradiation on complement titer reduction was observed. C3 and C4 concentrations were within the range of normal plasma in both plasma preparations. In S / D Plasma Powder and S / D-UV Plasma Powder, the plasma after reconstitution was 1.1 to 1.2 times more concentrated than FFP, resulting in a high protein content, and therefore the C3 and C4 concentrations were also 1.1 to 1.2 times more concentrated, but within the normal range.

[0109]

[0110] (3-3) Antibody Content As shown in Table 13, the antibody contents of IgG, IgA, and IgM in S / D-UV Plasma and S / D-UV Plasma Powder were measured by nephelometry (immunoturbidimetry), and the results were within the concentration range of IgG, IgM, and IgA in normal plasma. Because S / D Plasma Powder and S / D-UV Plasma Powder were 1.1- to 1.2-fold more concentrated than FFP, a concentrating effect was also observed in the antibody content (IgG, IgM, IgA). Abnormal values ​​of antibody content in plasma can be a cause of disease. For example, high IgG levels can lead to persistent antigen stimulation and abnormal activation of the immune system, and are seen in pathologies such as autoimmune diseases and collagen diseases. In addition, low IgG levels make the patient susceptible to infection and are seen in conditions such as multiple myeloma, immunodeficiency, and low γ-globulin plasma. When used as a plasma preparation, it is desirable that the antibody content (IgG, IgA, IgM) be within the concentration range of normal plasma.

[0111]

[0112] (3-4) Evaluation Results of Coagulation Factor Activity As shown in Tables 14 and 15, the coagulation factor activity of S / D-UV Plasma Powder included blood coagulation factors that met the EP standard, and the activities of various blood coagulation factors were maintained at higher levels than those required for hemostasis, making it possible to prepare dried plasma with physical properties similar to those of S / D Plasma.

[0113]

[0114]

[0115] (3-5) ROTEM Analysis Results Using Human Blood As shown in Table 16, compared to untreated human blood, 40% blood prolonged CT (clotting time) and CFT (clot formation time), and clot hardness (A25) 25 minutes after CT decreased. Compared to 40% blood (negative control), S / D-UV Plasma and S / D-UV Plasma Powder shortened CT and CFT, and increased clot hardness (A25). The CT, CFT, and A25 of S / D-UV Plasma and S / D-UV Plasma Powder were comparable to those of S / D Plasma.

[0116]

[0117] According to the production method of the present invention, viruses can be inactivated simply and easily while maintaining biological activity in plasma, and a safe plasma preparation with reduced complement value can be provided that can be used for plasma exchange and transfusion during organ transplantation.

[0118] This application is based on patent application No. 2023-142367 filed in Japan, the contents of which are incorporated in their entirety herein.

Claims

1. A method for producing a plasma preparation comprising the following steps: (1) subjecting plasma to a solvent / surfactant treatment (S / D treatment); and (2) irradiating the S / D-treated plasma with ultraviolet light having a central wavelength of 200-260 nm from an LED light source for 10 minutes or more so that ΔA352, calculated using a 1% potassium iodide aqueous solution equivalent to the cumulative exposure dose, is 0.2-0.

7.

2. The method of claim 1 further comprising an oil extraction step.

3. The method of claim 2, further comprising a purification step by solid phase extraction.

4. The method of claim 3, further comprising a vacuum freeze-drying step.

5. A plasma preparation obtained by the manufacturing method described in any one of claims 1 to 3.

6. A dried plasma preparation obtained by the manufacturing method described in claim 4.

7. The plasma preparation described in claim 5, having a complement value (CH50) of less than 25 CH50 / mL.

8. The plasma preparation according to claim 7, comprising one or more selected from the group consisting of factor I (fibrinogen) of 150 mg / dL to 400 mg / dL, factor VIII of 0.5 IU / mL to 1.5 IU / mL, factor V of 0.5 IU / mL to 1.3 IU / mL, α2-antiplasmin of 0.2 IU / mL to 1.2 IU / mL, protein C of 0.7 IU / mL to 1.5 IU / mL, factor II of 0.5 IU / mL to 1.3 IU / mL, and vWF (von Willebrand factor) of 0.6 IU / mL to 1.7 IU / mL.

9. The plasma preparation according to claim 7, which is a culture reagent.

10. A method for reducing the complement value (CH50) of a plasma product, comprising the following steps: (1) subjecting the plasma to a solvent / detergent treatment (S / D treatment); and (2) irradiating the S / D-treated plasma with ultraviolet light having a central wavelength of 200 to 260 nm from an LED light source for 10 minutes or more so that ΔA352, calculated using a 1% potassium iodide aqueous solution equivalent to the cumulative irradiation dose, is 0.2 to 0.

7.

11. A method for inactivating viruses in a plasma product, comprising the following steps: (1) subjecting the plasma to a solvent / detergent (S / D) treatment; and (2) irradiating the S / D-treated plasma with ultraviolet light having a central wavelength of 200 to 260 nm from an LED light source for 10 minutes or more so that ΔA352, calculated using a 1% potassium iodide aqueous solution equivalent to the cumulative exposure dose, is 0.2 to 0.7.

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