Method for producing blood products, and blood products

Abstract

To provide means capable of accomplishing inactivation of a pathogen in a blood sample containing red blood cells and platelets together, while sufficiently reducing the hemolysis rate of the red blood cell product finally obtained.SOLUTION: This means involves applying ultraviolet rays to a blood sample containing red blood cells and platelets in the presence of a hemolysis inhibitor and a surfactant having an HLB value of 13 or greater and having the number of oxyethylene groups of a hydrophilic portion in the molecular structure of 20 or greater when producing a blood product containing red blood cells and platelets.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a method for producing a blood product, and to a blood product. [Background technology]

[0002] In recent years, blood transfusion has become common in which components are separated from blood (whole blood) obtained through donations, etc., to provide only the components needed by the patient. Blood component transfusion has the advantage of reducing the burden on the circulatory system and side effects for the patient, as well as making effective use of donated blood.

[0003] Generally, blood (whole blood) collected from a donor in a blood bag is centrifuged to separate necessary blood products (red blood cell products, platelet products, and plasma products) and transferred to multiple bags for each blood product, and pathogens such as viruses are inactivated in each blood product. For example, Japanese Patent Application Laid-Open No. 2004-081692 discloses a technique in which a photoactivator (pathogen inactivator) is added to a bag containing a blood product, and light of a specific wavelength is irradiated to activate the pathogen inactivator and inactivate the pathogen. However, the technique described in Japanese Patent Application Laid-Open No. 2004-081692 separates blood collected in a blood bag into multiple blood products, and then inactivates pathogens in each blood product. Therefore, it is necessary to perform the inactivation process for each blood product, which is problematic in that it takes a lot of time and costs.

[0004] In addition, a technique has been proposed in which a blood sample containing both red blood cells and platelets is irradiated with ultraviolet light in the presence of a photosensitizer to inactivate pathogens, but in this case, there is a problem that most of the light required to activate the photosensitizer is absorbed by the red blood cell components in the blood sample and may not be effectively used to inactivate pathogens. On the other hand, if the blood sample is irradiated with a sufficient amount of light to inactivate pathogens in the red blood cells, damage is caused to the red blood cell components and components other than the red blood cell components, which causes a problem of a decrease in the quality of the obtained blood product.

[0005] With the aim of solving these problems, International Publication No. 2008 / 71541 (JP Patent Publication No. 2010-535235) discloses a technique for inactivating pathogens in a blood sample containing both red blood cells and platelets by irradiating the sample with ultraviolet light in the presence of an allosazine photosensitizer such as riboflavin before separation into each blood product.

[0006] For example, the product standard for red blood cell products in Europe requires that the hemolysis rate must be 0.8% or less for a storage period of at least six weeks. Currently, red blood cell products supplied to the market are discarded when the six-week expiration date expires, and even in Europe alone, there is a growing awareness that the fact that a significant amount of valuable blood resources collected through blood donations are being discarded must be improved (see Ayyalil et al., Transfusion 2017; 57 (12): 2870-7). Therefore, if the period during which red blood cell products meet the above condition could be extended, the loss of such valuable value could be significantly reduced, which would be highly desirable not only from an economic perspective but also from an ethical perspective. Summary of the Invention [Problem to be solved by the invention]

[0007] According to the inventors' investigations, it has been found that even the technology described in International Publication No. 2008 / 71541 (JP Patent Publication No. 2010-535235) cannot achieve inactivation of pathogens in a blood sample containing both red blood cells and platelets while sufficiently reducing the hemolysis rate of the final red blood cell preparation.

[0008] Therefore, an object of the present invention is to provide a means for achieving inactivation of pathogens in a blood sample containing both red blood cells and platelets while sufficiently reducing the hemolysis rate of the final red blood cell product. [Means for solving the problem]

[0009] The present inventors have conducted extensive research to solve the above problems. As a result, they have surprisingly found that the above problems can be solved by irradiating a blood sample containing both red blood cells and platelets with ultraviolet light in the presence of a hemolysis inhibitor and a specific surfactant. Based on this finding, they have completed the present invention.

[0010] That is, according to one embodiment of the present invention, there is provided a method for producing a blood product, which comprises irradiating with ultraviolet light a blood sample containing red blood cells, platelets, a hemolysis inhibitor, and a surfactant having an HLB value of 13 or more and having 20 or more oxyethylene groups in the hydrophilic moiety in its molecular structure.

[0011] According to another aspect of the present invention, there is provided a blood product containing red blood cells, platelets, a hemolysis inhibitor, and a surfactant having an HLB value of 13 or more and having 20 or more oxyethylene groups in the hydrophilic part in its molecular structure, in which pathogens have been inactivated.

[0012] Furthermore, according to yet another aspect of the present invention, there is provided a blood cell preservation solution which contains a hemolysis inhibitor and a surfactant having an HLB value of 13 or more and having 20 or more oxyethylene groups in the hydrophilic part of its molecular structure, and which is used by being mixed with the blood sample in the manufacturing method of the above-mentioned aspect. [Brief description of the drawings]

[0013] [Figure 1] FIG. 1 is an explanatory diagram for explaining one embodiment of a method for producing a blood product according to the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] Hereinafter, an embodiment of the present invention will be described with reference to the accompanying drawings.

[0015] <Blood product manufacturing method> One aspect of the present invention relates to a method for producing a blood product, which comprises irradiating with ultraviolet light a blood sample containing red blood cells, platelets, a hemolysis inhibitor, and a surfactant having an HLB value of 13 or more and a hydrophilic moiety having an oxyethylene group number of 20 or more in the molecular structure. The method for producing a blood product according to the present invention makes it possible to sufficiently reduce the hemolysis rate of the finally obtained red blood cell product, while achieving inactivation of pathogens in a blood sample containing both red blood cells and platelets.

[0016] Fig. 1 is an explanatory diagram for explaining one embodiment of the method for producing a blood product according to the present invention. In the method for producing a blood product according to the embodiment shown in Fig. 1, first, a whole blood sample 10 containing red blood cells 12, white blood cells 14, and platelets 16 in plasma 18 is prepared. Next, the whole blood sample 10 is treated using a white blood cell removal filter to remove the white blood cells 14. Thereafter, vitamin B 2 (riboflavin), vitamin E (tocopherol acetate) as a hemolysis inhibitor, and Tween (registered trademark, the same applies below) 80 (polyoxyethylene sorbitan monooleate) as a surfactant are added. After these additions, the sample is irradiated with ultraviolet light to inactivate pathogens in the sample. Then, by centrifugation, a red blood cell preparation 20 containing red blood cells 12 in plasma 18 and a platelet preparation 30 containing platelets 16 in plasma 18 are obtained.

[0017] The present invention will now be described in more detail.

[0018] [Blood sample] In the manufacturing method according to the present embodiment, first, a blood sample is prepared. The blood sample contains red blood cells and platelets, as well as a hemolysis inhibitor and a specified surfactant, which will be described later. The blood sample may be a whole blood sample or may be derived from a whole blood sample. However, from the viewpoint of reducing the occurrence of side effects during transfusion, a leukocyte removal process is performed on the whole blood sample, so that the number of leukocytes per bag is reduced to 1×10 6It is preferable that the leukocytes are derived from a whole blood sample from which leukocytes have been removed to a level of 100 or less. As a method for removing leukocytes from a whole blood sample, a conventionally known method can be used, for example, a method in which a leukocyte removal filter is used when collecting a whole blood sample such as a blood donation.

[0019] [Hemolysis prevention agent] The hemolysis inhibitor is intended to prevent the destruction of the red blood cell membrane by its antioxidant effect and affinity with lipids in the red blood cell membrane, and specifically, vitamin E or an unsaturated chain hydrocarbon compound selected from the group consisting of 7-tetradecene, 8-octadecene, 9-eicosene, and squalene is preferred. Vitamin E includes tocopherols such as α-tocopherol, β-tocopherol, γ-tocopherol, and δ-tocopherol, and tocotrienols such as α-tocotrienol, β-tocotrienol, γ-tocotrienol, and δ-tocotrienol, with α-tocopherol being preferred. Vitamin E may also be an ester compound of these tocopherols or tocotrienols, and acetate compounds, specifically tocopherol acetate, are preferred.

[0020] Since the concentration of the hemolysis inhibitor in the red blood cell preparation is preferably 25 to 100 ppm, when added together with a surfactant, the concentration of the hemolysis inhibitor in the blood sample is preferably 75 to 300 ppm. If the concentration is 75 ppm or more, the destruction of the red blood cell membrane can be sufficiently suppressed. Specifically, the hemolysis suppression rate is likely to decrease, in other words, the amount of Hb in the plasma is likely to increase. Also, if the concentration is 300 ppm or less, it is possible to prevent an increase in costs due to saturation of the effect of suppressing the destruction of the red blood cell membrane.

[0021] [Surfactants] The surfactant has the effect of promoting uniform dispersion of the hemolysis inhibitor in the blood sample and coating of the red blood cell membrane with the hemolysis inhibitor, and is also capable of suppressing destruction of the red blood cell membrane by itself. For this reason, it is essential that the surfactant has an HLB value of 13 or more. The HLB value of the surfactant is preferably 13 to 20, and the number of oxyethylene groups (EO number) in the hydrophilic part in the molecular structure is 20 or more, preferably 20 to 40. In addition, as for the molecular structure of the surfactant, a form in which the hydrophilic part is made of polyoxyethylene (PEO) or a form in which the hydrophilic part is made of polyoxyethylene sorbitan (PEO sorbitan) is mentioned as a preferable example.

[0022] Here, if the HLB value of the surfactant is less than 13, the surfactant may not be sufficiently dispersed in the blood sample, and therefore the destruction of the red blood cell membrane may not be suppressed. Also, if the EO number is less than 20, the molecular weight of the hydrophilic part of the surfactant is small, and therefore the destruction of the red blood cell membrane may not be suppressed. Also, if the HLB value exceeds 20 or the EO number exceeds 40, there is no significant improvement in the inhibition of the destruction of the red blood cell membrane, and it is likely to lead to an increase in costs. The HLB value is measured by the Griffin method and calculated as HLB value = (20 x sum of formula weights of hydrophilic parts) / (molecular weight of surfactant).

[0023] Examples of surfactants in which the hydrophilic portion is made of polyoxyethylene (PEO) include polyoxyethylene oleyl ether (Emulgen (registered trademark) 430) and polyoxyethylene lauryl ether (Emulgen 130K).

[0024] Examples of surfactants in which the hydrophilic portion is composed of polyoxyethylene sorbitan (PEO sorbitan) include polyoxyethylene sorbitan monooleate (Tween (registered trademark) 80), polyoxyethylene sorbitan monostearate (Tween 60), polyoxyethylene sorbitan monolaurate (Tween 20), etc. Among these, surfactants in which the hydrophilic portion is composed of polyoxyethylene sorbitan (PEO sorbitan) are preferred, with polyoxyethylene sorbitan monooleate (Tween (registered trademark) 80) being particularly preferred.

[0025] Since the concentration of the surfactant in the red blood cell preparation is preferably 100 to 300 ppm, when added together with the hemolysis inhibitor, the concentration of the surfactant in the blood sample is preferably 300 to 900 ppm. If the concentration is 300 ppm or more, the destruction of the red blood cell membrane can be sufficiently suppressed. Specifically, the decrease in the hemolysis inhibition rate can be prevented, in other words, the amount of Hb in the plasma is unlikely to increase. Also, if the concentration is 900 ppm or less, the destruction of the red blood cell membrane can be sufficiently suppressed and an increase in costs can be prevented.

[0026] The blood sample may further contain additives other than those mentioned above, for example, an allosazine photosensitizer such as riboflavin as disclosed in International Publication WO 2008 / 71541 (JP 2010-535235 A).

[0027] [Ultraviolet light irradiation process] The method for producing a blood product according to the present embodiment includes a step of irradiating the blood sample prepared above (containing red blood cells, platelets, an anti-hemolytic agent, and a surfactant having an HLB value of 13 or more and having 20 or more oxyethylene groups in the hydrophilic part in its molecular structure) with ultraviolet light.

[0028] There is no particular restriction on the specific form of the ultraviolet light used in this step. For example, the peak wavelength of the ultraviolet light is preferably in the range of 200 nm to 400 nm, more preferably in the range of 290 nm to 330 nm or in the range of 360 nm to 380 nm, and particularly preferably in the range of 300 nm to 315 nm. Here, when the peak wavelength is in the range of 300 nm to 315 nm, it is possible to suitably transmit the ultraviolet light through red blood cells.

[0029] There is no particular limitation on the means for irradiating ultraviolet light, and conventionally known methods can be appropriately adopted. For example, low pressure mercury lamps, medium pressure mercury lamps, high pressure mercury lamps, ultra-high pressure mercury lamps, chemical lamps, black light lamps, microwave-excited mercury lamps, metal halide lamps, etc. may be exemplified. In addition, the Mirasol (registered trademark) system available from Terumo BCT, Inc., Lakewood (Colorado) may be used. The amount of ultraviolet light irradiation (accumulated light amount) is not particularly limited, but from the viewpoint of both sufficient inactivation of pathogens and minimizing damage, particularly to red blood cell components, it is preferably 40 J / mL RBC or more and 120 J / mL RBC or less, more preferably 60 J / mL RBC or more and 90 J / mL RBC or less.

[0030] [Other processes] In the method for producing a blood product according to the present invention, other steps may be performed after the above-mentioned step of irradiating with ultraviolet light. For example, the method may further include separating the ultraviolet-irradiated blood sample into a red blood cell product and a platelet product after the ultraviolet light irradiation. There is no particular limitation on the specific method for separating the ultraviolet-irradiated blood sample into a red blood cell product and a platelet product, and a commonly used centrifugation process may be adopted as a preferred method. Note that a person skilled in the art can appropriately determine the specific device and conditions for the centrifugation process in consideration of the physical properties of each product.

[0031] <Blood products> According to the method for producing a blood product according to the above-mentioned embodiment, a blood product is obtained. Therefore, another embodiment of the present invention also provides a blood product. The blood product thus provided contains red blood cells, platelets, a hemolysis inhibitor, and a surfactant having an HLB value of 13 or more and a hydrophilic part having 20 or more oxyethylene groups in its molecular structure. The blood product is also characterized in that the pathogens are inactivated. In the blood product according to the present invention, "pathogens are inactivated" means that the inactivation effect of the pathogens is not significantly lower than that of a blood product obtained by inactivation treatment without using the hemolysis inhibitor and surfactant according to the present invention. Here, whether or not the pathogens in the blood product are inactivated can be confirmed by, for example, plaque assay.

[0032] ≪Blood cell preservation solution≫ In the above, the case where the hemolysis inhibitor and the predetermined surfactant are directly added to the blood sample for the production of the blood product according to the present invention has been described as an example, but the present invention is not limited to such a form. For example, the hemolysis inhibitor and the predetermined surfactant may be dissolved in a suitable solvent in advance to prepare a blood cell preservation solution. The blood cell preservation solution may be added to a blood sample containing red blood cells and platelets to mix the red blood cells and platelets with the hemolysis inhibitor and the predetermined surfactant. As a solvent for preparing the blood cell preservation solution, a conventionally known preservation solution used for long-term storage of blood products is used. Examples of such preservation solutions include ACD solution, CPD solution, MAP solution, SAGM solution, OPTISOL (registered trademark) (AS-5), ADSOL (AS-1), Nutricel (AS-3), PAGG-S, SAGP-maltose, etc. Among them, MAP solution, SAGM solution, or PAGG-S is preferable. Here, the MAP solution is a mixed solution containing mannitol, glucose, adenine, phosphate, citrate, and sodium chloride. The SAGM solution is a mixed solution containing mannitol, glucose, adenine, and sodium chloride, and the PAGG-S solution is a mixed solution containing sorbitol, glucose, adenine, guanosine, phosphate, and sodium chloride.

[0033] According to the present invention, as clearly demonstrated in the examples described below, it is possible to achieve inactivation of pathogens in a blood sample containing both red blood cells and platelets while sufficiently reducing the hemolysis rate of the finally obtained red blood cell product. By achieving such an effect, it is possible to extend the expiration date of the red blood cell product in particular. As a result, it is possible to save a large amount of value loss caused by the disposal of blood products due to the expiration date. In addition, according to the present invention, since pathogen inactivation of red blood cell products can be easily performed, it is possible to provide safe blood products to all patients who require transfusion. In particular, in the Middle East and emerging countries, blood contamination by pathogens such as hepatitis B virus (total treatment cost: about 800,000 yen / person) and HIV (annual treatment cost with anti-HIV drugs: about 3 million yen / person) is quite serious at 10 to 50%, but the benefit of being able to supply safe blood products to patients in these regions is immeasurable. EXAMPLES

[0034] The effects of the present invention will be described using the following examples and comparative examples, although the technical scope of the present invention is not limited to the following examples.

[0035] [Preparation of blood products] (Comparative Example) First, a whole blood sample derived from a human was prepared, and then leukocyte components were removed from the whole blood sample using a leukocyte removal filter.

[0036] After that, 5 × 10 bacteriophage φX174 was added to the sample from which the white blood cell components had been removed as a substitute microorganism. 6 The infected samples were treated with the Mirasol® system available from Terumo BCT, Inc., Lakewood, Colorado, using the instructions provided with the system. 2After adding riboflavin, the pathogens in the sample were inactivated by irradiating it with ultraviolet light. 2 The added concentration of (riboflavin) was 140 ppm, and the ultraviolet irradiation conditions were 80 J / mL RBC.

[0037] After the treatment with Mirasol, the sample was centrifuged to separate it into a preparation containing red blood cell components (red blood cell preparation) and a preparation containing platelets (platelet preparation). In this way, the blood preparations (red blood cell preparation and platelet preparation) of this comparative example were obtained.

[0038] (Example) Vitamin B 2 The blood products (red blood cell product and platelet product) of this example were obtained in the same manner as in the comparative example described above, except that a hemolysis inhibitor (vitamin E; tocopherol acetate) and a surfactant (polyoxyethylene sorbitan monooleate; Tween (registered trademark) 80) were further added in addition to the addition of riboflavin. The hemolysis inhibitor was added at a concentration of 50 ppm, and the surfactant was added at a concentration of 100 ppm.

[0039] [Effect on inactivation of pathogens] The pathogen (φX174) reduction effect was compared for the red blood cell preparations obtained in each of the above-mentioned Comparative Examples and Examples, compared to that before inactivation treatment using the Mirasol system. Specifically, the amount of pathogens (PFU / mL (plaque forming units per unit volume of sample)) before and after inactivation treatment was calculated by plaque assay, and the reduction amount was calculated as a log reduction value (log PFU / mL). For example, if the amount of pathogens is 1 / 10, the log reduction value is "1", and if it is 1 / 100, the log reduction value is "2". These results are shown in Table 1 below. The results shown in Table 1 were obtained by performing the same test using seven samples for both the Examples and Comparative Examples.

[0040] [Table 1]

[0041] As can be seen from the results shown in Table 1, it was confirmed that even when the present invention was applied (Example), there was no adverse effect on the pathogen reduction effect caused by ultraviolet irradiation.

[0042] [Effect on hemolysis] The change in hemolysis rate was monitored over time for the red blood cell preparations obtained in each of the above-mentioned Comparative Examples and Examples. Specifically, the hemolysis rate was measured using an absorbance method for the red blood cell preparations obtained in each of the Comparative Examples and Examples, in addition to the sample before the inactivation treatment, immediately after the inactivation treatment was set as day 0, and then 7, 14, 21, 28, 35, and 42 days thereafter. These results are shown in Table 2 below. Note that the results shown in Table 2 were also obtained from measurements of the same seven samples as those in Table 1 above, for both the Examples and Comparative Examples.

[0043] [Table 2]

[0044] From the results shown in Table 2, in the comparative red blood cell preparation, all samples showed a sufficiently low hemolysis rate to comply with the European (EU) and United States (US) guidelines up to 21 days after inactivation treatment. However, on the 28th day, one sample did not meet the European guidelines, and from the 35th day onwards, the majority of the samples did not comply with the European and US guidelines.

[0045] In contrast, in the red blood cell preparations of the embodiment, all samples showed a sufficiently low hemolysis rate to comply with the European and US guidelines up to 28 days after inactivation treatment, and most samples maintained a sufficiently low hemolysis rate to comply with the European and US guidelines even on days 35 and 42.

[0046] From the above, it can be seen that the present invention can achieve inactivation of pathogens in a blood sample containing both red blood cells and platelets while sufficiently reducing the hemolysis rate of the finally obtained red blood cell preparation. The mechanism by which such an effect is exhibited is presumably due to the hemolysis inhibitor and surfactant of the present invention exerting an effect of protecting red blood cells during the pathogen inactivation treatment (ultraviolet light irradiation treatment) of red blood cells.

[0047] This application is based on Japanese Patent Application No. 2018-209374, filed on November 7, 2018, the disclosure of which is incorporated by reference in its entirety. [Explanation of symbols]

[0048] 10 whole blood samples, 12 red blood cells, 14 white blood cells, 16 Platelets, 18 plasma, 20 Red blood cell products, 30 Platelet preparations.

Claims

1. A method for producing a blood product, comprising irradiating ultraviolet light to a blood sample containing red blood cells, platelets, vitamin E, and a surfactant having an HLB value of 13 or more and having 20 or more oxyethylene groups in the hydrophilic moiety in its molecular structure.

2. The method according to claim 1, wherein the blood product has a vitamin E concentration of 25 to 100 ppm by mass and a surfactant concentration of 100 to 300 ppm by mass.

3. The method according to claim 1 or 2, wherein the surfactant comprises a polyoxyethylene sorbitan fatty acid ester.

4. The method of claim 3, wherein the surfactant comprises polyoxyethylene sorbitan monooleate (Tween® 80).

5. The method according to any one of claims 1 to 4, wherein the blood sample is a leukocyte-removed blood sample.

6. The method according to any one of claims 1 to 5, further comprising separating the ultraviolet-irradiated blood sample into a red blood cell product and a platelet product after the ultraviolet irradiation.

7. The present invention comprises vitamin E and a surfactant having an HLB value of 13 or more and a hydrophilic moiety having 20 or more oxyethylene groups in its molecular structure, A blood cell preservation solution for use by being mixed with the blood sample in the manufacturing method according to any one of claims 1 to 6.

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