Additive solution for red blood cell concentrate

Abstract

The present invention relates to an additive solution for preserving red blood cell concentrates, a red blood cell concentrate comprising said additive solution, a process for producing a red blood cell concentrate diluted with said additive solution comprising a step of irradiating with UV light, and the use of said additive solution for preserving red blood cell concentrates.

Description

[Technical field]

[0001] The subject of the present invention is an additive solution for preserving a red blood cell concentrate, a red blood cell concentrate comprising said additive solution, and a method for producing a red blood cell concentrate diluted with said additive solution, said method comprising a step of irradiating with UV light in the range of 300 to 200 nm, as well as the use of said additive solution or components of said additive solution for preserving a red blood cell concentrate. [Background technology]

[0002] An erythrocyte concentrate (EC) is a "unit of stored blood" consisting of red blood cells (erythrocytes). In Germany, erythrocyte concentrates are marketable if they contain at least 40 g of hemoglobin per unit and have a hematocrit of 0.5 to 0.7. Hematocrit (abbreviation: hct) means the percentage of red blood cells (erythrocytes) in the blood volume, and is defined below in the unit L / L in all cases. According to the prior art, erythrocyte concentrates are usually diluted with an additive solution to make them more suitable for storage. The additive solution, often called a storage solution, serves the purpose of suspending and storing the red blood cells.

[0003] Red blood cell concentrates can be obtained in a variety of ways. They are typically obtained from whole blood donations or using apheresis, where red blood cells are separated from the donor's blood in a dialysis-like device using a continuous flow process and the remaining blood components are returned to the donor's circulatory system. For example, mechanical blood donations use the anticoagulant ACD-A.

[0004] Whole blood donation is performed by filling a predetermined amount of whole blood from each donor, for example 450 mL of whole blood by venous blood collection, into each blood bag made of plastic material, which contains a stabilizer solution or a stabilizer solution is added to the whole blood to increase the storage life of the whole blood and inhibit blood coagulation.

[0005] A CPD stabilizer solution containing citrate buffer, sodium dihydrogen phosphate, and D-glucose is a typical stabilizer solution. Generally, 63 mL of CPD stabilizer solution is used for 450 mL of whole blood, or 70 mL of CPD stabilizer solution is used for 500 mL of whole blood. The pH value of whole blood is stabilized to 7.1-7.2 by the stabilizer solution.

[0006] When donated whole blood is separated into its individual components, red blood cell concentrates (ECs) and the like are obtained.

[0007] Generally, leukoreduction is performed on whole blood or before the production of red blood cell concentrates. Leukoreduction is understood to mean the substantial removal of the donor's white blood cells. This is a legal requirement in many countries, for example in Germany. Leukoreduction is performed during apheresis, when blood passes through a filter that retains the white blood cells, for example after adding a stabilizer or additive solution, or when the white blood cells are separated from the red blood cells, for example by centrifugation. The filters used are often made of polyester fibers, which are compressed into a pack so that pores of a defined size are formed, or polyurethane sponges with a defined pore diameter are used.

[0008] Filtration for leukoreduction can be performed either directly after obtaining the blood and before obtaining the red blood cell concentrate, or after storage of the red blood cell concentrate, optionally first at the patient's bedside before administration to the patient. At least in Germany, it is common to perform leukoreduction before storage of the red blood cell concentrate.

[0009] To produce a red blood cell concentrate, whole blood is centrifuged, the supernatant containing the plasma and the so-called buffy coat of platelets and white blood cells is separated, and the red blood cells remaining as a centrifugal product or pellet are suspended in an additive solution.

[0010] The red blood cell concentrate is optionally once again pre-suspended in a nutrient solution, which may be different from the additive solution, and centrifuged again to separate and replace the remainder of the donor's plasma with the nutrient solution, before the red blood cells are suspended in the additive solution and stored. Such washed red blood cell concentrates are useful in cases of pre-transfusion intolerance (e.g., allergic reactions to proteins in the donor's plasma, e.g., in patients with IgA deficiency).

[0011] In order to adjust the red blood cell concentrate obtained from the centrifugation to a physiologically compatible viscosity, an additive solution must be added, which also provides the substances necessary to enhance the preservation properties, improving the viability and inhibiting the hemolysis of red blood cells that occurs during storage.

[0012] Additive solutions of red blood cell concentrates are known per se and have been proposed previously in various designs.

[0013] A solution containing sodium chloride, glucose or fructose, and adenine plus the sugar alcohol mannite is known from US Pat. No. 4,267,269. Similarly, a solution containing sodium chloride, glucose or fructose, and adenine, but with sorbitol or xylite, respectively, as sugar alcohol, and optionally further containing guanosine, is described in EP-A-0100419. EP-A-0301250 discloses an additive solution containing sodium chloride, disodium hydrogen phosphate and / or sodium hydrogen phosphate, glucose and / or fructose, sorbitol, mannite and / or xylite, adenine and / or guanosine, and optionally a colloid.

[0014] The additive solution SAG-M has become economically important, for example: it contains adenine, glucose, D-mannitol and sodium chloride (CF Hogman, K. Hedlund, Y. Sahlestrom: “Red cell preservation in protein-poor media. III. Protection against in vitro hemolysis” in Vox Sang. 1981 Nov-Dec; 41(5-6):274-81).

[0015] PAGGS-mannitol (PAGGS-M) is currently sold as an aqueous solution as follows and is a known excipient solution. 47.4mmol / L D-glucose monohydrate 8.0mmol / L Sodium dihydrogen phosphate dihydrate 8.0mmol / L disodium hydrogen phosphate dihydrate 1.4mmol / L adenine 1.4mmol / L guanosine 54.9mmol / L Mannitol 72mmol / L sodium chloride

[0016] In WH Walker, M. Netz, KH Ganshrit: “49 days of shelf life of erythrocyte concentrates in the presence of PAGGS-Mannitol”. Beitr. Infusionsther. 26(1990): 55-59, Walter et al. investigated the shelf life of red blood cell concentrates with the additive solution PAGGS-M and used CPD as a stabilizer solution.

[0017] The therapeutic application of blood products is known to be associated with the risk of viral and / or bacterial infection in the recipient of the blood product. These include, for example, the viruses Hepatitis B (HBV), West Nile (WNV) and Hepatitis C (HCV), as well as the AIDS viruses HIV-1 and HIV-2, or bacteria, such as Staphylococcus or Streptococcus. The risk is always present unless steps to inactivate or remove these pathogens are used during the manufacture of the product.

[0018] Inactivation of pathogens can be carried out, for example, by UV irradiation. Methods of this kind are known, for example, from WO 2007 / 076832. Ultraviolet (UV) light is differentiated by wavelength, which in the present application are defined as follows: UVA: 400-320 nm, UVB: 320-280 nm, UVC: 280-200 nm. It is known that bacteria, but also viruses, contained, for example, in blood plasma or in cellular blood products, can be inactivated by irradiation with short-wave ultraviolet (UV) light, i.e. in the wavelength range below 320 nm (UVB and UVC). Above 320 nm, the energy of the radiation is too low to effectively inactivate microorganisms and viruses. In contrast to chemical, photochemical and photodynamic pathogen inactivation methods, pure irradiation with UV light has the advantage that it is generally effective in itself and does not require the addition of reactive chemicals or photoactive substances. Summary of the Invention [Problem to be solved by the invention]

[0019] It is an object of the present invention to improve the preservation of red blood cell concentrates, in particular red blood cells that have been UV irradiated prior to being introduced into an additive solution, or to provide a medium for the UV irradiation and storage of red blood cells. [Means for solving the problem]

[0020] The invention is characterised by the independent claims, preferred embodiments are the subject of the dependent claims and / or are described below.

[0021] The additive solution according to the present invention contains the following components in addition to water: disodium hydrogen phosphate (Na2HPO4) at 12-50 mmol / L, especially 17-50 mmol / L or 20-25 mmol / L; adenine at 0.1-3.5 mmol / L, especially 1.5-2.5 mmol / L; D-glucose between 10 and 90 mmol / L, especially between 45 and 55 mmol / L; 0.1-3 mmol / L, especially 1.25-1.75 mmol / L guanosine; Sodium chloride from 10 to 80 mmol / L, in particular from 20 to 60 mmol / L, and more particularly from 35 to 45 mmol / L; and Trisodium citrate 10-50 mmol / L, especially 14-50 mmol / L or 25-35 mmol / L.

[0022] The additive solution consists in particular of the following components: disodium hydrogen phosphate (Na2HPO4) at 12-50 mmol / L, especially 17-50 mmol / L or 20-25 mmol / L; adenine at 0.1-3.5 mmol / L, especially 1.5-2.5 mmol / L; D-glucose between 10 and 90 mmol / L, especially between 45 and 55 mmol / L; 0.1-3 mmol / L, especially 1.25-1.75 mmol / L guanosine; Sodium chloride from 10 to 80 mmol / L, in particular from 20 to 60 mmol / L, and more particularly from 35 to 45 mmol / L; and trisodium citrate from 10 to 50 mmol / L, especially from 14 to 50 mmol / L or from 25 to 35 mmol / L; The remainder is water.

[0023] The above components may also be used as hydrates in any case. The components generally exist so as to dissociate in solution.

[0024] Concentrates or dilutions, especially dilutions of the above additive solutions, are likewise claimed.

[0025] The dye additive solution preferably has a pH of 7 or more, preferably 7.5 or more, especially a pH of 8-9, all at 22°C.

[0026] The osmotic pressure of the additive solution is preferably 260 to 300 mOsm / kg. The osmotic pressure is measured by the freezing point depression method (osmometer).

[0027] A method for producing a red blood cell concentrate diluted with an additive solution, comprising a step of irradiating a red blood cell concentrate and / or a precursor of a red blood cell concentrate, e.g. whole blood, with ultraviolet light in the range of 300 to 200 nm, in particular 280 to 220 nm, preferably 260 to 240 nm (hereinafter also usually referred to as "UV irradiation" or "UV-irradiation"), is a further subject of the present invention.

[0028] In the following three designs, the method for producing a red blood cell concentrate comprises the steps of: - obtaining a red blood cell concentrate from the whole blood so irradiated by exposing the whole blood or diluted whole blood to UV radiation and adding an additive solution or a component of an additive solution; or - UV-irradiating an additive solution or a red blood cell concentrate containing components of the additive solution, wherein the red blood cell concentrate is preferably a diluted red blood cell concentrate having an hct of less than 0.5, and is concentrated to an hct of 0.5 or more after UV-irradiation; or - UV-irradiating the diluted red blood cell concentrate containing the second additive solution, wherein the diluted red blood cell concentrate is preferably a diluted red blood cell concentrate having an hct of less than 0.5, and is concentrated to an hct of 0.5 or more after UV irradiation; wherein the second additive solution is substituted with the additive solution or components of the additive solution after irradiation at least 75% by weight or more, preferably completely, based on the second additive solution; The UV irradiation in each case is carried out at wavelengths of 300 to 200 nm, in particular 280 to 220 nm, preferably 260 to 240 nm. When the components of the additive solution are added, this means that they are added so that the same concentrations are obtained in each case as if the additive solution had been added. In this respect, it is the same as if the additive solution had been added.

[0029] The present invention therefore further relates to a diluted red blood cell concentrate, in particular a leukodepleted diluted red blood cell concentrate, with an hct of 0.1-0.4, preferably 0.25-0.35, comprising the above-mentioned additive solution. The diluted red blood cell concentrate is for example obtained by 1:2 dilution of the red blood cell concentrate with the additive solution. Red blood cell concentrates with an hct of less than 0.5 are also referred to herein as "diluted red blood cell concentrates".

[0030] The red blood cell concentrate preferably comprises (L = liters): Red blood cells: 0.40-0.80 L / L, especially 0.50-0.70 L / L Additive solution 0.10~0.60L / L, especially 0.25~0.50L / L and optionally Stabilizer solution 0.0001~0.10L / L, especially CPD stabilizer solution Human plasma 0.0001~0.2L / L The sum of the values ​​in each case is less than 1L / L, specifically 1L / L.

[0031] When UV irradiation is performed on whole blood (WB), the composition of the red blood cell concentrate comprises or consists of, among others: Red blood cells 0.5~0.7L / L Additive solution 0.27~0.48L / L CPD stabilizer solution 0.03~0.09L / L Human plasma 0.025~0.15L / L or 0.015~0.025L / L or after UV irradiation of the red blood cell concentrate, if UV irradiation was not performed on whole blood (WB), comprising or consisting of: Red blood cells 0.5~0.7L / L Additive solution 0.27~0.49L / L CPD stabilizer solution 0.001~0.009L / L or 0.001~0.014L / L Human plasma 0.005-0.05L / L.

[0032] According to one embodiment, a suitable CPD stabilizer solution comprises: Trisodium citrate dihydrate 80-100mmol / L, especially 89.4mmol / L Citric acid monohydrate 13-18mmol / L, especially 15.6mmol / L NaH2PO4 dihydrate 13-19mmol / L, especially 16.1mmol / L D-glucose monohydrate 115-140 mmol / L, especially 128.7 mmol / L.

[0033] According to one embodiment, the CPD stabilizer solution is added to the whole blood in a volume ratio of 1:6.1 to 1:8.1, in particular 1:7.14 (in each case V / V), for example 63 mL of CPD stabilizer solution for 450 mL of whole blood or 70 mL of CPD stabilizer solution for 500 mL of whole blood, i.e. before the additive solution is used. Upon reprocessing, the abovementioned components of the CPD stabilizer solution remain in the red blood cell concentrate afterwards at the defined concentrations.

[0034] Since glucose and trisodium citrate are also included in the stabilizer solution, their percentage in the red blood cell concentrate will increase accordingly if a CPD stabilizer solution is used, however, other stabilizer solutions can also be used.

[0035] According to one embodiment, the red blood cell concentrate comprises: D-glucose between 10 and 30 mmol / L, especially between 14 and 26 mmol / L; disodium hydrogen phosphate, for example as the dihydrate, at 5-12 mmol / L, in particular 6-10 mmol / L; adenine at 0.3-1.2 mmol / L, especially 0.5-0.8 mmol / L; 0.25-0.9 mmol / L, especially 0.4-0.7 mmol / L guanosine; sodium chloride between 8 and 25 mmol / L, especially between 11 and 20 mmol / L; trisodium citrate at 6-18 mmol / L, especially 8-16 mmol / L; and optionally, Sodium dihydrogen phosphate, for example as the dihydrate, from 0.01 to 1 mmol / L, in particular from 0.02 to 0.8 mmol / L; Citric acid, for example as the monohydrate, at 0.01-1 mmol / L, in particular 0.02-0.8 mmol / L.

[0036] This embodiment can be obtained, for example, when a red blood cell concentrate is obtained by addition of a CPD or CPDA-1 stabilizer solution for blood donation and an additive solution according to the present invention, and sodium dihydrogen phosphate and citric acid are subsequently introduced with the CPD stabilizer solution.

[0037] Thus, the present invention further relates to the use of the additive solution or components of the additive solution for preserving red blood cell concentrates.

[0038] The difference in concentration between the CPD stabilizer solution and human plasma is due to the dilution of EC with the additive solution and subsequent concentration, during which part of the supernatant is removed, along with some of the plasma and CPD stabilizer solution originally contained therein. [Brief description of the drawings]

[0039] [Figure 1] FIG. 1 shows the hemolysis rate over the storage period in days. [Diagram 2] FIG. 2 shows the decrease in glucose concentration during storage of red blood cell concentrates that were UVC irradiated in UG65 and stored in UG65. [Diagram 3] FIG. 3 shows the increase in lactate concentration during storage of red blood cell concentrates that were UVC irradiated in UG65 and stored in UG65. [Figure 4]FIG. 4 shows the hemolysis rate of red blood cell concentrates irradiated with UVC in the presence of NaCl, SAG-M or UG65 and stored in UG65, with EC stored in UG65 without UVC irradiation serving as a control. [Diagram 5] FIG. 5 shows the ATP content of red blood cell concentrates irradiated with UVC in the presence of NaCl, SAG-M or UG65 and stored in UG65, with EC stored in UG65 without UVC irradiation serving as a control. [Figure 6] FIG. 6 shows the hemolysis rate during storage of UVC-irradiated ECs and ECs stored in the same additive solution. [Figure 7] FIG. 7 shows the ATP content at the end of storage (week 5) of UVC-irradiated ECs and ECs stored in the same additive solution. [Figure 8] FIG. 8 shows the glucose content at the end of storage (week 5) of UVC-irradiated ECs and ECs stored in the same additive solution. [Figure 9] FIG. 9 shows the lactic acid content at the end of storage (5 weeks) of UVC-irradiated EC and EC stored in the same additive solution. [Figure 10] FIG. 10 shows the hemolysis rate when ECs obtained from UVC-irradiated whole blood were preserved in UG65 and SAG-M. [Figure 11] FIG. 11 shows the ATP content at the end of storage (4 weeks) when ECs obtained from UVC-irradiated whole blood were stored in UG65 and SAG-M. [Figure 12] FIG. 12 shows the glucose content at the end of storage (4 weeks) when ECs obtained from UVC-irradiated whole blood were stored in UG65 and SAG-M. [Figure 13] FIG. 13 shows the lactate content at the end of storage (4 weeks) when ECs obtained from UVC-irradiated whole blood were stored in UG65 and SAG-M. [Figure 14] FIG. 14 shows the hemolysis rate during storage of ECs obtained from UVC-irradiated whole blood when the ECs were stored with UG65 or PAGG-M. [Figure 15]FIG. 15 shows the ATP content at the end of storage (4 weeks) when ECs obtained from UVC-irradiated whole blood were preserved with UG65 and PAGG-M. [Figure 16] FIG. 16 shows the glucose content at the end of storage (4 weeks) when ECs obtained from UVC-irradiated whole blood were stored with UG65 and PAGG-M. [Figure 17] FIG. 17 shows the lactate content at the end of storage (4 weeks) when ECs obtained from UVC-irradiated whole blood were stored with UG65 and PAGG-M. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0040] According to one embodiment, red blood cell concentrates are isolated from individual blood donations or obtained from individual donors by mechanical apheresis. The volume of the preparations is generally about 200-350 mL. The volume of whole blood donations is often 400-500 mL. The preparations are each stored in flat plastic bags and are typically stored at about 4° C.-37° C. for whole blood and 4° C.±2° C. for red blood cell concentrates.

[0041] Leukoreduction can be performed at the level of whole blood, i.e. before the first centrifugation of the whole blood, or at the level of red blood cells obtained by centrifugation suspended and diluted in an additive solution, i.e. at the level of the red blood cell concentrate. Generally, leukoreduction is performed by filtration. When red blood cells are obtained by apheresis, separate leukoreduction is not necessary, as leukoreduction is already performed as part of the apheresis.

[0042] According to one embodiment, leukodepletion is carried out in a red blood cell concentrate diluted with an additive solution according to the invention, for example at 0.1 to 0.4 hct. Leukodepletion is optionally followed by UV irradiation, followed by concentration, in particular to 0.5 to 0.7 hct. Leukodepletion can be carried out before or after UV irradiation, preferably before.

[0043] UV irradiation can be performed on whole blood (WB), i.e., prior to preparation of the red blood cell concentrate, or on the red blood cell concentrate, and a UV-irradiated pathogen-free red blood cell concentrate, and optionally a leukoreduced red blood cell concentrate, are obtained as products in the additive solution of the present invention.

[0044] The starting material for UV irradiation of red blood cell concentrates is, for example, red blood cell concentrates with an hct of 0.8 to 1, in particular 0.8 to 0.98. According to one embodiment, they are adjusted to an hct of 0.5 to 0.7 with the additive solution according to the invention and UV irradiated at this concentration or preferably further diluted (diluted red blood cell concentrate) and UV irradiated. In the context of the present application, red blood cell concentrates with an hct of less than 0.5 are called diluted red blood cell concentrates.

[0045] The leukodepleted red blood cell concentrate may also be further diluted with additive solution, for example from a 1:2 dilution of red blood cell concentrate to additive solution, to an hct (diluted red blood cell concentrate) of 0.1-0.4, preferably 0.25-0.35. The thus diluted red blood cell concentrate is transferred to an irradiation bag via a sterile hose connection and UV irradiated in an agitated environment.

[0046] The irradiated red blood cell concentrate is transferred to an empty bag and concentrated, for example by centrifugation and squeezing of the supernatant, to an hct of 0.5-0.8, particularly 0.5-0.7.

[0047] It is known that pathogens in blood products can be inactivated by irradiation with short wavelength ultraviolet (UV) light, which, according to the method of the present invention, is achieved by irradiating the blood product with ultraviolet (UV) light of 300-200 nm (UVB-UVC range), particularly ultraviolet (UV) light of wavelengths in the UVC range of 280-220 nm, and particularly 260-240 nm.

[0048] The radiation energy is preferably 0.3 to 10 J / cm for whole blood. 2 , more preferably 2.5 to 5.5 J / cm 2 , and particularly preferably 3 to 5 J / cm 2and for red blood cell concentrates, preferably 1.5 to 4.5 J / cm 2 , and particularly preferably 2 to 4 J / cm 2 (in both cases based on the radiation energy impinging on the blood product). The radiation energy impinging on a container such as an irradiation bag will actually be greater, depending on the material, as irradiation bags generally absorb radiation energy at the relevant wavelengths.

[0049] If irradiation is carried out through a medium that absorbs UV light, the irradiation energy is increased accordingly. Irradiation bags made of EVA (ethylene vinyl acetate) for example absorb about 30-40% of the radiation energy. Irradiation is carried out at temperatures of the blood product in particular between 2 and 37 °C.

[0050] Methods of this kind for UV irradiation are known, for example, from WO 2007 / 076832, and are applied to the red blood cell concentrate according to the invention. According to this, the preparation, i.e. donor blood (whole blood) and / or red blood cell concentrate (EC), is moved in a suitable manner in an irradiation bag, such that a constant circulation of the sample takes place in the container. This results in a vigorous movement in the liquid or suspension, respectively, such that in some areas a layer is formed that is thin enough for the light used to be transparent. The movement is performed in such a way that the liquid or suspension, respectively, is effectively mixed in the bag. Both of these are in particular achieved, for example, if the following conditions are met: 1. The irradiation bag is flexible. 2. The irradiation bag is filled to 40% of the maximum filling volume, in particular to 30% of the maximum filling volume, in particular to 15% of the maximum filling volume. 3. The irradiation bag is vigorously moved, for example horizontally (linearly back and forth or in a circular or elliptical manner) and / or vertically (rocking). In conjunction with constant simultaneous mixing, the entire formulation (and the pathogens contained therein) is finally irradiated, resulting in pathogen reduction.

[0051] The irradiation bag can be shaken using an orbital shaker, platform shaker, rocking shaker or wobble shaker, and can be moved, preferably for at least three-quarters of the total irradiation time. The irradiation bag usually has a volume of up to 5000 mL. When the irradiation bag is placed on one side, the height of the irradiation bag constantly changes due to movement or shaking, respectively, over the entire upper surface of the irradiation bag in contact with the bag contents, based on the distance along the surface normal between the intersection of the surface on which the irradiation bag rests and the upper surface of the irradiation bag.

[0052] The irradiation bag is made of a UV-transparent plastic material. Suitable plastics are, for example, ethylene vinyl acetate and polyolefins, with a film thickness of, for example, 1 mm or less, in particular 0.5 mm or less. The irradiation bag is formed flat and preferably has no absorption maximum in the range of 200-320 nm. In the horizontally filled state, the irradiation bag has a thickness of only a few mm, for example less than 10 mm, in particular 5 mm, more preferably less than 3 mm, and is intended to accommodate, for example, a sample volume of up to 600 mL. However, the maximum capacity (volume) of the irradiation bag is larger than the actual sample volume accommodated therein, preferably at least 3 times, typically at least 5 times, or at least 10 times. For example, the base of the irradiation bag in the horizontal position is 19×38 cm, the filling volume is 500-600 mL, and the average filling height in the horizontal, stationary state is 6.9-8.3 mm.

[0053] Each UV irradiated unit is preferably traceable back to a donor.

[0054] Hematocrit (hct) indicates the percentage of cellular components in blood. Normal values ​​for hematocrit in blood are 0.42-0.5 in men and 0.37-0.45 in women. In this case, it is specified as L / L. Since red blood cells physiologically account for 99% of the total volume of blood cells, the hct value is approximately equal to the percentage of their cell volume.

[0055] hct is measured by centrifuging a non-coagulated blood sample in a tube according to DIN 58933-1:1995-01. An anticoagulant such as EDTA (ethylenediaminetetraacetate) or heparin is then added to prevent blood clotting. Once the heavy red blood cells have separated from the plasma, the height of the red blood cell mass relative to the total blood volume is measured. The boundaries between the red blood cells, the white blood cells / platelets and the plasma are visible to the naked eye. If the white blood cells and / or platelets have already separated from the plasma, the precipitated solids consist almost exclusively of red blood cells. EXAMPLES

[0056] The effects of additive solution UG65 on the quality of UVC-irradiated blood products and the pathogen inactivation effect of UV-irradiated blood products were examined.

[0057] (Production of ingredients) Whole blood donations (450–500 mL) were collected in 70 mL of anticoagulant CPD (day 0) (hereafter referred to as whole blood donations) and stored at room temperature overnight. The anticoagulant CPD contained the following components in addition to water: JPEG2024528034000002.jpg31154

[0058] On day 1, whole blood was either used directly for pathogen inactivation tests or further processed to red blood cell concentrates. For this purpose, red blood cells were obtained as "dry" red blood cell concentrates after centrifugation in a conventional bag centrifuge and automated component separation in a press, which were then suspended in the respective specific additive solutions (110 mL). Leukoreduction was performed by filtration at the whole blood level through leukoreduction filters ("whole blood filtration") or by filtration of the red blood cell concentrate (filtration after centrifugation and squeezing).

[0059] Additive solution UG65 composition: The aqueous solution contains the following components: 22.7mmol / L disodium hydrogen phosphate dihydrate 1.85mmol / L adenine 51.6mmol / L D-glucose monohydrate 1.44mmol / L guanosine 40mmol / L sodium chloride 28.4mmol / L Trisodium Citrate Dihydrate The rest is water.

[0060] Additive solution SAG-M composition: The aqueous solution contains the following components: 1.25mmol / L adenine 45.4mmol / L D-glucose monohydrate 28.8mmol / L D-mannitol 150mmol / L sodium chloride Remaining Water

[0061] (Pathogen inactivation by UV irradiation) Whole blood (520-570 mL) or red blood cell concentrate (600 mL, hct approximately 0.3) diluted with additive solution was filled into a UV-transparent bag (bottom surface 19 × 38 cm, made of EVA) and irradiated with UVC light (254 nm) at 4.5 J / cm using a UV irradiation system (Macotronic UV). 2 (EC) or 6J / cm 2 (WB) Irradiated and shaken (300 rpm) at the same time.

[0062] Then, normal red blood cell concentrates with hct of 0.5–0.7 were obtained from whole blood or diluted red blood cell concentrates by centrifugation and automated separation.

[0063] Information about the irradiated energy affects the outside of the irradiation bag. It is estimated that about 50-75% of the irradiated energy, in this embodiment about 60%, passes through the irradiation bag. The irradiation bag is irradiated from above and below.

[0064] (Measurement of quality parameters) The percent hemolysis is defined as the ratio of free hemoglobin in the supernatant of the red blood cell concentrate to the total content. Hemolysis rate (%) = ((100-hematocrit * 100) × free hemoglobin in the supernatant / total hemoglobin).

[0065] hct was measured using a hematocrit centrifuge (Haematokrit210, Hettich). Free hemoglobin in the supernatant was measured optically by the three-wavelength method by Harboe (see M. Harboe, A method for determination of hemoglobin in plasma by near-ultraviolet spectrophotometry. Scand J Clin Lab Invest, 1959. 11(1): p. 66-70). Total hemoglobin was measured by a hematology analyzer (XS1000i or XN550, sysmex).

[0066] Measurements of glucose and lactate concentrations were performed using a blood gas analyzer ABL90FLEX (Radiometer). Red blood cell ATP content was measured using a commercial kit ATP Hexokinase FS (Diasys Greiner). pH was measured at 22°C using a conventional pH meter. Volume was determined by weighing, taking into account the specific gravity of the red blood cell concentrate.

[0067] (Test 1) Quality parameters of red blood cell concentrates UVC-irradiated in UG65 and stored in UG65 Erythrocyte concentrates (n=9) in additive solution UG65 were UVC irradiated and concentrated again as described above. The finished erythrocyte concentrates, with an hct of approximately 0.6, were then stored at 4±2°C and samples were taken weekly to determine in vitro quality.

[0068] (result) The hct of the UVC-irradiated red blood cell concentrates was 0.59-0.64, which was in accordance with the Council of Europe guidelines. The hemolysis rate increased during the storage period. However, after 36 days of storage, all nine red blood cell concentrates showed hemolysis rates below 0.8%, which met the quality requirements of the Council of Europe guidelines.

[0069] The pH value of the UVC-irradiated erythrocyte concentrate was 7.14 ± 0.06 on day 2, but decreased during the storage period to 6.54 ± 0.05 on day 36. In parallel, the glucose content of the erythrocyte concentrate decreased from 37.8 ± 1.6 to 23.7 ± 1.9, and the lactate concentration increased from 6.9 ± 0.6 to 30.4 ± 1.6.

[0070] Figure 1 shows the hemolysis rate over the storage period by days.

[0071] FIG. 2 shows the decrease in glucose concentration of red blood cell concentrates that were UVC irradiated in UG65 and stored in UG65, and FIG. 3 shows the increase in lactate concentration of red blood cell concentrates that were UVC irradiated in UG65 and stored in UG65 during storage.

[0072] All values ​​are the average of nine samples.

[0073] The newly developed additive was found to be suitable for producing UVC-irradiated red blood cell concentrates of good quality.

[0074] (Test 2) The effect of different additive solutions on UVC irradiation of red blood cell concentrates stored in additive solution UG65. The four dried red blood cell concentrates were pooled and split again. The red blood cell concentrate was suspended in 110 mL UG65 and filtered for leukocyte depletion (untreated control without UVC irradiation).

[0075] For the remaining three red blood cell concentrates, the pooled red blood cell concentrates were suspended in 110 mL of isotonic saline (NaCl 0.9%), 110 mL of additive solution SAG-M, and 110 mL of additive solution UG65, respectively, filtered, and then diluted to approximately 0.3 hct with the respective same additive solutions. UVC irradiation was 4.5 J / cm 2The UVC-irradiated red blood cell concentrate was then centrifuged, the supernatant removed, and all red blood cells were resuspended in UG65. Storage of the red blood cell concentrate in additive solution UG65 was at 4±2°C, and samples for in vitro quality determination were taken weekly.

[0076] (result) As shown in Figure 4, UVC irradiation increased the rate of hemolysis compared to the non-irradiated control. The rate of hemolysis was highest when red blood cells were irradiated in the presence of NaCl or SAG-M. The quality of the UVC-irradiated red blood cell concentrate was best when the additive solution UG65 was present during irradiation.

[0077] ATP content, a parameter of the energy state of red blood cells, was similarly affected by UVC irradiation. ATP content at the end of storage was highest when red blood cells were irradiated in the presence of UG65 (Figure 5). Irradiation of red blood cell concentrates in the presence of NaCl or SAG-M reduced ATP content compared to non-irradiated controls.

[0078] Figure 4 shows the hemolysis rate of red blood cell concentrates irradiated with UVC in the presence of NaCl, SAG-M or UG65 and stored in UG65, and Figure 5 shows the ATP content in these cases. ECs stored in UG65 without UVC irradiation served as a control.

[0079] A series of tests showed that diluting red blood cell concentrate with additive solution UG65 during UVC irradiation has a positive effect on the quality of red blood cells. The newly developed additive solution offers advantages compared to other possible dilution solutions such as saline or conventional additive solutions.

[0080] (Test 3) Quality comparison of UVC-irradiated red blood cell concentrates irradiated and stored in additive solution UG65 with red blood cell concentrates irradiated and stored in conventional additive solution SAG-M "Dried" erythrocyte concentrates (hematocrit >0.8) were obtained as described above. Two dried erythrocyte concentrates were pooled and split again, after which one was suspended in 110 mL of the commercial additive solution SAG-M (control) and one in 110 mL of the newly developed additive solution UG65 (test). Leukoreduction was performed by filtering these erythrocyte concentrates using a conventional leukoreduction filter. After filtration, each erythrocyte concentrate was mixed with an equal volume of the respective additive solution (w / w). 600 g of the diluted erythrocyte concentrate was transferred to a UVC-transparent irradiation bag. UVC irradiation was performed as described above. The conventional erythrocyte concentrate was subsequently obtained from the diluted erythrocyte concentrate by centrifugation and automated separation. The finished erythrocyte concentrates (n=3, test and control) were stored at 4±2°C and samples were taken weekly to determine in vitro quality.

[0081] (result) After manufacture, the test and control red blood cell concentrates showed comparable values ​​for volume, hct and hemoglobin per unit (Table 1). [Table 1]

[0082] The hemolysis rate, the most important quality parameter of the red blood cell concentrates, was significantly lower in the red blood cell concentrates UVC-irradiated and stored in UG65 compared to the red blood cell concentrates UVC-irradiated and stored in the conventional additive solution SAG-M (Figure 6). All other quality parameters also showed significant differences between the control and test red blood cell concentrates during the storage period (Figures 7-9).

[0083] FIG. 6 shows the hemolysis rate during storage of UVC-irradiated ECs and ECs stored in the same additive solution.

[0084] FIG. 7 shows the ATP content, FIG. 8 shows the glucose content, and FIG. 9 shows the lactate content at the end of storage (5 weeks) for ECs that had been irradiated with UVC and then stored in the same additive solution.

[0085] During the storage process, the significant advantage of the newly developed additive solution UG65 in UVC irradiation of red blood cell concentrates was revealed: ECs irradiated with UVC and stored in UG65 were of better quality than ECs stored in the conventional additive solution SAG-M.

[0086] (Test 4) Comparison of the quality of red blood cell concentrates after UVC irradiation at the whole blood level with additive solution UG65 and conventional additive solution SAG-M Whole blood donations (approximately 570 nm) were irradiated with UVC as described above, followed by automated component separation with a press to obtain "dried" red blood cell concentrates (hematocrit >0.8). The two dried red blood cell concentrates were pooled and split again, after which one was suspended in 110 mL of a commercial additive solution SAG-M (control) and one in 110 mL of a newly developed additive solution UG65 (test). Leukoreduction was performed by filtration of these red blood cell concentrates through a commercial leukoreduction filter. The red blood cell concentrates (n=4, test and control) were then stored at 4±2°C and samples were taken weekly to determine in vitro quality.

[0087] (result) After manufacture, the test and control red blood cell concentrates showed comparable values ​​for volume, hct and hemoglobin per unit (Table 2). [Table 2]

[0088] As the most important quality parameter of the red blood cell concentrate, the hemolysis rate of the red blood cell concentrate obtained from whole blood irradiated with UVC in UG65 was significantly lower than that of the red blood cell concentrate obtained from whole blood irradiated with UVC in the conventional additive solution SAG-M (Figure 10). During the storage process, further quality parameters also showed significant differences between the control and test red blood cell concentrates (Figures 11-13).

[0089] FIG. 10 shows the hemolysis rate when ECs obtained from UVC-irradiated whole blood were preserved in UG65 and SAG-M.

[0090] At the end of the storage period (4 weeks), FIG. 11 shows the ATP content, FIG. 12 shows the glucose content, and FIG. 13 shows the lactate content.

[0091] During the storage process, the newly developed additive solution UG65 demonstrated superior advantages in the production of red blood cell concentrates from UVC-irradiated whole blood. The quality of ECs from UVC-irradiated whole blood supplemented with UG65 was superior to that supplemented with the conventional additive solution SAG-M.

[0092] (Test 5) bacterial inactivation The bacterial strains Klebsiella pneumoniae (PEI-BP-08-01), Serratia marcescens (PEI-BP-56), and Pseudomonas fluorescens (PEI-BP-77) were grown in CASA broth, mixed with human albumin, and stored frozen until use (see U. Gravemann, et al., Bacterial inactivation of platelet concentrates with the THERAFLEX UV-Platelets pathogen inactivation system. Transfusion, 2019. 59(4): p. 1324-1332.). Approximately 1 × 10 6 The samples were spiked with a bacterial suspension of KBE / mL (n=3 for each bacteria used) followed by irradiation with UVC light and shaking. Diluted erythrocyte concentrates or conventional erythrocyte concentrates were then obtained from whole blood. Samples were taken at different time points and bacterial counts were determined by plating on agar plates.

[0093] (result) Bacteria were inactivated in a dose-dependent manner with log reduction values ​​ranging from 4 to 6 log levels in red blood cell concentrates (Table 3) and whole blood (Table 4). This study demonstrated the bacterial inactivation efficiency of the method. [Table 3] [Table 4]

[0094] (Test 6) Viral inactivation EMVC (EMC strain, ATCC VR129B), Sindbis virus (Ar339 strain, ATCC VR-68), and VSV (Indiana strain, ATCC VR-158) were propagated and titrated on Vero cells (African green monkey cell line derived from kidney tissue, ATCC, Bio Whittaker number BE76-108B). Culture and titration were performed as described by Mohr et al. (H. Mohr et al., A novel approach to pathogen reduction in platelet concentrates using short-wave ultraviolet light. Transfusion, 2009. 49(12): p. 2612-24).

[0095] Whole blood or red blood cell concentrate diluted with additive solution UG65 with hct of about 0.3 was spiked with virus suspension (10% v / v, n=3 for each virus used) and then irradiated with UVC light and shaking. Conventional red blood cell concentrate was then obtained from the diluted red blood cell concentrate or whole blood. Samples were taken at different time points and virus titer was measured by titration endpoint. When the detection limit was reached, the large volume plate method was applied instead of titration endpoint.

[0096] Virus was inactivated in a dose-dependent manner with log reductions of 3 to 5 logs in red blood cell concentrates (Table 5) and whole blood (Table 6). This study demonstrated the virus inactivation efficiency of this method. [Table 5] [Table 6]

[0097] (Test 7) Comparison of additive solution UG65 and additive solution PAGGS-M on the quality of red blood cell concentrates after UVC irradiation at the whole blood level Two whole blood donations (approximately 500 mL whole blood + 70 mL CPD stabilizer solution in each case) were pooled and split again. Whole blood was irradiated with UVC as described above, followed by automated component separation with a squeezer to obtain a "dried" red blood cell concentrate (hematocrit >0.8).

[0098] The two dried erythrocyte concentrates were pooled and divided again, after which one was suspended as described above in 110 mL of the commercial additive solution PAGGS-M (control, composition as described above) and one was suspended in 110 mL of the newly developed additive solution UG65 (test). Leukoreduction was performed by filtration of these erythrocyte concentrates through a conventional leukoreduction filter. The erythrocyte concentrates (n=4, test and control) were then stored at 4±2° C. and samples were taken weekly to determine the in vitro quality. With the same amount of CPD, the erythrocyte concentrates differed in their composition in each case at least in that the erythrocyte concentrates with the additive solution according to the invention did not contain mannitol and the contents of disodium hydrogen phosphate and trisodium citrate were significantly greater in the erythrocyte concentrates according to the invention compared to those with CPD and PAGGS-M. [Table 7] [Table 8]

[0099] (result) After manufacture, the test and control red blood cell concentrates showed comparable values ​​for volume, hct, and hemoglobin per unit (Table 9). [Table 9]

[0100] The hemolysis rate, which is the most important quality parameter of the red blood cell concentrate, was significantly lower in the red blood cell concentrate obtained from UVC-irradiated whole blood in UG65 compared to the red blood cell concentrate obtained from UVC-irradiated whole blood in the conventional additive solution PAGG-M (Figure 14). During the storage process, further quality parameters also showed significant differences between the control and test red blood cell concentrates (Figures 15-17).

[0101] FIG. 14 shows the hemolysis rate during storage when ECs obtained from UVC-irradiated whole blood were stored with UG65 and PAGG-M.

[0102] At the end of the storage period (four weeks), FIG. 15 shows the ATP content, FIG. 16 shows the glucose content, and FIG. 17 shows the lactate content.

[0103] During the storage process, the significant advantage of the newly developed additive solution UG65 was revealed in the production of red blood cell concentrates from UVC-irradiated whole blood. The quality of ECs from UVC-irradiated whole blood in UG65 was superior to that using the additive solution PAGGS-M.

Claims

1. A method for producing a red blood cell concentrate (RBC concentrate), wherein the red blood cell concentrate contains UV-irradiated red blood cells, the method comprising: - irradiating whole blood or diluted whole blood with UV radiation and adding an additive solution or components of the additive solution to obtain a red blood cell concentrate from the irradiated whole blood; or - irradiating a red blood cell concentrate or a diluted red blood cell concentrate having an hct of less than 0.5, which contains an additive solution or components of the additive solution, and concentrating to an hct of 0.5 or more after the UV irradiation; or - irradiating a diluted red blood cell concentrate containing a second additive solution, wherein at least 75% by weight or more of the second additive solution, based on the second additive solution, is replaced by the additive solution or components of the additive solution after the UV irradiation; the UV irradiation is performed at a wavelength of 300 to 200 nm, According to Option A, the additive solution contains at least the following components together with water: 12 to 50 mmol / L of disodium hydrogen phosphate, 0.1 to 3.5 mmol / L of adenine, 10 to 90 mmol / L of D-glucose, 0.1 to 3 mmol / L of guanosine, 10 to 80 mmol / L of sodium chloride, and 10 to 50 mmol / L of trisodium citrate, Or according to Option B, the components of the additive solution contain the following components: Disodium hydrogen phosphate, adenine, D-glucose, guanosine, sodium chloride and trisodium citrate, The components in Option B are 10 to 30 mmol / L of D-glucose, 5 to 12 mmol / L of disodium hydrogen phosphate, 0.3 to 1.2 mmol / L of adenine, 0.25 to 0.9 mmol / L of guanosine, 8 to 25 mmol / L of sodium chloride, and 6 to 18 mmol / L of trisodium citrate A method used in an amount to obtain the red blood cell concentrate containing the same.

2. The method according to claim 1, wherein the concentrations of disodium hydrogen phosphate, adenine, D-glucose, guanosine, sodium chloride and trisodium citrate in the additive solution are each individually or simultaneously as follows: 17 to 50 mmol / L of disodium hydrogen phosphate, 1.5 to 2.5 mmol / L of adenine, 45 to 55 mmol / L of D-glucose, 1.25 to 1.75 mmol / L of guanosine, 20 to 60 mmol / L of sodium chloride, 14 to 50 mmol / L of trisodium citrate.

3. The additive solution consists of the component at the said concentration and the balance of water, the method according to claim 1 or 2.

4. The additive solution has a pH of 7 or more at 22 °C, the method according to claim 1 or 2.

5. The osmotic pressure of the additive solution is 260 to 300 mOsm / kg, the method according to claim 1 or 2.

6. The additive solution does not contain mannitol or sorbitol, or does not contain both mannitol and sorbitol, the method according to claim 1 or 2.

7. The red blood cell concentrate contains 0.40 to 0.80 L / L of red blood cells and 0.10 to 0.60 L / L of the additive solution, and the total of the volume ratios of L / L in each of the above cases is a numerical value of 1 L / L or less, the method according to claim 1 or 2.

8. The red blood cell concentrate contains 0.0001 to 0.1 L / L of a stabilizer solution, or 0.0001 to 0.2 L / L of human plasma, or both of them, the method according to claim 1 or 2.

9. The red blood cell concentrate contains red blood cells irradiated with UV during whole blood, the method according to claim 1 or 2.

10. The red blood cell concentrate contains red blood cells irradiated with UV during diluted red blood cell concentrate, and the diluted red blood cell concentrate is concentrated after UV irradiation, the method according to claim 1 or 2.

11. The red blood cell concentrate has an hct of 0.4 to 0.8, the method according to claim 1 or 2.

12. The red blood cell concentrate does not contain mannitol or sorbitol, or does not contain both mannitol and sorbitol, the method according to any one of claims 1 or 2.

13. The red blood cell concentrate, as option A, contains 10 to 30 mmol / L of D-glucose, 5 to 12 mmol / L of disodium hydrogen phosphate, 0.3 to 1.2 mmol / L of adenine, 0.25 to 0.9 mmol / L of guanosine, 8 to 25 mmol / L of sodium chloride, 6 to 18 mmol / L of trisodium citrate, optionally, 0.01 to 1 mmol / L of sodium dihydrogen phosphate, 0.01 to 1 mmol / L of citric acid, and or, as option B, contains 14 to 26 mmol / L of D-glucose, 6 to 10 mmol / L of disodium hydrogen phosphate, 0.5 to 0.8 mmol / L of adenine, 0.4 to 0.7 mmol / L of guanosine 11 to 20 mmol / L of sodium chloride, and 8 to 16 mmol / L of trisodium citrate, and Optionally, 0.01 to 1 mmol / L of sodium dihydrogen phosphate, and 0.01 to 1 mmol / L of citric acid, the method according to claim 1 or 2.

14. The method according to claim 1 or 2, wherein the red blood cell concentrate contains less than 0.6 mmol / L of sodium dihydrogen phosphate.

15. - UV irradiating a red blood cell concentrate containing the additive solution or a component of the additive solution, wherein the red blood cell concentrate is a diluted red blood cell concentrate with an hct of less than 0.5, and the hct is concentrated to 0.5 or more after UV irradiation; Or - UV irradiating a diluted red blood cell concentrate containing a second additive solution, wherein the diluted red blood cell concentrate is a diluted red blood cell concentrate with an hct of less than 0.5, and the hct is concentrated to 0.5 or more after UV irradiation, wherein the second additive solution is at least 75% by weight or more replaced by the additive solution or a component of the additive solution after UV irradiation, based on the second additive solution; the method according to claim 1 or 2.

16. The method according to claim 1 or 2, wherein the UV irradiation is performed at a wavelength of 280 to 220 nm.

17. The method according to claim 1 or 2, wherein the additive solution or a component of the additive solution is added to the red blood cell concentrate having an hct of 0.5 or more.

18. Use of an additive solution for storing a red blood cell concentrate (RBC concentrate) containing UV-irradiated red blood cells, wherein the additive solution contains at least the following components together with water: Use: 12 to 50 mmol / L of disodium hydrogen phosphate, 0.1 to 3.5 mmol / L of adenine, 10 to 90 mmol / L of D-glucose, 0.1 to 3 mmol / L of guanosine, 10 to 80 mmol / L of sodium chloride, and 10 to 50 mmol / L of trisodium citrate.

19. The use according to claim 18, wherein the concentrations of the disodium hydrogen phosphate, adenine, D-glucose, guanosine, sodium chloride and trisodium citrate are as follows individually or simultaneously: 17 to 50 mmol / L of disodium hydrogen phosphate, 1.5 to 2.5 mmol / L of adenine, 45 to 55 mmol / L of D-glucose, 1.25 to 1.75 mmol / L of guanosine, 20 to 60 mmol / L of sodium chloride, 14 to 50 mmol / L of trisodium citrate.

20. The use according to claim 18 or 19, wherein the additive solution consists of the component at the said concentration and the balance of water.

21. The use according to claim 18 or 19, wherein the additive solution has a pH of 7 or more at 22°C.

22. The use according to claim 18 or 19, wherein the osmotic pressure of the additive solution is 260 to 300 mOsm / kg.

23. The use according to claim 18 or 19, wherein the additive solution does not contain mannitol or sorbitol, or does not contain both mannitol and sorbitol.

24. The use according to claim 18 or 19, wherein the additive solution is a dilution of the additive solution with water or a concentrated solution of the additive solution.

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