Methods for preservation of reagent red blood cells using carbon monoxide

Abstract

To provide a method for increasing a shelf-life of a preserved red blood cell composition for drug delivery.SOLUTION: Provided is a method for increasing the shelf-life of a preserved red blood cell composition for drug delivery, comprising: obtaining a red blood cell comprising a pharmaceutical agent; treating the red blood cell comprising the pharmaceutical agent with a chemical agent to prepare a red blood cell comprising a hemoglobin derivative; and storing the red blood cell comprising the hemoglobin derivative under a storage atmosphere comprising an oxygen pressure of less than 20 millimeters of mercury (mmHg) to prepare a preserved red blood cell composition for drug delivery.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 896,360, filed September 5, 2019, which is incorporated herein by reference in its entirety.

[0002] The present invention relates to the field of blood typing and to the preparation of improved red blood cell-containing reagents for use in blood typing prior to its use in transfusion medicine. The present invention also relates to improved storage and in vivo circulation of red blood cells for drug delivery. [Background technology]

[0003] Red blood cell (RBC) reagents are manufactured from blood collected from established donors with well-characterized phenotypes. The RBCs are diluted, packaged in a diluent solution, and used as standards for determining the blood type of processed RBCs at blood centers / blood banks. In most cases, automated devices are used to characterize blood typing and other important RBC quality parameters. To preserve surface antigens, RBCs are not fixed and are therefore unstable due to the accumulation of storage defects. These time-dependent changes limit the shelf life of these important RBC preparations to approximately 9 weeks (63 days).

[0004] RBCs evolved to provide oxygen transport throughout the body, where diffusion from the surrounding air is not an option, except at the surface. RBCs accomplish this function by packing a very high concentration of hemoglobin, containing oxidizable ferrous iron, into their cytosol. During their approximately 120-day circulation, RBCs are maintained to transport oxygen by a dense network of metabolic / oxidoreductase enzymes. However, these meticulous evolutionary optimizations are no longer viable once RBCs are removed from the circulation and stored at low temperatures, resulting in storage-induced damage (storage damage) that accumulates over the shelf life of stored RBCs. Oxidative damage is one of two major driving forces in the development of storage damage, which, although known, has not been comprehensively addressed until recently.

[0005] Chemical oxidation of iron in hemoglobin is the central reaction initiating oxidative stress in stored RBCs, a major factor in the development of storage injury. RBCs contain high concentrations of reactive ferrous iron in the prosthetic group of hemoglobin, along with high concentrations of dissolved oxygen. The four iron moieties in hemoglobin (ferrous state) chemically react with oxygen to form methemoglobin (ferric state). As a by-product, superoxide anion is generated, which is converted by superoxide dimutase to form H2O2, the major reactive oxygen species (ROS) and the substrate for hydroxyl radical (OH·) generation. In vivo, methemoglobin can be reduced back to hemoglobin by reductase enzymes, but the activity of these enzymes is inhibited under cryopreservation conditions. This phenomenon, coupled with higher dissolved oxygen concentrations due to increased solubility at low temperatures, leads to increased production of methemoglobin and superoxide anion. ROS molecules react with lipids and structural proteins in RBCs, compromising their integrity and reducing their circulating lifespan. ROS also attack important enzymes or surface molecules that make the "product" RBCs valuable, reducing their effectiveness or usefulness.

[0006] Hemoglobin has a 400-fold higher affinity for CO than O2, and CO does not readily react chemically with heme. The heme-CO2 complex is highly stable, significantly reducing oxidative RBC storage injury, where oxygen-induced hemoglobin oxidation is the primary cause of oxidative stress during cryogenic RBC storage. Unlike O2, CO2 does not readily react with ferrous iron, but it prevents Hb oxidation by stabilizing it as Hb-CO2, significantly reducing the occurrence of oxidative storage injury in RBCs stored at low temperatures (i.e., 1–6°C). ROS damage can be further reduced by storing RBCs under anaerobic conditions, either CO2 or an inert gas such as nitrogen.

[0007] Although the high-affinity binding of CO to Hb renders Hb and RBCs containing Hb-CO useless as oxygen carriers until CO is released, stabilization of Hb with CO is beneficial during storage, extending the shelf life of reagent RBCs not only before they are opened or reconstituted for use, but also after they are opened. Much more than transfusion RBCs, reagent RBCs are highly characterized, highly valuable, and carefully managed reagents. Even small improvements in shelf life can significantly reduce the costs of blood banking. CO treatment of reagent RBCs reduces storage damage accumulation and extends shelf life.

[0008] In addition to their ability to transport oxygen, red blood cells also serve as biocompatible carriers of various drugs, peptide molecules, and enzymes. Red blood cells can be engineered to treat a variety of diseases through the expression of biotherapeutic proteins on the cell surface or within the cytosol. These red blood cells can be used to treat cancer, autoimmune diseases, gastrointestinal disorders, and to replace defective enzymes in patients with enzyme deficiencies.Extensive reviews of cells for drug delivery include Shi, J., et al., “Engineered red blood cells as carriers for systemic delivery of a wide array of functional probes,” PNAS 111(28):10131-10136(2014); Deshmukhe A and Shetty S, “Resealed erythrocytes: a novel and promising drug carrier,” Int J Pharm Sci Res 8(8):3242-51(2017); Hamadi and Tajerzadeh, “Carrier Erythrocytes: An Overview,” Drug Delivery 10:9-20(2003); Harisa et al., “Application and safety of erythrocytes as a novel drug delivery system,” Asian Journal of Biochem.6(4):309-321(2011); Ravilla et al., “Erythrocytes as Carriers for Drugs, Enzymes, and See, for example, Sprandel and Zollner, "Osmotic fragility of drug carrier erythrocytes," Journ. of Applied Pharm. Sci. 2(04):166-176 (2012); Sprandel and Zollner, "Osmotic fragility of drug carrier erythrocytes," Res. Exp. Med. 185:77 (1985); U.S. Patent Publication Nos. 2017 / 0020926, 2018 / 0085402, 2018 / 0153989, 2018 / 0135012, and U.S. Patent No. 9,644,180, each of which is incorporated by reference in its entirety.

[0009] One problem with red blood cells for drug delivery arises during storage. Oxidative damage initiates red blood cell storage failure in conventionally stored red blood cells, regardless of whether a pharmaceutical agent is used for drug delivery. Furthermore, nonoxidative damage also reduces the circulating lifespan of drug-delivering RBCs. When delivering therapeutic agents, maintaining high quality during storage and ensuring consistent circulating lifespan is important. Given the dose-dependence of active agents, methods are needed to ensure predictable and consistent quality of RBCs for drug delivery. In contrast to RBCs for transfusion, the drug-delivery ability of RBCs to deliver oxygen is of secondary importance. Rather, the primary goal is to reduce and stabilize red blood cell damage during long-term circulation. Therefore, additional methods are needed for drug delivery to reduce oxidative damage and stabilize red blood cells to extend their shelf life and circulating lifespan in treated patients.

[0010] Here, we provide, for the first time, a storage method for red blood cells used for drug delivery that improves their shelf life and increases their circulation time by both reducing oxidative damage and stabilizing hemoglobin. This is achieved by reducing oxygen levels and preparing stable derivatives of hemoglobin during storage. In one embodiment, carbon monoxide is added to hemoglobin-containing cells to form stable carboxyhemoglobin. As mentioned above, hemoglobin has an affinity for carbon monoxide approximately 400 times greater than oxygen, and carbon monoxide does not readily react chemically with the ferrous iron in hemoglobin. We also provide several alternatives to carbon monoxide, such as cyanide and azide. Cyanide reacts with oxidized hemoglobin (methemoglobin) to form highly stable cyano-methemoglobin. This complex does not readily decompose. The cyano-methemoglobin complex can be formed with a number of oxidizing agents, such as methylene blue, phenylmethylsulfate, and potassium ferricyanide. Azide also reacts with methemoglobin to form a stable azide-hemoglobin complex. Summary of the Invention

[0011] The present disclosure provides and includes a method for storing reagent red blood cells (RBCs), comprising obtaining red blood cells, flushing the red blood cells with a gas containing carbon monoxide to prepare carbon monoxide-saturated hemoglobin in the RBCs (CO-Hb RBCs), and storing the CO-Hb RBCs under anaerobic conditions in the presence of carbon monoxide (CO), wherein surface antigens of the CO-Hb RBCs are stabilized.

[0012] The present disclosure provides and includes a method for storing reagent red blood cells (RBCs), comprising obtaining red blood cells, treating the red blood cells with a chemical agent to prepare red blood cells comprising a hemoglobin derivative, and storing the red blood cells comprising the hemoglobin derivative under anaerobic conditions to form reagent red blood cells, wherein surface antigens of the reagent red blood cells comprising the hemoglobin derivative are stabilized.

[0013] The present disclosure provides and includes a kit comprising one or more vials of carbon monoxide saturated RBCs (CO-Hb RBCs) having a plurality of CO-Hb RBCs with a common set of surface antigens in a buffer solution, and instructions for use.

[0014] The present disclosure provides a vial of carbon monoxide saturated RBCs (CO-Hb RBCs) comprising a buffer solution and CO-Hb RBCs, wherein the CO-Hb RBCs are blood group O cells and are selected from the group consisting of D, C, c, E, e, CW, K, k, P1, Fy a ,Fy b , J.K. a , J.K. b , Le a , Le b CO-Hb RBCs that are positive for surface antigens selected from the group consisting of M, N, S, and s, and D, C, c, E, e, CW, K, k, P1, Fy a ,Fy b , J.K. a , J.K. b , Le a , Le b ,M,N,S,s,I,Lu a , Lu b , Js b, Kp b , and Yt a CO-Hb RBCs, which are blood group O cells, positive for a surface antigen selected from the group consisting of D, C, c, E, e, CW, K, k, P1, and Fy a ,Fy b , J.K. a , J.K. b , Le a , Le b ,M,N,S,s,I,Lu a , Lu b , Js b , Kp b , and Yt a and the surface antigen Js is positive for a surface antigen selected from the group consisting of a , Kp a , Wr a , Di a , V w ,V.,Lu a , and C w CO-Hb RBCs, which are blood group O cells, are negative for surface antigens D, C, c, E, e, f, CW, K, k, P1, and Fy. a ,Fy b , J.K. a , J.K. b , Le a , Le b ,M,N,S,s,Lu a , and Lu b CO-Hb RBCs are type O cells that are negative for Rh antigens D, C, and e, and CO-Hb RBCs are type O cells that are positive for Rh antigens D, C, and e, I, Lu b , Js b , Kp b , and Yt a CO-Hb RBCs, which are type O cells, positive for Rh antigens D, C, and e, I, Lu b , Js b , Kp b , and Yt a , and the surface antigen Js a , Kp a , Wr a , Di a V w ,V.,Lu a , and Cw CO-Hb RBCs that are type O cells and negative for surface antigen A1; CO-Hb RBCs that are type A cells and positive for surface antigen A1 and negative for surface antigens D, C, and E; CO-Hb RBCs that are type A cells and positive for surface antigen A2; CO-Hb RBCs that are type A cells and positive for surface antigen A2 and negative for surface antigens D, C, and E; CO-Hb RBCs that are type B cells and positive for surface antigen B and negative for surface antigens D, C, and E; CO-Hb RBCs that are type B cells and positive for surface antigen D, C, and e and have the Rh phenotype R1R1; w , and e positive, and Rh phenotype R1 w CO-Hb RBCs that are type O cells and have R1, positive for surface antigens D, c, and E and have the Rh phenotype R2R2, CO-Hb RBCs that are type O cells and have the Rh phenotype rr, positive for surface antigens D, C, and e and have the Rh phenotype R1R1 and have the surface antigen Lu b , Js b , Kp b , and Yt a CO-Hb RBCs, which are type O cells, are positive for surface antigens D and C. w , and e positive, and Rh phenotype R1 w R1 and surface antigen Lu b , Js b , Kp b , and Yt a CO-Hb RBCs that are type O cells, positive for surface antigens D, c, and E, have an Rh phenotype of R2R2, and have surface antigen Lu b , Js b , Kp b , and Yt aCO-Hb RBCs that are type O cells and positive for surface antigens d, c, and e, have the Rh phenotype rr, and have surface antigen Lu b , Js b , Kp b , and Yt a CO-Hb RBCs that are type O cells and positive for surface antigens D, C, and e, have Rh phenotype R1R1, and have surface antigen Js a , Kp a , Wr a , Di a ,Vw,V,Lu a , and C w CO-Hb RBCs, which are type O cells and negative for surface antigens D and C w , and e positive, and Rh phenotype R1 w R1 and surface antigen Js a , Kp a , Wr a , Di a ,Vw,V,Lu a , and C w CO-Hb RBCs, which are type O cells and negative for surface antigens D, c, and E, have the Rh phenotype R2R2, and have surface antigen Js a , Kp a , Wr a , Di a ,Vw,V,Lu a , and C w CO-Hb RBCs that are type O cells and negative for surface antigens d, c, and e, have the Rh phenotype rr, and have surface antigen Js a , Kp a , Wr a , Di a ,Vw,V,Lu a , and C w CO-Hb RBCs are type O cells that are negative for surface antigens D, C, and e and have the Rh phenotype R1, or are positive for surface antigens D, c, and e and have the Rh heterotype R2. CO-Hb RBCs are type O cells that are positive for surface antigens D, C, and e and have the Rh heterotype R1 and have the surface antigen Lub , Js b , Kp b , and Yt a or positive for surface antigens D, c, and e, have Rh Habsburg R2, and have surface antigen Lu b , Js b , Kp b , and Yt a CO-Hb RBCs, which are type O cells, positive for surface antigens D, C, and e, have Rh haplotype R1, and have surface antigen Js a , Kp a , Wr a , Di a ,Vw,V,Lu a , and C w Negative for or positive for surface antigens D, c, and e, have Rh haplotype R2, and have surface antigen Js a , Kp a , Wr a , Di a ,Vw,V,Lu a , and C w CO-Hb RBCs, which are type O cells and negative for Hb, and ficin-treated RBCs, which have surface antigens M, N, and Fy. a ,Fy b , S, s, Xg a , Pr, Ch a , Rg a , and Yk a CO-Hb RBCs, which are type O cells and negative for Hb, and ficin-treated RBCs, which have surface antigens M, N, and Fy. a ,Fy b , S, s, Xg a , Pr, Ch a , Rg a , and Yk a Negative for D, C, E, c, e, f, Jk a , J.K. b , Le a , Le b ,P1,I,IH,Vel,PP1P k CO-Hb RBCs, which are type O cells and positive for antibody binding to the , and P antigens, and CO-Hb RBCs, which are ficin-treated and positive for surface antigens M, N, Fya ,Fy b , S, s, Xg a , Pr, Ch a , Rg a Negative for Fya, Fyb, S, s, M, N, Xg a , Pr, Ch a , Rg a , and Yk a CO-Hb RBCs are type O cells that have reduced or no binding of antibodies to the D antigen and are sensitized with anti-D(Rh0) serum. CO-Hb RBCs are type O cells that are positive for binding of antibodies to the A2 antigen. CO-Hb RBCs are type A cells that are positive for binding of antibodies to the B antigen and negative for binding of anti-D(Rh0) antibodies. CO-Hb RBCs are type B cells that are positive for binding of antibodies to the A1 antigen and negative for binding of D(Rh0) antibodies. CO-Hb RBCs are type A cells that are positive for binding of antibodies to the A1 antigen and negative for binding of D(Rh0) antibodies. CO-Hb RBCs are type AB cells that are positive for binding of antibodies to the A1, B antigens, and Rh antigens d, c, and e of the Rh serotype rr(dce / dec). CO-Hb RBCs, which are type O cells, positive for binding of antibodies to Rh antigens D, d, C, c, and e and have the Rh phenotype R1r(DCe / dce), and D(RH1), C(RH2), E(RH3), c(RH4), e(RH5), M, N, S, s, P1, K, k, Fy a ,Fy b , J.K. a , J.K. b , Le a , and Le b CO-Hb RBCs, which are type O cells, positive for binding of antibodies to D(RH1), C(RH2), E(RH3), c(RH4), e(RH5), M, N, S, s, P1, K, k, Fy a ,Fy b , J.K. a , J.K. b , Le a , and Le b positive for binding of antibodies to Lu b , Js b , Kp b, and Yt a CO-Hb RBCs, which are type O cells positive for binding of antibodies to antigens, and D(RH1), C(RH2), E(RH3), c(RH4), e(RH5), M, N, S, s, P1, K, k, Fy a ,Fy b , J.K. a , J.K. b , Le a , and Le b positive for binding of antibodies to Js a , Kp a , Wr a , Di a ,Vw,V,Lu a , and C w and CO-Hb RBCs, which are type O cells that are negative for binding of antibodies to the antigen.

[0015] The present disclosure provides and includes a method for increasing the shelf life of a red blood cell composition for drug delivery, the method comprising obtaining red blood cells containing a pharmaceutical agent, treating the red blood cells with a chemical agent to prepare red blood cells containing a hemoglobin derivative, and storing the red blood cells containing the pharmaceutical agent under a preservation atmosphere.

[0016] The present disclosure also provides and includes a method for increasing the shelf life of a red blood cell composition for drug delivery, the method comprising purging red blood cells containing a pharmaceutical agent with carbon monoxide and storing the purged red blood cells for a period of time.

[0017] The present disclosure provides and includes pharmaceutical compositions comprising leukocyte-depleted red blood cells (RBC+PA) expressing a pharmaceutical agent, the pharmaceutical composition comprising a non-oxyhemoglobin binder and less than 25% SO2.

[0018] The present disclosure provides and includes a storage vial containing a carbon monoxide saturated pharmaceutical composition comprising red blood cells containing a pharmaceutical agent.

[0019] The present disclosure also provides and includes a method of packaging a predetermined dose of red blood cell drug, the method including depleting oxygen by gas exchange with carbon monoxide, filling a drug container with the predetermined dose of red blood cell drug, and sealing the drug container.

[0020] The present disclosure further provides and includes a method for increasing the circulation life of red blood cells for drug delivery, the method comprising obtaining red blood cells (RBC+PA) containing a pharmaceutical agent, treating the red blood cells with a chemical agent to prepare red blood cells containing a hemoglobin derivative, and storing the red blood cells containing the pharmaceutical agent under a preservation atmosphere. [Brief explanation of the drawings]

[0021] [Figure 1] 1 is a graph showing deoxygenation and carbon monoxide conversion of a red blood cell concentrate in an embodiment according to Example 2(a). DETAILED DESCRIPTION OF THE INVENTION

[0022] Blood group serology requires the determination of blood cell compatibility between a blood donor and a patient-recipient prior to a blood transfusion or organ transplant involving the patient. Blood cell compatibility is determined by the absence of an immunological reaction between antibodies contained in the patient's serum and antigens present on blood cells from the donor. Tests for blood cell typing and compatibility are generally of two types: (1) agglutination tests, which determine whether specific antibodies added to cells cause their agglutination, and (2) cytolytic tests, which determine whether specific antibodies added to test cells with serum complement cause hemolysis. In commonly used blood cell typing and compatibility testing procedures, both agglutination and cytolytic tests are performed manually by trained technicians or using automated devices. The presence of an immunological reaction indicates incompatibility for transfusion and transplant therapy.

[0023] The International Society of Blood Transfusion lists 33 blood group systems, representing over 300 antigens. See Logdberg et al., "Human blood group genes 2004: Chromosomal locations and cloning strategies," Transfus Med Rev. 19:45-57 (2005) and Logdberg et al., "Human blood group genes 2010: Chromosomal locations and cloning strategies revisited," Transfus Med Rev. 25:36-46 (2011). Cloning and sequencing have demonstrated that the genes for these blood group systems are autosomal, with the exception of XG and XK, which are X-derived, and MIC2, which is present on both the X and Y chromosomes. Many different blood group antigens are found on the surface of red blood cells in all individuals. These antigens, which are the products of inherited genes, occur in combinations that are unique between all individuals except identical twins.

[0024] A number of important blood group systems are known in the art and are shown in Table 1. [Table 1]

[0025] Blood typing is generally the process of testing red blood cells to determine which antigens are present and which are absent, usually utilizing antibodies against the antigen being tested. In addition, if a person does not have a particular red blood cell antigen on their red blood cells, their serum may contain antibodies against that antigen. Whether antibodies are present in the serum depends on whether their immune system has previously been challenged with and responded to that particular antigen or one closely related to it. For example, a person whose red blood cells are type A, i.e., who has the "A" antigen on their red blood cells, will have anti-B antibodies in their serum. Therefore, if such a person is given type B blood, an immunological response will occur, with potentially serious clinical consequences.

[0026] Prior to transfusion therapy, collected blood is tested for compatibility by analyzing blood types. Blood type testing is performed by testing RBCs for various antigens (A, B, etc.) and serum for antibodies to the antigens. In the former test, RBCs from a sample of blood are incubated with antibodies that recognize each of the blood group antigens and scored for binding using agglutination or one of several other known immunological approaches. Serum testing can be performed using a variety of methods, such as ELISA, in which the antigen is provided and antibody binding is indirectly detected via an enzyme or fluorescent reporter. A more traditional approach is to use RBCs that have already been characterized to express a specific antigen in an agglutination assay called a hemagglutination assay. Briefly, agglutination assays involve mixing reagent RBCs with serum or plasma from a test sample of blood. Antibodies in the test sample, typically IgM with five antigen-binding sites, cross-link the cells together, causing agglutination that can be seen macroscopically. Bivalent IgG antibodies are also suitable for agglutination assays. Agglutination assays, including those for blood typing, are well known in the art. See Low et al., "Antiglobulin test in low-ionic strength salt solution for rapid antibody screening and cross-matching," Vox Sang 26:53-61 (1974); Malyska et al., "The gel test," Laboratory Medicine 25:81 (1994); and Technical manual. 14th ed. Bethesda, MD: American Association of Blood Banks, 2002.

[0027] The more common reagent RBCs used specifically for reverse typing either have A, A, or B antigens or no ABO antigens (A1, A2, B, O) on their surface. These cells are useful for detecting preformed antibodies that cause agglutination of the reagent RBCs. Front typing tests use monoclonal anti-A and anti-B antibodies to detect the presence of their respective antigens on the surface of sample red blood cells. Another well-known blood group is the Rh blood group, which has 58 different antigen types, although nine types are the most common. Blood group and antigen designations are shown in Tables 2 and 3. Byrne et al., “Review: other blood group systems--Diego, Yt, Immunol.14(3-4):143-53(2005), Daniels G., “Functions of red cell surface proteins,” Vox Sang.93(4):331-40(2007), Eyler and Telen, “The Lutheran glycoprotein: a multifunctional adhesion receptor.” Transfusion 46(4):668-77(2006), Palacajornsuk P.,“Review:molecular basis of MNS blood group Immunohematology,”22(4):171-82(2006);Quill E., “Medicine.Blood-matching goes genetic,”Science 14;319(5869):1478-9(2008), Westhoff CM., "The structure and function of the Rh antigen complex," Semin Hematol 44(1):42-50 (2007); Westhoff and Reid, "Review: the Kell, Duffy, and Kidd blood group systems," Immunohematology 20(1):37-49 (2004); and Yamamoto F., "Review: ABO blood group system--ABH oligosaccharide antigens, anti-A and anti-B, A and B glycosyltransferases, and ABO genes," Immunohematology 20(1):3-22 (2004), each of which is incorporated herein by reference in its entirety. [Table 2] TIFF2025179084000004.tif130130 [Table 3]

[0028] The present disclosure provides and includes methods for reducing the degradation of blood group antigens during storage. More specifically, the present disclosure provides for reducing the formation of storage damage in RBCs during storage, including, but not limited to, ROS-induced damage. Briefly, RBCs are collected and exposed to an atmosphere of carbon monoxide (CO) for a period sufficient to remove oxygen (O) bound to the heme group of hemoglobin. As shown in Example 2 and Figure 1, the exchange of oxygen with CO is rapid and essentially complete within 30 minutes and three gas exchanges. Methods for contacting CO with blood, particularly those that increase the surface area of ​​the CO / liquid interface relative to the volume ratio, will significantly reduce the length of time required for the exchange of bound oxygen with carbon monoxide. As discussed above, carbon monoxide has a significantly higher affinity for hemoglobin than oxygen (e.g., 400 times greater), so exposure of RBCs to CO at any level and time initiates the exchange process, and sufficient CO is provided to drive the exchange to completion.

[0029] The present disclosure provides and includes, but is not limited to, exchanging oxygen for carbon monoxide according to the method shown in Example 2. In one embodiment, red blood cell concentrate (RCC, also known as packed red blood cells) is held in a container, CO2 is introduced, and the container is gently shaken or mixed for a period of time. In one embodiment, the container is a standard blood storage bag containing polyvinyl chloride. In one embodiment, CO2 gas is substituted and mixing is repeated one or more times until hemoglobin is saturated with CO2 (e.g., Hb-CO2 RBCs are produced). In another embodiment, the container containing the RCC is provided with a volume of CO2 and left overnight, with or without occasional mixing. Mixing improves the exchange rate but is not required. In another embodiment, the blood and CO2 are separated by a gas-permeable membrane. This can be done using methods known in the art, for example, using a Sorin D100 oxygenator, providing CO2 rather than oxygen. The advantage of a membrane-based approach is that gas can be exchanged continuously, thereby maintaining a concentration gradient and increasing the exchange rate.

[0030] In another embodiment, CO gas can be bubbled through a container or bag of RBCs. Without being limited by theory, it is believed that maintaining bubbles less than 1 μm in diameter can prevent or minimize erythrocyte lysis. The "bubbling" method shares the same advantage as the membrane-based approach, in that CO bubbles provide the maximum concentration difference, thereby promoting the kinetics of the exchange reaction. The bubble approach also offers the added benefit of mixing the RBCs.

[0031] While carbon monoxide exchange may be preferred with RBCs, the present disclosure provides and includes exchanging CO with blood oxygen at any stage of the process. In embodiments, CO is exchanged with whole blood, for example, before platelet or white blood cell removal. In embodiments, CO is exchanged with leukocyte-reduced blood. The methods herein can be applied to RBCs prepared by apheresis or collected using conventional methods. The methods can also be performed after processing the RBCs into a suitable buffer for reagent use and storage. See, for example, U.S. Patent Publication No. 2011 / 0045455, published February 26, 2001, and International Patent Publication No. 1983 / 003477, published October 13, 1983. Other storage solutions compatible with blood typing methods are known in the art.

[0032] The present specification provides and includes a method for preparing CO-Hb RBCs, further comprising storing the CO-Hb RBCs under anaerobic conditions in the presence of CO. In an embodiment, the cells are prepared as described above and transferred to a vial under an atmosphere containing carbon monoxide. In another embodiment, the CO-Hb RBCs are transferred to a container for storage having a nitrogen atmosphere. As provided herein, any suitable non-oxygen-containing gas is suitable during storage. In certain embodiments, additional CO is provided before sealing the container for CO-Hb RBC storage.

[0033] The present specification provides and includes a method for preparing CN-Hb RBCs, further comprising storing the CN-Hb RBCs under anaerobic conditions. In one embodiment, the cells are prepared as described above and transferred to a vial under an atmosphere containing ambient air. In another embodiment, the CN-Hb RBCs are transferred to a container for storage having a nitrogen atmosphere. As provided herein, any suitable non-oxygen-containing gas is suitable during storage. In certain embodiments, additional non-oxygen-containing gas is provided before sealing the container for CN-Hb RBC storage.

[0034] The present disclosure provides and includes a method for preparing N3-Hb RBCs, further comprising storing the N3-Hb RBCs under anaerobic conditions. In one embodiment, the cells are prepared as described above and transferred to a vial under an atmosphere containing ambient air. In another embodiment, the N3-Hb RBCs are transferred to a container for storage having a nitrogen atmosphere. As provided herein, any suitable non-oxygen-containing gas is suitable during storage. In certain embodiments, additional non-oxygen-containing gas is provided before sealing the container for N3-Hb RBC storage.

[0035] The present disclosure provides and includes a method for storing reagent red blood cells (RBCs), comprising obtaining red blood cells, treating the red blood cells with a chemical agent to prepare red blood cells comprising a hemoglobin derivative, and storing the reagent red blood cells comprising the hemoglobin derivative under anaerobic conditions to form reagent red blood cells, wherein surface antigens of the reagent red blood cells comprising the hemoglobin derivative are stabilized.

[0036] In one embodiment, the chemical agent is carbon monoxide (CO) and the hemoglobin derivative is carboxy-hemoglobin (CO-Hb). In another embodiment, the chemical agent is cyanide and the hemoglobin derivative is cyano-methemoglobin (CN-Hb). In another embodiment, the chemical agent is azide (N3) and the hemoglobin derivative is azido-methemoglobin (N3-Hb), which is prepared by reacting with an aqueous solution of sodium azide.

[0037] The present disclosure provides and includes a kit comprising one or more vials of cyanide- or azide-saturated RBCs (CN-Hb RBCs or N3-Hb RBCs) having a plurality of CN-Hb RBCs or N3-Hb RBCs with a common set of surface antigens in a buffer solution, and instructions for use.

[0038] The present disclosure provides a vial of CN-Hb RBCs or N3-Hb RBCs containing a buffer and cyanide or azide saturated RBCs (CN-Hb RBCs or N3-Hb RBCs), wherein the CN-Hb RBCs or N3-Hb RBCs are blood group O cells and are selected from the group consisting of D, C, c, E, e, CW, K, k, P1, Fy a ,Fy b , J.K. a , J.K. b , Le a , Le b CN-Hb RBCs or N3-Hb RBCs that are positive for a surface antigen selected from the group consisting of D, C, c, E, e, CW, K, k, P1, Fy a ,Fy b , J.K. a , J.K. b , Le a , Le b ,M,N,S,s,I,Lu a , Lu b , Js b , Kp b , and Yt a CN-Hb RBCs or N3-Hb RBCs that are blood group O cells positive for a surface antigen selected from the group consisting of D, C, c, E, e, CW, K, k, P1, and Fy a ,Fy b , J.K. a , J.K. b , Le a , Le b ,M,N,S,s,I,Lu a , Lu b , Js b , Kp b , and Yt a and the surface antigen Js is positive for a surface antigen selected from the group consisting of a , Kpa , Wr a , Di a , V w ,V.,Lu a , and C w CN-Hb or N3-Hb RBCs, which are blood group O cells, negative for surface antigens D, C, c, E, e, f, CW, K, k, P1, and Fy a ,Fy b , J.K. a , J.K. b , Le a , Le b ,M,N,S,s,Lu a , and Lu b CN-Hb RBCs or N3-Hb RBCs that are type O cells and negative for Rh antigens D, C, and e, and CN-Hb RBCs or N3-Hb RBCs that are type O cells and positive for Rh antigens D, C, and e, I, Lu b , Js b , Kp b , and Yt a CN-Hb RBCs or N3-Hb RBCs that are type O cells and positive for Rh antigens D, C, and e, I, and Lu b , Js b , Kp b , and Yt a positive for surface antigen Js a , Kp a , Wr a , Di a V w ,V.,Lu a , and C wCN-Hb RBCs or N3-Hb RBCs that are type O cells and negative for surface antigen A1; CN-Hb RBCs or N3-Hb RBCs that are type A cells and positive for surface antigen A1 and negative for surface antigens D, C, and E; CN-Hb RBCs or N3-Hb RBCs that are type A cells and positive for surface antigen A2; CN-Hb RBCs or N3-Hb RBCs that are type A cells and positive for surface antigen A2 and negative for surface antigens D, C, and E; CN-Hb RBCs or N3-Hb RBCs that are type B cells and positive for surface antigen B; and CN-Hb RBCs or N3-Hb RBCs that are type B cells and positive for surface antigen B and negative for surface antigens D, C, and E. RBCs and CN-Hb RBCs or N3-Hb RBCs, which are type O cells and are positive for surface antigens D, C, and e and have the Rh phenotype R1R1. w , and e positive, and Rh phenotype R1 w CN-Hb RBCs or N3-Hb RBCs that are type O cells and have R1, positive for surface antigens D, c, and E and have the Rh phenotype R2R2, CN-Hb RBCs or N3-Hb RBCs that are type O cells and have the Rh phenotype rr, positive for surface antigens D, C, and e and have the Rh phenotype R1R1, and positive for surface antigen Lu b , Js b , Kp b , and Yt a CN-Hb RBCs or N3-Hb RBCs, which are type O cells, positive for surface antigens D and C w , and e positive, and Rh phenotype R1 w R1 and surface antigen Lu b , Js b , Kp b , and Yt aCN-Hb RBCs or N3-Hb RBCs that are type O cells and positive for surface antigens D, c, and E, have an Rh phenotype of R2R2, and have surface antigen Lu b , Js b , Kp b , and Yt a CN-Hb RBCs or N3-Hb RBCs that are type O cells and are positive for surface antigens d, c, and e, have the Rh phenotype rr, and have the surface antigen Lu b , Js b , Kp b , and Yt a CN-Hb RBCs or N3-Hb RBCs that are type O cells and positive for surface antigens D, C, and e, have an Rh phenotype of R1R1, and have surface antigen Js a , Kp a , Wr a , Di a ,Vw,V,Lu a , and C w CN-Hb RBCs or N3-Hb RBCs, which are type O cells and negative for surface antigens D and C w , and e positive, and Rh phenotype R1 w R1 and surface antigen Js a , Kp a , Wr a , Di a ,Vw,V,Lu a , and C w CN-Hb RBCs or N3-Hb RBCs that are type O cells and negative for surface antigens D, c, and E, have an Rh phenotype of R2R2, and have surface antigen Js a , Kp a , Wr a , Di a ,Vw,V,Lu a , and C w CN-Hb RBCs or N3-Hb RBCs that are type O cells and are negative for surface antigens d, c, and e, have the Rh phenotype rr, and have surface antigen Js a , Kp a , Wr a , Di a,Vw,V,Lu a , and C w and CN-Hb RBCs or N3-Hb RBCs that are type O cells and negative for surface antigens D, C, and e and have Rh haplotype R1, or that are type O cells and positive for surface antigens D, c, and e and have Rh haplotype R2, and CN-Hb RBCs or N3-Hb RBCs that are type O cells and positive for surface antigens D, C, and e and have Rh haplotype R1 and have Rh haplotype R2, or that are type O cells and positive for surface antigens D, C, and e and have Rh haplotype R1 and have Rh haplotype R2, b , Js b , Kp b , and Yt a or positive for surface antigens D, c, and e, have Rh haplotype R2, and have surface antigen Lu b , Js b , Kp b , and Yt a CN-Hb RBCs or N3-Hb RBCs that are type O cells and positive for surface antigens D, C, and e, have Rh haplotype R1, and have surface antigen Js a , Kp a , Wr a , Di a ,Vw,V,Lu a , and C w Negative for or positive for surface antigens D, c, and e, have Rh haplotype R2, and have surface antigen Js a , Kp a , Wr a , Di a ,Vw,V,Lu a , and C w CN-Hb RBCs or N3-Hb RBCs, which are type O cells and negative for the surface antigens M, N, and Fy, were used. a ,Fy b , S, s, Xg a , Pr, Ch a , Rg a , and Yk a CN-Hb RBCs or N3-Hb RBCs, which are type O cells and negative for the surface antigens M, N, and Fy, were treated with ficin. a ,Fyb , S, s, Xg a , Pr, Ch a , Rg a and Yk a Negative for D, C, E, c, e, f, Jk a , J.K. b , Le a , Le b ,P1,I,IH,Vel,PP1P k CN-Hb RBCs or N3-Hb RBCs, which are type O cells and positive for antibody binding to the α, β, and P antigens, and ficin-treated RBCs with surface antigens M, N, and Fy. a ,Fy b , S, s, Xg a , Pr, Ch a , Rg a Negative for Fya, Fyb, S, s, M, N, Xg a , Pr, Ch a , Rg a , and Yk aand CN-Hb RBCs or N3-Hb RBCs, which are type O cells, positive for antibody binding to D antigen and sensitized with anti-D(Rh0) serum, and CN-Hb RBCs or N3-Hb RBCs, which are type A cells, positive for antibody binding to B antigen and negative for anti-D(Rh0) antibody binding, and CN-Hb RBCs or N3-Hb RBCs, which are type B cells, positive for antibody binding to A1 antigen and negative for anti-D(Rh0) antibody binding, and CN-Hb RBCs, which are type A cells, positive for antibody binding to A1 antigen and negative for anti-D(Rh0) antibody binding, and CN-Hb RBCs, which are type AB cells, positive for antibody binding to A1, B antigen, and Rh antigens d, c, and e of the Rh blood group rr(dce / dec). CN-Hb RBC or N3-Hb RBC, which are type O cells, positive for binding of antibodies to Rh antigens D, d, C, c, and e and have the Rh phenotype R1r(DCe / dce), and D(RH1), C(RH2), E(RH3), c(RH4), e(RH5), M, N, S, s, P1, K, k, Fy a ,Fy b , J.K. a , J.K. b , Le a and Le b CN-Hb RBC or N3-Hb RBC, which are type O cells, positive for antibody binding to D(RH1), C(RH2), E(RH3), c(RH4), e(RH5), M, N, S, s, P1, K, k, Fy a ,Fy b , J.K. a , J.K. b , Le a , and Le b positive for binding of antibodies to Lu b , Js b , Kp b , and Yt aCN-Hb RBC or N3-Hb RBC, which are type O cells positive for antibody binding to antigen, and D(RH1), C(RH2), E(RH3), c(RH4), e(RH5), M, N, S, s, P1, K, k, Fy a ,Fy b , J.K. a , J.K. b , Le a , and Le b positive for binding of antibodies to Js a , Kp a , Wr a , Di a ,Vw,V,Lu a , and C w and CN-Hb RBCs or N3-Hb RBCs that are type O cells and are negative for binding of antibodies to the antigen.

[0039] The present disclosure provides and includes methods for storing reagent red blood cells (RBCs), including obtaining red blood cells selected from, but not limited to, the following 40 immunological types: 1. Blood type O cells, D, C, c, E, e, CW, K, k, P1, Fy a ,Fy b , J.K. a , J.K. b , Le a , Le b blood group O cells positive for a surface antigen selected from the group consisting of: M, N, S, and s; 2.D, C, c, E, e, CW, K, k, P1, Fy a ,Fy b , J.K. a , J.K. b , Le a , Le b ,M,N,S,s,I,Lu a , Lu b , Js b , Kp b , and Yt a blood group O cells positive for a surface antigen selected from the group consisting of: 3.D, C, c, E, e, CW, K, k, P1, Fy a ,Fy b , J.K. a , J.K. b , Le a , Le b ,M,N,S,s,I,Lu a , Lu b , Js b , Kp b , and Yt a and the surface antigen Js is positive for a surface antigen selected from the group consisting of a , Kp a , Wr a , Di a , V w ,V.,Lu a , and C w blood type O cells, which are negative for 4. Surface antigens D, C, c, E, e, f, CW, K, k, P1, Fy a ,Fy b , J.K. a , J.K. b , Le a , Le b ,M,N,S,s,Lu a , and Lu b blood type O cells, which are negative for 5. Blood type O cells, positive for Rh antigens D, C, and e, 6. Rh antigens D, C, and e, I, Lu b , Js b , Kp b , and Yt a positive for blood type O cells, 7. Rh antigens D, C, and e, I, Lu b , Js b , Kp b , and Yt a positive for surface antigen Js a , Kp a , Wr a , Di a V w ,V.,Lu a , and C w blood type O cells, which are negative for 8. Blood group A cells positive for surface antigen A1, 9. Blood group A cells, positive for surface antigen A1 and negative for surface antigens D, C, and E, 10. Blood group A cells, positive for surface antigen A2, 11. Blood group A cells, positive for surface antigen A2 and negative for surface antigens D, C, and E, 12. Blood group B cells positive for surface antigen B, 13. Blood group B cells positive for surface antigen B and negative for surface antigens D, C, and E, 14. Blood group O cells, positive for surface antigens D, C, and e and with Rh phenotype R1R1, 15. Surface antigens D, C w , and e positive, and Rh phenotype R1 w Blood type O cells with R1, 16. Blood group O cells, positive for surface antigens D, c, and E and with Rh phenotype R2R2, 17. Blood group O cells, positive for surface antigens d, c, and e and with Rh phenotype rr, 18. Positive for surface antigens D, C, and e, have Rh phenotype R1R1, and have surface antigen Lu b , Js b , Kp b , and Yt a positive for blood type O cells, 19. Surface antigens D, C w , and e positive, and Rh phenotype R1 w R1 and surface antigen Lu b , Js b , Kp b , and Yt a positive for blood type O cells, 20. Positive for surface antigens D, c, and E, have Rh phenotype R2R2, and have surface antigen Lu b , Js b , Kp b , and Yt a positive for blood type O cells, 21. Positive for surface antigens d, c, and e, have Rh phenotype rr, and have surface antigen Lu b , Js b , Kp b , and Yt a positive for blood type O cells, 22. Positive for surface antigens D, C, and e, have Rh phenotype R1R1, and have surface antigen Js a , Kp a , Wr a , Di a ,Vw,V,Lu a , and C w blood type O cells, which are negative for 23. Surface antigens D, C w , and e positive, and Rh phenotype R1 w R1 and surface antigen Js a , Kp a , Wr a , Di a ,Vw,V,Lu a , and C w blood type O cells, which are negative for 24. Positive for surface antigens D, c, and E, have Rh phenotype R2R2, and have surface antigen Js a , Kp a , Wr a , Di a ,Vw,V,Lu a , and C w blood type O cells, which are negative for 25. Positive for surface antigens d, c, and e, have Rh phenotype rr, and have surface antigen Js a , Kp a , Wr a , Di a ,Vw,V,Lu a , and C w blood type O cells, which are negative for 26. Blood type O cells, positive for surface antigens D, C, and e and with Rh haplotype R1, or positive for surface antigens D, c, and e and with Rh haplotype R2, 27. Tests positive for surface antigens D, C, and e, has Rh haplotype R1, and has surface antigen Lu b , Js b , Kp b , and Yt a or positive for surface antigens D, c, and e, have Rh haplotype R2, and have surface antigen Lu b , Js b , Kp b , and Yt a positive for blood type O cells, 28. Positive for surface antigens D, C, and e, have Rh haplotype R1, and have surface antigen Js a , Kp a , Wr a , Di a ,Vw,V,Lu a , and C w Negative for or positive for surface antigens D, c, and e, have Rh haplotype R2, and have surface antigen Js a , Kp a , Wr a , Di a ,Vw,V,Lu a , and C w blood type O cells, which are negative for 29. Ficin-treated and detects surface antigens M, N, and Fy. a ,Fy b , S, s, Xg a , Pr, Ch a , Rg a , and Yk a blood type O cells, which are negative for 30. Ficin-treated and detects surface antigens M, N, and Fy. a ,Fy b , S, s, Xg a , Pr, Ch a , Rg a , and Yk a Negative for D, C, E, c, e, f, Jk a , J.K. b , Le a , Le b ,P1,I,IH,Vel,PP1P k, and blood group O cells, which are positive for binding of antibodies to the P antigen; 31. Ficin-treated and detects surface antigens M, N, and Fy. a ,Fy b , S, s, Xg a , Pr, Ch a , Rg a Negative for Fya, Fyb, S, s, M, N, Xg a , Pr, Ch a , Rg a , and Yk a blood group O cells, with reduced or no binding of antibodies to 32. Blood group O cells that are positive for antibody binding to the D antigen and the CO-Hb RBCs are sensitized with anti-D (Rh0) serum; 33. Blood group A cells positive for binding of antibodies to the A2 antigen; 34. Blood group B cells, positive for binding of antibodies to B antigens and negative for binding of anti-D (Rh0) antibodies; 35. CO-Hb RBCs, which are type A cells, positive for binding of antibodies to the A1 antigen and negative for binding of anti-D (Rh0) antibodies. 36. Blood group AB cells positive for binding of antibodies to A1 antigen, B antigen, and Rh antigens d, c, and e of Rh blood group rr(dce / dec), 37. Blood group O cells positive for binding of antibodies to Rh antigens D, d, C, c, and e and having the Rh phenotype R1r (DCe / dce), 38.D(RH1), C(RH2), E(RH3), c(RH4), e(RH5), M, N, S, s, P1, K, k, Fy a ,Fy b , J.K. a , J.K. b , Le a , and Le b blood group O cells, positive for binding of antibodies to 39.D(RH1), C(RH2), E(RH3), c(RH4), e(RH5), M, N, S, s, P1, K, k, Fy a ,Fyb , J.K. a , J.K. b , Le a , and Le b positive for binding of antibodies to Lu b , Js b , Kp b , and Yt a blood type O cells, which are positive for binding of antibodies to antigens; and 40.D(RH1), C(RH2), E(RH3), c(RH4), e(RH5), M, N, S, s, P1, K, k, Fy a ,Fy b , J.K. a , J.K. b , Le a , and Le b positive for binding of antibodies to Js a , Kp a , Wr a , Di a ,Vw,V,Lu a , and C w Blood type O cells, which are negative for binding of antibodies to antigens.

[0040] In embodiments of the present disclosure, carbon monoxide treatment can be replaced with cyanide (CN) or azide (N3). In embodiments, the present disclosure provides and includes a method for reducing the degradation of blood group antigens during storage. More specifically, the present disclosure provides for reducing the formation of storage damage in RBCs during storage, including but not limited to ROS-induced damage. Briefly, RBCs are collected and exposed to cyanide (CN) or azide (N3) for a period sufficient to remove oxygen (O2) bound to the heme group of hemoglobin. The present disclosure provides a method for reacting cyanide with oxidized hemoglobin (methemoglobin) to form a highly stable cyano-methemoglobin. The cyano-methemoglobin complex can be formed with a number of oxidizing agents, such as methylene blue, phenylmethyl sulfate, and potassium ferricyanide. In another embodiment, the present disclosure provides a method for reacting azide with methemoglobin to form a stable azide-hemoglobin complex.

[0041] As will be appreciated by those skilled in the art, the selection of RBCs suitable for preparation of reagent RBCs is not limited to the blood type combinations provided above. Those skilled in the art will recognize that RBCs having any of the antigen type combinations listed in Tables 2 and 3 are useful for the methods of the present application. Indeed, those skilled in the art will recognize that any immunologically characterized RBCs are suitable for preparation of reagent RBCs stored with carbon monoxide (Hb-CO RBCs), cyanide (Hb-CN RBCs), or azide (Hb-N RBCs).

[0042] The present specification provides and includes exchanging oxygen bound to hemoglobin with carbon monoxide using any method known in the art, including but not limited to the methods shown in the Examples. Thus, as used herein, the term "flushing" refers to any method of contacting carbon monoxide gas with RBCs, including bubbling or passing it through a membrane.

[0043] The present disclosure provides and includes kits containing carbon monoxide-stabilized red blood cells (Hb-CO RBCs). The kits herein may include, but are not limited to, one or more of the 40 specific types of Hb-CO RBCs described above. Other suitable Hb-CO RBCs can be prepared as needed. In certain embodiments, the kits provide cells suspended in a suitable buffer, and cell preparation may include various washing steps either before or after carbon monoxide exchange. Additional reagents and buffers necessary for performing immunological assays may be included in the kit. In many embodiments, the kits include instructions for performing the assay, lot characterization of Hb-CO RBCs, and other materials relevant to those skilled in the art. In some embodiments, the kits herein may include various testing equipment, such as tubes, pipettes, and buffers.

[0044] The present specification provides and includes a container containing CO-Hb RBCs as described herein. In an embodiment, the present specification provides and includes a container containing CN-Hb RBCs as described herein. In another embodiment, the present specification provides and includes a container containing N3-Hb RBCs as described herein. In an embodiment, the container is a vial. In another embodiment, the container is a tube. In yet another embodiment, the container is an ampoule.

[0045] The present disclosure provides and includes a method for increasing the shelf life of a red blood cell composition for drug delivery, the method comprising obtaining red blood cells containing a pharmaceutical agent, treating the red blood cells with a chemical agent to prepare red blood cells containing a hemoglobin derivative, and storing the red blood cells containing the pharmaceutical agent under a preservation atmosphere.

[0046] The present disclosure provides and includes a method for increasing the shelf life of a red blood cell composition for drug delivery, the method comprising obtaining red blood cells containing a pharmaceutical agent and storing the red blood cells containing the pharmaceutical agent in liquid nitrogen.

[0047] The present disclosure provides red blood cells comprising a pharmaceutical agent. In one embodiment of the present disclosure, the pharmaceutical agent comprises a transgene expressed on the cell surface of the red blood cell. In another embodiment, the pharmaceutical agent is localized in the cytosol of the red blood cell. In another embodiment, the pharmaceutical agent is localized in an intracellular membrane. In yet another embodiment, the pharmaceutical agent is a fusion protein. In a further embodiment, the pharmaceutical agent comprises a protein selected from the group consisting of proteins provided by Tables B and C of U.S. Patent Publication No. 2017 / 0020926, published January 26, 2019, and those listed in Tables 4, 5, 6, and 7. In another embodiment, the pharmaceutical agent comprises an antigen. In another embodiment, the antigen is expressed by fusion with a red blood cell protein. In yet another embodiment, the antigen is selected from the tables provided by Tables F and G of U.S. Patent Publication No. 2017 / 0020926, published January 26, 2019, and antigens listed in Tables 8, 9, and 10. In another embodiment, the medicament is a protein selected from the class of proteins listed in Table 11. In a further embodiment, the medicament comprises an antigen or antigen target listed in Table 12. In yet another embodiment, the medicament treats a disease and comprises an exogenous antigen or target, or is adapted to target an exogenous antigen or target provided by U.S. Patent Publication No. 2018 / 0085402, published March 29, 2018, and listed in Table 13. In another embodiment, the medicament comprises an antibody molecule. In another embodiment, the medicament inhibits an immune checkpoint molecule. In yet another embodiment, the medicament comprises a first polypeptide on the surface of an erythrocyte that promotes fusion of the erythrocyte with a target cell, and a second polypeptide selected from any one of the polypeptides listed in Tables 14, 15, and 16. In another embodiment, the second polypeptide is formulated to activate or inhibit T cells as provided in Tables 15 and 16. [Table 4] [Table 5] TIFF2025179084000008.tif218170 TIFF2025179084000009.tif161170 Table 6 Table 7 TIFF2025179084000012.tif47170 Table 8 TIFF2025179084000014.tif72153 Table 9 Table 10 Table 11 Table 12 TIFF2025179084000019.tif186170 TIFF2025179084000020.tif161170 TIFF2025179084000021.tif186170 TIFF2025179084000022.tif185170 TIFF2025179084000023.tif174170 TIFF2025179084000024.tif170170 TIFF2025179084000025.tif181170 TIFF2025179084000026.tif181170 TIFF2025179084000027.tif182170 TIFF2025179084000028.tif54170 Table 13 TIFF2025179084000030.tif177170 TIFF2025179084000031.tif189170 TIFF2025179084000032.tif181170 TIFF2025179084000033.tif169170 TIFF2025179084000034.tif183170 TIFF2025179084000035.tif178170 TIFF2025179084000036.tif181170 TIFF2025179084000037.tif169170 TIFF2025179084000038.tif182170 TIFF2025179084000039.tif179170 TIFF2025179084000040.tif103170 Table 14 TIFF2025179084000042.tif219162 Table 15 Table 16

[0048] In an embodiment of the present disclosure, the pharmaceutical agent is an anti-cancer therapy. In another embodiment, the pharmaceutical agent is a therapeutic anti-cancer antibody provided in U.S. Patent Publication No. 2017 / 0020926, published on January 26, 2019, and Table 17 below. In another embodiment, the pharmaceutical agent is an agent that binds to an immune checkpoint molecule or a costimulatory molecule, as provided in Tables 4 and 5, and Tables 18 and 19 of U.S. Patent Publication No. 2017 / 0020926, published on January 26, 2019. In a further embodiment, the pharmaceutical agent targets any of the diseases and targets provided in U.S. Patent Publication No. 2018 / 0135012, published on May 17, 2018, and U.S. Patent No. 9,664,180.

[0049] In an embodiment of the present disclosure, methods for loading red blood cells with pharmaceutical agents include electroporation, endocytosis, osmotic-based methods such as hypnotic dilution, dialysis, osmotic lysis, lipid fusion, and chemical perturbation. In another embodiment, the method for incorporating pharmaceutical agents is selected from the group consisting of electroporation, osmotic shock, hypotonic and hypertonic cycling, cell surface labeling by heterobifunctional crosslinking, and cell surface labeling by click chemistry. Methods for loading pharmaceutical agents are understood by those skilled in the art. See Dischmukhe A and Shetty S: Resealed erythrocytes: a novel and promising drug carrier. Int J Pharm Sci Res 8(8):3242-51(2017). [Table 17] TIFF2025179084000046.tif211166 [Table 18] [Table 19]

[0050] In an embodiment of the present disclosure, red blood cells are treated with a chemical agent to prepare red blood cells containing a hemoglobin derivative. In an embodiment, the chemical agent is carbon monoxide, and the hemoglobin derivative is carboxyhemoglobin. In another embodiment, the chemical agent is cyanide, and the hemoglobin derivative is cyanomethemoglobin. In an embodiment, hemoglobin is first oxidized with an oxidizing agent such as methylene blue or potassium ferricyanide, and then reacted with NaCN solution or HCN gas to form cyanomethemoglobin. In a further embodiment, the agent is azide (N3), and the hemoglobin derivative is azidomethemoglobin formed by reacting with an aqueous solution of sodium azide. In another embodiment, the agent is an agent that can bind to and stabilize hemoglobin molecules.

[0051] In an embodiment of the present disclosure, increasing the shelf life of a red blood cell composition comprising carboxyhemoglobin comprises increasing the maximum length of refrigerated or room temperature storage of the red blood cell composition while maintaining the ability of the red blood cell composition to circulate in a patient in need thereof. In another embodiment, increasing the shelf life of a red blood cell composition comprising carboxyhemoglobin comprises increasing the maximum length of refrigerated or ambient temperature storage while minimizing the formation of Heinz bodies. In yet another embodiment, increasing the shelf life of a red blood cell composition comprising carboxyhemoglobin comprises reducing the number of Heinz bodies formed by at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70%, or more, compared to RBCs stored under similar conditions containing oxyhemoglobin. In another embodiment, Heinz bodies are reduced by 5-10, 10-20, 20-30, 30-40, 40-50, 50-60, or 60-70% in red blood cell compositions comprising carboxyhemoglobin compared to RBCs containing oxyhemoglobin and stored under similar conditions. In another embodiment, increasing the shelf life of a red blood cell composition comprising carboxyhemoglobin comprises reducing the number of lysed cells compared to RBCs containing oxyhemoglobin and stored under similar conditions. In an embodiment, the number of lysed cells is reduced by at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70%, or more in red blood cell compositions comprising carboxyhemoglobin compared to RBCs containing oxyhemoglobin and stored under similar conditions.

[0052] In an embodiment of the present disclosure, increasing the shelf life of a red blood cell composition comprising cyano-methemoglobin comprises increasing the maximum length of refrigerated or room temperature storage of the red blood cell composition while retaining the ability of the red blood cell composition to circulate in a patient in need thereof. In another embodiment, increasing the shelf life of a red blood cell composition comprising cyano-methemoglobin comprises increasing the maximum length of refrigerated or ambient temperature storage without forming Heinz bodies. In yet another embodiment, increasing the shelf life of a red blood cell composition comprising cyano-methemoglobin comprises reducing the number of Heinz bodies formed by at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70%, or more, compared to RBCs containing oxyhemoglobin and stored under similar conditions. In another embodiment, Heinz bodies are reduced by 5-10, 10-20, 20-30, 30-40, 40-50, 50-60, or 60-70% in red blood cell compositions comprising cyano-methemoglobin compared to RBCs containing oxyhemoglobin and stored under similar conditions. In another embodiment, increasing the shelf life of a red blood cell composition comprising cyano-methemoglobin comprises reducing the number of lysed cells compared to RBCs containing oxyhemoglobin and stored under similar conditions. In an embodiment, the number of lysed cells is reduced by at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70%, or more in red blood cell compositions comprising cyano-methemoglobin compared to RBCs containing oxyhemoglobin and stored under similar conditions.

[0053] In an embodiment of the present disclosure, increasing the shelf life of a red blood cell composition comprising azido-methemoglobin comprises increasing the maximum length of refrigerated or room temperature storage of the red blood cell composition while retaining the ability of the red blood cell composition to circulate in a patient in need thereof. In another embodiment, increasing the shelf life of a red blood cell composition comprising azido-methemoglobin comprises increasing the maximum length of refrigerated or ambient temperature storage without forming Heinz bodies. In yet another embodiment, increasing the shelf life of a red blood cell composition comprising azido-methemoglobin comprises reducing the number of Heinz bodies formed by at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70%, or more, compared to RBCs stored under similar conditions containing oxyhemoglobin. In another embodiment, Heinz bodies are reduced by 5-10, 10-20, 20-30, 30-40, 40-50, 50-60, or 60-70% in red blood cell compositions comprising azido-methemoglobin compared to RBCs stored under similar conditions containing cyano-methemoglobin. In another embodiment, increasing the shelf life of red blood cell compositions comprising azido-methemoglobin comprises reducing the number of lysed cells compared to RBCs stored under similar conditions containing oxyhemoglobin. In an embodiment, the number of lysed cells is reduced by at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70% or more in red blood cell compositions comprising azido-methemoglobin compared to RBCs stored under similar conditions containing oxyhemoglobin.

[0054] In an embodiment of the present disclosure, the shelf life is measured by quantifying the effectiveness of red blood cells containing the pharmaceutical agent and carboxyhemoglobin. In another embodiment of the present disclosure, the shelf life is measured by quantifying the effectiveness of red blood cells containing the pharmaceutical agent and cyano-methemoglobin. In another embodiment of the present disclosure, the shelf life is measured by quantifying the effectiveness of red blood cells containing the pharmaceutical agent and azido-hemoglobin. In another embodiment, the shelf life is measured by quantifying the effectiveness of red blood cells containing the pharmaceutical agent by combining red blood cells containing the pharmaceutical agent with biotin, 51 Cr, or99m It is measured by labeling with Tc and quantifying the length of time it remains in the circulation.

[0055] In an embodiment of the present disclosure, the shelf life of RBCs comprising carboxyhemoglobin is increased by one week or more compared to RBCs comprising oxyhemoglobin and stored under similar conditions. In another embodiment, the shelf life of RBCs comprising carboxyhemoglobin is increased by two weeks or more compared to RBCs comprising oxyhemoglobin and stored under similar conditions. In another embodiment, the shelf life of RBCs comprising carboxyhemoglobin is increased by three weeks or more compared to RBCs comprising oxyhemoglobin and stored under similar conditions. In another embodiment, the shelf life of RBCs comprising carboxyhemoglobin is increased by four weeks or more compared to RBCs comprising oxyhemoglobin and stored under similar conditions. In another embodiment, the shelf life of RBCs comprising carboxyhemoglobin is increased by five weeks or more compared to RBCs comprising oxyhemoglobin and stored under similar conditions. In another embodiment, the shelf life of RBCs comprising carboxyhemoglobin is increased by two days or more compared to RBCs comprising oxyhemoglobin and stored under similar conditions. In another embodiment, the shelf life of RBCs containing carboxyhemoglobin is increased by 4 days or more compared to RBCs containing oxyhemoglobin and stored under similar conditions. In another embodiment, the shelf life of RBCs containing carboxyhemoglobin is increased by 3-10 days, 5-10 days, 7-14 days, or 5-30 days compared to RBCs containing oxyhemoglobin and stored under similar conditions. In yet another embodiment, the shelf life of RBCs containing carboxyhemoglobin is increased by 1-2 weeks, 1-3 weeks, 1-4 weeks, 1-5 weeks, or 2-10 weeks compared to RBCs containing oxyhemoglobin and stored under similar conditions. In another embodiment, the shelf life of RBCs containing carboxyhemoglobin is increased by 5-10%, 10-20%, 20-40%, 40-60%, 60-80%, or 80-100% compared to RBCs containing oxyhemoglobin and stored under similar conditions. In another embodiment, the shelf life is increased by at least 10% compared to RBCs containing oxyhemoglobin and stored under similar conditions, hi another embodiment, the shelf life is increased by at least 20% compared to RBCs containing oxyhemoglobin and stored under similar conditions.In another embodiment, the shelf life is increased by at least 30% compared to RBCs containing oxyhemoglobin and stored under similar conditions. In another embodiment, the shelf life is increased by at least 40% compared to RBCs containing oxyhemoglobin and stored under similar conditions. In another embodiment, the shelf life is increased by at least 50% compared to RBCs containing oxyhemoglobin and stored under similar conditions.

[0056] In another embodiment, the shelf life of RBCs comprising cyano-methemoglobin is increased by one week or more compared to RBCs comprising oxyhemoglobin and stored under similar conditions. In another embodiment, the shelf life of RBCs comprising cyano-methemoglobin is increased by two weeks or more compared to RBCs comprising oxyhemoglobin and stored under similar conditions. In another embodiment, the shelf life of RBCs comprising cyano-methemoglobin is increased by three weeks or more compared to RBCs comprising oxyhemoglobin and stored under similar conditions. In another embodiment, the shelf life of RBCs comprising cyano-methemoglobin is increased by four weeks or more compared to RBCs comprising oxyhemoglobin and stored under similar conditions. In another embodiment, the shelf life of RBCs comprising cyano-methemoglobin is increased by five weeks or more compared to RBCs comprising oxyhemoglobin and stored under similar conditions. In another embodiment, the shelf life of RBCs comprising cyano-methemoglobin is increased by two days or more compared to RBCs comprising oxyhemoglobin and stored under similar conditions. In another embodiment, the shelf life of RBCs containing cyano-methemoglobin is increased by 4 days or more compared to RBCs containing oxyhemoglobin and stored under similar conditions. In another embodiment, the shelf life is increased by 3-10 days, 5-10 days, 7-14 days, or 5-30 days compared to RBCs containing oxyhemoglobin and stored under similar conditions. In yet another embodiment, the shelf life of RBCs containing cyano-methemoglobin is increased by 1-2 weeks, 1-3 weeks, 1-4 weeks, 1-5 weeks, or 2-10 weeks compared to RBCs containing oxyhemoglobin and stored under similar conditions. In another embodiment, the shelf life of RBCs containing cyano-methemoglobin is increased by 5-10%, 10-20%, 20-40%, 40-60%, 60-80%, or 80-100% compared to RBCs containing oxyhemoglobin and stored under similar conditions. In another embodiment, the shelf life is increased by at least 10% compared to RBCs containing oxyhemoglobin and stored under similar conditions, hi another embodiment, the shelf life is increased by at least 20% compared to RBCs containing oxyhemoglobin and stored under similar conditions.In another embodiment, the shelf life is increased by at least 30% compared to RBCs containing oxyhemoglobin and stored under similar conditions. In another embodiment, the shelf life is increased by at least 40% compared to RBCs containing oxyhemoglobin and stored under similar conditions. In another embodiment, the shelf life is increased by at least 50% compared to RBCs containing oxyhemoglobin and stored under similar conditions.

[0057] In an embodiment of the present disclosure, the shelf life of RBCs comprising azido-methemoglobin is increased by one week or more compared to RBCs comprising oxyhemoglobin and stored under similar conditions. In another embodiment, the shelf life of RBCs comprising azido-methemoglobin is increased by two weeks or more compared to RBCs comprising oxyhemoglobin and stored under similar conditions. In another embodiment, the shelf life of RBCs comprising azido-methemoglobin is increased by three weeks or more compared to RBCs comprising oxyhemoglobin and stored under similar conditions. In another embodiment, the shelf life of RBCs comprising azido-methemoglobin is increased by four weeks or more compared to RBCs comprising oxyhemoglobin and stored under similar conditions. In another embodiment, the shelf life is increased by five weeks or more compared to RBCs comprising oxyhemoglobin and stored under similar conditions. In another embodiment, the shelf life is increased by two days or more compared to RBCs comprising oxyhemoglobin and stored under similar conditions. In another embodiment, the shelf life is increased by 4 days or more compared to RBCs containing oxyhemoglobin and stored under similar conditions. In another embodiment, the shelf life is increased by 3 to 10 days, 5 to 10 days, 7 to 14 days, or 5 to 30 days compared to RBCs containing oxyhemoglobin and stored under similar conditions. In yet another embodiment, the shelf life is increased by 1 to 2 weeks, 1 to 3 weeks, 1 to 4 weeks, 1 to 5 weeks, or 2 to 10 weeks compared to RBCs containing oxyhemoglobin and stored under similar conditions. In another embodiment, the shelf life of RBCs containing azido-methemoglobin is increased by 5 to 10%, 10 to 20%, 20 to 40%, 40 to 60%, 60 to 80%, or 80 to 100% compared to RBCs containing oxyhemoglobin and stored under similar conditions. In another embodiment, the shelf life is increased by at least 10% compared to RBCs containing oxyhemoglobin and stored under similar conditions. In another embodiment, the shelf life is increased by at least 20% compared to RBCs containing oxyhemoglobin and stored under similar conditions, hi another embodiment, the shelf life is increased by at least 30% compared to RBCs containing oxyhemoglobin and stored under similar conditions.In another embodiment, the shelf life is increased by at least 40% compared to RBCs containing oxyhemoglobin and stored under similar conditions, hi another embodiment, the shelf life is increased by at least 50% compared to RBCs containing oxyhemoglobin and stored under similar conditions.

[0058] The present disclosure provides a storage atmosphere for storing red blood cell compositions for drug delivery. In some embodiments, the storage atmosphere contains a minimum partial pressure of oxygen. In some embodiments, the storage atmosphere contains less than 20 mmHg, 15 mmHg, or 10 mmHg. In other embodiments, the storage atmosphere contains a minimum partial pressure of oxygen relative to the partial pressure of nitrogen, argon, helium, or carbon monoxide. In some embodiments, the red blood cell composition for drug delivery contains carboxyhemoglobin, cyanomethemoglobin, or azidomethemoglobin.

[0059] In embodiments, the storage atmosphere is contained in a vial, container, syringe, or bag. In another embodiment, storage is under ambient pressure. In another embodiment, the storage atmosphere comprises ambient air, carbon monoxide, N2, or a combination thereof. In further embodiments, the storage atmosphere comprises less than 20, 15, 10, 5, or 3% saturated oxygen. In another embodiment, the storage atmosphere comprises 3-5, 5-10, 10-15, or 15-20% saturated oxygen.

[0060] The present disclosure provides for treating red blood cells containing a pharmaceutical agent with gas exchange. In an embodiment, the present disclosure provides for treating red blood cells containing a pharmaceutical agent and oxyhemoglobin with gas exchange. In an embodiment, the gas exchange comprises rapid gas exchange. In another embodiment, the gas exchange comprises overnight gas exchange. In yet another embodiment, the gas exchange comprises membrane gas exchange. In a further embodiment, the gas exchange comprises microbubble gas exchange. In another embodiment, the gas exchange is with carbon monoxide, HCN gas, or NAN3 solution.

[0061] In embodiments of the present disclosure, the storage temperature is 0.1 to 6°C, -4 to 6°C, 6 to 24°C, or 24 to 38°C. In other embodiments, the storage temperature is greater than -4°C, greater than 4°C, greater than 10°C, greater than 15°C, greater than 20°C, greater than 25°C, greater than 30°C, greater than 35°C, or greater than 40°C. In other embodiments, the storage temperature is about 37°C. In yet other embodiments, the storage temperature is about 27°C. In some embodiments, the composition further comprises a cryoprotectant. In yet other embodiments, the storage temperature is about -80°C.

[0062] In an embodiment of the present disclosure, the red blood cell composition includes an additive solution. In another embodiment, the additive solution includes one or more of glucose, phosphate, citrate, bicarbonate, or sodium chloride (NaCl). In another embodiment, the red blood cell composition including the additive solution has a pH of 5.5 to 8. In another embodiment of the present disclosure, the red blood cell composition includes at least 2 red blood cells per 10 microliters of liquid. In another embodiment, the red blood cell composition includes at least 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 cells per 10 microliters of liquid.

[0063] The present disclosure also provides and includes a method for increasing the shelf life of a red blood cell composition for drug delivery, the method comprising purging red blood cells containing a pharmaceutical agent with carbon monoxide and storing the purged red blood cells for a period of time. In an embodiment of the present disclosure, the red blood cells are purged with ambient air, carbon monoxide, N2, or a combination thereof.

[0064] In embodiments of the present disclosure, the red blood cell composition containing carboxyhemoglobin is stored for 1, 2, 3, 4, 5, 6, 7, 8, 9 weeks, or more. In other embodiments, the red blood cell composition is stored for 1-3 weeks, 3-6 weeks, 6-9 weeks, or 9-12 weeks. In other embodiments, the red blood cell composition is stored for at least 1, 2, 3, 4, 5, 6, 7, or 8 weeks.

[0065] In embodiments of the present disclosure, the red blood cell composition containing cyano-methemoglobin is stored for 1, 2, 3, 4, 5, 6, 7, 8, 9 weeks, or more. In other embodiments, the red blood cell composition is stored for 1-3 weeks, 3-6 weeks, 6-9 weeks, or 9-12 weeks. In other embodiments, the red blood cell composition is stored for at least 1, 2, 3, 4, 5, 6, 7, or 8 weeks.

[0066] In embodiments of the present disclosure, the red blood cell composition containing azido-methemoglobin is stored for 1, 2, 3, 4, 5, 6, 7, 8, 9 weeks, or more. In other embodiments, the red blood cell composition is stored for 1-3 weeks, 3-6 weeks, 6-9 weeks, or 9-12 weeks. In other embodiments, the red blood cell composition is stored for at least 1, 2, 3, 4, 5, 6, 7, or 8 weeks.

[0067] The present disclosure provides and includes a pharmaceutical composition comprising red blood cells expressing a pharmaceutical agent, the pharmaceutical composition comprising a deoxyhemoglobin binder and less than 25% SO. In another embodiment, the pharmaceutical composition comprises less than 20, 15, 10, 5, or 3% SO. In another embodiment, the pharmaceutical composition comprises 25-50, 50-75, or 75-100% carbon monoxide.

[0068] The present disclosure provides and includes a storage vial containing a carbon monoxide-saturated pharmaceutical composition comprising red blood cells containing a pharmaceutical agent. In an embodiment, the storage vial contains a headspace with carbon monoxide, nitrogen, or argon. In an embodiment, the storage vial contains a headspace with carbon monoxide. In another embodiment, the storage vial contains a headspace with argon. In an embodiment, the storage vial contains a headspace with nitrogen.

[0069] The present disclosure also provides and includes a method of packaging a predetermined dose of red blood cell drug, the method including depleting oxygen by gas exchange with carbon monoxide, filling a drug container with the predetermined dose of red blood cell drug, and sealing the drug container.

[0070] In an aspect of the present disclosure, the red blood cell medicinal product comprises a pharmaceutical agent and a hemoglobin derivative selected from carbon monoxide, cyanide, or azide.

[0071] In an embodiment of the present disclosure, the deoxyhemoglobin binder and the chemical binder are the same compound.

[0072] In embodiments of the present disclosure, the predetermined volume is 1 to 100 microliters, 1 to 500 microliters, 500 to 1000 microliters, or 1 to 2 milliliters. In other embodiments, the predetermined volume is at least 1, 10, 100, or 500 microliters.

[0073] In an embodiment of the present disclosure, the red blood cell drug product is red blood cells containing a pharmaceutical agent provided in the present disclosure. In an embodiment of the present disclosure, the drug container is a vial, syringe, tube, bag, or ampoule.

[0074] The present disclosure further provides and includes a method for increasing the circulation life of red blood cells for drug delivery, the method comprising obtaining red blood cells containing a pharmaceutical agent, treating the red blood cells with the pharmaceutical agent to prepare red blood cells containing a hemoglobin derivative, and storing the red blood cells containing the pharmaceutical agent under a preservation atmosphere.

[0075] In an embodiment of the present disclosure, the circulating life of red blood cells is increased by at least 2, 4, 5, 8, 16, or 32 days in a patient in need thereof compared to red blood cells stored under similar conditions with oxyhemoglobin. In another embodiment, the circulating life of red blood cells for drug delivery is increased by at least 1, 2, or 3 weeks compared to red blood cells stored under similar conditions with oxyhemoglobin.

[0076] In embodiments of the present disclosure, "similar conditions" are defined by all conditions that are consistent except for oxygen saturation, hemoglobin derivatives, or oxygen saturation and hemoglobin derivatives. For example, similar conditions include the length of storage time, storage temperature, and storage container. In embodiments, similar conditions do not include headspace gas composition (i.e., CO vs. ambient air). In embodiments, similar conditions include storage temperature, length of storage time, and additive solution.

[0077] In embodiments of the present disclosure, "RBCs containing oxyhemoglobin" comprise a mixture of oxyhemoglobin and deoxyhemoglobin, with greater than 50% oxyhemoglobin. In another embodiment, RBCs containing oxyhemoglobin comprise greater than 60% oxyhemoglobin. In another embodiment, RBCs containing oxyhemoglobin comprise greater than 70% oxyhemoglobin. In another embodiment, RBCs containing oxyhemoglobin comprise greater than 80% oxyhemoglobin. In another embodiment, RBCs containing oxyhemoglobin comprise greater than 90% oxyhemoglobin. In another embodiment, RBCs containing oxyhemoglobin comprise greater than 95% oxyhemoglobin. In another embodiment, RBCs containing oxyhemoglobin comprise greater than 98% oxyhemoglobin. In another embodiment, RBCs containing oxyhemoglobin comprise 100% oxyhemoglobin. In yet another embodiment, the RBCs containing oxyhemoglobin contain less than 50, 40, 30, 20, 10, 5, or 2% deoxyhemoglobin.

[0078] As used herein, the term "reagent red blood cells" refers to antigenically characterized red blood cells. In an embodiment of the present disclosure, the reagent red blood cells are antigenically characterized red blood cells stabilized with a hemoglobin derivative selected from the group consisting of carbon monoxide, cyanide, and azide. In another embodiment, the reagent red blood cells are packed red blood cells. In another embodiment, the reagent red blood cells comprise a 2-3% suspension of pooled washed red blood cells. In another embodiment, the reagent red blood cells comprise a 2-3% suspension of pooled washed red blood cells in modified Alsever solution. In yet another embodiment, the reagent red blood cells comprise a storage solution. In an embodiment, the storage solution comprises trisodium citrate, citric acid, dextrose, inosine, neomycin sulfate, chloramphenicol, or a combination thereof. In another embodiment, the storage solution comprises trisodium citrate, citric acid, dextrose, inosine, neomycin sulfate (0.103 grams / liter), chloramphenicol (0.349 grams / liter), or a combination thereof.

[0079] As used herein, the terms "reduced," "reduced," "reduced," "reduced," or "reduced" refer to a final amount that is lower than the initial amount or lower than the control sample. In one embodiment, the control sample comprises RBCs containing oxyhemoglobin and stored under similar conditions. In another embodiment, the control sample comprises RBCs containing a mixture of oxyhemoglobin and deoxyhemoglobin.

[0080] As used herein, the term "increasing" or "increased" is meant to refer to a final amount that is higher than the initial amount or higher compared to a control sample.

[0081] The present specification provides and includes the following embodiments.

[0082] Embodiment 1. A method for storing reagent red blood cells (RBCs), comprising: a) obtaining red blood cells; b) flushing the red blood cells with a gas containing carbon monoxide to prepare carbon monoxide saturated RBCs (CO-Hb RBCs); c) storing the CO-Hb RBCs under anaerobic conditions in the presence of carbon monoxide (CO), wherein the surface antigens of the CO-Hb RBCs are stabilized. Embodiment 2. The method of embodiment 1, wherein the gas does not contain oxygen. Embodiment 3. The CO-Hb RBCs are selected from the group consisting of D, C, c, E, e, CW, K, k, P1, Fy a ,Fy b , J.K. a , J.K. b , Le a , Le b 10. The method of embodiment 1, wherein the cells are blood group O cells that are positive for a surface antigen selected from the group consisting of: M, N, S, and s. Embodiment 4. The CO-Hb RBCs contain surface antigen I, Lu a , Lu b , Js b , Kp b , and Yt a 4. The method of embodiment 3, wherein the blood group is blood group O cells, further positive for . Embodiment 5. The CO-Hb RBCs have surface antigen Js a , Kp a , Wr a , Di a , V w ,V.,Lu a , and C w 5. The method of embodiment 4, wherein the cells are blood group O cells that are negative for HIV-1. Embodiment 6. The CO-Hb RBCs contain surface antigens D, C, c, E, e, f, CW, K, k, P1, Fy a ,Fy b , J.K. a , J.K. b , Le a , Le b ,M,N,S,s,Lu a , and Lu b 2. The method of embodiment 1, wherein the cells are type O cells that are negative for HIV-1. Embodiment 7. The method of embodiment 1, wherein said CO-Hb RBCs are type O cells positive for Rh antigens D, C, and e. Embodiment 8. The CO-Hb RBCs contain surface antigen I, Lu b , Js b , Kp b , and Yt a 8. The method of embodiment 7, wherein the cells are type O cells positive for HIV-1. Embodiment 9. The CO-Hb RBCs have surface antigen Js a , Kp a , Wr a , Di a V w ,V.,Lu a , and C w 9. The method of embodiment 8, wherein the cells are type O cells that are negative for HIV-1. Embodiment 10. The method of embodiment 1, wherein said CO-Hb RBCs are type A cells that are positive for surface antigen A1. Embodiment 11. The method of embodiment 10, wherein said CO-Hb RBCs are type A cells that are negative for surface antigens D, C, and E. Embodiment 12 The method of embodiment 1, wherein said CO-Hb RBCs are type A cells that are positive for surface antigen A2. Embodiment 13 The method of embodiment 12, wherein said CO-Hb RBCs are type A cells that are negative for surface antigens D, C, and E. Embodiment 14 The method of embodiment 1, wherein said CO-Hb RBCs are type B cells that are positive for surface antigen B. Embodiment 15. The method of embodiment 14, wherein said CO-Hb RBCs are type B cells that are negative for surface antigens D, C, and E. Embodiment 16. The CO-Hb RBCs are positive for surface antigens D, C, and e and have the Rh phenotype R1R1, or are positive for surface antigens D, C, and e and have the Rh phenotype R1R1. w , and e positive, and Rh phenotype R1 w R1 or positive for surface antigens D, c, and E and have an Rh phenotype of R2R2, or 2. The method of embodiment 1, wherein the cells are type O cells that are positive for surface antigens d, c, and e and have an Rh phenotype rr. Embodiment 17. The CO-Hb RBCs have the surface antigen Lu b , Js b , Kp b , and Yt a 17. The method of embodiment 16, wherein the cells are type O cells positive for HIV-1. Embodiment 18. The CO-Hb RBCs have surface antigen Js a , Kp a , Wr a , Di a ,Vw,V,Lu a , and C w 17. The method of embodiment 16, wherein the cells are type O cells that are negative for HIV-1. Embodiment 19. The method of embodiment 1, wherein the CO-Hb RBCs are type O cells that are positive for surface antigens D, C, and e and have Rh haplotype R1, or positive for surface antigens D, c, and e and have Rh haplotype R2. Embodiment 20. The CO-Hb RBCs have the surface antigen Lu b , Js b , Kp b , and Yt a 20. The method of embodiment 19, wherein the cells are type O cells positive for HIV-1. Embodiment 21. The CO-Hb RBCs have surface antigen Js a , Kp a , Wr a , Di a ,Vw,V,Lu a , and C w 20. The method of embodiment 19, wherein the cells are type O cells that are negative for HIV-1. Embodiment 22. The CO-Hb RBCs are ficin-treated and contain surface antigens M, N, Fy a ,Fy b , S, s, Xg a , Pr, Ch a , Rg a , and Yk a 2. The method of embodiment 1, wherein the cells are type O cells that are negative for HIV-1. Embodiment 23. The CO-Hb RBCs are selected from the group consisting of D, C, E, c, e, f, Jk a , J.K. b , Le a , Le b ,P1,I,IH,Vel,PP1P k 23. The method of embodiment 22, wherein the cells are type O cells, and positive for binding of an antibody to the P antigen. Embodiment 24. The CO-Hb RBCs are selected from the group consisting of Fya, Fyb, S, s, M, N, and Xg. a , Pr, Ch a , Rg a , and Yk a 23. The method of embodiment 22, wherein the cells are type O cells that have reduced or no binding of antibodies to Embodiment 25. The method of embodiment 1, wherein the CO-Hb RBCs are type O cells that are positive for binding of antibodies to D antigen, and the CO-Hb RBCs are sensitized with anti-D (Rh0) serum. Embodiment 26 The method of embodiment 1, wherein the CO-Hb RBCs are type A cells that are positive for binding of antibodies to the A2 antigen. Embodiment 27. The method of embodiment 1, wherein said CO-Hb RBCs are type B cells that are positive for binding of antibodies to B antigens and negative for binding of anti-D (Rh0) antibodies. Embodiment 28. The method of embodiment 1, wherein the CO-Hb RBCs are type A cells that are positive for binding of antibodies to the A1 antigen and negative for binding of anti-D (Rh0) antibodies. Embodiment 29. The method of embodiment 1, wherein the CO-Hb RBCs are type AB cells that are positive for binding of antibodies to A1 antigen, B antigen, and Rh antigens d, c, and e of the Rh blood type rr(dce / dec). Embodiment 30. The method of embodiment 1, wherein the CO-Hb RBCs are type O cells that are positive for binding of antibodies to Rh antigens D, d, C, c, and e and have an Rh phenotype R1r (DCe / dce). Embodiment 31. The CO-Hb RBC is selected from the group consisting of D(RH1), C(RH2), E(RH3), c(RH4), e(RH5), M, N, S, s, P1, K, k, Fy a ,Fy b , J.K. a , J.K. b , Le a , and Le b 2. The method of embodiment 1, wherein the cells are type O cells positive for binding of an antibody to Embodiment 32. The CO-Hb RBCs are b , Js b , Kp b , and Yt a 32. The method of embodiment 31, wherein the cells are type O cells that are positive for binding of an antibody to the antigen. Embodiment 33. The CO-Hb RBCs are a , Kp a , Wr a , Di a ,Vw,V,Lu a , and C w 32. The method of embodiment 31, wherein the cells are type O cells that are negative for binding of an antibody to the antigen. Embodiment 34. The method of embodiment 1, wherein said CO-Hb RBCs are type O cells with no profile in the Resolve® Panel A instructions. Embodiment 35. The method of embodiment 1, wherein said CO-Hb RBCs are type O cells with no profile in the Resolve® Panel B Instructions.

[0083] Embodiment 36. The method of embodiment 1, wherein said CO-Hb RBCs are type O cells with no profile in the Resolve® Panel C instructions. Embodiment 37. A kit comprising: a) one or more vials of carbon monoxide saturated RBCs (CO-Hb RBCs), the vials containing a buffer; b) a plurality of CO-Hb RBCs having a common set of surface antigens, the CO-Hb RBCs comprising: (i) Blood type O cells, including D, C, c, E, e, CW, K, k, P1, and Fy a ,Fy b , J.K. a , J.K. b , Le a , Le b CO-Hb RBCs that are positive for a surface antigen selected from the group consisting of M, N, S, and s; (ii)D, C, c, E, e, CW, K, k, P1, Fy a ,Fy b , J.K. a , J.K. b , Le a , Le b ,M,N,S,s,I,Lu a , Lu b , Js b , Kp b , and Yt a CO-Hb RBCs, which are blood group O cells, positive for a surface antigen selected from the group consisting of: (iii).D, C, c, E, e, CW, K, k, P1, Fy a ,Fy b , J.K. a , J.K. b , Le a , Le b ,M,N,S,s,I,Lu a , Lu b , Js b , Kp b , and Yt a and the surface antigen Js is positive for a surface antigen selected from the group consisting of a , Kp a , Wr a , Di a , V w ,V.,Lu a , and C w CO-Hb RBCs, which are blood type O cells and negative for (iv) Surface antigens D, C, c, E, e, f, CW, K, k, P1, Fy a ,Fy b , J.K. a , J.K. b , Le a , Le b ,M,N,S,s,Lua , and Lu b CO-Hb RBCs, which are type O cells and negative for (v) CO-Hb RBCs, which are type O cells and positive for Rh antigens D, C, and e; (vi) Rh antigens D, C, and e, I, Lu b , Js b , Kp b , and Yt a CO-Hb RBCs, which are type O cells, positive for (vii) Rh antigens D, C, and e, I, Lu b , Js b , Kp b , and Yt a positive for surface antigen Js a , Kp a , Wr a , Di a V w ,V.,Lu a , and C w CO-Hb RBCs, which are type O cells and negative for (viii) CO-Hb RBCs that are type A cells and are positive for surface antigen A1; (ix) CO-Hb RBCs, which are type A cells, positive for surface antigen A1 and negative for surface antigens D, C, and E; (x) CO-Hb RBCs that are type A cells and are positive for surface antigen A2; (xi) CO-Hb RBCs, which are type A cells, positive for surface antigen A2 and negative for surface antigens D, C, and E; (xii) CO-Hb RBCs, which are type B cells and are positive for surface antigen B; (xiii) CO-Hb RBCs, which are type B cells, positive for surface antigen B and negative for surface antigens D, C, and E; (xiv) CO-Hb RBCs that are type O cells, positive for surface antigens D, C, and e and have the Rh phenotype R1R1; (xv) Surface antigens D, C w , and e positive, and Rh phenotype R1w CO-Hb RBC, which is a type O cell with R1; (xvi) CO-Hb RBCs that are type O cells positive for surface antigens D, c, and E and have the Rh phenotype R2R2; (xvii) CO-Hb RBCs that are type O cells, positive for surface antigens d, c, and e and have the Rh phenotype rr; (xviii) positive for surface antigens D, C, and e, have Rh phenotype R1R1, and have surface antigen Lu b , Js b , Kp b , and Yt a CO-Hb RBCs, which are type O cells, positive for (xix) Surface antigens D, C w , and e positive, and Rh phenotype R1 w R1 and surface antigen Lu b , Js b , Kp b , and Yt a CO-Hb RBCs, which are type O cells, positive for (xx) positive for surface antigens D, c, and E, have Rh phenotype R2R2, and have surface antigen Lu b , Js b , Kp b , and Yt a CO-Hb RBCs, which are type O cells, positive for (xxi) positive for surface antigens d, c, and e, have Rh phenotype rr, and have surface antigen Lu b , Js b , Kp b , and Yt a CO-Hb RBCs, which are type O cells, positive for (xxii) positive for surface antigens D, C, and e, Rh phenotype R1R1, and surface antigen Js a , Kp a , Wr a , Di a ,Vw,V,Lu a , and C w CO-Hb RBCs, which are type O cells and negative for (xxiii) Surface antigens D, C w , and e positive, and Rh phenotype R1 w R1 and surface antigen Js a , Kp a , Wr a , Di a ,Vw,V,Lu a , and C w CO-Hb RBCs, which are type O cells and negative for (xxiv) positive for surface antigens D, c, and E, Rh phenotype R2R2, and surface antigen Js a , Kp a , Wr a , Di a ,Vw,V,Lu a , and C w CO-Hb RBCs, which are type O cells and negative for (xxv) positive for surface antigens d, c, and e, have Rh phenotype rr, and have surface antigen Js a , Kp a , Wr a , Di a ,Vw,V,Lu a , and C w CO-Hb RBCs, which are type O cells and negative for (xxvi) CO-Hb RBCs that are type O cells that are positive for surface antigens D, C, and e and have Rh haplotype R1, or positive for surface antigens D, c, and e and have Rh haplotype R2; (xxvii) positive for surface antigens D, C, and e, have Rh haplotype R1, and have surface antigen Lu b , Js b , Kp b , and Yt a or positive for surface antigens D, c, and e, have Rh haplotype R2, and have surface antigen Lu b , Js b , Kp b , and Yt a CO-Hb RBCs, which are type O cells, positive for (xxviii) positive for surface antigens D, C, and e, have Rh haplotype R1, and have surface antigen Js a , Kp a , Wr a , Di a ,Vw,V,Lu a , and C w Negative for or positive for surface antigens D, c, and e, have Rh haplotype R2, and have surface antigen Js a , Kp a , Wr a , Di a ,Vw,V,Lu a , and C w CO-Hb RBCs, which are type O cells and negative for (xxix) ficin-treated surface antigens M, N, and Fy a ,Fy b , S, s, Xg a , Pr, Ch a , Rg a , and Yk a CO-Hb RBCs, which are type O cells and negative for (xxx) Ficin-treated surface antigens M, N, and Fy a ,Fy b , S, s, Xg a , Pr, Ch a , Rg a , and Yk a Negative for D, C, E, c, e, f, Jk a , J.K. b , Le a , Le b ,P1,I,IH,Vel,PP1P k , and CO-Hb RBCs, which are type O cells positive for binding of antibodies to the P antigen; (xxxi) ficin-treated surface antigens M, N, and Fy a ,Fy b , S, s, Xg a , Pr, Ch a , Rg a Negative for Fya, Fyb, S, s, M, N, Xg a , Pr, Ch a , Rga , and Yk a CO-Hb RBCs, which are type O cells, have reduced or no binding to (xxxii) CO-Hb RBCs that are positive for antibody binding to D antigen and are type O cells, wherein the CO-Hb RBCs are sensitized with anti-D (RhO) serum; (xxxiii) CO-Hb RBCs, which are type A cells and are positive for antibody binding to the A2 antigen; (xxxiv) CO-Hb RBC, which is a type B cell that is positive for antibody binding to B antigen and negative for anti-D (Rh0) antibody binding; (xxxv) CO-Hb RBCs, which are type A cells and positive for antibody binding to A1 antigen and negative for anti-D (Rh0) antibody binding; (xxxvi) CO-Hb RBCs, which are type AB cells positive for binding of antibodies to A1 antigen, B antigen, and Rh antigens d, c, and e of the Rh blood type rr(dce / dec); (xxxvii) CO-Hb RBCs that are type O cells and positive for antibody binding to Rh antigens D, d, C, c, and e and have the Rh phenotype R1r (DCe / dce); (xxxviii)D(RH1), C(RH2), E(RH3), c(RH4), e(RH5), M, N, S, s, P1, K, k, Fy a ,Fy b , J.K. a , J.K. b , Le a , and Le b CO-Hb RBCs, which are type O cells and positive for binding of antibodies to (xxxix)D(RH1), C(RH2), E(RH3), c(RH4), e(RH5), M, N, S, s, P1, K, k, Fy a ,Fy b , J.K. a , J.K. b , Le a , and Le b positive for binding of antibodies to Lu b , Jsb , Kp b , and Yt a CO-Hb RBCs, which are type O cells and positive for antibody binding to antigens; (xl)D(RH1), C(RH2), E(RH3), c(RH4), e(RH5), M, N, S, s, P1, K, k, Fy a ,Fy b , J.K. a , J.K. b , Le a , and Le b positive for binding of antibodies to Js a , Kp a , Wr a , Di a ,Vw,V,Lu a , and C w a plurality of CO-Hb RBCs selected from the group consisting of CO-Hb RBCs that are type O cells and are negative for binding of an antibody to an antigen; and c) instructions. Embodiment 38. A vial of carbon monoxide saturated RBCs (CO-Hb RBCs) comprising a buffer and CO-Hb RBCs, wherein the CO-Hb RBCs are: (i) Blood type O cells, including D, C, c, E, e, CW, K, k, P1, and Fy a ,Fy b , J.K. a , J.K. b , Le a , Le b CO-Hb RBCs that are positive for a surface antigen selected from the group consisting of: M, N, S, and s; (ii)D, C, c, E, e, CW, K, k, P1, Fy a ,Fy b , J.K. a , J.K. b , Le a , Le b ,M,N,S,s,I,Lu a , Lu b , Js b , Kp b , and Yt a CO-Hb RBCs, which are blood group O cells, positive for a surface antigen selected from the group consisting of: (iii).D, C, c, E, e, CW, K, k, P1, Fy a ,Fy b , J.K. a , J.K. b , Le a , Le b ,M,N,S,s,I,Lu a , Lu b , Js b , Kp b , and Yt a and the surface antigen Js is positive for a surface antigen selected from the group consisting of a , Kp a , Wr a , Di a , V w ,V.,Lu a , and C w CO-Hb RBCs, which are blood type O cells, negative for (iv) Surface antigens D, C, c, E, e, f, CW, K, k, P1, Fy a ,Fy b , J.K. a , J.K. b , Le a , Le b ,M,N,S,s,Lu a , and Lu b CO-Hb RBCs, which are type O cells and negative for Hb; (v) CO-Hb RBCs, which are type O cells positive for Rh antigens D, C, and e; (vi) Rh antigens D, C, and e, I, Lu b , Js b , Kp b , and Yt a CO-Hb RBCs, which are type O cells, positive for (vii) Rh antigens D, C, and e, I, Lu b , Js b , Kp b , and Yt a positive for surface antigen Js a , Kp a , Wr a , Di a V w ,V.,Lua , and C w CO-Hb RBCs, which are type O cells and negative for Hb; (viii) CO-Hb RBCs that are type A cells and are positive for surface antigen A1; (ix) CO-Hb RBCs, which are type A cells that are positive for surface antigen A1 and negative for surface antigens D, C, and E; (x) CO-Hb RBCs that are type A cells and are positive for surface antigen A2; (xi) CO-Hb RBCs, which are type A cells that are positive for surface antigen A2 and negative for surface antigens D, C, and E; (xii) CO-Hb RBCs that are type B cells and are positive for surface antigen B; (xiii) CO-Hb RBCs, which are type B cells positive for surface antigen B and negative for surface antigens D, C, and E; (xiv) CO-Hb RBCs that are type O cells that are positive for surface antigens D, C, and e and have the Rh phenotype R1R1; (xv) Surface antigens D, C w , and e positive, and Rh phenotype R1 w CO-Hb RBCs, which are type O cells having R1; (xvi) CO-Hb RBCs that are type O cells that are positive for surface antigens D, c, and E and have the Rh phenotype R2R2; (xvii) CO-Hb RBCs that are positive for surface antigens d, c, and e and have the Rh phenotype rr, and are type O cells; (xviii) positive for surface antigens D, C, and e, have Rh phenotype R1R1, and have surface antigen Lu b , Js b , Kp b , and Yt a CO-Hb RBCs, which are type O cells, positive for (xix) Surface antigens D, C w , and e positive, and Rh phenotype R1 w R1 and surface antigen Lub , Js b , Kp b , and Yt a CO-Hb RBCs, which are type O cells, positive for (xx) positive for surface antigens D, c, and E, have Rh phenotype R2R2, and have surface antigen Lu b , Js b , Kp b , and Yt a CO-Hb RBCs, which are type O cells, positive for (xxi) positive for surface antigens d, c, and e, have Rh phenotype rr, and have surface antigen Lu b , Js b , Kp b , and Yt a CO-Hb RBCs, which are type O cells, positive for (xxii) positive for surface antigens D, C, and e, Rh phenotype R1R1, and surface antigen Js a , Kp a , Wr a , Di a ,Vw,V,Lu a , and C w CO-Hb RBCs, which are type O cells and negative for Hb; (xxiii) Surface antigens D, C w , and e positive, and Rh phenotype R1 w R1 and surface antigen Js a , Kp a , Wr a , Di a ,Vw,V,Lu a , and C w CO-Hb RBCs, which are type O cells that are negative for Hb; (xxiv) positive for surface antigens D, c, and E, Rh phenotype R2R2, and surface antigen Js a , Kp a , Wr a , Di a ,Vw,V,Lu a , and C w CO-Hb RBCs, which are type O cells and negative for Hb; (xxv) positive for surface antigens d, c, and e, have Rh phenotype rr, and have surface antigen Js a , Kp a , Wr a , Di a ,Vw,V,Lu a , and C w CO-Hb RBCs, which are type O cells and negative for Hb; (xxvi) CO-Hb RBCs that are type O cells that are positive for surface antigens D, C, and e and have Rh haplotype R1, or positive for surface antigens D, c, and e and have Rh haplotype R2; (xxvii) positive for surface antigens D, C, and e, have Rh haplotype R1, and have surface antigen Lu b , Js b , Kp b , and Yt a or positive for surface antigens D, c, and e, have Rh haplotype R2, and have surface antigen Lu b , Js b , Kp b , and Yt a CO-Hb RBCs, which are type O cells, positive for (xxviii) positive for surface antigens D, C, and e, have Rh haplotype R1, and have surface antigen Js a , Kp a , Wr a , Di a ,Vw,V,Lu a , and C w Negative for or positive for surface antigens D, c, and e, have Rh haplotype R2, and have surface antigen Js a , Kp a , Wr a , Di a ,Vw,V,Lu a , and C w CO-Hb RBCs, which are type O cells and negative for Hb; (xxix) ficin-treated surface antigens M, N, and Fy a ,Fy b , S, s, Xg a, Pr, Ch a , Rg a , and Yk a CO-Hb RBCs, which are type O cells and negative for Hb; (xxx) Ficin-treated surface antigens M, N, and Fy a ,Fy b , S, s, Xg a , Pr, Ch a , Rg a , and Yk a Negative for D, C, E, c, e, f, Jk a , J.K. b , Le a , Le b ,P1,I,IH,Vel,PP1P k and CO-Hb RBCs, which are type O cells positive for binding of antibodies to the P antigen; (xxxi) ficin-treated surface antigens M, N, and Fy a ,Fy b , S, s, Xg a , Pr, Ch a , Rg a Negative for Fya, Fyb, S, s, M, N, Xg a , Pr, Ch a , Rg a , and Yk a CO-Hb RBCs, which are type O cells, have reduced or no binding to (xxxii) CO-Hb RBCs that are type O cells and are positive for antibody binding to D antigen, and the cells are sensitized with anti-D (RhO) serum; (xxxiii) CO-Hb RBCs, which are type A cells and positive for antibody binding to the A2 antigen; and (xxxiv) CO-Hb RBCs, which are type B cells that are positive for antibody binding to B antigen and negative for anti-D (Rh0) antibody binding; (xxxv) CO-Hb RBCs, which are type A cells that are positive for antibody binding to A1 antigen and negative for anti-D (Rh0) antibody binding; (xxxvi) CO-Hb RBCs, which are type AB cells positive for binding of antibodies to A1 antigen, B antigen, and Rh antigens d, c, and e of the Rh blood type rr(dce / dec); (xxxvii) CO-Hb RBCs that are type O cells that are positive for antibody binding to Rh antigens D, d, C, c, and e and have the Rh phenotype R1r(DCe / dce); (xxxviii)D(RH1), C(RH2), E(RH3), c(RH4), e(RH5), M, N, S, s, P1, K, k, Fy a ,Fy b , J.K. a , J.K. b , Le a , and Le b CO-Hb RBCs, which are type O cells positive for binding of antibodies to (xxxix) CO-Hb RBC is D(RH1), C(RH2), E(RH3), c(RH4), e(RH5), M, N, S, s, P1, K, k, Fy a ,Fy b , J.K. a , J.K. b , Le a , and Le b positive for binding of antibodies to Lu b , Js b , Kp b , and Yt a CO-Hb RBCs, which are type O cells that are positive for binding of antibodies to antigens; (xl)D(RH1), C(RH2), E(RH3), c(RH4), e(RH5), M, N, S, s, P1, K, k, Fy a ,Fy b , J.K. a , J.K. b , Le a , and Le b positive for binding of antibodies to Js a , Kp a , Wr a , Di a ,Vw,V,Lu a , and C wand CO-Hb RBCs, which are type O cells that are negative for binding of antibodies to the antigen. Embodiment 39. A method for storing reagent red blood cells (RBCs), comprising: a) obtaining antigenically characterized red blood cells; b) treating the red blood cells with a chemical agent to prepare red blood cells containing a hemoglobin derivative; c) storing the red blood cells containing the hemoglobin derivative under anaerobic conditions to form reagent red blood cells, wherein the surface antigens of the reagent red blood cells containing the hemoglobin derivative are stabilized. Embodiment 40 The method of embodiment 39, wherein the chemical agent is carbon monoxide (CO) and the hemoglobin derivative is carboxyhemoglobin. Embodiment 41 The method of embodiment 39, wherein the chemical agent is cyanide and the hemoglobin derivative is cyanomethemoglobin. Embodiment 42 The method of embodiment 39, wherein the chemical agent is azide (N3) and the hemoglobin derivative is azido-methemoglobin prepared by reacting with an aqueous solution of sodium azide. Embodiment 43 The method of embodiment 39, further comprising characterizing the red blood cells. Embodiment 44. A method for increasing the shelf life of a red blood cell composition for drug delivery, comprising: obtaining red blood cells containing a pharmaceutical agent; treating the red blood cells with a chemical agent to prepare red blood cells containing a hemoglobin derivative; storing the red blood cells containing the pharmaceutical agent under a preservation atmosphere. Embodiment 45. The method of embodiment 44, wherein the storage atmosphere contains less than 10 mmHg of oxygen. Embodiment 46. The method of embodiment 44, wherein said storing is under ambient pressure. Embodiment 47. The method of embodiment 44, wherein the chemical agent is carbon monoxide (CO) and the hemoglobin derivative is carboxyhemoglobin. Embodiment 48 The method of embodiment 44, wherein the chemical agent is cyanide and the hemoglobin derivative is cyanomethemoglobin. Embodiment 49. The method of embodiment 44, wherein the chemical agent is azide (N3) and the hemoglobin derivative is azido-methemoglobin prepared by reacting with an aqueous solution of sodium azide. Embodiment 50. The method of embodiment 47, wherein the storage atmosphere comprises carbon monoxide. Embodiment 51. The method of embodiment 48, wherein the atmosphere comprises: the storage atmosphere comprises ambient air, carbon monoxide, N2, or a mixture thereof. Embodiment 52. The method of embodiment 49, wherein the atmosphere comprises nitrogen. Embodiment 53 The method of embodiment 47, wherein the shelf life is increased by more than one week compared to RBCs containing oxyhemoglobin and stored under similar conditions. Embodiment 54 The method of embodiment 53, wherein the RBCs containing oxyhemoglobin comprise a mixture of oxyhemoglobin and deoxyhemoglobin and have greater than 50% oxyhemoglobin. Embodiment 55. The method of embodiment 54, wherein the RBCs containing oxyhemoglobin contain more than 60, 70, 80, 90, or 95% oxyhemoglobin. Embodiment 56 The method of embodiment 48, wherein the shelf life is increased by one week or more compared to RBCs containing oxyhemoglobin and stored under similar conditions. Embodiment 57. The method of embodiment 49, wherein the shelf life is increased by one week or more compared to RBCs containing oxyhemoglobin and stored under similar conditions. Embodiment 58. The method of embodiments 47-49, wherein the shelf life is increased by one or more days compared to RBCs containing oxyhemoglobin and stored under similar conditions. Embodiment 59. The method of any one of embodiments 45 to 58, wherein the development of Heinz bodies in said red blood cells is reduced compared to RBCs containing oxyhemoglobin and stored under similar conditions. Embodiment 60. The method of any one of embodiments 45 to 58, wherein cell lysis is reduced in the red blood cell composition compared to RBCs containing oxyhemoglobin and stored under similar conditions. Embodiment 61. The method of embodiment 44, wherein the pharmaceutical agent is a transgene expressed on the cell surface of the RBC. Embodiment 62 The method of embodiment 44, wherein the pharmaceutical agent is localized within the cytosol of the RBC. Embodiment 63 The method of embodiment 44, wherein the pharmaceutical agent is localized on the cell surface of the RBC. Embodiment 64. The method of embodiment 44, wherein the pharmaceutical agent is localized to an intracellular membrane. Embodiment 65. The method of embodiment 44, wherein said treating comprises gas exchange. Embodiment 66. The method of embodiment 65, wherein the gas exchange is rapid gas exchange, overnight gas exchange, membrane gas exchange, or microbubble gas exchange. Embodiment 67. The method of embodiment 65 or 66, wherein the gas exchange is by carbon monoxide. Embodiment 68 The method of embodiment 65 or 66, wherein the gas exchange is carried out with cyanide by treatment with HCN. Embodiment 69. The method of embodiment 65 or 66, wherein the gas exchange is by treating the red blood cells with a sodium azide (NaN3) solution. Embodiment 70. The method of embodiment 44, wherein the pharmaceutical agent is a fusion protein. Embodiment 71. The method of embodiment 70, wherein the fusion protein is selected from the group consisting of those listed in Table 4 and Table 5. Embodiment 72. The method of embodiment 44, wherein the pharmaceutical agent is a protein selected from the classes of proteins listed in Table 11. Embodiment 73. The method of embodiment 44, wherein the pharmaceutical agent is selected from the pharmaceutical agents listed in Table 12. Embodiment 74. The method of embodiment 44, wherein said storing is at a temperature of 0.1 to 6°C. Embodiment 75. The method of embodiment 44, wherein said storing is at a temperature above 6°C. Embodiment 76. The method of embodiment 44, wherein the storing is at a temperature of 24 to 38°C. Embodiment 77. The method of embodiment 44, wherein said storing is at 37°C. Embodiment 78. The method of embodiment 44, further comprising mixing the red blood cells with an additive solution having a pH of 5.5 to 8. Embodiment 79. The method of embodiment 78, wherein the additive solution comprises one or more of glucose, phosphate, citrate, bicarbonate, or sodium chloride (NaCl). Embodiment 80. The method of embodiment 44, wherein the red blood cell composition comprises at least 100 red blood cells per microliter. A method for increasing the shelf life of a red blood cell composition for drug delivery, comprising purging red blood cells containing a pharmaceutical agent with carbon monoxide; and storing the purged red blood cells for a period of time. Embodiment 81. The method of embodiment 80, wherein the shelf life is increased by more than 1 week compared to RBCs containing oxyhemoglobin and stored under similar conditions. Embodiment 82. The method of embodiment 80, wherein the shelf life is increased by one or more days compared to RBCs containing oxyhemoglobin and stored under similar conditions. Embodiment 83. The method of embodiment 80, wherein said storing is under reduced oxygen conditions of less than 25% oxygen. Embodiment 84. The method of embodiment 80, wherein the pharmaceutical agent is one or more expressed proteins. Embodiment 85. The method of embodiment 80, wherein the pharmaceutical agent is localized within the cytoplasm of the RBC. Embodiment 86 The method of embodiment 80, wherein the pharmaceutical agent is localized on the cell surface of the RBC. Embodiment 87. The method of embodiment 80, wherein the pharmaceutical agent is localized in the RBC membrane. Embodiment 88. The method of embodiment 80, wherein said purging is rapid, overnight, or by gas exchange with carbon monoxide. Embodiment 89. The method of embodiment 80, wherein said storing is at 37°C. Embodiment 90. The method of embodiment 80, wherein said storing is at a temperature of 0.1 to 6°C. Embodiment 91. The method of embodiment 80, wherein said storing is at a temperature above 6°C. Embodiment 92. The method of embodiment 80, further comprising mixing the red blood cells with an additive solution having a pH of 5.5 to 8. Embodiment 93. The method of embodiment 92, wherein the additive solution comprises one or more of glucose, phosphate, citrate, bicarbonate, or sodium chloride (NaCl). Embodiment 94 The method of embodiment 80, wherein the red blood cell composition comprises at least 100 red blood cells per microliter. Embodiment 95. The method of embodiment 44, wherein the headspace atmosphere contains less than 5 mmHg of oxygen. Embodiment 96. A pharmaceutical composition comprising red blood cells expressing a pharmaceutical agent, the pharmaceutical composition comprising a non-oxyhemoglobin binder and less than 25% SO2. Embodiment 97 The pharmaceutical composition of embodiment 96, wherein the non-oxyhemoglobin binder is carbon monoxide. Embodiment 98. The method of embodiment 96, wherein the non-oxyhemoglobin binding agent is cyanide. Embodiment 99. The method of embodiment 96, wherein the non-oxyhemoglobin binding agent is azide (N3). Embodiment 100. A storage vial containing a carbon monoxide saturated pharmaceutical composition comprising red blood cells containing a pharmaceutical agent. Embodiment 101. The pharmaceutical composition of embodiment 100, wherein the medicament comprises an antigen expressed by fusion with an erythrocyte protein selected from the group consisting of those listed in Table 5, Table 6, and Table 7. Embodiment 102. The pharmaceutical composition of embodiment 100, wherein the pharmaceutical agent is a protein selected from the classes of proteins listed in Table 11. Embodiment 103. The pharmaceutical composition of embodiment 101, wherein the antigen is selected from the antigens listed in Table 8, Table 9, or Table 10. Embodiment 104. The pharmaceutical composition of embodiment 101, wherein the pharmaceutical comprises an antibody molecule. Embodiment 105. The pharmaceutical composition of embodiment 101, wherein the pharmaceutical agent is an agent that inhibits an immune checkpoint molecule. Embodiment 106 The pharmaceutical composition of embodiment 101, wherein the antigen is selected from the antigens in Table 12. Embodiment 107 The pharmaceutical composition of embodiment 104, wherein the antibody is selected from the antibodies listed in Table 17. Embodiment 108. A method of packaging a predetermined dose of a red blood cell drug, comprising: depleting oxygen by gas exchange with carbon monoxide; filling a drug container with the predetermined dose of the red blood cell drug; and sealing the drug container. Embodiment 109. The method of embodiment 108, wherein the packaging step is carried out under reduced oxygen conditions. Embodiment 110. A method for increasing the in vivo circulation time of red blood cells for drug delivery, comprising: obtaining red blood cells containing a pharmaceutical agent; treating the red blood cells with a chemical agent to prepare red blood cells containing a hemoglobin derivative; storing the red blood cells containing the pharmaceutical agent under a preservation atmosphere. Embodiment 111 The method of embodiment 110, wherein the chemical agent is cyanide and the hemoglobin derivative is cyanomethemoglobin. Embodiment 112. The method of embodiment 110, wherein the chemical agent is azide (N3) and the hemoglobin derivative is azido-methemoglobin prepared by reacting the red blood cells with an aqueous solution of sodium azide. Embodiment 113. The method of embodiment 110, wherein the shelf life is increased by at least 5 days compared to RBCs containing oxyhemoglobin and stored under similar conditions.

[0084] Having now generally described the invention, the same will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to limit the invention unless specified.

[0085] Each journal, patent, and other document or reference cited herein is incorporated herein by reference in its entirety. [Example]

[0086] Example 1: Blood collection and preparation of red blood cell concentrate (RCC) Blood for preparation of reagent RBCs is collected from established donors in an anticoagulant solution using standard methods. Various known anticoagulants suitable for use in transfusion medicine are suitable, including citrate phosphate dextrose (CPD) and acid citrate dextrose (ACD). However, if the blood is not to be transfused, other anticoagulants, such as ethylenediaminetetraacetic acid (EDTA) and ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), can be used as needed. The collected blood is centrifuged or filtered to separate white blood cells (WBCs) and excess plasma, producing packed red blood cells (pRBCs) or red blood cell concentrates (RCCs).

[0087] Example 2: Preparation of Hb-CO-containing RCC (Hb-CO RCC) Preferably, without delay, the red blood cell concentrate (RCC) prepared in Example 1 is converted to Hb-CO by one of the following methods:

[0088] a. Rapid gas exchange: The RCCs are held in polyvinyl chloride or other suitable bags (≥150 ml), carbon monoxide is introduced, and the bag containing the RCCs and CO is placed in a platelet shaker for approximately 10 minutes. After incubation, gas containing unexchanged CO, residual released oxygen, and carbon dioxide is allowed to evolve and replaced with fresh CO. After a second incubation in the platelet shaker for approximately 10 minutes, the gas is again allowed to evolve and replaced with a third volume of CO. After a final incubation by shaking in the platelet shaker, excess gas is removed, and the Hb-CO RCCs are transferred to suitable vials for further characterization, quality control, and storage under anaerobic conditions.

[0089] b. Overnight gas exchange: The RCCs are kept in polyvinyl chloride or other suitable bags (150 ml or larger), carbon monoxide is introduced, and the bag containing the RCCs and CO is placed at 4 °C overnight with constant gentle shaking or agitation at regular intervals (e.g., 3-5 times over 8 h). After incubation, the excess gas containing CO is removed, and the Hb-CO RCCs are transferred under anaerobic conditions to suitable vials for further characterization, quality control, and storage. C. membrane gas exchange RCCs held in polyvinyl chloride or other suitable bags (≥150 ml) are pumped through a Sorin D100 oxygenator using carbon monoxide as the source gas. RCCs are pumped through the D100 for 30 minutes using either a centrifugal or peristaltic pump. Oxygen and CO levels are monitored until a Hb-CO level of >95% is achieved. Alternatively, the pH of the suspension is optimized by using a mixture of CO and CO2.

[0090] d. Microbubble gas exchange: The RCCs are placed in a polyvinyl chloride or other suitable ventilated bag (≥150 ml) and carbon monoxide is bubbled through the RCCs. Care is taken to ensure that the bubbles are less than 1 μm in diameter to prevent erythrocyte lysis. The resulting Hb-CO RCCs are transferred under anaerobic conditions to suitable vials for further characterization, quality control, and storage. Alternatively, the pH of the suspension can be optimized by using a mixture of CO and CO2 gases.

[0091] e. Modified HEMANEXT® Oxygen Reduction Bag (ORB) A HEMANEXT® oxygen reduction bag (ORB), as described in U.S. Patent No. 10,058,091, issued August 28, 2018, is modified by removing the adsorbent pack and filling the headspace (100-200 ml) with CO gas. The CO2-containing ORB bag is agitated at room temperature for 30 minutes on a platelet shaker. Alternatively, the CO2-containing ORB bag is placed at 4°C with either constant shaking or agitation at regular intervals (e.g., 3-5 times over an 8-hour period).

[0092] Example 3: Preparation and packaging of HB-CO Reagent RBC The CO-treated RBCs are used to continue the further manufacturing process.

[0093] The reagent RBCs are packaged in a reagent bottle head, the headspace is filled with CO under positive pressure, and stored at 4 °C.

[0094] Example 4: Long-term incubation of RBCs with carbon monoxide at 37°C. Preliminary studies are being conducted to estimate the behavior of stored RBCs after transfusion into recipients. Experiments are set up to incubate fresh and stored RBCs in tissue culture medium at 37°C for extended periods, with RBC morphology used as the outcome measure. When RBCs are placed in the medium under ambient air, within 1–3 days, small dark nodules (Heinz bodies) appear on the surface and continue to grow in size over several days. Over a week, the Heinz bodies continue to grow and become larger (estimated to exceed 1 μm in diameter), resulting in the rupture of most RBCs. This results in the development of RBC ghosts (clear cytosol, little or no hemoglobin) with large dark nodules attached.

[0095] Without being limited by theory, the development of Heinz bodies and RBC ghosts is likely caused by hemoglobin oxidation products. For example, the ferrous (+2) iron in hemoglobin is oxidized to the ferric (+3) state by reacting with oxygen during incubation. When hemoglobin is oxidized to methemoglobin, it becomes unstable and readily decomposes into hemichrome, which then decomposes into globin and hemin, all of which are hydrophobic, causing RBC membrane-forming aggregates (Heinz bodies). Furthermore, these hemoglobin oxidation products bind to proteins and the RBC cytoskeleton, disrupting normal morphology and function. Normally, in the circulation, small Heinz bodies are removed by macrophages, maintaining normal RBC morphology, albeit with a reduction in cell size. In combination with optimized nutrients and mechanical deformation in the circulation, RBCs have a circulatory lifespan of approximately 120 days. In macrophage-free media, Heinz body growth was not inhibited, resulting in rapid cell destruction.

[0096] Stabilization of hemoglobin by carbon monoxide (Hb-CO complex) prevents hemoglobin oxidation and inhibits Heinz body formation and cell destruction. Hb-CO complex RBCs maintain their normal biconcave morphology for 2-3 weeks during incubation in a 37°C cell culture environment when the ambient air is purged with 100% carbon monoxide or 5% CO2 / 95% CO2 in the culture bottle.

Claims

1. 1. A method for storing reagent red blood cells (RBCs), comprising: a) obtaining antigenically characterized red blood cells; b) treating the red blood cells with a chemical agent to prepare red blood cells containing a hemoglobin derivative; c) storing the red blood cells containing the hemoglobin derivative under anaerobic conditions to form reagent red blood cells, wherein the surface antigens of the reagent red blood cells containing the hemoglobin derivative are stabilized.

2. 2. The method of claim 1, wherein the chemical agent is carbon monoxide (CO) and the hemoglobin derivative is carboxy-hemoglobin.

3. 2. The method of claim 1, wherein the chemical agent is cyanide and the hemoglobin derivative is cyano-methemoglobin.

4. 2. The method of claim 1, wherein the chemical agent is azide (N3) and the hemoglobin derivative is azido-methemoglobin prepared by reacting with an aqueous solution of sodium azide.

5. The method of claim 1 further comprising characterizing the red blood cells.

6. 1. A method for increasing the shelf life of a red blood cell composition for drug delivery, comprising: obtaining red blood cells containing a pharmaceutical agent; treating the red blood cells with a chemical agent to prepare red blood cells containing a hemoglobin derivative; storing the red blood cells containing the pharmaceutical agent under a preservation atmosphere.

7. 7. The method of claim 6, wherein the storage atmosphere contains less than 10 mmHg of oxygen.

8. The method of claim 6 , wherein the storage is under ambient pressure.

9. 7. The method of claim 6, wherein the chemical agent is carbon monoxide (CO) and the hemoglobin derivative is carboxy-hemoglobin.

10. 7. The method of claim 6, wherein the chemical agent is cyanide and the hemoglobin derivative is cyano-methemoglobin.

11. The chemical agent is azide (N 3 7. The method of claim 6, wherein the hemoglobin derivative is azido-methemoglobin prepared by reacting with an aqueous solution of sodium azide.

12. 10. The method of claim 9, wherein the storage atmosphere comprises carbon monoxide.

13. The atmosphere may be ambient air, carbon monoxide, N 2 11. The method of claim 10, comprising including:

14. The method of claim 11 , wherein the atmosphere comprises nitrogen.

15. 10. The method of claim 9, wherein the shelf life is increased by more than one week compared to RBCs containing oxyhemoglobin and stored under similar conditions.

16. 1. A pharmaceutical composition comprising red blood cells expressing a pharmaceutical agent, said pharmaceutical composition comprising a non-oxygenated hemoglobin binder and less than 25% SO 2 A pharmaceutical composition comprising:

17. A storage vial containing a carbon monoxide saturated pharmaceutical composition comprising red blood cells containing a pharmaceutical agent.

18. 1. A method of packaging a dose of a red blood cell drug, the method comprising: depleting oxygen by gas exchange with carbon monoxide; filling a drug container with the dose of the red blood cell drug; and sealing the drug container.

19. 1. A method for increasing the in vivo circulation time of red blood cells for drug delivery, comprising: obtaining red blood cells containing a pharmaceutical agent; treating the red blood cells with a chemical agent to prepare red blood cells containing a hemoglobin derivative; storing the red blood cells containing the pharmaceutical agent under a preservation atmosphere.

20. 10. The invention substantially as herein described.

Images