HK1157140B - Improved quenching methods for red blood cell pathogen inactivation - Google Patents
Improved quenching methods for red blood cell pathogen inactivation Download PDFInfo
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Description
CROSS-REFERENCE TO RELATED APPLICATIONS
Priority benefits of U.S. provisional application No.61/043,666 entitled "quenching method for inactivation of red blood cell pathogens" filed on 9/4/2008 and U.S. provisional application No.61/087,034 entitled "quenching method for inactivation of red blood cell pathogens" filed on 7/8/2008 of the present application. The entire contents of both applications are hereby incorporated by reference as if fully set forth below.
Technical Field
The field of the invention relates to methods of quenching reactive electrophilic compounds used in the treatment of blood products to inactivate possible pathogen contaminants. In particular, an elevated concentration of a nucleophilic compound, such as a thiol, is used to quench the reactive electrophilic compound in the red blood cell composition, and then the concentration is reduced to reduce Red Blood Cell (RBC) dehydration.
Background
The transmission of diseases through blood products and other biological materials remains a serious health problem. Despite significant advances in donor screening and blood testing, viruses such as Hepatitis B Virus (HBV), Hepatitis C Virus (HCV), and Human Immunodeficiency Virus (HIV) may still escape detection in blood products during testing due to low levels of virus or viral antibodies. In addition to the viral hazard, there is currently no adequate approved test for screening blood destined for transfusion for the presence of non-viral microorganisms (such as bacteria or protozoa). There is also a risk of: the pathogens unknown to date may be prevalent in blood supplies and present a threat of disease transmission, as occurs before the risk of HIV transmission through blood transfusions is recognized.
Chemical agents have been introduced into blood or plasma to inactivate pathogens prior to clinical use of the blood product. Typically, for blood products with little or no red blood cell content (e.g., platelets and plasma), photochemically activated compounds, such as psoralen, are used. For blood products containing red blood cells, compounds for pathogen inactivation have been developed that do not require photoactivation. These compounds generally have electrophilic groups that react with pathogens, more specifically with pathogen nucleic acids. For example, the use of aryl diol epoxides to inactivate viruses in cells and protein-containing compositions is described in US 5,055,485. Other compounds that generate electrophiles in situ can be used. LoGrippo et al evaluated the CH of nitrogen mustard3-N(CH2CH2Cl)2The application in virus inactivation. LoGrippo et al, Proceedings of the six Congress of the International Society of Blood transfusions (Sixth conference of the International Society of Blood Transfusion), Bibliotheca Haematologica (edited by Hollander), 1958, pp.225-230. More significantly, U.S. patent 5,691,132; 6,177,441, respectively; 6,410,219, respectively; 6,143,490 and 6,093,725, the disclosures of which are incorporated herein by reference, describe the use of compounds having a nucleic acid targeting component and an electrophilic component that reacts with the nucleic acid to inactivate pathogens. Similar compounds are described in U.S. Pat. Nos. 6,093,725 and 6,514,987, the disclosures of which are incorporated herein by reference, wherein the nucleic acid targeting component of the compound is linked to the reactive electrophilic component by a hydrolyzable linker. The use of oligomers of ethyleneimine and related compounds for pathogen inactivation is described in US patent 6,136,586 and US 6,617,157The disclosures of these documents are hereby incorporated by reference. These ethyleneimine-derived compounds typically have an aziridine group to provide a reactive electrophilic component and a polyamine component to provide nucleic acid targeting of the compound. The general class of nucleic acid targeting compounds with electrophilic or similar groups reactive with nucleic acids are used to inactivate pathogens in blood, blood products and various samples of biological origin.
It is of some concern that although these compounds are designed to react specifically with target nucleic acids, they may still react with other components in the blood, such as proteins or cell membranes. These side reactions are undesirable and may lead to adverse effects such as protein and cell membrane modifications that can be recognized by the immune system. When such treated blood products are used repeatedly, they may cause the recipient to develop an immune response to the treated blood product. Us patent 6,270,952; 6,709,810, respectively; and 7,293,985, the disclosures of which are incorporated herein by reference, describe methods of quenching such pathogen inactivating compounds in order to reduce the level of any such adverse side reactions. U.S. patent publication No.2006/0115466 describes an improvement in these quenching conditions that addresses the immune response generated against pathogen inactivating compounds. However, despite the improved quenching effectiveness, in some cases, the treated red blood cells were found to have a reduced lifespan due to increased cell dehydration, as measured by reduced osmotic fragility and reduced self-selected hematocrit.
Thus, there is a need for a method of reducing the adverse electrophilic side reactions of a pathogen-inactivating compound while maintaining the ability of the pathogen-inactivating compound to inactivate harmful pathogens without adversely affecting the viability and longevity of the treated blood product. In particular, there is a need for improved methods of quenching pathogen-inactivating compounds in red blood cells.
Summary of The Invention
The present invention provides various methods for treating red blood cell compositions containing pathogen-inactivating compounds and quenchers under conditions that provide suitable pathogen inactivation and reduction of adverse side effects (e.g., alteration of red blood cells), while reducing or minimizing adverse effects, such as reduced cell dehydration. In some embodiments, the quencher is glutathione neutralized with an appropriate amount of base. In some embodiments, the method involves reducing the concentration of glutathione after pathogen inactivation.
In one aspect, the present invention provides a method of treating a red blood cell composition comprising: a) providing i) an effective amount of a pathogen-inactivating compound to inactivate a pathogen (if present), wherein the pathogen-inactivating compound comprises a functional group that is or forms a reactive electrophilic group, ii) an effective amount of a quencher comprising a thiol group, wherein the thiol is capable of reacting with the reactive electrophilic group of the pathogen-inactivating compound, and iii) a composition comprising red blood cells; b) mixing a pathogen-inactivating compound and a quencher with a composition comprising red blood cells; and c) reducing the concentration of the quencher in the mixture sufficiently to an amount that reduces the level of red blood cell dehydration resulting from storage of the mixture relative to the level of red blood cell dehydration resulting from storage of the mixture at the initial concentration of the quencher. In some of these embodiments, reducing the concentration of the quencher includes removing the solution used during inactivation and addition of the final additive solution (e.g., any solution described herein, such AS SAG-M, AS-5, or any solution of tables 2, 3, or 4).
In some embodiments, step (a) further comprises providing a suitable base, step (b) further comprises mixing the base with a composition comprising red blood cells, and the base is sufficient to reduce the level of adverse reaction of the pathogen-inactivating compound with the red blood cells in the mixture relative to a mixture without the base. In some embodiments, the adverse reaction of the pathogen-inactivating compound with the red blood cells is a modification of the surface of the red blood cells by the pathogen-inactivating compound. In some embodiments, step (a) further comprises providing a suitable base, step (b) further comprises mixing the base with the composition comprising red blood cells, and the base is sufficient to reduce the level of anti-pathogen inactivating compound antibody binding to the treated red blood cell composition in the resulting mixture by at least about 5% (or at least about 10%, at least about 25%, at least about 50%, at least about 75%, or at least about 90%) relative to the mixture without the base. In some embodiments, the base and the quencher are mixed with the red blood cell composition prior to, simultaneously with, or no more than about 30 minutes after the pathogen-inactivating compound is mixed with the red blood cell composition. In some embodiments, the base and the quencher are mixed together prior to mixing the base or the quencher with the red blood cell composition. In some embodiments, the base is NaOH. In some embodiments, the base is a base buffer. In some embodiments, the base comprises about 0.5 to 1.5 equivalents of base, where equivalent means a molar amount equivalent to the molar amount of quencher in the mixture. In some embodiments, the base comprises about 0.75 to 1.25 equivalents of base. In some embodiments, the base comprises about 1 equivalent of base. In some embodiments, the resulting mixture of step (b) has a pH of about 6.0 to 7.5 at 37 ℃. In some embodiments, the pH is about 6.5 to 7.1. In some embodiments, the pH is about 6.8 or 6.9.
In some embodiments, the quencher comprises cysteine or a cysteine derivative. In some embodiments, the quencher is glutathione or a pharmaceutically acceptable salt thereof. In some embodiments, the concentration of quencher in the resulting mixture of step (b) is greater than 2 mM. In some embodiments, the quencher concentration is about 5mM to about 30 mM. In some embodiments, the quencher concentration is about 15mM to about 25 mM. In some embodiments, the quencher concentration is about 20 mM.
In some embodiments, reducing the concentration of quencher in step (c) comprises centrifuging the mixture followed by removing the supernatant. In some embodiments, step (c) comprises size exclusion separation. In some embodiments, the quencher in the resulting mixture of step (c) is at a concentration of less than about 10 mM. In some embodiments, the quencher concentration is less than about 8 mM. In some embodiments, the quencher concentration is less than about 6mM (or less than about 4mM, or less than about 2 mM). In some embodiments, storage of the mixture is storage of the mixture at 4 ℃ for more than 10 days. In some embodiments, storage of the mixture is storage of the mixture at 4 ℃ for more than 42 days (or 28 days). In some embodiments, the method includes adding an additive solution (e.g., any of the additive solutions described in table 2, and/or an additive solution including sodium chloride, adenine, glucose, phosphate, guanosine, citrate, and/or mannitol). In some embodiments, the mixture is stored in an additive solution (e.g., any of the additive solutions described in table 2, and/or an additive solution comprising sodium chloride, adenine, glucose, phosphate, guanosine, citrate and/or mannitol). In some embodiments, the method further comprises replacing the treatment solution (e.g., any of the solutions described in tables 2, 3, or 4 and/or a solution comprising sodium chloride, adenine, glucose, phosphate, guanosine, citrate and/or mannitol) with an additive solution (e.g., any of the additive solutions described in table 2, and/or an additive solution comprising sodium chloride, adenine, glucose, phosphate, guanosine, citrate and/or mannitol). In some embodiments, the chloride concentration of the composition is less than about 100mM (or about 75mM) prior to reducing the quencher concentration. In some embodiments, the chloride concentration of the composition after reducing the quencher concentration and/or adding the additive solution is greater than about 100mM (or about 125 mM).
In some embodiments of each of the above methods, as well as other methods described herein, the functional group is a mustard, a mustard intermediate, or a mustard equivalent. In some embodiments, the functional group is or is capable of forming an aziridinium ion. In some embodiments, the reactive electrophilic group is capable of reacting with a nucleic acid. In some embodiments, the pathogen inactivating compound further comprises a nucleic acid binding ligand. In some embodiments, the nucleic acid binding ligand is an intercalator. In some embodiments, the intercalator is acridine. In some embodiments, the pathogen-inactivating compound comprises a cleavable linker that links the functional group and the nucleic acid-binding ligand. In some embodiments, the pathogen-inactivating compound is β -alanine, N- (acridin-9-yl), 2- [ bis (2-chloroethyl) amino ] ethyl ester. In some embodiments, the concentration of the pathogen inactivating compound in the mixture resulting from step (b) is from about 0.1 μ M to about 5 mM. In some embodiments, the concentration is sufficient to inactivate at least 1log of a pathogen in the red blood cell composition, if present. In some embodiments, the concentration is sufficient to inactivate at least 3log of the pathogen. In some embodiments, the time between step (b) and step (c) is about 1 to 48 hours. In some embodiments, the time is about 10 to 30 hours. In some embodiments, the time is about 15 to 25 hours. In some embodiments, the treatment inactivates at least 1log of pathogen contaminants, if any, in the red blood cell composition. In some embodiments, the treatment inactivates at least 3 logs of the red blood cell composition. In some embodiments, the method further comprises the step of reducing the concentration of the pathogen-inactivating compound in the mixture. In some embodiments, the steps of reducing the concentration of the quencher in the mixture and reducing the concentration of the pathogen-inactivating compound in the mixture are performed simultaneously.
In some embodiments of any of the methods described above, and other methods described herein, at 20 hours after step (b), the Red Blood Cells (RBCs) of the resulting mixture have a 65% lower anti-pathogen inactivating compound Antibody Binding Capacity (ABC) as compared to the ABC value of red blood cells from the same method under the same conditions but without the use of base. In some embodiments, the RBCs have an average ABC of less than about 50,000. In some embodiments, the RBCs have an average ABC of less than about 40,000. In some embodiments, the RBCs have an average ABC of about 25,000 to 70,000. In some embodiments, the RBCs have an average ABC of about 35,000 to 45,000. In some embodiments, the RBCs of the mixture obtained after step (c) have less than 1% hemolysis. In some embodiments, RBCs have less than 1% hemolysis when stored for 42 days (or 28 days) at 4 ℃ after step (c). In some embodiments, the RBCs have a Packed Cell Volume (PCV) of greater than 50% after step (c). In some embodiments, RBCs have greater than 50% PCV when stored at 4 ℃ for 42 days (or 28 days) after step (c). In some embodiments, the RBCs of the resulting mixture have an average red blood cell friability value greater than 140 (or 150) after storage at 4 ℃ for 42 days (or 28 days) after step (c). In some embodiments, the level of the pathogen inactivating compound is not reduced and/or the pathogen inactivating compound is not contacted with a compound uptake device (CAD).
In other aspects, the invention provides methods of reducing dehydration of red blood cells, comprising: a) a red blood cell composition comprising a mixture of: i) a quencher, wherein the quencher is capable of reacting with the pathogen-inactivating compound, and ii) red blood cells; and b) reducing the concentration of the quencher (and optionally the concentration of the pathogen-inactivating compound and/or its by-products) in the mixture sufficiently to reduce the level of red blood cell dehydration resulting from storage of the mixture relative to the level of red blood cell dehydration resulting from storage of the mixture at the initial concentration of the quencher. In some embodiments, the quencher comprises cysteine or a cysteine derivative. In some embodiments, the quencher is glutathione or a pharmaceutically acceptable salt thereof. In some embodiments, the concentration of quencher in the resulting mixture of step (b) is less than about 10 mM. In some embodiments, the quencher concentration is less than about 8 mM. In some embodiments, the quencher concentration is less than about 6mM, or less than about 2 mM. In some embodiments, the Red Blood Cells (RBCs) of the mixture obtained after step (b) have less than 1% hemolysis. In some embodiments, RBCs have less than 1% hemolysis when stored for 42 days (or 28 days) at 4 ℃ after step (b). In some embodiments, RBCs of the resulting mixture after step (b) have a Packed Cell Volume (PCV) of greater than 50%. In some embodiments, the RBCs of the resulting mixture have greater than 50% PCV when stored at 4 ℃ for 42 days (or 28 days) after step (b). In some embodiments, the RBCs of the resulting mixture have an average red blood cell friability value greater than 140 (or 150) after storage at 4 ℃ for 42 days (or 28 days) after step (b). In some embodiments, storage of the mixture is storage of the mixture at 4 ℃ for more than 10 days. In some embodiments, storage of the mixture is storage at 4 ℃ for more than 42 days (or 28 days). In some embodiments, the level of the pathogen inactivating compound is not reduced and/or the pathogen inactivating compound is not contacted with a compound uptake device (CAD).
Red Blood Cells (RBCs) produced by each of the above methods are provided. RBC compositions preparable by each of the above methods are also provided.
In a further aspect, the invention provides a composition comprising a) red blood cells, wherein the red blood cells have been covalently reacted with an electrophilic group of a pathogen-inactivating compound; and b) a quencher comprising a thiol group capable of reacting with the pathogen-inactivating compound; wherein the composition is suitable for infusion into a human after storage at 4 ℃ for up to 42 days (or 28 days). In some embodiments, at least 1log of pathogens, if present, are inactivated. In some embodiments, inactivation is at least 3 log. In some embodiments, the electrophilic group is a mustard, a mustard intermediate, or a mustard equivalent. In some embodiments, the electrophilic group is or is capable of forming an aziridinium ion. In some embodiments, the electrophilic group is capable of reacting with a nucleic acid. In some embodiments, the electrophilic group is covalently reactive with the cell surface of a red blood cell. In some embodiments, the pathogen inactivating compound further comprises a nucleic acid binding ligand. In some embodiments, the nucleic acid binding ligand is an intercalator. In some embodiments, the intercalator is acridine. In some embodiments, the pathogen inactivating compound comprises a cleavable linker connecting the electrophilic group and the nucleic acid binding ligand. In some embodiments, the pathogen-inactivating compound is β -alanine, N- (acridin-9-yl), 2- [ bis (2-chloroethyl) amino ] ethyl ester. In some embodiments, the quencher comprises cysteine or a cysteine derivative. In some embodiments, the quencher is glutathione or a pharmaceutically acceptable salt thereof. In some embodiments, the quencher concentration is sufficiently low to avoid or minimize dehydration of the red blood cells during storage. In some embodiments, the quencher concentration is less than about 10 mM. In some embodiments, the quencher concentration is less than about 8 mM. In some embodiments, the quencher concentration is less than about 6mM, or less than about 2 mM. In some embodiments, the Red Blood Cells (RBCs) have a Packed Cell Volume (PCV) of greater than 55%. In some embodiments, RBCs have greater than 60% PCV. In some embodiments, the RBCs have an average Antibody Binding Capacity (ABC) of less than about 50,000. In some embodiments, the RBCs have an average ABC of less than about 40,000. In some embodiments, the RBCs have an average ABC of about 25,000 to 60,000. In some embodiments, the RBCs have an average ABC of about 25,000 to 70,000. In some embodiments, the RBCs have an average ABC of about 35,000 to 45,000. In some embodiments, the composition further comprises an additive solution (e.g., any of the additive solutions described in table 2, and/or an additive solution comprising sodium chloride, adenine, glucose, phosphate, guanosine, citrate and/or mannitol). In some embodiments, the chloride concentration of the additive solution and/or composition is greater than about 100mM (or about 125 mM).
In other aspects, the invention provides methods of infusing red blood cells into an individual, comprising: a) providing any of the red blood cell compositions described above or a red blood cell composition made by any of the methods described herein, and b) infusing the red blood cell composition into a subject.
In one aspect, the present invention provides a method of treating a red blood cell composition comprising: (a) mixing the following: (i) a pathogen-inactivating compound comprising a functional group (e.g., an effective amount of a pathogen-inactivating compound to inactivate a pathogen, if present), the functional group being a reactive electrophilic group; (ii) a quencher (e.g., an effective amount of a quencher) comprising a thiol group, wherein the thiol group is capable of reacting with the reactive electrophilic group of the pathogen-inactivating compound; and (iii) a composition comprising red blood cells; and (b) reducing the concentration of the quencher in the mixture sufficiently to an amount that reduces the level of red blood cell dehydration resulting from storage of the mixture relative to the level of red blood cell dehydration resulting from storage of the mixture at the initial concentration of the quencher. In some of these embodiments, reducing the concentration of the quencher includes removing the solution used during inactivation and addition of the final additive solution (e.g., any solution described herein, such AS SAG-M, AS-5, or any solution of tables 2, 3, or 4). The method includes any one or more of the embodiments described above and/or herein.
In some embodiments, the method further comprises mixing a suitable base with the composition comprising red blood cells, and the base is in sufficient amount to reduce an adverse reaction of the pathogen-inactivating compound with the red blood cells in the mixture relative to a mixture without the base. In some embodiments, the adverse reaction of the pathogen-inactivating compound with red blood cells is a change in the surface of red blood cells caused by the pathogen-inactivating compound. In some embodiments, the method further comprises mixing a suitable base with the composition comprising red blood cells, and the base is sufficient to reduce the level of anti-pathogen inactivating compound antibody binding to the treated red blood cell composition in the resulting mixture by at least about 5% (or at least about 10%, at least about 25%, at least about 50%, at least about 75%, or at least about 90%) relative to the mixture without the base.
In one aspect, the invention provides a method of reducing dehydration of a red blood cell composition, wherein the composition is a mixture comprising a quencher capable of reacting with a pathogen-inactivating compound and red blood cells; the method includes reducing the concentration of the quencher in the mixture sufficiently to an amount that reduces the level of red blood cell dehydration resulting from storage of the mixture relative to the level of red blood cell dehydration resulting from storage of the mixture at the initial concentration of the quencher.
In another aspect, the invention provides a method of red blood cell infusion comprising infusing into an individual a red blood cell composition as described herein.
Brief Description of Drawings
FIG. 1 shows the red blood cell osmotic fragility at various base levels after initial quencher dosing as described in example 6.
FIG. 2 shows red blood cell densities at various alkali levels, at 20 hours of culture.
FIG. 3 shows red blood cell densities at various alkali levels after culturing and storage for 36 days.
FIG. 4 shows the osmotic fragility of red blood cells after culturing and storage for 36 days with and without a reduced quencher concentration (i.e., with/without an exchange step).
FIG. 5 shows the osmotic fragility of red blood cells after culture and storage for 42 days using varying initial quencher concentrations (using the exchange step) compared to moderate initial quencher concentrations (without the exchange step).
FIG. 6 shows the osmotic fragility of red blood cells after 36 days of culture and storage with and without pathogen inactivating compounds.
FIG. 7 shows Antibody Binding Capacity (ABC) values for several red blood cell preparations using the methods of the present invention.
Detailed Description
The present invention provides methods of treating red blood cell compositions to inactivate pathogens that may be present, while reducing or minimizing adverse side effects (such as red blood cell changes leading to an adverse immune response) and while reducing or minimizing adverse effects on cell viability (e.g., reduced osmotic fragility and/or increased dehydration) and/or longevity during or after treatment. We have found that proper control of pH in combination with proper amounts of quencher during the pathogen inactivation treatment can reduce the initial dehydration of red blood cells treated with the pathogen inactivation compound. The initial quencher concentration is then reduced after the treatment to provide healthy pathogen inactivated red blood cells that are capable of cell storage without significant changes in osmotic fragility. These methods are particularly useful for preparing red blood cell compositions in which pathogens have been inactivated for clinical use, particularly when the compositions are stored for a period of time prior to clinical use.
Thus, in one aspect, a pathogen-inactivating compound and a quencher are provided by (1) mixing the pathogen-inactivating compound and the quencher with a composition comprising red blood cells; and (2) reducing the concentration of the quencher sufficiently to reduce the level of red blood cell dehydration resulting from storage of the mixture relative to the level of red blood cell dehydration resulting from storage of the mixture at the initial concentration of the quencher, the present invention provides a method of treating a red blood cell composition comprising a pathogen-inactivating compound and the quencher.
In other aspects, the invention provides methods of reducing red blood cell dehydration and/or increasing osmotic fragility, and methods of infusing red blood cells into a human. Treated red blood cell compositions are also provided.
Red blood cell
The red blood cell compositions of the present invention include, but are not limited to, any blood product (e.g., human blood) that contains red blood cells, wherein the blood product is provided, or processed to provide, red blood cells suitable for use in a human, mammalian and/or vertebrate body, such as for transfusion. Red blood cell compositions include, for example, whole blood and red blood cell concentrates, such as packed red blood cells (pRBC; e.g., red blood cells with increased hematocrit and/or red blood cells without additive solutions). Red blood cell compositions can be described by their hematocrit or Packed Cell Volume (PCV), which is a measure of the concentration of red blood cells in the composition. The red blood cell composition may have a hematocrit in the range of about 1 to 100%, more preferably about 10 to 90%, and about 35 to 80%, or about 40 to 70%. Such red blood cell compositions may include chemicals, such as pathogen inactivating compounds and quenchers. They may also include buffers and other solutions, such AS red blood cell additive solutions (e.g., any of the solutions described herein, such AS SAG-M, AS-5 or any of the solutions of tables 2, 3 or 4), including salts or buffer solutions. In some embodiments, the red blood cell compositions described herein are packed red blood cells having a hematocrit in the range of about 70 to 90%, or about 75 to 85%, or about 80%, prior to use in the treatment methods described herein. In some embodiments, the red blood cell composition is non-packed red blood cells having a hematocrit in the range of about 50 to 70%, or about 55 to 65%, or about 60%, prior to and/or during use in the treatment methods described herein. In some embodiments, the red blood cell composition is diluted with a dilution solution and has a hematocrit in the range of about 30 to 50%, or about 35 to 45%, or about 40%, before and/or during use in the treatment methods described herein. In some embodiments, the red blood cell compositions described herein have been leukoreduced prior to use in the treatment methods described herein. In some embodiments, the white blood cells of the red blood cell composition are not reduced. Any red blood cell composition that will contact or be introduced into a living human, mammal, or vertebrate body, where such contact carries a risk of transmitting a disease due to a contaminating pathogen, can be treated as disclosed herein.
Blood pathogen
Pathogen contaminants inactivated in the methods of the invention include any nucleic acid-containing material capable of causing disease in humans, other mammals, or vertebrates, if present. The pathogen may be unicellular or multicellular. Examples of pathogens are bacteria, viruses, protozoa, fungi, yeasts, molds and mycoplasmas, which cause diseases in humans, other mammals or vertebrates. The genetic material of the pathogen may be DNA or RNA, and the genetic material may be present as single-stranded or double-stranded nucleic acid. Table 1 lists examples of viruses and is not intended to limit the invention in any way.
TABLE 1 non-limiting examples of viruses
| Family: | virus: |
| adenoviridae (Adeno) | Adenovirus type 2 |
| Canine hepatitis | |
| Mosaic virus family (Arena) | Pichinde |
| Lasha | |
| Bunyviridae (Bunya) | Turlock |
| California encephalitis | |
| Herpesviridae (Herpes) | Herpes simplex type 1 |
| Herpes simplex type 2 | |
| Cytomegalovirus | |
| Pseudorabies | |
| Orthomyxovirus (Orothomyxo) | Influenza virus |
| Papovaviridae (Papova) | SV-40 |
| Paramyxoviridae (Paramyxo) | Measles, measles and other diseases |
| Epidemic parotitis | |
| Parainfluenza types 2 and 3 | |
| Picorna virus family (Picorna) | Poliomyelitis types 1 and 2 |
| Family: | virus: |
| coxsackie A-9 | |
| Echo 11 | |
| Poxviridae (Pox) | Vaccinia virus |
| Fowl pox | |
| Reoviridae (Reo) | Blue tongue |
| Colorado tick fever | |
| Retroviridae (Retro) | HIV |
| Sarcoma of birds | |
| Mouse sarcoma | |
| Murine leukemia | |
| Rhabdoviridae (Rhabdo) | Herpetic stomatitis virus |
| Toga virus family | Western equine encephalitis |
| Dengue fever type 2 | |
| Dengue fever type 4 | |
| St Louis encephalitis | |
| Hepadnaviridae (Hepadna) | Hepatitis B |
| Bacteriophage | λ |
| R17 | |
| T2 | |
| (Rickettsia's body) | Akari (Rickett pox) |
In addition to inactivating potential pathogen contaminants, the methods of the invention can also inactivate leukocytes present in red blood cell compositions. Leukocyte reduction methods are used to preferentially remove a majority of leukocytes from red blood cell compositions determined for infusion, as they may result in an adverse immune response in the recipient. However, not all blood is leukoreduced, or the leukoreduction method does not remove all leukocytes. Thus, the risk of these immune responses may be further reduced by the inactivation of any residual leukocytes by the methods of the invention described herein.
Pathogen inactivating compounds
Inactivation of pathogens in the red blood cell composition may be accomplished by contacting the pathogens in the red blood cell composition with a pathogen-inactivating compound. In any of the embodiments described herein, the pathogen-inactivating compound (e.g., S-303 described herein) can be present in an effective amount (e.g., an effective amount to inactivate a pathogen, such as an effective amount sufficient to inactivate a pathogen, if present, in, for example, at least 1log, 2log, or 3log of a red blood cell composition). Pathogen inactivating compounds which may be used in the methods of the invention include compounds which comprise a functional group which is or is capable of forming and which has formed, for example, a reactive group (e.g., an electrophilic group) in situ. In some cases, the pathogen-inactivating compounds of the invention do not require photoactivation to be reactive. For example, the functional group can be a mustard group, a mustard group intermediate, a mustard group equivalent, an epoxide, formaldehyde, or formaldehyde synthetic fiber. Such functional groups are capable of forming reactive groups in situ, such as electrophilic aziridines, aziridinium, thiirane or thiiranium ions. The mustard group may be a mono-or bis- (haloethyl) amine group or a mono (haloethyl) sulfide group. Mustard equivalents are groups that react by a similar mechanism to mustard, for example, by forming reactive intermediates such as aziridinium and aziridinium groups, or thiirane and thiiranium groups. Examples include aziridine derivatives, mono-or bis- (methylsulfonylethyl) amine groups, mono- (methylsulfonylethyl) sulfide groups, mono-or bis- (toluenesulfonylethyl) amine groups and mono- (toluenesulfonylethyl) sulfide groups. Formaldehyde synthetic fibers are any compound that decomposes to formaldehyde, including hydroxylamines, such as hydroxymethylglycine. The reactive group of the pathogen inactivating compound is capable of reacting with a nucleic acid of the pathogen, e.g., reacting with a nucleophilic group on the nucleic acid. The reactive group may also include components that target the compound to the nucleic acid, such as an anchor moiety. An anchoring moiety includes a moiety capable of non-covalently binding a nucleic acid biopolymer (such as DNA or RNA) and is also referred to as a nucleic acid binding ligand, a nucleic acid binding group or a nucleic acid binding moiety. Examples of such compounds are described in U.S. Pat. Nos. 5,691,132, 6,410,219, 6,136,586, 6,617,157 and 6,709,810, which are incorporated herein by reference. Another class of pathogen-inactivating compounds that may be quenched by the methods of the present invention include the above-described reactive groups linked to the nucleic acid-binding group via a hydrolyzable linker, as described in U.S. Pat. No.6,514,987, which is incorporated herein by reference. The anchoring site of the pathogen inactivating compound has an affinity for the nucleic acid. This affinity may be due to any of several non-covalent binding nucleic acid means, including, but not limited to, intercalation, minor groove binding, major groove binding, and electrostatic binding (e.g., phosphate backbone binding). The affinity may also be due to a mixed binding pattern (e.g., intercalation and minor groove binding). Binding may be sequence specific (i.e., increased binding affinity for one or more particular nucleic acid sequences over other nucleic acid sequences) or non-sequence specific. Detailed examples of these nucleic acid binding moieties can be found in the above-mentioned patents.
In some embodiments of each of the methods, compositions, and kits described herein, the pathogen-inactivating compound may comprise a functional group that is or forms a reactive electrophilic group that reacts with a nucleophile of the selected quencher. In some embodiments, the pathogen inactivating group comprises a nucleic acid binding ligand and a functional group that is or forms an electrophilic group.
Specific examples of suitable pathogen-inactivating compounds for use in the present invention are beta-alanine, N- (acridin-9-yl), 2- [ bis (2-chloroethyl) amino ] ethyl ester (or also referred to herein as "S-303"), the structure of which is as follows, including salts thereof.
In some embodiments, the concentration of the pathogen-inactivating compound (e.g., S-303) in the mixture with the red blood cell composition is about 0.05mM to 4mM, about 0.05mM to 2mM, about 0.05mM to 0.5mM, about 0.1mM to 0.3mM, or about 0.2 mM. In some embodiments, once the two components are mixed with the red blood cell composition, the molar ratio of quencher to pathogen-inactivating compound is from about 10: 1 to about 400: 1, also from about 10: 1 to about 200: 1, also from about 20: 1 to about 200: 1, also from about 50: 1 to about 200: 1, and also about 100: 1.
Quenching agent
The quencher used in the method of the invention is intended to reduce adverse side reactions of the reactive electrophilic species used to inactivate pathogens (e.g., binding of a pathogen inactivating compound to the surface of RBCs, which may result in an undesirable immune response). In any of the embodiments described herein, a quencher (e.g., glutathione as described herein) can be present in an effective amount (e.g., an effective amount to reduce adverse side reactions, such as the amounts described herein). Suitable quenchers include nucleophilic groups capable of reacting with electrophilic groups of the pathogen inactivating compound. Non-limiting examples are described in detail in U.S. patent 6,709,810, which is incorporated herein by reference in its entirety. In some embodiments, the quencher is capable of substantially reducing an adverse side reaction in the red blood cell composition while allowing the pathogen-inactivating compound to substantially inactivate pathogens that may contaminate the red blood cell composition. In some embodiments, the improved methods of the invention provide an effective amount of a quencher in combination with an effective amount of a pathogen-inactivating compound under conditions that provide optimal reduction of adverse side reactions (e.g., binding of the pathogen-inactivating compound) and sufficient inactivation of the bound pathogen without significantly altering (e.g., not reducing) the osmotic fragility of the cells and without significantly altering (e.g., not increasing) dehydration. Various adverse side reactions, such as the reaction of pathogen inactivating compounds with proteins and/or red blood cell components, may be reduced. In some embodiments, the quencher provides an optimal reduction of red blood cell alterations, such as binding of IgG to red blood cells or binding of a pathogen-inactivating compound to red blood cells. Although the methods of the invention involve in vitro treatment of red blood cell compositions, some quencher remains in the composition when introduced into the body of an individual. Thus, in some embodiments, the quenchers of the invention are suitable for infusion. Suitable quenchers include, but are not limited to, compounds comprising a thiol group, such as quenchers comprising the amino acid cysteine or suitable cysteine derivatives (such as N-acetylcysteine). Examples of such quenchers include cysteine and peptides comprising at least one cysteine, such as glutathione. In some embodiments, suitable quenchers include cysteine derivatives that can form thiol groups in situ, with or without other chemicals or added enzymes, such as S-acetylcysteine or other suitable thiol-derivatized cysteine prodrugs, or peptides comprising S-acetylcysteine or other suitable thiol-derivatized cysteine prodrugs. Suitable cysteine derivatives are those which comprise or are capable of forming cysteinyl thiol in situ, which cysteinyl thiol is capable of reacting with an electrophilic group of a pathogen-inactivating compound.
In some embodiments, the quencher is a 2 to 10 amino acid peptide, wherein at least one amino acid is cysteine, N-acetylcysteine, S-acetylcysteine, or other suitable cysteine derivative. In some embodiments, the quenching agent is at least 3 amino acid peptide, such as about 3-10 amino acids, and also about 3-6 amino acids, at least one of which is cysteine, N-acetyl cysteine, S-acetyl cysteine or other suitable cysteine derivatives. In some embodiments, quenchers are peptides of at least 3 amino acids, such as about 3-10 amino acids, and also about 3-6 amino acids, wherein at least one amino acid is cysteine, N-acetylcysteine, S-acetylcysteine, or other suitable cysteine derivative, and also wherein at least 2 or at least 3 amino acids are cysteine, N-acetylcysteine, S-acetylcysteine, or other suitable cysteine derivative.
In a preferred embodiment, the quencher is neutral glutathione (also known as L-glutathione and γ -L-glutamyl-L-cysteinyl-glycine). Glutathione has a number of properties that make it particularly useful as a quencher. It is usually present in all cell types. Passive penetration into pathogens is not thought to be possible, such as through the cell membrane or lipid layer of bacteria and lipid enveloped viruses, or through the viral capsid of non-enveloped viruses. At about neutral pH, glutathione is charged and does not penetrate the lipid bilayer to any extent in the absence of active transport. This is consistent with the inactivation of lipid enveloped viruses that are substantially unaffected by glutathione (e.g., HIV and VSV), including the use of neutral glutathione concentrations above 2 mM. The use of glutathione has certain effects on the inactivation of, for example, Yersinia enterocolitica (Yersinia enterocolitica), Staphylococcus epidermidis (Staphylococcus epidermidis) and Serratia marcescens (Serratia marcescens). However, this can be controlled by using effective amounts of neutral glutathione and a pathogen inactivating compound. Thus, a preferred quenching method is provided wherein a red blood cell composition is inactivated by at least 2 logs, preferably at least 3 logs, of contamination with a viral or bacterial pathogen. In some embodiments, staphylococcus epidermidis can be inactivated up to at least 3 logs, also about 4logs, or about 5 logs, and VSV can be inactivated up to at least 4logs, also about 5 logs, or about 6 logs. In some embodiments, inactivation of Staphylococcus epidermidis with S-303 is within about 3 logs, and also about 2 logs, or about 1log of a similar composition inactivated with 2mM acidic glutathione and 0.2mM S-303. In some embodiments, inactivation of VSV with S-303 is within about 2 logs, and also about 1log, or substantially equivalent, of a similar composition inactivated with 2mM acidic glutathione and 0.2mM S-303. Under appropriate conditions, glutathione is also compatible with in vitro storage of red blood cells, as described herein, and the resulting red blood cell composition is suitable for in vivo introduction.
In some embodiments, the quencher is a reduced form of glutathione. Glutathione disulfide, which is an oxidized form of glutathione, may also be used, provided that glutathione disulfide is substantially reduced in solution prior to addition of the solution to the mixture comprising the red blood cell composition, or substantially reduced after addition of the mixture comprising the red blood cell composition.
In some embodiments, the quencher is a glutathione derivative, such as a mono-or diacyl glutathione ester, wherein the alkyl group is a straight or branched chain group having 1 to 10 carbon atoms. Specific examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, isopentyl, neopentyl, tert-pentyl, 1-methylbutyl, hexyl, isohexyl, 2-methylpentyl, 1-ethylbutyl, heptyl, octyl, nonyl, and decyl. For example, non-limiting examples of glutathione derivatives include glutathione methyl ester, glutathione monoethyl ester, and glutathione monoisopropyl ester. In some embodiments, glutathione oxidized diethyl ester (GSSG- (glycyl) -diethyl-ester) is used. In some embodiments, the thioester of the glutathione is hydrolyzed to form a thiol upon addition of the red blood cell composition.
It is understood that in some embodiments, the quencher will be provided in the free acid or base form, while in other embodiments, the quencher will be provided in the salt form. If the quencher is in the form of a salt, the salt is preferably a pharmaceutically acceptable salt. Pharmaceutically acceptable salts of the compounds (in the form of water-or oil-soluble or dispersible products) include the conventional non-toxic salts or quaternary ammonium salts, formed, for example, from inorganic or organic acids or bases. Examples of such acid addition salts include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, and undecanoate. The basic salt comprises: an ammonium salt; alkali metal salts such as sodium and potassium salts; alkaline earth metal salts such as calcium and magnesium salts; salts with organic bases, such as dicyclohexylamine salts, N-methyl-D-glucamine, and salts with amino acids, such as arginine, lysine, and the like. In addition, the basic nitrogen-containing groups may be quaternized with such agents as: such as alkyl halides, such as methyl, ethyl, propyl and butyl chlorides, bromides and iodides; dialkyl sulfates such as dimethyl, diethyl, dibutyl and diamyl sulfates; long chain halides, such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides; aralkyl halides such as benzyl bromide and phenethyl bromide and the like. Other pharmaceutically acceptable salts include ethanolate and sulfate salts.
For example, in some embodiments, the quencher is in the form of a pharmaceutically acceptable salt formed from glutathione. In some embodiments, the quencher is in the form of a pharmaceutically acceptable salt formed from glutathione and one or more cations, such as sodium, aluminum, calcium, lithium, magnesium, zinc, or tetramethylammonium. In some embodiments, the quencher is glutathione (reduced) and is provided in the form of glutathione monosodium salt (e.g., available from BiomedicaFoscama, italy). In some other embodiments, the glutathione (reduced) is provided as glutathione hydrochloride. In some other embodiments, the glutathione is provided in the form of glutathione (reduced) disodium salt. In a further embodiment, glutathione monoalkyl ester sulfate is used. In some embodiments, the glutathione is provided in the form of glutathione disodium oxide.
Methods of inactivation and quenching
The methods of the present invention involve mixing the red blood cell composition with the pathogen-inactivating compound and the quencher under conditions in which, when the composition is mixed with the pathogen-inactivating compound and the quencher, the pH of the resulting composition is within a suitable range to provide adequate pathogen inactivation and reduction of adverse side reactions (e.g., alteration of red blood cells) with limited or no effect on the viability (e.g., osmotic fragility and dehydration) and/or longevity of the treated blood product. In addition, the present invention describes reducing the concentration of quencher in the red blood cell composition after a period of pathogen inactivation to help maintain the viability and longevity of the red blood cells during storage. As described herein, the additive solution may also be used for red blood cells during storage and may be used to replace treatment and/or dilution solutions used during pathogen inactivation.
The improved process includes several features that are important for quenching. The first feature is a thiol group, or other suitable nucleophilic group. The second is the adjustment of the pH of the solution. A certain level of quenching can be provided by suitably adjusting the pH of the solution. Thus, the quenchers of the invention provide some buffering capacity to the composition comprising red blood cells, wherein the buffering capacity itself provides improved quenching. For example, using a cysteine analog (e.g., methionine) as a quencher, appropriately adjusted to provide a suitable pH change in the red blood cell composition, will result in a level of quenching of the binding of the pathogen-inactivating compound to the red blood cells. Since the sulfur source in methionine is not nucleophilic in nature, methionine does not provide any quenching, but rather provides the desired pH of the solution. Thus, the combination of pH adjustment and thiol groups provides improved quenching. Appropriate adjustment of pH and alkali equivalents may also reduce the level of dehydration of red blood cells during the inactivation phase. In some embodiments, to provide improved quenching may be important third feature is basically not infiltration of pathogens (virus and bacteria) inside the preferred quenching agent selection. These quenchers provide suitable quenching in the extracellular environment, where detrimental reactions occur, such as binding to the surface of red blood cells, without additional quenching of pathogen inactivating compounds once inside the pathogen is permeated. Finally, the improved quenching method of the invention involves reducing the quencher concentration after inactivation and, in some cases, adding an additive solution for storage. It has been shown that red blood cells have an increased lifespan and reduced dehydration levels during storage when the overall concentration of quencher is reduced to a suitable level.
In one aspect, the present invention provides a method of treating a red blood cell composition comprising: a) providing i) a pathogen-inactivating compound comprising a functional group that is or forms a reactive electrophilic group (e.g., an effective amount of a pathogen-inactivating compound to inactivate a pathogen, if present), ii) a quencher comprising a thiol group (e.g., an effective amount of a quencher as described herein), wherein the thiol group is capable of reacting with the reactive electrophilic group of the pathogen-inactivating compound; and iii) a composition comprising red blood cells; and b) mixing the pathogen-inactivating compound and the quencher with the composition comprising red blood cells; and c) reducing the concentration of the quencher in the mixture sufficiently to an amount that reduces the level of red blood cell dehydration resulting from storage of the mixture relative to the level of red blood cell dehydration resulting from storage of the mixture at the initial concentration of the quencher. In some embodiments, step (a) further comprises providing a suitable base, step (b) further comprises mixing the base with the composition comprising red blood cells, and the base is sufficient to reduce the level of adverse reaction of the pathogen-inactivating compound with the red blood cells in the mixture relative to a mixture without the base. In some embodiments, the adverse reaction of the pathogen-inactivating compound with the red blood cells is a modification of the surface of the red blood cells by the pathogen-inactivating compound. In some embodiments, step (a) further comprises providing a suitable base, step (b) further comprises mixing the base with the composition comprising red blood cells, and the base is sufficient to reduce the level of anti-pathogen inactivating compound antibody binding to the treated red blood cell composition in the resulting mixture by at least about 5% (or at least about 10%, at least about 25%, at least about 50%, at least about 75%, or at least about 90%) relative to the mixture without the base. In some embodiments, storage of the mixture is more than, equal to, or less than 7, 10, 14, 21, 28, 35, or 42 days at 4 ℃ or room temperature. In some embodiments, the mixture is stored in an additive solution (e.g., any of the additive solutions described in table 2, and/or an additive solution comprising sodium chloride, adenine, glucose, phosphate, guanosine, citrate and/or mannitol). In some embodiments, the method further comprises replacing the solution used during the treatment with an additive solution (e.g., any of the additive solutions described in table 2, and/or an additive solution comprising sodium chloride, adenine, glucose, phosphate, guanosine, citrate and/or mannitol).
In other aspects, the invention provides methods of reducing red blood cell dehydration comprising: a) providing a red blood cell composition comprising i) a quencher, wherein the quencher is capable of reacting with the pathogen-inactivating compound, and ii) red blood cells; and b) reducing the concentration of the quencher in the mixture sufficiently to an amount that reduces the level of red blood cell dehydration resulting from storage of the mixture relative to the level of red blood cell dehydration resulting from storage of the mixture at the initial concentration of the quencher. In some embodiments, storage of the mixture is more than, equal to, or less than 7, 10, 14, 21, 28, 35, or 42 days at 4 ℃ or room temperature. In some embodiments, the method further comprises adding an additive solution (e.g., any of the additive solutions described in table 2, and/or an additive solution comprising sodium chloride, adenine, glucose, phosphate, guanosine, citrate and/or mannitol) prior to storage.
The quencher and/or added base used in the methods described herein (or neutralizing the quencher) can be mixed with the red blood cell composition prior to, simultaneously with, or after the pathogen-inactivating compound is added to the red blood cell composition. If the quencher and base (or neutralization quencher) are mixed with the red blood cell composition after the pathogen-inactivating solution is mixed with the red blood cell composition, the quencher and/or base (or neutralization quencher) is preferably added to the red blood cell composition before a significant amount of the side reaction of the pathogen-inactivating compound with the red blood cells occurs so that proper quenching of the adverse side reaction can be achieved. In some embodiments, the quencher and/or base (or neutralization quencher) is mixed with the red blood cell composition within about one hour, within about 30 minutes, within about 20 minutes, within about 10 minutes, within about 5 minutes, within about 2 minutes, or within about 1 minute after the pathogen-inactivating compound is mixed with the red blood cell composition. In some embodiments, the quencher and the base are mixed with the red blood cell composition at the same time as the pathogen inactivating compound.
In some embodiments of any of the methods described herein, the quencher and added base (or neutralizing quencher) are pretreated with the red blood cell composition for a suitable time interval prior to addition of the pathogen-inactivating compound (e.g., S-303), such as less than about one hour, less than about 30 minutes, less than about 20 minutes, less than about 10 minutes, less than about 5 minutes, less than about 2 minutes, or less than about 1 minute prior to mixing the pathogen-inactivating compound with the red blood cell composition. In some embodiments, the pretreatment is at a temperature of about 1 ℃ to 30 ℃, also about 18 ℃ to 25 ℃, or about 37 ℃, or about room temperature.
In some embodiments of each of the methods described herein, the pathogen-inactivating compound (e.g., S-303) is incubated with the red blood cell composition in the presence of the quencher and added base (or neutralization quencher) for a suitable interval of time, such as from about 30 minutes to 48 hours, and also from about 2 to 36 hours, and also from about 8 to 24 hours, and also about 20 hours. In some further embodiments, the incubation is at a temperature in the range of about 1 ℃ to 30 ℃, also about 18 ℃ to 25 ℃, or about 37 ℃, or about room temperature.
With respect to the characteristics of adjusting the pH of red blood cell compositions, previous quenching methods (e.g., pathogen inactivating compounds) have failed to recognize the importance of the pH of the resulting mixture for quenching effectiveness and cell viability in the inactivation process. While previous methods demonstrated the need for sufficient base and appropriate pH levels to properly quench the adverse side reactions of pathogen-inactivating compounds (e.g., by increasing the level of non-protonated glutathione to reduce binding of pathogen-inactivating compounds to RBC surfaces), these methods did not achieve and describe the effect of increased base on cell dehydration during the inactivation process. Accordingly, one aspect of the invention relates to adjusting the pH of the red blood cell composition to a suitable level for incubation with the pathogen inactivating compound and the quencher (e.g., to avoid adversely affecting dehydration).
In some embodiments, when the pathogen-inactivating compound and the quencher are mixed with the red blood cell composition, the pH of the mixture is at a suitable level to reduce adverse side reactions of the pathogen-inactivating compound during inactivation (e.g., binding of the pathogen-inactivating compound to the surface of RBCs, which can lead to an unwanted immune response) and to substantially reduce cell dehydration. In some embodiments, the adverse side effect is a modification of the surface of the red blood cells by the pathogen inactivating compound. In some embodiments, the alteration is covalent binding of the pathogen-inactivating compound to the surface of the red blood cells. In other embodiments, the alteration is non-covalent binding of the pathogen-inactivating compound to the surface of the red blood cells.
As described herein, in some embodiments of each method, adverse (also referred to herein as "unwanted") side reactions of the pathogen-inactivating compound with red blood cells are reduced. In some embodiments, the reduced adverse side effect is alteration of the surface of the red blood cells by the pathogen inactivating compound. In some embodiments, the level of side reactions is reduced by at least about 5%, at least about 10%, at least about 25%, at least about 50%, at least about 75%, or at least about 90%. For example, a reduction in side reactions can be demonstrated by measuring the amount of antibody specific for the pathogen inactivating compound that binds to the treated red blood cells and/or measuring the lifetime of the treated red blood cells in vivo, and comparing these measurements to red blood cells treated by a second, different method (e.g., a method in which no sufficient amount of quencher and/or base is added to the reaction mixture, a method in which no quencher and/or base is added to the reaction mixture, and/or treatment at a lower pH). For example, in some embodiments of the methods described herein, the level of antibody binding of the anti-pathogen inactivating compound to the treated red blood cells is reduced by at least about 10%, at least about 25%, at least about 50%, at least about 75%, or at least about 90% relative to the second method (e.g., a method in which no sufficient amount of quencher and/or base is added to the reaction mixture, a method in which no quencher and/or base is added to the reaction mixture, and/or treatment at a lower pH).
In some embodiments, when the pathogen-inactivating compound and the quencher are mixed with the red blood cell composition, the pH of the mixture is in the range of about 6.0 to 8.5, about 6.0 to 7.5, about 6.5 to 7.1, about 6.5 to 7.0, about 6.6 to 6.8, or about 6.6, 6.7, 6.8, or 6.9. Although the pH of the red blood cell composition may change over time, it is desirable that the pH when the quencher is added to the red blood cell composition is in the desired range, regardless of whether the composition already contains a pathogen-inactivating compound. The methods of the invention involve adding a pathogen-inactivating compound and a quencher to the red blood cell composition. When the pathogen-inactivating compound and/or quencher is added to the red blood cell composition, a desired pH range is required, regardless of the order in which the pathogen-inactivating compound and/or quencher is added to the red blood cell composition. In other words, once all three ingredients are mixed, the pH is within the desired range. In some embodiments, the quencher is added prior to the pathogen-inactivating compound. In some embodiments, the pathogen-inactivating compound is added prior to the quencher. In some embodiments, the quencher and the pathogen-inactivating compound are added substantially simultaneously. Thus, the addition of the pathogen-inactivating compound and the quencher represents the point at which the quencher and the pathogen-inactivating compound are mixed with the red blood cell composition. The desired pH can be achieved in several ways and is not a limitation in adjusting the pH of the red blood cell composition or, in some embodiments, is not adjusted to a greater extent from the natural pH of the blood product. For example, the desired pH of the red blood cell composition can be achieved by adjusting the pH. For example, the pH adjustment can be performed by adding a suitable additive solution, such as a buffer solution, prior to adding the pathogen-inactivating compound and the quencher. In some embodiments, the red blood cell composition is washed one or more times with a suitable buffer before being suspended in the same or other suitable buffer. Alternatively, the pH of the red blood cell composition can be adjusted while adding the pathogen-inactivating compound, the quencher, or both. In some embodiments, the pH is adjusted while adding the quencher. In some embodiments, the quencher is neutral such that the addition of the neutral additive provides the desired pH range to the red blood cell composition. As an example, neutralization of glutathione may be used to achieve the desired pH adjustment. In some embodiments, neutralization of glutathione can be used at a suitable level, for example, by adding 1 equivalent of base, to provide a quencher that will provide the desired pH adjustment of the composition when added to the red blood cell composition. Suitable neutralization will depend on the quencher used. For example, when a peptide is used, this may depend on the amino acid composition of the peptide. In some embodiments, quenchers that do not significantly affect the pH of the red blood cell composition may be used. For example, the use of a peptide comprising cysteine that may further comprise one or more amino acids results in a more neutral pH of the solution of the naturally isolated peptide. In some embodiments, the peptide further comprises at least one basic amino acid, such as arginine or lysine.
In some embodiments of the methods described herein, the base is a basic salt in which case the base is mixed with the red blood cell composition and the pathogen inactivation compound and quencher to increase the pH of the mixture to a desired level and/or improve the quenching of adverse side reactions. The alkaline salt is first dissolved in an aqueous solution prior to mixing with the red blood cell composition. In other embodiments, the salt in solid form is added directly to the red blood cell composition. In some embodiments, the basic salt includes a quencher and provides the mixture with the quencher and the base. In some embodiments, the base used in the method is a strong base, such as NaOH. Typically, a strong base (e.g., NaOH) is first dissolved in the aqueous solution prior to mixing with the red blood cell composition. In some embodiments, a strong base (e.g., in solution or solid form) is mixed with the quencher prior to mixing the quencher with the red blood cell composition. In some embodiments, the base is an alkaline buffer (added in sufficient quantity and having a suitable pKa to give the mixture a desired pH range). If an alkaline buffer is used, in some embodiments, the buffer is a pharmaceutically acceptable buffer. In some embodiments, the buffer will have titratable protons with pKa in the range of about 7 to 8. Examples of buffers that can be used as alkaline buffers include, but are not limited to, N- (2-hydroxyethyl) -piperazine-N' -2-ethanesulfonic acid (HEPES), Phosphate Buffered Saline (PBS), and sodium phosphate buffer. Other suitable alkaline buffers are readily identified by those skilled in the art.
In some embodiments of each of the methods and compositions described herein, the pH of the mixture of red blood cells, quencher, pathogen-inactivating compound, and any added base is above about 5.5, above about 5.7, above about 6.0, above about 6.3, above about 6.5, above about 6.7, above about 7.0, or above about 7.2. In some embodiments of each of the methods and compositions described herein, the pH of the mixture of red blood cells, quencher, pathogen-inactivating compound, and base (if added) is in the range of about 6.0 to 8.5, about 6.0 to 7.5, about 6.5 to 7.1, about 6.5 to 7.0, or about 6.6 to 6.8, or about 6.6, 6.7, 6.8, or 6.9. In some embodiments, the pH indicated is a pH at room temperature. In some embodiments, the pH is a pH at 37 ℃. For example, in some embodiments, a composition comprising red blood cells is treated with a pathogen-inactivating compound in the presence of a quencher and any added base, wherein the pH of the mixture is in the range of about 6.5 to about 7.0 (or 7.1) at 37 ℃.
In some embodiments, the pH of the mixture of red blood cells, quencher, and base (if base is added as part of the process) prior to mixing the pathogen-inactivating compound with the red blood cell composition is in the range of about 6.0 to 8.5, about 6.0 to 7.5, about 6.5 to 7.1, about 6.5 to 7.0, or about 6.6 to 6.8, or about 6.5, 6.7, 6.8, or 6.9. In some other embodiments, the pH is obtained at the same time or within about 1 hour, within about 30 minutes, within about 20 minutes, within about 10 minutes, within about 5 minutes, or within about 2 minutes of mixing the pathogen-inactivating compound with the composition comprising red blood cells. In some embodiments of those methods in which pH is adjusted, the pH is adjusted to the desired pH range prior to, simultaneously with, or within about 1 hour, within about 30 minutes, within about 20 minutes, within about 10 minutes, within about 5 minutes, or within about 2 minutes of mixing the pathogen-inactivating compound with the composition comprising red blood cells. In those embodiments in which the quencher is glutathione and the pathogen-inactivating compound is S-303, the pH of the mixture comprising the red blood cell composition and the quencher is preferably adjusted to a desired pH range (e.g., pH6.5 to 7.0) prior to mixing S-303 with the red blood cell composition.
In some embodiments, the pH of the resulting composition after mixing the red blood cells, quencher, and base is not necessarily an adjustment of the pH of the starting red blood cell composition. For example, the red blood cell composition can have a pH in the desired range of 6.0-7.5, and the pH of the composition does not change significantly upon addition of the quencher and subsequent addition of the pathogen-inactivating compound. In such embodiments, the quencher naturally provides the desired pH, or neutralizes it, thus providing the desired pH. In combination with the addition of a high initial level of quencher, e.g., about 5mM to about 40mM, and importantly a desired range of the resulting pH. For example, known methods of using these glutathione concentrations have not been used in conjunction with other methods of the present invention in conjunction with the desired pH range. Thus, for peptides, regardless of the peptide quencher, neutralization can be effected as needed to provide a suitable amount of buffering in the desired pH range. Thus, by neutralizing quencher is meant that the quencher is appropriately titrated with an acid or base as needed such that when added to the red blood cell composition, the resulting mixture has a pH that provides better quenching of adverse side reactions (e.g., binding of pathogen inactivating compounds to the RBC surface, which may lead to an unwanted immune response), while avoiding cell dehydration during inactivation, such as a pH in the range of about 6.0 to 8.5, about 6.0 to 7.5, about 6.5 to 7.0, about 6.5 to 7.1, or about 6.6 to 6.8, or about 6.6, 6.7, 6.8, or 6.9. In some embodiments, the naturally isolated peptide is suitably neutralized, i.e., no acid or base is required to be added, to provide the desired pH in the final mixture. In addition, preferred quenchers will provide buffering capacity to maintain the pH in the desired range and for the time required to quench the adverse side reactions.
In some embodiments of each of the methods and compositions described herein, the quencher is neutralized. The quencher is "neutralized" by the base, such that quenching of adverse side reactions between the pathogen-inactivating compound and red blood cells in a mixture of a composition comprising red blood cells, the pathogen-inactivating compound, and the quencher is enhanced if a sufficient amount of base is mixed with the quencher. The "neutralization quencher" need not have a neutral pH, nor need it be uncharged. In some embodiments, the neutralization quencher is neither its most protonated form nor its most deprotonated form. In some embodiments, where the quencher is very acidic, the pH of the neutralization quencher is still below 7.0 (e.g., about 6.6, 6.7, 6.8, or 6.9). In some embodiments, the pH of the neutralization quencher solution can be greater than 7.0. In some embodiments, the pH of the neutralization quencher solution will be detectably higher than the pH of the quencher prior to addition of the base. In some embodiments, the quencher is neutralized with at least about 0.25 equivalents, at least about 0.5 equivalents, at least about 0.75 equivalents, at least about 1 equivalent, at least about 1.25 equivalents, at least about 1.5 equivalents, or at least about 2 equivalents of base. In some embodiments, the quencher is neutralized with less than about 2 equivalents, less than about 1.5 equivalents, less than about 1.25 equivalents, less than about 1 equivalent, or less than about 0.75 equivalents of base. In some embodiments, the quencher is neutralized with about 0.25 to about 2 equivalents, about 0.5 to about 1.5 equivalents, or about 0.75 to about 1.25 equivalents of base. In some embodiments, about 0.75 equivalents of base is used to neutralize the quencher. In other embodiments, the quencher is neutralized with about 1 equivalent of base. In other embodiments, the quencher is neutralized with about 1.25 equivalents of base. For example, in some embodiments of the invention, 1 equivalent of a suitable base (e.g., sodium hydroxide) is used to neutralize glutathione. In this case, the solution of protonated glutathione has a pH of about 3, the solution neutralized with 1 equivalent of sodium hydroxide has a pH of about 4.5, and the solution neutralized with 2 equivalents of sodium hydroxide has a pH of about 9.5. Suitable peptide quenchers comprising at least one cysteine may be suitably adjusted to provide the desired pH when added to the red blood cell composition.
Suitable methods for neutralizing glutathione and other quenchers will be apparent to those skilled in the art. In some embodiments, sodium hydroxide is used to neutralize or partially neutralize the quencher. In some embodiments, solid granular NaOH is first dissolved in water to produce a concentrated base solution, such as a 1N, 5N, 10N, or 20N NaOH solution. In some embodiments, an appropriate amount of NaOH solution is then added to the quencher before, simultaneously with, or after the quencher is added to the mixture. Alternatively, NaOH is added to the red blood cell composition or the pathogen inactivating compound, or a mixture of both, prior to adding the quencher to the mixture.
In addition to providing a suitably pH-adjusted or neutralized quencher, in some embodiments, preferred quenchers are unable to significantly enter the pathogen such that they optimally quench adverse reactions in the extracellular environment without the pathogen-inactivating compound interfering with pathogen inactivation once permeated into the interior of the pathogen.
In some embodiments of each of the methods described herein, the quencher is an acidic compound. In some embodiments, the quencher is provided in the free acid form. In some embodiments, the quencher is acidic and at least about 1 equivalent of base is added to neutralize the quencher. In some implementations, solutions containing such neutralization quenchers can be basic, neutral, or even acidic. In some embodiments, about 1 equivalent of base is added to neutralize or partially neutralize the quencher. In some embodiments, about 2 equivalents of base are added. In some embodiments, the quencher is acidic, and about 0.5 to about 1.5 equivalents of base are used to neutralize the quencher. In some embodiments, about 0.75 to about 1.25 equivalents of base are used. In some embodiments, about 1 equivalent of base is used.
In some embodiments, the quencher is neutralized prior to addition of the red blood cell composition and/or the pathogen-inactivating compound. In other embodiments, the quencher is neutralized after mixing the quencher with the red blood cell composition and/or the pathogen-inactivating compound. In some embodiments, the pH of the neutralization quencher is in the range of about 2.5 to 7.5, about 3.0 to 6.5, about 3.5 to 5.5, about 4.0 to 5.0, or about 4.3 to 4.5, or about 4.4 prior to addition of the red blood cell composition and/or the pathogen-inactivating compound.
In some embodiments, the quencher is glutathione and is provided in the form of glutathione monosodium salt and is neutralized with about 1 equivalent of a base, or is not neutralized with a base. In some other embodiments, the quencher is glutathione and is provided in the form of glutathione hydrochloride and neutralized with about 1 equivalent of base.
In some embodiments of each of the methods described herein, the initial concentration of the quencher in the mixture comprising the red blood cell composition, the quencher, the pathogen-inactivating compound, and any added base is increased during the inactivation stage and then decreased to a lower concentration after the inactivation stage. In some embodiments, the initial concentration of the quencher is adapted to substantially reduce adverse side reactions of the pathogen-inactivating compound (e.g., binding of the pathogen-inactivating compound to the surface of RBCs) and then to a lower concentration sufficient to reduce adverse effects of viability (e.g., osmotic fragility and dehydration) and/or longevity during storage of the cells.
The present invention includes a number of methods for reducing the concentration of the quencher following the pathogen inactivation stage. In some embodiments, the concentration of the quencher (e.g., glutathione) is reduced by centrifuging the mixture comprising the red blood cell composition, the quencher, and the pathogen-inactivating compound, followed by removing the mixed supernatant, and then adding a fresh solution, such as an additive solution (e.g., any of the additive solutions described in table 2, and/or an additive solution comprising sodium chloride, adenine, glucose, phosphate, guanosine, citrate, and/or mannitol) for cell resuspension (e.g., by washing the cells). In some embodiments, the process of centrifugation, supernatant removal, and addition of fresh solution (e.g., any of the additive solutions described in table 2, and/or additive solutions including sodium chloride, adenine, glucose, phosphate, guanosine, citrate, and/or mannitol) may be repeated an additional 1, 2, 3,4, or 5 or more times. In some embodiments, the method for reducing the concentration of the quencher is automated. In some embodiments, the fresh solution contains no quencher or a lower concentration of quencher. In some embodiments, the concentration of the quencher (e.g., glutathione) is reduced by chemically inactivating the quencher. In some embodiments, the concentration of the quencher (e.g., glutathione) is reduced by size exclusion using a membrane (e.g., a hollow fiber membrane or dialysis membrane) or size exclusion beads in a batch or flow removal method or flow process. In some embodiments, the quencher is not reduced and/or does not contact a Compound Adsorption Device (CAD).
In some embodiments of each of the methods and compositions described herein, the initial concentration of the quencher (e.g., glutathione) in the mixture comprising the red blood cell composition, the quencher, the pathogen-inactivating compound, and any added base is greater than about 2mM, greater than about 4mM, greater than about 6mM, greater than about 8mM, greater than about 10mM, greater than about 15mM, or greater than about 20 mM. In some embodiments, the initial concentration of quencher in the mixture is about 2mM to 100mM, about 2mM to 40mM, about 4mM to 40mM, about 5mM to 30mM, or about 10mM to 30mM, or at most 2mM, 5mM, 10mM, 15mM, 20mM, 25mM, 30mM, 35mM, 40mM, 45mM, 50mM, or 100 mM. In some embodiments, the initial concentration of quencher in the mixture is about 20 mM.
In some embodiments of each of the methods and compositions described herein, the initial concentration of the quencher (e.g., glutathione) in the mixture of red blood cells, quencher and pathogen-inactivating compound is greater than about 2mM, greater than about 4mM, greater than about 6mM, greater than about 8mM or greater than about 10mM, and the pH of the mixture is greater than about 5.5, greater than about 5.7, greater than about 6.0, greater than about 6.3, greater than about 6.5, greater than about 6.7, greater than about 7.0 or greater than about 7.2. In some embodiments of each of the methods and compositions described herein, the initial concentration of the quencher in the mixture is in the range of about 2mM to 40mM, about 4mM to 40mM, about 5mM to 30mM, or about 10mM to 30mM, or about 20mM, and the pH of the mixture is in the range of about 6.0 to 8.5, about 6.0 to 7.5, about 6.5 to 7.1, about 6.5 to 7.0, or about 6.6 to 6.8, or about 6.6, 6.7, 6.8, or 6.9. In some embodiments, the initial concentration of quencher in the mixture is above about 2mM, above about 4mM, above about 6mM, above about 8mM or above about 10mM, and the pH of the mixture is in the range of about 6.0 to 8.5, about 6.0 to 7.5, about 6.5 to 7.1, about 6.5 to 7.0, or about 6.6 to 6.8, or about 6.6, 6.7, 6.8 or 6.9. In some embodiments, the quencher (e.g., glutathione) concentration of the mixture is in the range of about 10mM to about 30mM, and the pH of the mixture is in the range of about 6.0 to 7.5. In some embodiments, the concentration of the quencher (e.g., glutathione) in the mixture is about 20mM, and the pH of the mixture is in the range of about 6.5 to 7.0 (or 7.1).
In some embodiments of each of the methods and compositions described herein, the initial concentration of the quencher (e.g., glutathione) in the mixture comprising the red blood cell composition, the quencher, the pathogen-inactivating compound, and any added base is reduced by greater than 2-fold, or 3-fold, or 4-fold, or 5-fold, or 6-fold, or 7-fold, or 8-fold, or 9-fold, or 10-fold, or 15-fold, or 20-fold, or 25-fold, or 30-fold, or 35-fold, or 40-fold, or 50-fold, or 100-fold, or 500-fold, or 1000-fold relative to the initial concentration of the quencher (e.g., glutathione) in the mixture after the inactivation stage.
In some embodiments of each of the methods and compositions described herein, the concentration of the quencher (e.g., glutathione) in the mixture comprising the red blood cell composition, the quencher, the pathogen-inactivating compound, and any added base decreases after the inactivation stage by less than about 15mM, less than about 10mM, less than about 8mM, less than about 6mM, less than about 5mM, less than about 4mM, less than about 3mM, less than about 2mM, less than about 1mM, less than about 0.75mM, less than about 0.5mM, or less than about 0.25 mM. In some embodiments, the concentration of quencher in the mixture that decreases after the inactivation stage is in the range of about 1mM to 20mM, about 2mM to 15mM, about 3mM to 10mM, about 4mM to 8mM, or about 5mM to 6 mM. In some embodiments, the concentration at which the quencher in the mixture is reduced after the inactivation stage is at most about a concentration of 0.25mM, or 0.5mM, or 0.75mM, or 1mM, or 1.5mM, or 2mM, or 3mM, or 4mM, or 5mM, or 6mM, or 7mM, or 8mM, or 9mM, or 10mM, or 12.5mM, or 15mM, or 20 mM.
In some embodiments of each of the methods and compositions described herein, the initial concentration of the quencher (e.g., glutathione) in the mixture comprising the red blood cell composition, the quencher, the pathogen-inactivating compound, and any added base is greater than 2mM, greater than about 4mM, greater than about 6mM, greater than about 8mM, or greater than about 10mM, and the concentration of the quencher in the mixture decreases after the inactivation stage to less than about 15mM, less than about 10mM, less than about 8mM, less than about 6mM, less than about 5mM, less than about 4mM, less than about 3mM, less than about 2mM, less than about 1.5mM, less than about 1mM, less than about 0.75mM, less than about 0.5mM, or less than about 0.25 mM. In some embodiments, the initial concentration of the quencher is in the range of about 2mM to 100mM, about 2mM to 40mM, about 4mM to 40mM, about 5mM to 30mM, or about 10mM to 30mM, or about 20mM, and the concentration of the quencher in the mixture decreases after the inactivation stage is in the range of about 1mM to 20mM, about 2mM to 15mM, about 3mM to 10mM, about 4mM to 8mM, or about 5mM to 6 mM. In some embodiments, the initial concentration of the quencher (e.g., glutathione) is in the range of about 10mM to 30mM, and the concentration of the quencher in the mixture that decreases after the inactivation stage is in the range of about 2mM to 15 mM. In some embodiments, the initial concentration of the quencher (e.g., glutathione) is about 20mM, and the concentration of the quencher in the mixture that decreases after the inactivation stage is in the range of about 4mM to 8 mM.
In some embodiments, the initial concentration of the quencher (e.g., glutathione) in the mixture comprising the red blood cell composition, the quencher (e.g., glutathione), the pathogen-inactivating compound, and any added base is greater than 2mM, greater than about 4mM, greater than about 6mM, greater than about 8mM, or greater than about 10 mM; the pH of the mixture is above about 5.5, above about 5.7, above about 6.0, above about 6.3, above about 6.5, above about 6.7, above about 7.0 or above about 7.2; and the quencher in the mixture is reduced in concentration after the inactivation stage by less than about 15mM, less than about 10mM, less than about 8mM, less than about 6mM, less than about 5mM, less than about 4mM, less than about 3mM, less than about 2mM, less than about 1.5mM, less than about 1mM, less than about 0.75mM, less than about 0.5mM, or less than about 0.25 mM. In some embodiments, the initial concentration of the quencher is in the range of about 2mM to 100mM, about 2mM to 40mM, about 4mM to 40mM, about 5mM to 30mM, or about 10mM to 30mM, or about 20 mM; the pH of the mixture is in the range of about 6.0 to 8.5, about 6.0 to 7.5, about 6.5 to 7.0, about 6.5 to 7.1, or about 6.6 to 6.8, or about 6.6, 6.7, 6.8 or 6.9; and the concentration of the quencher in the mixture decreases after the inactivation stage in the range of about 1mM to 20mM, about 2mM to 15mM, about 3mM to 10mM, about 4mM to 8mM, or about 5mM to 6 mM. In some embodiments, the initial concentration of the quencher (e.g., glutathione) is in the range of about 10mM to 30 mM; the pH of the mixture is in the range of about 6.0 to 7.5; and the quencher in the mixture decreases in concentration after the inactivation stage in the range of about 2mM to 15 mM. In some embodiments, the initial concentration of quencher (e.g., glutathione) is about 20 mM; the pH of the mixture is in the range of about 6.5 to 7.0 (or 7.1); and the quencher in the mixture decreases in concentration after the inactivation stage in the range of about 4mM to 8 mM.
In some embodiments of each of the methods and compositions described herein, the time period between the point of addition of the initial concentration of quencher and the point of lowering the concentration of quencher in the mixture comprising the red blood cell composition, quencher, pathogen-inactivating compound, and any added base to a lower concentration is sufficient to reduce adverse side reactions of the pathogen-inactivating compound (e.g., binding of the pathogen-inactivating compound to the surface of RBCs, which may result in an unwanted immune response). In some embodiments, the period of time is sufficient to reduce adverse side reactions of the pathogen-inactivating compound during the inactivation treatment and avoid or reduce cell dehydration.
In some embodiments, the time period between the point of addition of the initial concentration of quencher and the point of reducing the concentration of quencher in the mixture comprising the red blood cell composition, quencher, pathogen-inactivating compound, and any added base to a lower concentration is greater than, about equal to, or less than 5 hours, 10 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, or 50 hours. In some embodiments, the time period is about 1 to 96 hours, or about 1 to 72 hours, or about 1 to 48 hours, or about 10 to 30 hours, or about 15 to 25 hours, or about 20 hours.
In some embodiments of each of the methods and compositions described herein, the initial concentration of the quencher (e.g., glutathione) in the mixture comprising the red blood cell composition, the quencher, the pathogen-inactivating compound, and any added base is greater than 2mM, greater than about 4mM, greater than about 6mM, greater than about 8mM, greater than about 10mM, or greater than about 15 mM; the quencher in the mixture decreases in concentration after the inactivation stage by less than about 25mM, less than about 20mM, less than about 15mM, less than about 10mM, less than about 8mM, less than about 6mM, less than about 5mM, less than about 4mM, less than about 3mM, less than about 2mM, less than about 1.5mM, less than about 1mM, less than about 0.75mM, less than about 0.5mM, or less than about 0.25 mM; the time period between the point of addition of the initial concentration of quencher and the point of decreasing the concentration of quencher to the lower concentration is greater than, about the same as, or less than 5 hours, 10 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, or 50 hours.
In some embodiments, the initial concentration of the quencher is in the range of about 2mM to 100mM, about 2mM to 40mM, about 4mM to 40mM, about 5mM to 30mM, or about 10mM to 30mM, or about 20 mM; the quencher in the mixture decreases in concentration after the inactivation stage in a range of about 1mM to 20mM, about 2mM to 15mM, about 3mM to 10mM, about 4mM to 8mM, or about 5mM to 6 mM; and the time period between the point of addition of the initial concentration of quencher and the point of reducing the concentration of quencher to a lower concentration is about 1 to 96 hours, or about 1 to 72 hours, or about 1 to 48 hours, or about 10 to 30 hours, or about 4 to 30 hours, or about 10 to 25 hours, or about 15 to 25 hours, or about 20 hours.
In some embodiments, the initial concentration of the quencher (e.g., glutathione) is in the range of about 10mM to 30 mM; the quencher in the mixture decreases in concentration after the inactivation stage in the range of about 2mM to 15 mM; and the time period between the point of addition of the initial concentration of quencher and the point of decreasing the concentration of quencher to a lower concentration is about 10 to 30 hours. In some embodiments, the initial concentration of quencher (e.g., glutathione) is about 20 mM; the quencher in the mixture decreases in concentration after the inactivation stage in the range of about 4mM to 8 mM; and the time period between the point of addition of the initial concentration of quencher and the point of decreasing the concentration of quencher to a lower concentration is about 15 to 25 hours. In some of these embodiments, the pH of the mixture is in the range of about 6.5 to 7.0 (or 7.1). In still other of these embodiments, the pH of the mixture is in the range of about 6.0 to 7.5.
In any of these embodiments, the temperature of the mixture comprising the red blood cell composition and the quencher is in the temperature range of about 1 ℃ to 30 ℃, and also about 18 ℃ to 25 ℃, or about 37 ℃, or about room temperature during the time period between the point of addition of the initial concentration of the quencher and the point of reducing the concentration of the quencher to a lower concentration.
In some embodiments, the present invention provides methods of treating a red blood cell composition comprising: a) providing i) a pathogen-inactivating compound (e.g., S-303) comprising a labile linker linking a mustard group and a nucleic acid binding ligand (e.g., an effective amount of a pathogen-inactivating compound to inactivate a pathogen, if present), ii) a quencher comprising a thiol group (e.g., an effective amount of a quencher) wherein the thiol group is capable of reacting with a reactive electrophilic group of a pathogen-inactivating compound (e.g., glutathione), iii) a composition comprising red blood cells and iv) a suitable base (e.g., NaOH); b) mixing a pathogen-inactivating compound, a quencher, and a suitable base with a composition comprising red blood cells; and c) reducing the concentration of the quencher in the mixture sufficiently to an amount that reduces the level of red blood cell dehydration resulting from storage of the mixture (e.g., after 10, 28, or 42 days at 4 ℃) relative to the level of red blood cell dehydration resulting from storage of the mixture at the initial concentration of the quencher. In some embodiments, the mixture comprises about 0.5 to 1.5 equivalents of base (or about 0.75 to 1.25 equivalents), where equivalent means a molar amount equivalent to the molar amount of quencher in the mixture, and/or the resulting mixture of step (b) has a pH of about 6.0 to 7.5 (or about 6.5 to 7.0, or 7.1) at 37 ℃. In some embodiments, the base of step (a) is sufficient to reduce the level of anti-pathogen-inactivating compound antibody binding to the treated red blood cell composition in the resulting mixture by at least about 5% (or at least about 10%, at least about 25%, at least about 50%, at least about 75%, or at least about 90%) relative to the mixture without the base. In some embodiments, the quencher concentration is about 5mM to about 30mM (or about 15mM to about 25mM) and/or the quencher concentration in the resulting mixture of step (c) is less than about 10mM (or less than about 6mM, or less than about 2 mM). In some embodiments, the concentration of the pathogen-inactivating compound in the resulting mixture of step (b) is from about 0.1 μ M to about 5mM and/or sufficient to inactivate at least 1log (or 3log) of the pathogen in the red blood cell composition, if present. In some embodiments, the time between step (b) and step (c) is about 1 to 48 hours (or 15 to 25 hours). In some embodiments, at 20 hours after step (b), the Red Blood Cells (RBCs) of the resulting mixture have an Antibody Binding Capacity (ABC) of less than 65% and/or have an average ABC of less than about 50,000 (or between about 25,000 and 70,000), and/or have a hemolysis of less than 1% after step (c) (or after 28 or 42 days of storage at 4 ℃) and/or have a collected cell volume (PCV) of greater than 50% after step (c) (or after 28 or 42 days of storage at 4 ℃) and/or have a median corrcualarial frailty of greater than 140 (or 150) after step (c) and after 28 (or 42) days of storage at 4 ℃), as compared to the ABC value of red blood cells from the same method under the same conditions but without the use of a base. In some of these embodiments, reducing the concentration of the quencher in step (c) comprises removing the solution used in the inactivation process and adding a final additive solution (e.g., any of the solutions described herein, such as SAG-M, AS-5, the solutions in tables 2, 3, or 4, and/or an additive solution comprising sodium chloride, adenine, glucose, phosphate, guanosine, citrate, and/or mannitol).
In some embodiments, the present invention provides a method of treating a red blood cell composition comprising (a) mixing (i) a pathogen-inactivating compound (e.g., S-303) comprising a functional group that is or forms a reactive electrophilic group (e.g., an effective amount of a pathogen-inactivating compound to inactivate a pathogen, if present), (ii) a quencher (e.g., glutathione) comprising a thiol group (e.g., an effective amount of a quencher), wherein the thiol group is capable of reacting with the reactive electrophilic group of the pathogen-inactivating compound; (iii) a composition comprising red blood cells; and (iv) a suitable base (e.g., NaOH), and; b) the concentration of the quencher in the mixture is sufficiently reduced to an amount that reduces the level of red blood cell dehydration caused by storage of the mixture (e.g., after 10, 28, or 42 days at 4 ℃) relative to the level of red blood cell dehydration caused by storage of the mixture at the initial concentration of the quencher. In some embodiments, the mixture comprises about 0.5 to 1.5 equivalents of base (or about 0.75 to 1.25 equivalents), where equivalent means a molar amount equivalent to the molar amount of quencher in the mixture, and/or the resulting mixture of step (a) has a pH of about 6.0 to 7.5 (or about 6.5 to 7.0, or 7.1) at 37 ℃. In some embodiments, the base of step (a) is sufficient to reduce the level of anti-pathogen-inactivating compound antibody binding to the treated red blood cell composition in the resulting mixture by at least about 5% (or at least about 10%, at least about 25%, at least about 50%, at least about 75%, or at least about 90%) relative to the mixture without the base. In some embodiments, the quencher concentration is about 5mM to about 30mM (or about 15mM to about 25mM) and/or the quencher concentration in the resulting mixture of step (b) is less than about 10mM (or less than about 6mM, or less than about 2 mM). In some embodiments, the concentration of the pathogen-inactivating compound in the resulting mixture of step (a) is from about 0.1 μ M to about 5mM and/or is sufficient to inactivate at least 1log (or 3log) of the pathogen in the red blood cell composition, if present. In some embodiments, the time between step (a) and step (b) is about 1 to 48 hours (or 15 to 25 hours). In some embodiments, at 20 hours after step (a), the Red Blood Cells (RBCs) of the resulting mixture have an Antibody Binding Capacity (ABC) of less than 65% and/or have an average ABC of less than about 50,000 (or between about 25,000 and 70,000), and/or have a hemolysis of less than 1% after step (b) (or after 28 or 42 days of storage at 4 ℃) and/or have a Packed Cell Volume (PCV) of greater than 50% after step (b) (or after 28 or 42 days of storage at 4 ℃) and/or have an average red blood cell friability of greater than 140 (or 150) after step (b) after 28 (or 42 days of storage at 4 ℃), as compared to the ABC value of red blood cells from the same method under the same conditions but without the use of a base. In some of these embodiments, reducing the concentration of the quencher in step (b) comprises removing the solution used in the inactivation process and adding a final additive solution (e.g., any of the solutions described herein, such as SAG-M, AS-5, the solutions in tables 2, 3, or 4, and/or an additive solution comprising sodium chloride, adenine, glucose, phosphate, guanosine, citrate, and/or mannitol).
In some embodiments, the present invention provides methods of reducing dehydration of red blood cells, comprising: a) providing a red blood cell composition comprising i) a quencher (e.g., glutathione), wherein the quencher is capable of reacting with the pathogen-inactivating compound, and ii) red blood cells; and b) reducing the concentration of the quencher in the mixture sufficiently to an amount that reduces the level of red blood cell dehydration caused by storage of the mixture (e.g., after 10, 28, or 42 days at 4 ℃) relative to the level of red blood cell dehydration caused by storage of the mixture at the initial concentration of the quencher. In some embodiments, the concentration of quencher in the resulting mixture of step (b) is less than about 10mM (or less than about 6mM, or less than about 2 mM). In some embodiments, the Red Blood Cells (RBCs) of the resulting mixture have less than 1% hemolysis after step (b) (or after 28 or 42 days of storage at 4 ℃) and/or have greater than 50% Packed Cell Volume (PCV) after step (b) (or after 28 or 42 days of storage at 4 ℃) and/or have greater than 140 (or 150) average red blood cell friability after 28 (or 42) days of storage at 4 ℃ after step (b).
The methods of the invention comprise the use of a pathogen-inactivating compound and a quencher in vivo. In vivo use involves the use of the compound for treating red blood cell compositions in vitro in a living human, mammal, or vertebrate, where the treated biological material is intended for use in vivo in a living human, mammal, or vertebrate. For example, blood is removed from a human and a compound is introduced into the blood to inactivate pathogens, which would be limited to in vivo use of the compound if it was determined to reintroduce the blood into the human or another person. Reintroduction of human blood into the human or another person will be an in vivo use of the blood as opposed to an in vivo use of the compound. Upon reintroduction into the human body, the compound is introduced in vivo, in addition to in vivo use, if the compound is still present in the blood. Some embodiments of the invention relate to the in vivo use of a quencher, wherein the red blood cell composition is determined to be for in vivo use. In some cases, some level of quencher is still present in the red blood cell composition, such that the quencher is also introduced in vivo. The in vivo use of a material or compound involves the use of the material or compound in vitro of a living human, mammal, or vertebrate, where the material or compound is not intended to be reintroduced into the body of the living human, mammal, or vertebrate. An example of in vivo use is the diagnostic analysis of red blood cell sample components. The methods of the invention are suitable for use in vitro with red blood cell compositions, as alterations in red blood cells or other components may affect the in vitro analysis of blood sample constituents. Thus, the methods of the invention can provide safety in handling in vitro samples whose changes that might otherwise affect the diagnostic testing of the sample have been properly quenched.
Additive solutions, including salts and/or buffer solutions, can be used with the methods and red blood cell compositions described herein. For example, a selected buffer (e.g., SAG-M, AS-5, or any of the solutions described in tables 2, 3, and/or 4) can be added to the red blood cell composition before, during, and/or after the inactivation stage, and/or while reducing the concentration of the quencher.
Inactivation method using packed red blood cells
In some embodiments, packed red blood cells (pRBC) (e.g., red blood cells lacking the additive solution and/or having a hematocrit of about 70 to 90%, or about 75 to 85%, or about 80%) are subjected to the inactivation method described herein (e.g., a method wherein the composition comprises about 20mM GSH with 1 equivalent of base and about 0.2mM S-303), and then to the additive solution (in some cases, preserved with the additive solution) (e.g., SAG-M, AS-5, or any solution described herein or in table 2). Examples of additive solutions are shown in table 2 and described herein. In some of these embodiments, the additive solution (e.g., any of the solutions described herein or in table 2) is added to the red blood cell composition comprising the quencher, the pathogen-inactivating compound, and any added base at about 5 minutes to 20 hours after the addition of the pathogen-inactivating compound (e.g., S-303) and/or the quencher (e.g., GSH). In some embodiments, the additive solution is added to the RBC composition at about 5 minutes to 10 hours, or about 5 minutes to 5 hours, or about 5 minutes to 60 minutes, or about 5 minutes to 30 minutes, or about 10 minutes to 20 minutes, or about 15 minutes after the addition of the pathogen-inactivating compound (e.g., S-303) and/or the quencher (e.g., GSH). In some embodiments, after addition of the additive solution (e.g., SAG-M, AS-5, or any solution described herein or in table 2), the quencher concentration is reduced as described herein. For example, pRBC is treated with an inactivation method as described herein (e.g., a treatment wherein the composition comprises about 20mM GSH, about 1 equivalent of base, and about 0.2mM s-303), then treated with an additive solution (e.g., SAG-M, AS-5 or any solution described herein or in table 2) at a specified time (e.g., about 5 minutes to 5 hours, or about 10 minutes to 20 minutes, or about 15 minutes) after addition of the pathogen-inactivating compound and/or quencher, followed by reducing the quencher concentration as described herein (e.g., to less than about 10mM, or less than about 5 mM). In some embodiments, reducing the quencher concentration includes removing the treatment solution and/or the additive solution, followed by adding a final additive solution (e.g., SAG-M, AS-5, or any solution described herein or in table 2) to provide a red blood cell composition having, for example, about 50 to 70%, or about 55 to 65%, or about 60% hematocrit. In some embodiments, the chloride ion concentration in the red blood cell composition prior to and/or during inactivation is less than or greater than about 150mM, or about 120mM, or about 100mM, or about 90mM, or about 80mM, or about 70mM, or about 60mM, or about 50mM, about 40mM, about 30mM, or about 20mM, about 10mM, or between about 25 and 250mM, or about 40 to 100mM, or about 50 to 75mM, or about 60 to 70mM, or about 65 mM.
In some embodiments, an additive solution as referred to herein (e.g., an additive solution administered before and/or after reducing the concentration of a quencher) comprises one or more of the following: glucose, adenine, guanosine, mannitol, citrate (e.g. sodium citrate), citric acid, phosphoric acidSalt (e.g. Na)2HPO4And/or NaH2PO4) And chlorides (e.g., from sodium chloride). In some embodiments, the additive solution has a glucose concentration and/or a final concentration of glucose in the RBC composition after exchange (e.g., prior to transfusion) of from about 10mM to about 150mM, or from about 20mM to about 120mM, or from about 25mM to about 100mM, or from about 30mM to about 75mM, or from about 40mM to about 50 mM. In some embodiments, the concentration of adenine in the additive solution and/or the final concentration of adenine in the RBC composition after exchange (e.g., prior to transfusion) is from about 0.5mM to about 5mM, or from about 0.75mM to about 3mM, or from about 1mM to about 2.5 mM. In some embodiments, the additive solution has a guanosine concentration and/or a final guanosine concentration in the RBC composition after exchange (e.g., prior to transfusion) of from about 0.5mM to about 5mM, or from about 0.75mM to about 3mM, or from about 1mM to about 2.5mM, or from about 1.5mM to about 2 mM. In some embodiments, the mannitol concentration of the additive solution and/or the final concentration of mannitol in the RBC composition after exchange (e.g., prior to transfusion) is from about 10mM to about 150mM, or from about 20mM to about 120mM, or from about 25mM to about 100mM, or from about 30mM to about 75mM, or from about 40mM to about 50mM, or from about 35mM to about 45 mM. In some embodiments, the additive solution has a citrate (e.g., sodium citrate) concentration and/or a final citrate concentration in the RBC composition after exchange (e.g., prior to transfusion) of from about 5mM to about 100mM, or from about 10mM to about 75mM, or from about 15mM to about 50mM, or from about 15mM to about 35mM, or from about 20mM to about 30 mM. In some embodiments, the phosphate salt (e.g., Na) of the additive solution2HPO4And/or NaH2PO4) The concentration and/or final phosphate concentration in the RBC composition after exchange (e.g., prior to transfusion) is from about 1mM to about 150mM, or from about 2mM to about 100mM, or from about 3mM to about 75mM, or from about 4mM to about 50mM, or from about 5mM to about 25mM, or from about 10mM to about 20 mM. In some embodiments, the chloride concentration of the additive solution and/or the final chloride concentration in the RBC composition after exchange (e.g., prior to transfusion) is less than or greater than about 500mM, or about 250mM, or about 200mM, or about 150mM, or about 100mM, about 75mM, or about 50mM, or about 25 to about 250mM, or about 40 to about 100mM, or about 50 to about 75mM, or about 60 to about 70mM, or about 100 to about 200mMmM, or about 125mM to about 175mM, or about 150 mM.
In some embodiments, the additive solution referred to herein (e.g., the additive solution administered before and/or after the reduction in quencher concentration) and/or the final RBC composition after exchange (e.g., before transfusion) comprises 10mM to about 150mM (or about 50mM to about 90mM) glucose, 0.5mM to about 5mM (or about 0.75mM to about 3mM) adenine, about 10mM to about 150mM (or about 25mM to about 100mM) mannitol, about 10mM to about 75mM (or about 15mM to about 50mM) citrate (e.g., sodium citrate), about 3mM to about 75mM (or about 5mM to about 25mM) phosphate (e.g., Na)2HPO4And/or NaH2PO4) And about 50 to about 250mM or (about 100 to about 175mM) chloride.
Table 2: exemplary additive solutions
Inactivation method using diluted erythrocytes
The red blood cell compositions described herein can be diluted prior to inactivation. Diluting erythrocytes can dissolve the dissolved substances (e.g., salts, such as Cl)-) To a level suitable for inactivation using the methods described herein. Examples of the diluted solutions are described herein and shown in table 3. In some embodiments, prior to the inactivation methods described herein (e.g., methods wherein the composition comprises about 20mM GSH with about 1 equivalent of base and about 0.2mM S-303), non-packed red blood cells (e.g., red blood cells having about 50 to 70%, or about 55 to 65%, or about 60% and optionally comprising SAG-M or Optisol) are subjected to a dilution solution (e.g., any solution described herein or in table 3), followed by a reduction in the quencher concentration (e.g., to less than about 10mM, or less than about 5mM) as described herein. In some of these embodiments, reducing the quencher concentration includes removing the treatment solution (e.g., diluted treatment solution), followed by addition of the final additiveA solution (e.g., SAG-M, AS-5, or any of the solutions described above or in table 2) to provide a red blood cell composition, e.g., having a hematocrit of about 50 to 70%, or about 55 to 65%, or about 60%. In some embodiments, prior to inactivation, the chloride ion concentration in the red blood cell composition is diluted to less than or greater than about 150mM, or about 120mM, or about 100mM, or about 90mM, about 80mM, or about 70mM, or about 60mM, or about 50mM, about 40mM, about 30mM, or about 20mM, about 10mM, or about 25 to 250mM, or about 40 to 100mM, or about 50 to 75mM, or about 60 to 70mM, or about 65 mM. In some embodiments, the amount of the diluted solution added to the RBC solution (by volume) is about 0.2 to 2 times, or about 0.3 to 1.5 times, or about 0.4 to 1 times, or about 0.5 to 0.75 times the amount of the RBC solution. In some of these embodiments, the red blood cell composition is diluted with a dilution solution (e.g., any of the solutions described herein or in table 3) to a hematocrit level of about 30 to 50%, or about 35 to 45%, or about 40%.
In some embodiments, the dilute solutions referred to herein comprise one or more of the following ingredients: glucose, adenine, mannitol, citrate (e.g., sodium citrate), citric acid, phosphate (e.g., Na)2HPO4And/or NaH2PO4) And chlorides (e.g., from sodium chloride). In some embodiments, the glucose concentration of the dilution solution and/or the final concentration of glucose in the RBC composition after dilution with the dilution solution is from about 10mM to about 150mM, or from about 20mM to about 120mM, or from about 25mM to about 100mM, or from about 30mM to about 75mM, or from about 40mM to about 50mM, or from about 50mM to about 60 mM. In some embodiments, the concentration of adenine in the diluted solution and/or the final concentration of adenine in the RBC composition after dilution with the diluted solution is from about 0.5mM to about 5mM, or from about 0.75mM to about 3mM, or from about 1mM to about 2.5 mM. In some embodiments, the mannitol concentration of the dilution solution and/or the final concentration of mannitol in the RBC composition after dilution with the dilution solution is from about 10mM to about 150mM, or from about 20mM to about 120mM, or from about 25mM to about 100mM, or from about 30mM to about 75mM, or from about 40mM to about 60mM, or from about 25mM to about 35 mM. In some embodiments, the citrate salt of the solution is diluted (e.g.Sodium citrate) concentration and/or the final concentration of citrate in the RBC composition after dilution with a dilution solution is from 5mM to about 100mM, or from about 10mM to about 75mM, or from about 15mM to about 50mM, or from about 15mM to about 35mM, or from about 20mM to about 30 mM. In some embodiments, the phosphate salt of the solution is diluted (e.g., Na)2HPO4And/or NaH2PO4) The concentration and/or the final phosphate concentration in the RBC composition after dilution with the dilution solution is from about 1mM to about 150mM, or from about 2mM to about 100mM, or from about 3mM to about 75mM, or from about 4mM to about 50mM, or from about 5mM to about 25mM, or from about 10mM to about 20 mM. In some embodiments, the chloride concentration of the diluted solution and/or after dilution with the diluted solution is less than or greater than about 500mM, or about 250mM, or about 200mM, or about 150mM, or about 120mM, or about 100mM, or about 90mM, or about 80mM, or about 70mM, or about 60mM, or about 50mM, or about 40mM, or about 30mM, or about 20mM, or about 10mM or about 25mM to about 250mM, or about 40mM to about 100mM, or about 50mM to about 75mM, or about 60mM to about 70mM, or about 100mM to about 200mM, or about 125mM to about 175 mM.
In some embodiments, the diluted solutions referred to herein and/or RBC compositions after dilution with the diluted solutions comprise 10mM to about 150mM (or about 35mM to about 65mM) glucose, 0.5mM to about 5mM (or about 0.75mM to about 3mM) adenine, about 10mM to about 150mM (or about 25mM to about 75mM) mannitol, about 10mM to about 75mM (or about 15mM to about 50mM) citrate (e.g., sodium citrate), about 3mM to about 75mM (or about 5mM to about 25mM) phosphate (e.g., Na)2HPO4And/or NaH2PO4) And about 5 to about 50mM, or (about 10 to about 25mM) chloride.
In some embodiments, prior to the inactivation method described herein, the non-packed red blood cells are subjected to a dilution solution (e.g., any of the solutions described above and in table 3), followed by a reduction in quencher concentration AS described herein, and then treated with a final additive solution (e.g., SAG-M, AS-5, or any of the solutions described above or in table 2) to provide RBC compositions suitable for use (e.g., suitable for transfusion). In some embodiments, the final additive solution may be any additive solution described herein, for example, wherein the chloride concentration (and/or the final concentration of chloride in the RBC composition after exchange (e.g., prior to transfusion) is less than about 500mM, or about 250mM, or about 200mM, or about 150mM, or about 100mM, about 75mM, or about 50mM, or about 25 to 250mM, or about 40 to 100mM, or about 50 to 75mM, or about 60 to 70mM, or about 100 to 200mM, or about 125mM to 175mM, or about 150 mM.
Table 3: exemplary Diluent solutions
Inactivation reaction using reconstituted packed red blood cells
In some embodiments, packed red blood cells (pRBC) (e.g., red blood cells having a hematocrit of about 70 to 90%, or about 75 to 85%, or about 80%) are subjected to the inactivation methods described herein (e.g., methods in which the composition comprises about 20mM GSH with about 1 equivalent of base and about 0.2mM S-303). Examples of treatment solutions are shown in table 4. In some embodiments, a treatment solution (e.g., any of the solutions described in table 4) is added to the pRBC prior to addition of the quencher, the pathogen-inactivating compound, and any added base. In some of these embodiments, the pRBC composition is treated with the treatment solution to form non-packed red blood cells (e.g., red blood cells having a hematocrit of about 50 to 70%, or about 55 to 65%, or about 60%). In some embodiments, (a) a treatment solution is added to pRBC, (b) an inactivation method described herein (e.g., a method wherein the composition comprises about 20mM GSH with about 1 equivalent of base and about 0.2mM S-303) is performed, and (c) the concentration of the quencher is reduced (e.g., to less than about 10mM, or less than about 5mM) as described herein. In some of these embodiments, step (c) includes removing the treatment solution and adding a final additive solution (e.g., any of the solutions described herein, such AS SAG-M, AS-5, or any of the solutions of tables 2, 3, or 4) to provide a red blood cell composition having, for example, about 50 to 70%, or about 55 to 65%, or about 60% hematocrit. In some of these embodiments, the chloride ion concentration in the red blood cell composition prior to and/or during inactivation is less than or greater than about 150mM, or about 120mM, or about 100mM, or about 90mM, about 80mM, or about 70mM, or about 60mM, or about 50mM, about 40mM, about 30mM, or about 20mM, about 10mM, or about 25 to 250mM, or about 40 to 100mM, or about 50 to 75mM, or about 60 to 70mM, or about 65 mM.
In some embodiments, the treatment solutions referred to herein comprise one or more of the following: dextrose, adenine, mannitol, citrate (e.g., sodium citrate), citric acid, phosphate (e.g., Na)2HPO4And/or NaH2PO4) And chlorides (e.g., from sodium chloride). In some embodiments, the glucose concentration of the treatment solution and/or the glucose concentration in the additive solution after removal of the treatment solution in the RBC composition is from about 10mM to about 150mM, or from about 20mM to about 120mM, or from about 25mM to about 100mM, or from about 30mM to about 75mM, or from about 40mM to about 50mM, or from about 50mM to about 60 mM. In some embodiments, the concentration of adenine in the treatment solution and/or the concentration of adenine in the additive solution after removal of the treatment solution in the RBC composition is from about 0.5mM to about 5mM, or from about 0.75mM to about 3mM, or from about 1mM to about 2.5 mM. In some embodiments, the mannitol concentration of the treatment solution and/or the mannitol concentration of the additive solution after removal of the treatment solution from the RBC composition is from about 10mM to about 150mM, or from about 20mM to about 120mM, or from about 25mM to about 100mM, or from about 30mM to about 75mM, or from about 40mM to about 60 mM. In some embodiments, the citrate (e.g., sodium citrate) concentration of the treatment solution and/or the citrate concentration in the additive solution after removal of the treatment solution in the RBC composition is from about 1mM to about 100mM, or from about 2mM to about 75mM, or from about 5mM to about 50mM, or from about 7.5mM to about 25mM, or from about 10mM to about 15 mM. In some embodiments, the phosphate (e.g., Na) of the treatment solution2HPO4And/or NaH2PO4) The concentration and/or phosphate concentration in the additive solution after removal of the treatment solution in the RBC composition is from about 1mM to about 150mM, or from about 2mM to about 100mMmM, or about 3mM to about 75mM, or about 4mM to about 50mM, or about 5mM to about 25mM, or about 10mM to about 20 mM. In some embodiments, the chloride concentration of the treatment solution and/or the chloride concentration in the additive solution after removal of the treatment solution in the RBC composition is from about 250mM, or about 200mM, or about 150mM, or about 120mM, or about 100mM, or about 90mM, or about 80mM, or about 70mM, or about 60mM, or about 50mM, or about 40mM, or about 30mM, or about 20mM, about 10mM, or about 25 to about 250mM, or about 40 to about 100mM, or about 50 to about 75mM, or about 60 to about 70mM, or about 100 to about 200mM, or about 125mM to about 175 mM.
In some embodiments, the additive solution after removal of the treatment solution in the treatment solution and/or RBC composition comprises 10mM to about 150mM (or about 35mM to about 65mM) glucose, 0.5mM to about 5mM (or about 0.75mM to about 3mM) adenine, about 10mM to about 150mM (or about 25mM to about 75mM) mannitol, about 5mM to about 75mM (or about 10mM to about 20mM) citrate (e.g., sodium citrate), about 3mM to about 75mM (or about 5mM to about 25mM) phosphate (e.g., Na)2HPO4And/or NaH2PO4) And about 5 to about 100mM, or (about 25 to about 75mM) chloride.
Table 4: exemplary treatment solutions
| Solution 1 | Solution 2 | Solution 3 | Solution 4 | Solution 5 | |
| Glucose (mM) | 45.4 | 45.4 | 45.4 | 45.4 | 45.4 |
| Adenine/adenine HCl (mM) | 1.3 | 1.3 | 1.3 | 1.3 | 1.3 |
| Mannitol (mM) | 55 | 44.5 | 44.5 | 44.5 | 30 |
| Sodium citrate dihydrate or anhydrous (mM) | 12 | 12 | 12 | ||
| Na2HPO4(mM) | 15 | ||||
| NaH2PO4(mM) | |||||
| NaCl(mM) | 70 | 60 | 60 | 60 | 70 |
| Penetration degree (mOsm) | |||||
| pH (adjusted with citric acid) | 7 | 6.5 | 6.5 |
Evaluation method validation
In addition to comparing log inactivation as described above, the efficacy of the improved quenching method can be evaluated by several other methods, such as described in U.S. patent publication No.2006/0115466, the contents of which are incorporated herein by reference in their entirety. For example, quenching methods can be evaluated by assessing changes in the composition of red blood cells with respect to their function, morphology, and hydration state, as well as with respect to the reactivity of the treated red blood cells with the immune system (e.g., antibodies). If the treated red blood cell composition is intended for human use, such as transfusion, the quenching method should not substantially impair red blood cell function (e.g., by dehydration). The absence of substantial impairment of red blood cell function can be measured by methods known in the art for testing red blood cell function. In particular, the level of dehydration can be measured, for example, by hematocrit (collected cell volume, PCV), osmotic fragility, Mean Corpuscular Hemoglobin Concentration (MCHC), percent hemolysis, and red cell deformation metrics. Levels of other functional indicators, such as total ATP (adenosine 5' -triphosphate), total 2, 3-DPG (2, 3-diphosphoglycerol) or extracellular potassium, can be measured and compared to untreated controls. In addition, intracellular and extracellular pH, hemoglobin, glucose consumption and lactate production can be measured. The improved methods of the invention can be compared to the treatment conditions described previously in U.S. patent publication No.2006/0115466 (e.g., a fully quenched (2 base equivalents) 20mM glutathione bound S-303/red blood cell mixture, where there is no decrease in quencher concentration after incubation).
In some embodiments of the invention, the red blood cells of the methods and compositions described herein have minimal or no damage (e.g., dehydration, hemolysis, etc.) after treatment. In some embodiments, the red blood cells of the resulting mixture (either before or after the reduction in the concentration of the quencher) comprising the red blood cell composition, the quencher, the pathogen-inactivating compound, and any added base have less than 4%, or less than 3%, or less than 2%, or less than 1% hemolysis, or less than 0.5% hemolysis. In some embodiments, the red blood cells of the resulting mixture have less than 4%, or less than 3%, or less than 2%, or less than 1%, or less than 0.5% hemolysis at 4 ℃ for about 10 days at 4 ℃, or about 28 or 42 days at 4 ℃, or about 42 days at 4 ℃ after the concentration of the quencher (e.g., glutathione) is reduced.
In some embodiments, the red blood cells of the resulting mixture (either before or after the reduction in the concentration of the quencher) comprising the red blood cell composition, the quencher, the pathogen-inactivating compound, and any added base have a Packed Cell Volume (PCV) of greater than 50%, or greater than 55%, or greater than 60%, or greater than 65%. In some embodiments, the red blood cells of the resulting mixture have a Packed Cell Volume (PCV) of greater than 50%, or greater than 55%, or greater than 60%, or greater than 65% at 4 ℃ for about 10 days, or about 28 or 42 days at 4 ℃, or about 42 days at 4 ℃ after the concentration of the quencher (e.g., glutathione) is reduced.
In some embodiments, the red blood cells of the resulting mixture comprising the red blood cell composition, the quencher, the pathogen-inactivating compound, and any added base (either before or after the quencher concentration is reduced) have an average red blood cell friability (MCF; permeability to produce 50% hemolysis) of greater than 130, or greater than 135, or greater than 140, or greater than 145, or greater than 150, or greater than 155. In some embodiments, the red blood cells of the resulting mixture have an average red blood cell friability (MCF) greater than 130, or greater than 135, greater than 140, or greater than 145, or greater than 150, or greater than 155 at 4 ℃ for about 10 days at 4 ℃, or about 28 or 42 days at 4 ℃, or about 42 days at 4 ℃ after the concentration of the quencher (e.g., glutathione) is reduced.
Methods for measuring ATP, 2, 3-DPG, glucose, hemoglobin, hemolysis and potassium are available in the art and described in the experimental section. See, e.g., Davey et al, Transfusion, 32: 525-528(1992), the disclosure of which is incorporated herein. Methods for determining erythrocyte function are described in Greenwalt et al, Vox Sang, 58: 94-99 (1990); hogman et al, Vox Sangg, 65: 271, 278 (1993); and Beutler et al, Blood, Vol.59(1982), the disclosure of which is incorporated herein by reference. For example, total ATP and total 2, 3-DPG can be measured using a Sigma ATP kit or a 2, 3-DPG kit (Sigma, st. The ATP kit can be used according to Sigma procedure No.366-UV, the disclosure of which is incorporated herein by reference. Luciferase-based enzyme assays or assays as described by Beutler (1984) may also be usedTotal ATP was measured. Ciba Corning Model 614K may be used+/Na+An analyzer (Ciba Corning Diagnostics corp., Medford, MA) to measure extracellular potassium levels. Extracellular pH can be measured by centrifuging the cells at 4 ℃ and 12,000xg for 15 minutes and removing the supernatant, for which purpose the pH can be measured at room temperature using a standard pH meter (e.g., Beckman, mercuric oxide electrode). For intracellular pH, the remaining precipitate can be capped in a centrifuge tube and stored at about-80 ℃ for at least 2 hours. It is then cleaved by the addition of deionized water. The lysed samples were mixed well and the pH of the solution could be measured at room temperature using a standard pH meter or at room temperature using a Ciba Corning Model 238 blood gas analyzer (Ciba Corning Diagnostics corp., Medford, MA). Measurements may be made immediately after treatment and as a function of storage after treatment, for example for up to 42 days. The method of the present invention provides a red blood cell composition wherein the hemolysis of treated red blood cells is less than 3% after 28 days storage, more preferably less than 2% after 42 days storage, and most preferably less than or equal to about 1% after 42 days storage at 4 ℃. In some embodiments, a red blood cell composition (e.g., using any of the methods described herein) is provided, wherein the level of total ATP is higher when compared to a red blood cell composition treated with 2mM acidic glutathione and 0.2mM S-303. In some embodiments, the quenching methods described herein provide a red blood cell composition that has an ATP level that is about 20%, also 30%, also 40% or about 50% higher when compared to a composition using the method of 2mM acidic glutathione and 0.2mM S-303. In some embodiments, higher levels of ATP are maintained after 7, 14, 21, 28, 35, or 42 days of storage. In some embodiments, the higher level of ATP decreases during storage.
In some embodiments of the invention, the methods and compositions described herein include red blood cell compositions in which the red blood cells have a reduced number of adverse side reactions caused by the pathogen inactivating compound (e.g., binding of the pathogen inactivating compound to the surface of RBCs). In some embodiments, the side effect is a change in the surface of the red blood cells by the pathogen inactivating compound. The reduction of red blood cell changes in the methods of the present invention can be evaluated by several assays known in the art, such as those described in U.S. patent publication No.2006/0115466, the contents of which are incorporated herein by reference. Acridine content bound to the RBC surface can also be determined using the sensitive fluorescence activated immune flow cytometry assay (IFC) described herein.
With respect to fluorescence detection assays, the quenching methods of the invention can result in a decrease in mean fluorescence of at least 10%, also at least 25%, also at least 50%, also at least 75%, or at least 90% when compared to the same treatment without the use of a base (e.g., using a method that neutralizes glutathione compared to the same method that uses unneutralized glutathione). For example, the quenching methods of the invention using any of the compositions and using a base (e.g., a red blood cell composition comprising about 15-25mM glutathione, about 0.5 to 1.5 equivalents base, and about 0.2mM S-303) may result in a lower level of mean fluorescence when compared to the same composition without the base (e.g., a red blood cell composition comprising about 15-25mM glutathione and about 0.2mM S-303, but without the base).
The level of pathogen-inactivating compound binding to the surface of RBC of the quenching methods and compositions of the invention can also be measured with respect to antibody binding capacity (ABC; number of molecules of pathogen-inactivating compound or derivative thereof per red blood cell, as determined using calibration beads from Bangs Laboratories, Inc; Fishers, IN; see examples 5 and 9), which involves Allophycocyanin (APC) -conjugated murine monoclonal anti-acridine antibodies and FACS-Caliber flow cytometers (BD Biosciences). In some embodiments of any of the methods and compositions of the present invention, the RBCs have an average ABC value of less than about 75,000, or less than about 70,000, or less than about 60,000, or less than about 55,000, or less than about 52,500, or less than about 50,000, or less than about 47,500, or less than about 45,000, or less than about 42,500, or less than about 40,000, or less than about 37,500, or less than about 35,000, or less than about 32,500, or less than about 30,000, or less than about 27,500, or less than about 25,000. In some embodiments, the RBCs have an average ABC value of from about 10,000 to 80,000, or from about 20,000 to 70,000, or from about 25,000 to 60,000, or from about 30,000 to 50,000, or from about 35,000 to 45,000. In some embodiments of the methods described herein, ABC values of less than 90%, also less than 75%, also less than 65%, also less than 55%, also less than 45%, also less than 35%, also less than 25%, or less than 10% may result when compared to similar treatment with a quencher and a base (e.g., neutralizing glutathione) compared to the same method using RBC compositions that are not treated with a base.
The quenching methods of the invention can also be compared to existing methods by determining the level of nucleic acid alteration in a sample. Generally, the red blood cell composition may contain white blood cells, and the nucleic acid of the white blood cells may be isolated. Upon reaction of the compound with the nucleic acid, the pathogen-inactivating compound having the radioisotope will still bind to the nucleic acid. This can be used to determine the amount of compound that reacts with the nucleic acid, for various quenching methods, and provides a measurement that is directly related to the expected leukocyte inactivation. The amount of S-303 adduct formed per 1,000 nucleic acid base pairs can be used as a model to determine the expected effect of various methods on pathogen inactivation. Alternatively, a suitable amount of the pathogen may be added to the red blood cell composition and the nucleic acid of the pathogen isolated after treatment. However, in this case, the sample needs to be leukoreduced so that any residual leukocyte levels do not affect the measurement of pathogen nucleic acids.
In addition to providing suitable pathogen inactivation while reducing the level of adverse side reactions (e.g., binding of pathogen inactivating compounds to the RBC surface, which may lead to an unwanted immune response) and dehydration, in at least some embodiments, the quenching methods of the present invention also provide a reduction in the concentration of reactive electrophilic species after pathogen inactivation. If the red blood cell composition is determined for transfusion, it is important that the level of reactive electrophilic species is as low as possible, preferably substantially no longer detectable. The presence of reactive electrophilic species can be determined using methods available in the artIn, e.g., chromatographic methods, including liquid chromatography-mass spectrometry (LC-MS). In addition, the residual activity of a sample can be determined by assessing the ability to react with guanine residues of nucleic acids, as tested using the general alkylating agents described in Mattes (Mattes, WR, anal. biochem, 10.1992; 206 (1): 161-7). In this assay, RBCs are extracted after incubation with a pathogen inactivating compound and a quencher for a suitable time. Any residual pathogen inactivating compounds, as well as quenchers and other small substances, are separated from the proteins. Then use 8-3Double-stranded (ds) DNA synthesized from H guanine residues. Residual pathogen inactivating compound reacts with ds DNA at the N7 position of guanine, which acidifies the 8-H position and will bind the DNA to the site3H is released into solution where it can be isolated and measured. The amount of tritium released can be quantified and correlated to the amount of residual alkylating agent present in the extraction sample tested by a 1: 1 relationship. The level of electrophilic species determined by these methods can be assessed using the improved methods of the present invention and compared to known methods.
In some embodiments of each of the methods described herein, the method further comprises the step of reducing the concentration of a compound in the mixture, wherein the compound is selected from the group consisting of a pathogen-inactivating compound and a degradation product of the pathogen-inactivating compound. In some embodiments, the method comprises reducing the concentration of the pathogen-inactivating compound in the mixture. In some embodiments, the method comprises reducing the concentration of the electrophilic species in the mixture. The concentration of pathogen-inactivating compounds in the biological material (e.g., blood product) can be reduced after treatment, for example, by batch adsorption or flow removal methods. Methods and devices that may be used are described in us patent 6,544,727; 6,331,387, respectively; 6,951,713, respectively; and 7,037,642; and U.S. patent applications 2002/0192632 (disclaimer) and 2001/0009756 (disclaimer), the disclosures of each of which are incorporated herein by reference in their entirety. Thus, in some embodiments, the concentration of the pathogen-inactivating compound is reduced by contacting the mixture with an adsorbent media comprising adsorbent particles having an affinity for the pathogen-inactivating compound. In some embodimentsThe adsorption system is configured to remove pathogen inactivating compounds in a batch process. In some embodiments, the concentration of the pathogen-inactivating compound in the mixture is reduced by washing the red blood cells using techniques known in the art. In some embodiments, by methods described herein and/or known in the art (e.g., using a centrifuge and a pressing apparatus, or a combined centrifugation and pressing, such as byManufactured TACSI) to remove some or all of the treatment solution (e.g., SAG-M, AS-5, or any of the solutions described in tables 2, 3, and/or 4) to reduce the concentration of pathogen-inactivating compounds in the mixture. In some embodiments, the concentration of the pathogen-inactivating compound in the mixture is reduced by removing some or all of the treatment solution (e.g., SAG-M, AS-5, or any of the solutions described in tables 2, 3, and/or 4) followed by adding an additive solution (e.g., SAG-M, AS-5, or any of the solutions described in table 2) to the mixture. In some embodiments, the concentration of the pathogen-inactivating compound is reduced while the concentration of the quencher is reduced.
Treated blood compositions
In some embodiments, the invention also provides red blood cell compositions obtained from each of the treatment methods described herein. In some embodiments, the invention also provides red blood cell compositions that can be prepared by each of the treatment methods described herein. In one aspect, the invention provides a composition comprising a) red blood cells, wherein the red blood cells are covalently reacted with an electrophilic group of a pathogen-inactivating compound; and b) a quencher comprising a thiol group capable of reacting with the pathogen-inactivating compound; wherein the composition is suitable for infusion into a human after 28 or 42 days storage at 4 ℃.
In some embodiments of each of the methods and compositions described herein, the red blood cells in the red blood cell composition are mammalian blood cells. For example, the red blood cell can be a rodent (e.g., mouse or rat), a dog, a lagomorph (e.g., rabbit), a non-human primate (e.g., chimpanzee), or a human red blood cell. For example, in some embodiments, the red blood cells are human. In some embodiments, the red blood cells have reduced white blood cells. In some other embodiments, the red blood cells do not decrease white blood cells. In some embodiments, there is a possibility that the composition comprising red blood cells is contaminated with a pathogen. In some embodiments, the red blood cell composition is contaminated with a pathogen. In some embodiments, at least 1log, or at least 2log, or at least 3log, or at least 4log of pathogens in the composition are inactivated, if present.
In some embodiments, the invention encompasses red blood cell compositions in which the red blood cells have been altered by a pathogen-inactivating compound (e.g., S-303), as described herein. In some embodiments, the red blood cell compositions produced by these treatment methods comprise a degradation product of the pathogen-inactivating compound (e.g., a reaction product of a quencher and the pathogen-inactivating compound). In some embodiments, the change is a reaction of an electrophilic group of the pathogen-inactivating compound with the surface of a red blood cell. In some embodiments, the pathogen-inactivating compound is covalently bound to the surface of the red blood cells. In some embodiments, the pathogen-inactivating compound is covalently bound to one or more proteins on the surface of the red blood cells. In some embodiments, the change is a reaction of a nucleophilic group of the red blood cell with an electrophilic group of the pathogen-inactivating compound, wherein the electrophilic group is a mustard group, and the nucleophilic group replaces one or more chlorine atoms of the mustard group. In some embodiments, the pathogen-inactivating compound is non-covalently bound to the surface of the red blood cells. In some embodiments, the RBC compositions have an average ABC value of less than 75,000, or less than 70,000, or less than 60,000, or less than 55,000, or less than 52,500, or less than 50,000, or less than 47,500, or less than 45,000, or less than 42,500, or less than 40,000, or less than 37,500, or less than 35,000, or less than 32,500, or less than 30,000, or less than 27,500, or less than 25,000. In some embodiments, the RBCs have an average ABC value of from about 10,000 to 80,000, or from about 20,000 to 70,000, or from about 25,000 to 60,000, or from about 30,000 to 50,000, or from about 35,000 to 45,000.
In some embodiments, the red blood cell composition comprises a reduced level of modification of the surface of red blood cells by the pathogen-inactivating compound relative to red blood cells produced by other methods involving treatment with the pathogen-inactivating compound. In some embodiments, the red blood cell compositions produced by the treatment methods described herein comprise a reduced content of a pathogen-inactivating compound comprising a reactive electrophilic group after treatment is complete relative to red blood cells produced by another method involving treatment with a pathogen-inactivating compound (e.g., a method in which no sufficient amount of quencher and/or base is added to the reaction mixture, a method in which no quencher and/or base is added to the reaction mixture, and/or a treatment at a lower pH). In some embodiments, the amount of pathogen-inactivating compound comprising a reactive electrophilic group in the composition is reduced by about 10%, about 25%, about 50%, about 75%, about 90%, about 95%, or about 99% relative to a composition treated with another method involving treatment with a pathogen-inactivating compound (e.g., a method in which no sufficient amount of quencher and/or base is added to the reaction mixture, a method in which no quencher and/or base is added to the reaction mixture, and/or a treatment at a lower pH).
In some of these embodiments, the red blood cell composition comprises residual quencher compounds (e.g., glutathione). In some embodiments, the composition comprises a osmotic fragility quencher in which the concentration of the quencher is sufficiently reduced to maintain RBC viability and longevity during storage and avoid red blood cell dehydration and/or reduction. In some embodiments, the composition comprises a quencher at a much lower concentration than the concentration of quencher previously used in the composition. In some embodiments, a higher concentration of the quencher previously used reduces RBC viability and longevity during storage and/or improves red blood cell dehydration and reduces osmotic fragility, while a lower concentration is much lower than the quencher previously used in the composition. In some embodiments, the quencher in the composition is at a concentration of less than about 25mM, less than about 20mM, less than about 15mM, less than about 10mM, less than about 8mM, less than about 6mM, less than about 5mM, less than about 4mM, less than about 3mM, less than about 2mM, or less than about 1 mM. In some embodiments, the concentration of the quencher in the composition is about 1mM to 20mM, about 2mM to 15mM, about 3mM to 10mM, about 4mM to 8mM, or about 5mM to 6 mM.
In some embodiments, the composition comprises an additive solution (e.g., a solution described in table 2, or a solution comprising any combination of ingredients described in table 2). In some embodiments, the composition comprises sodium chloride, adenine, glucose, phosphate, guanosine, citrate and/or mannitol. In some embodiments, the final concentration of chloride ion in the RBC composition (e.g., prior to transfusion) is less than about 500mM, or about 250mM, or about 200mM, or about 150mM, or about 100mM, about 75mM, or about 50mM, or about 25 to 250mM, or about 40 to 100mM, or about 50 to 75mM, or about 60 to 70mM, or about 100 to 200mM, or about 125mM to 175mM, or about 150 mM.
In some embodiments, the composition is suitable for infusion into an individual (e.g., a human) after storage at 4 ℃ for about 2 days, or about 5 days, or about 10 days, or about 15 days, or about 20 days, or about 28 days, or about 35 days, or about 42 days.
In some of these embodiments, the composition comprises a) red blood cells that covalently react with electrophilic groups of a pathogen-inactivating compound (e.g., S-303) on the surface of the cells and i) have a Packed Cell Volume (PCV) of greater than 60% and/or ii) have an average Antibody Binding Capacity (ABC) of about 25,000 to 70,000 (or about 35,000 to 45,000) and b) a glutathione quencher at a concentration of less than about 8mM (or less than 6mM, or less than about 2 mM). In some embodiments, at least 3 logs (or at least 1log) of the pathogen is inactivated, if present. In some embodiments, the composition is suitable for delivery into a human when stored at 4 ℃ for up to 28 or 42 days.
Reagent kit
In addition to the improved quenching method, the present invention provides a disposable kit for treating red blood cell compositions, wherein the treatment can be performed manually or automatically. In some embodiments, the invention provides kits comprising a pathogen-inactivating compound, a quencher, and/or a base for use in each of the methods described herein. In some embodiments, the kit provides a fresh solution (such as a buffer for cell resuspension) for use after the quencher concentration is reduced as described herein.
In some embodiments, the kit comprises S-303, including any salt thereof, and neutralizing glutathione, including any salt thereof. S-303 can be in solid form or in solution. Similarly, the neutralized glutathione may be in solid form or in solution. These solids or solutions may further comprise acceptable excipients, adjuvants, diluents or stabilizers. In some embodiments, S-303 is a hydrochloride salt and the neutralized glutathione is neutralized with about 1 equivalent of sodium hydroxide. In some embodiments, S-303 and neutralized glutathione are in solid form and the kit further comprises a suitable solution for solubilizing S-303 and a suitable solution for solubilizing neutralized glutathione. In some embodiments, the invention provides a kit comprising a pathogen-inactivating compound, a quencher, and a solution for solubilizing the quencher, wherein the solution neutralizes or partially neutralizes the quencher. The methods and kits described herein include any suitable pharmaceutical preparation of the pathogen inactivation compound and the quencher, which may be formulated as a mixture or separately. Pharmaceutically acceptable formulations are known to those skilled in The art, and examples of suitable excipients, adjuvants, diluents or stabilizers can be found, for example, in Gennaro editors, Remington's The Science and Practice of Pharmacy, 20 th edition, Lippincott Williams & Wilkins. The invention also includes a composition resulting from the above-described method comprising red blood cells, a pathogen-inactivating compound, and a quencher, wherein the composition is within a suitable pH range to achieve improved quenching of the pathogen-inactivating compound.
In another aspect, the invention provides a kit, e.g., for treating red blood cell compositions to inactivate a pathogen, comprising a pathogen inactivating compound comprising a nucleic acid binding ligand and a functional group that is or forms an electrophilic group (including any salt thereof), a quencher comprising a thiol group (including any salt thereof), and about 0.75 to about 1.25 equivalents of a base, wherein an equivalent means a molar amount equivalent to the molar amount of quencher in the kit. In some embodiments, the kit comprises about 1 equivalent of a suitable base.
In yet another aspect, the invention provides a kit for treating red blood cell compositions to inactivate a pathogen, comprising a nucleic acid binding ligand and a functional group that is or forms an electrophilic group (e.g., S-303), including any salt thereof, a neutralization quencher comprising a thiol group (e.g., neutralizing glutathione), including any salt thereof, and optionally a fresh solution (e.g., a buffer for cell resuspension) for use after the quencher concentration is reduced as described herein. In some embodiments, the solution is an additive solution, diluent solution, and/or treatment solution described herein (e.g., SAG-M, AS-5, or any of the solutions described above and in tables 2, 3, and/or 4).
Examples, materials & methods
The invention is further illustrated by the following non-limiting examples.
Example 1: organic formulation
Examples, materials & methods
The bacteria and viral strains used in these studies were clinical servings obtained from the public health california department or the american type culture center.
Bacteria: frozen working stocks of bacteria were inoculated into 500mL flasks containing 50% yeast extract without added glucose and 50% fetal bovine serum medium. The flask was incubated overnight in a shaking water bath set at 37 ℃. Gram positive bacteria harvested from overnight cultures were added to blood products. Overnight cultures of gram-negative bacteria were further subcultured by dilution 1: 1000 into fresh medium and cultured as above until they reached log phase as determined by optical density. This log phase growth was added to blood products for PI experiments.
Virus: cell-free virus stocks were prepared using appropriate cell lines for the respective viruses. These stocks were frozen at-80 ℃ until they were thawed and added directly to blood products for PI experiments.
Example 2: preparation of RBC fractions
Blood was received at Cerus as 450mL or 500mL units of whole blood on the day of collection or up to 3 days after collection. In most cases, whole blood is subjected to leukocyte filtration prior to processing into RBC fractions. Occasional segments may not be successful in leukocyte filtration (e.g., blood from a consistently trained donor) and use these non-leukocyte filtered segments for PI studies of organisms for which internal leukocyte survival is unknown.
After leukocyte filtration, the blood was centrifuged and the plasma was expressed. The desired RBC additive solution, such AS-3(Nutricel), is then added and the resulting RBC fraction is used immediately or stored at 4 ℃ until use.
Example 3: pathogen Inactivation (PI)
The PI method involves inoculating the RBC fraction with a culture of the organism to be tested. The typical organism target input titer in the RBC fraction is approximately 10 for RBC6cfu or pfu/mL. In most cases, the organism volume (including any culture medium) is aboutIs 1% of the volume of the RBC fraction and is usually not higher than 10%. To evaluate lower inactivation, higher physiological relevance, an input of 10 to 10 was used5Bacterial levels of cfu/unit. For low level infusion studies, two RBC fractions were combined, added, and then divided into a test fraction treated as described herein and a control fraction to which only quencher (GSH) was added (no pathogen inactivator, e.g., S-303) and maintained at the same temperature conditions as the test fraction.
After adding the organisms to the RBC segment, the segment is mixed by grasping the end of the vessel and moving the end 10 times in a splayed or cycling motion.
The contained RBCs are then transferred to the mixing vessel of the RBC PI processing disposable. The device consists of a series of plastic containers and ports connected by plastic tubing. The mixing vessel was a two-port 600mL capacity PL1813 plastic vessel. Attached to each port is a Y-tube set with a Luer-adapted pediatric filter attached to one lead. The unused tubing on one port was connected to another 600mL capacity PL1813 plastic container (culture container). The remaining unused lead is the line connecting the original RBC units.
Feed solutions were prepared and added to the fractions as follows: a600 mM Glutathione (GSH) solution containing 1 equivalent of NaOH was prepared by dissolving 2.8g of GSH in 12mL of 0.9% saline and 0.9mL of 10N NaOH. An appropriate volume of GSH solution was introduced into a 20mL capacity syringe. The volume used was typically 10mL GSH/280mL RBC, plus 2mL line loss, to produce 20mM GSH in the fed RBC fraction. The syringe containing GSH was connected to the mixing vessel using a filtered lead sharing a Y set with the lead connected to the culture vessel. The portions were placed on a shaker to facilitate top to bottom mixing during addition of the feed solution. GSH was added to the fractions while the fractions were mixed on a shaker. The parts are then manually mixed using the figure-of-eight mixing method as described above. After addition of GSH, the portions were allowed to stand at room temperature for 5 minutes.
After the resting stage, a small sample of RBC was removed and cultured to determine the pretreatment titer. The standard plate assay was used for bacterial samples, while the cell culture assay was used for viruses.
6mM amustaline hydrochloride (S-303) was prepared by dissolving 46mg S-303 in-15 mL 0.9% saline. The appropriate dose of S-303 was introduced into a 20mL syringe. The volume used was typically 10mL S-303 solution/280 mL RBC, plus 2mL line loss to produce 0.2mM S-303 in the fed RBC fraction. The parts are then manually mixed using the figure-of-eight mixing method as described above. After addition of S-303, the treated RBC fraction was incubated at room temperature for a minimum of 3 hours to ensure complete pathogen inactivation before sampling for post-treatment titration.
At 3 hours post-PI, samples were taken and incubated as described above to determine post-treatment titers. For studies evaluating inactivation of low levels of bacterial input, after treatment, the test and control sections were incubated at RT for-20 hours, then at 37 ℃ overnight. After incubation at 37 ℃, samples were taken from each treated test fraction and the same untreated control fraction and incubated to obtain a quantitative determination of bacterial titer. The untreated portion exhibited growth.
The log reduction of each fraction was determined by taking the log of the ratio of pre-treatment titer to post-treatment titer, where the titer was expressed as 10 xcfu or pfu/mL.
Example 4: RBC in vitro functional assay
Human RBC fractions were prepared in additive solutions (e.g., AS-3(Nutricel)) according to the manufacturer's instructions. RBC fractions were treated with GSH at various concentrations to obtain final concentrations ranging from 2mM to 30 mM. In some cases, the pH of the GSH is adjusted with 1 or 2 base equivalents of sodium bicarbonate or sodium hydroxide prior to treatment. After treatment with GSH, RBCs were treated with S-303 dissolved in 0.9% sodium chloride to obtain a concentration of 0.2mM S-303 in RBCs, or to simulate dosing with 0.9% sodium chloride. After treatment, the fractions were incubated at 20-25 ℃ for 20 hours. After incubation, fractions were centrifuged at 21 ℃ at 4100 Xg for 6 minutes, the supernatant was squeezed out and 100mL of fresh additive solution was added to the RBC. All fractions were stored at 4 ℃. After preparation in the additive solution, the untreated control was placed at 4 ℃.
In vitro function was tested at different time points during storage. Extracellular pH at 37 ℃ was determined by measuring RBC pH of each fraction in a ChironDiagnostics blood gas analyzer. Total ATP is measured using a luciferase-based enzyme assay or the protocol described by Beutler (1984). Cell-free supernatants were prepared to evaluate extracellular potassium, glucose and lactate. K measurement of cell-free supernatants by Using a Chiron Diagnostics Na/K Analyzer (model #614) or similar Analyzer+The content is used for measuring the extracellular potassium. Extracellular glucose and lactate were evaluated on a NexCT analyzer. The red blood cell indices were collected using an Advia hematology analyzer (Siemens).
RBCs were washed three times in 0.9% sodium chloride and incubated at RT for a minimum of 1 hour before analyzing osmotic fragility and density characteristics. The method for osmotic fragility was described by Beutler et al, 1982, (Blood Journal 59: 1141-. Density profiles were obtained according to Danon and Marikovsky, 1964(J Lab Clin Med 6: 668-674) using phthalate esters in differential hemograph tubes.
Example 5: quantification of pathogen inactivating compounds bound to RBC surfaces
The level of acridine bound to the RBC surface was detected with a sensitive fluorescence-activated immune flow cytometry assay (IFC) using an Allophycocyanin (APC) -conjugated murine anti-acridine monoclonal antibody and a FACS-Caliber flow cytometer (BD Biosciences). Briefly, RBCs were washed three times in 0.9% saline and in flow culture buffer (HBSS, 1% BSA, 0.1% NaN)31mM EDTA, 3% BSA) weightSuspended to 4% hematocrit. Then, the APC-conjugated murine monoclonal anti-acridine antibody was added and incubated at 4 ℃ for 30 minutes; cells were washed in flow wash buffer (HBSS 1% BSA, 0.1% NaN)31mM EDTA) and resuspended in the same buffer, and a total of 30,000 events were evaluated in a FACS-Caliber under appropriate control. Quantification of the amount of S-303 molecules bound to the cell surface of human RBC (ABC) was performed using the Quantum simple Cellular bead kit (Bang Laboratories, Inc; Fishers, IN).
Example 6: glutathione pH Effect on immediate and storage-related RBC dehydration and function
GSH quenching was enhanced by treating the RBC fraction with S-303(0.2mM) and GSH (20mM) adjusted with NaOH pH (different base equivalents (b.e.)). The pH of the feed solutions tested were 2.9, 4.5 and 8.9 for 0b.e., 1b.e., and 2b.e., respectively. Following treatment, RBCs were stored at 4 ℃ and periodically tested for extracellular pH, glucose, lactate, potassium, total ATP, and hemolysis. RBC physical parameters were measured by optical flow cytometry (MCV, MCH, MCHC, HDW, etc.), and osmotic fragility measurements were performed as modified by Beutler et al (1982) and Lew et al (2003).
Exposure of RBCs to basic GSH results in reduced Hct, as well as immediate and sustained reduced osmotic fragility and increased RBC density (e.g., see fig. 1). This immediate dehydration is corrected by a reduction in base equivalents, resulting in a lower pH of the GSH solution (see table 5). Although immediate dehydration is mitigated, the osmotic fragility of RBCs continues to be altered in a concentration-dependent manner by the presence of GSH (Table 6; more examples in FIGS. 2 and 3). MCHC measurements using an optical flow cytometer (Adiva hematology analyzer, Siemens) and MCH distribution correlate with changes in osmotic fragility and density. Dehydration is S-303 independent, as this effect is also demonstrated by pH and GSH in the absence of S-303.
Table 5: effect of sodium hydroxide base-adjusted GSH levels on RBC dehydration immediately after treatment
*MCF (mean erythrocyte fragility) is the degree of penetration at which 50% hemolysis occurs
Table 6: effect of GSH base equivalent on RBC dehydration and function during storage
*MCF (mean erythrocyte fragility) is the degree of penetration at which 50% hemolysis occurs
**Days after dosing. The blood feed was 5 days old.
RBC dehydration changes are related to GSH, pH and concentration, but not to S-303 or biochemical tests (ATP, lactate, glucose) commonly used to measure RBC function. Limited exposure to GSH at high pH prevents initial dehydration. The limited sustained exposure of high levels of GSH prevents storage dehydration. These studies indicate that the determination of the hydration status of stored RBCs should be included as a predictor of RBC quality, since substantial changes in dehydration have no effect on conventional criteria, but contribute to modest changes in red blood cell life.
Example 7: improved quenching methods using subsequent quencher reduction lead to reduced RBC dehydration after storage
RBC fractions were treated with S-303(0.2mM) and GSH (20mM) pH adjusted with 1 equivalent NaOH to enhance GSH quenching. After treatment with GSH, RBCs were treated with S-303 dissolved in 0.9% sodium chloride to obtain a concentration of 0.2mM S-303 in RBCs, or a simulated feed with 0.9% sodium chloride was used. After treatment, the fractions were incubated at 20-25 ℃ for 20 h. After incubation, fractions were centrifuged at 21 ℃ at 4100 Xg for 6min, the supernatant was squeezed out and 100mL of fresh additive solution was added to the RBC. All fractions were stored at 4 ℃. After preparation with the additive solution, the untreated control was placed at 4 ℃. RBC osmotic fragility measurements were performed following the modifications of Beutler et al (1982) and Lew et al (2003).
Prolonged exposure of RBCs to high concentrations of GSH results in increased RBC density and reduced osmotic fragility (see, e.g., table 5, fig. 3 and 4). This storage-related dehydration was corrected by removing GSH prior to RBC storage (see, e.g., fig. 4 and 5). Time and GSH concentration dependent dehydration is S-303 independent, as this effect is also demonstrated by GSH in the absence of S-303 (see, e.g., fig. 6).
RBC dehydration changes are associated with GSH. Limiting exposure to high concentrations of GSH by replacing the treatment solution with fresh additive solution prevents storage-induced dehydration.
Example 8: pathogen inactivation for improved quenching methods
Leukocyte-reduced RBC fractions with approximately 60% hematocrit were prepared in AS-3 storage media. RBC sections were inoculated with approximately 6logs/ml of living organisms and aliquots were taken to serve as untreated input controls. GSH in 1 equivalent NaOH solution was added to the inoculated fraction to a final concentration of 20mM and mixed well. S-303 was added to a final concentration of 0.2mM, the fractions were thoroughly mixed again, and incubated at 20 to 25 ℃ for three hours. After incubation, samples were removed and tested to detect residual living organisms. The control sample was titrated immediately after preparation and again after the 3-hour incubation period. This was repeated at least twice for each organism.
In addition to pseudomonas, gram-negative, gram-positive and one example virus were effectively inactivated by treatment with GSH neutralized with 1 equivalent NaOH, compared to neutralization with 2 equivalents NaOH (see table 7).
Pathogen inactivation of pseudomonas aeruginosa using a total vaccination titer of up to 4.4logs/RBC units resulted in complete inactivation.
Table 7: pathogen inactivation improving conditions before quenching conditions vs
Example 9: surface-bound acridine levels using improved quenching of exchange steps
The method described in example 5 was used to determine the ability of anti-acridine antibodies to bind to erythrocytes treated with 20mM GSH neutralized with 1 equivalent NaOH and 0.2mM S-303. The antibody binding capacity measured in several RBC formulations was approximately 39,000 per red blood cell (see fig. 7). This level of binding was compared to 18,407 + -1195 ABC when RBC were treated with 20mM GSH neutralized with 2 equivalents NaOH, and 123,714 + -5123 ABC when RBC were treated with 2mM acidic GSH.
Example 10: pathogen inactivation with S-303 treatment at different Hematocrits (Hct)
RBC fractions from 450 to 500mL WB collections were prepared without the use of additive solution (80% rotary hematocrit (Hct)) or prepared in additive solution (60% Hct). RBCs were leukoreduced prior to treatment, unless otherwise indicated. The test portion of 40% Hct was diluted with a dilution solution. RBC fractions were seeded with either a high input level of-106 organisms/ml or a low input level of 10 to 105 organisms/fraction. For high level input, a 28mL control sample was taken prior to S-303 treatment. For low levels of bacterial input, test and control fractions were prepared by pooling and dividing the entire RBC fraction, and the control fraction was inoculated with-10 organisms/fraction. Test fractions with 80% and 60% Hct were treated with 200 μ M S-303 and 20mM GSH, which was neutralized with one base equivalent of sodium hydroxide (1 b.e.). Test fractions with 40% Hct were treated with 130 μ M S-303 and 13mM GSH (1, b.e.). Control samples or fractions were treated with 20mM GSH or 13mM GSH (1, b.e.) based on Hct. For the high input level portion, the control sample is assayed for living organisms while processing the test portion. After 3 hours of incubation at RT, the test and control samples were assayed for viable organisms, which were quantified by enrichment of growth on agar plates (bacteria) or by plaque assay on Vero cells (VSV). For the low level input fractions, the control and test fractions were incubated at RT for 20 hours, then at 37 ℃ for-20 hours. The samples were then coated to detect bacterial growth. The results are shown in table 8.
Table 8: pathogen inactivation data for different hematocrit value samples
aThe Log reduction was calculated as Log (untreated titer/post-treatment titer), the titer was expressed as 10x/mL
bPathogen reduction without leukocyte filtration
cn＝3
dIn all cases, the control fraction of the lowest input level was positive for bacterial growth
Example 11: s-303 treated RBC hydration at different Hematocrits (Hct)
RBCs of 40% or 60% Hct were prepared from leukoreduced whole blood in additive solution, Hct was measured by spinning hematocrit, and 80% Hct was taken as packed red blood cells. These fractions were treated with GSH (sodium salt, Biomedical Foscama, Italy) and S-303 at respective final concentrations of 20mM and 0.2 mM. All treated fractions were incubated at RT for up to 20 hours. The treatment solution was replaced with SAG-M and these fractions were adjusted to 60% Hct for storage at 4 ℃. Control RBC fractions were prepared in SAG-M and stored at 4 ℃. Measuring all the physical parameters periodically; MCHC was measured manually and the penetration brittleness measurement was performed by standard methods (Beutler et al and Lew et al, 2003). Mean red cell friability (MCF) was defined as the NaCl concentration with 50% RBC hemolysis. The changes in MCF are surface to volume ratio (S/V) and RBC hydration index during storage. After approximately 6 weeks of storage, all treated fractions had MCF values comparable to untreated controls, regardless of Hct at the time of treatment. At the end of storage, MCHC (another index of RBC hydration) was similar between the test and control fractions. The results are shown in table 9. In the wide range of hcts used in conventional practice for preparing RBC concentrates, treated RBCs stored for up to 6 weeks did not significantly alter RBC hydration and S/V.
Table 9: hydration data for different hematocrit value samples
Example 12: in vitro quality of S-303 treated RBCs stored at different Hematocrits (Hct)
RBCs of 40% or 60% Hct were prepared from leukoreduced whole blood in additive solution, Hct was measured by spinning hematocrit, and 80% Hct was taken as packed red blood cells. These fractions were treated with GSH (sodium salt) and S-303 at respective final concentrations of 20mM and 0.2 mM. All treated fractions were incubated at RT for up to 20 hours. Replacing the treatment solution with SAG-M and subjecting these toThe fractions were adjusted to 60% Hct for storage at 4 ℃. Control RBC fractions were prepared in SAG-M and stored at 4 ℃. In vitro function was determined at regular time intervals before and after treatment and during storage for up to 6 weeks. Parameters measured for RBC function in vitro include pH, total ATP, hemolysis, and extracellular potassium, glucose, and lactate. After approximately 6 weeks of storage (38 to 44 days), all test fractions had total ATP levels above 2 μmol ATP/gHb, and hemolysis and MCHC were comparable to the control fraction, independent of treatment Hc t. Throughout storage, the test fraction had higher extracellular glucose than the control fraction for 40% and 60% Hct, while the fraction with 80% Hct was more similar to the control. Extracellular lactate was lower in all tested fractions compared to control, independent of Hct. Extracellular K in the test fractions at the end of storage for 40% and 60% Hct fractions+Slightly lower than the control, and 80% Hct fraction was comparable to the control. The pH of all test parts was similar to the control throughout storage. The hemoglobin yield from this process meets AABB requirements, independent of handling Hct. During 6 weeks storage, the activity of all metabolic parameters was similar to the control after S-303 treatment in a broad range of Hct. The results are shown in table 10.
Table 10: metabolic parameters of pathogen inactivation of different hematocrit values RBC
Example 13: in vitro function and pathogen inactivation of diluted RBC
SAG-M RBC fractions were prepared from 500mL of collected leukocyte-reduced whole blood fractions. For RBC function studies, SAG-M RBC fractions were pooled by ABO blood grouping and assigned to appropriate test and control fractions. Prior to treatment, 150mL of a dilute solution containing 28.8mM mannitol, 1.3mM adenine, 16.2mM sodium phosphate, 20mM sodium citrate, ph7.5 was added to the test portion. The test fractions were treated with GSH sodium salt and S-303 at final concentrations of 20mM and 0.2mM, respectively. The test portions were incubated at Room Temperature (RT) for up to 20 hours. After RT incubation, the fractions were centrifuged and the supernatant was exchanged for 100mL SAG-M, which was added before storage at 4 ℃. Control RBC fractions were prepared in SAG-M and stored at 4 ℃. All fractions were evaluated during approximately 6 weeks storage at 4 ℃ by sampling at different time points. For the RBC pathogen inactivation study, SAG-M RBCs were split in half and RBC sections were inoculated with the pathogen prior to addition of treatment solution and GSH. After addition of the treatment solution and GSH, a control sample (5mL to 7mL) was removed from this portion to determine the input pathogen titer, and the remaining portion was treated with S-303. After 3 hours of incubation at room temperature, the treated fraction was sampled for residual live pathogen titration.
In vitro assays were used to assess in vitro metabolic and physical indices at various time points during storage. The pH at 37 ℃ was measured in a Siemens Diagnostics blood gas analyzer. Total ATP was measured using a luciferase-based enzyme assay. Cell-free supernatants were prepared to evaluate extracellular potassium (K)+) Glucose and lactate. Use ofNa/K Analyzer by measuring K of cell-free supernatant+The content is used for measuring the extracellular potassium. In NexCTTMExtracellular glucose and lactate were evaluated on the analyzer. Mean Corpuscular Hemoglobin Concentration (MCHC) and spun hematocrit were measured manually. The penetration brittleness measurement was performed by standard methods (Beutler et al and Lew et al, 2003). Mean red cell friability (MCF) was defined as the NaCl concentration at 50% RBC hemolysis.
For bacterial inactivation studies, RBCs were inoculated with approximately 6.5log cfu/mL E.coli, Serratia marcescens, Staphylococcus aureus, Yersinia enterocolitica, or Pseudomonas aeruginosa. For virus inactivation studies, RBCs were seeded at approximately 4.1log pfu/mL to 6.4log pfu/mL depending on the virus. GSH dissolved in saline was added to the fractions to a final concentration of 20 mM. Bacterial titres were determined by counting colony forming units (cfu) on agar plates and viral titres were determined by spot forming units (pfu) on appropriate cell lines. Untreated samples were serially diluted prior to counting. The treated samples were not diluted prior to titrimetry.
The results shown in tables 11 and 12 below demonstrate acceptable RBC metabolic function and physiological parameters and acceptable pathogen inactivation during storage duration.
Table 11: hydration and metabolic parameters for pathogen inactivation of diluted RBC fractions
N＝4
Table 12: pathogen inactivation data for diluted RBC fraction samples
Claims (79)
1. A method of treating a red blood cell composition, comprising:
(a) mixing in a treating solution or diluent solution
(i) An effective amount of a pathogen-inactivating compound which is β -alanine, N- (acridin-9-yl), 2- [ bis (2-chloroethyl) amino ] ethyl ester;
(ii) an effective amount of a quencher that is glutathione;
(iii) a composition comprising red blood cells; and
(iv)0.5 to 1.5 equivalents of base, wherein equivalent means a molar amount equivalent to the molar amount of quencher in the mixture;
wherein the treatment solution or diluent solution comprises one or more of the following: dextrose, adenine, mannitol, citrate, citric acid; and is
Wherein between 40mM and 100mM chloride ions are contained in the mixture of step (a) after addition of the treatment solution or diluent solution;
and
(b) replacing the solution used during the treatment of the red blood cell composition in step (a) with a fresh additive solution such that the concentration of quencher in the mixture is reduced to less than 10mM, wherein the additive solution comprises one or more of the following components: 10mM to 150mM glucose, 0.5mM to 5mM adenine, 10mM to 150mM mannitol, 5mM to 75mM citrate, 3mM to 75mM phosphate and 50mM to 250mM chloride.
2. The method of treating a red blood cell composition of claim 1, wherein the base is present in an amount sufficient to reduce the level of adverse reactions of the pathogen-inactivating compound with red blood cells in the mixture relative to the mixture without the base.
3. The method of treating a red blood cell composition of claim 1, wherein the treatment solution comprises one or more of the following: dextrose, adenine, mannitol, citrate, citric acid, phosphate and chloride.
4. The method of treating a red blood cell composition of claim 1, wherein the diluent solution comprises one or more of the following: dextrose, adenine, mannitol, citrate, citric acid, phosphate and chloride.
5. The method of treating a red blood cell composition of claim 1, wherein composition (iii) comprising red blood cells further comprises an additive solution.
6. The method of treating a red blood cell composition of claim 2, wherein the adverse reaction of the pathogen-inactivating compound with the red blood cells is a modification of the surface of the red blood cells by the pathogen-inactivating compound.
7. The method of treating a red blood cell composition of claim 1, wherein the base and the quencher are mixed with the red blood cell composition no more than 30 minutes before, simultaneously with, or after the pathogen-inactivating compound is mixed with the red blood cell composition.
8. The method of treating a red blood cell composition of claim 1, wherein the base and the quencher are mixed together prior to mixing the base or the quencher with the red blood cell composition.
9. The method of treating a red blood cell composition of claim 1, wherein the base is NaOH.
10. The method of treating a red blood cell composition of claim 1, wherein the base is an alkaline buffer.
11. The method of treating a red blood cell composition of claim 1, wherein the base comprises 0.75 to 1.25 equivalents of base, wherein an equivalent means a molar amount equivalent to the molar amount of quencher in the mixture of step (a).
12. The method of treating a red blood cell composition of claim 1, wherein the base comprises 1 equivalent of base, wherein equivalent means a molar amount equivalent to the molar amount of quencher in the mixture of step (a).
13. The method of treating a red blood cell composition of claim 1, wherein the resulting mixture of step (a) has a pH of 6.5 to 7.1 at 37 ℃.
14. The method of treating a red blood cell composition of claim 1, wherein the quencher is glutathione monosodium salt.
15. The method of treating a red blood cell composition according to claim 1, wherein the quencher in the resulting mixture of step (a) is at a concentration greater than 2 mM.
16. The method of treating a red blood cell composition of claim 15, wherein the quencher in the resulting mixture of step (a) is at a concentration of 5mM to 30 mM.
17. The method of treating a red blood cell composition of claim 15, wherein the quencher in the resulting mixture of step (a) is at a concentration of 15mM to 25 mM.
18. The method of treating a red blood cell composition according to claim 15, wherein the quencher in the resulting mixture of step (a) is at a concentration of 20 mM.
19. The method of treating a red blood cell composition of claim 1, wherein step (b) comprises centrifugation of the mixture followed by removal of the supernatant of the mixture.
20. The method of treating a red blood cell composition of claim 1, wherein step (b) comprises size-exclusion separation.
21. The method of treating a red blood cell composition of claim 1, wherein step (b) comprises the use of a pressing device.
22. The method of treating a red blood cell composition according to claim 1, wherein the quencher in the resulting mixture of step (b) is at a concentration of less than 8 mM.
23. The method of treating a red blood cell composition according to claim 1, wherein the quencher in the resulting mixture of step (b) is at a concentration of less than 6 mM.
24. The method of treating a red blood cell composition according to claim 1, wherein the concentration of the pathogen inactivating compound in the mixture obtained in step (a) is from 0.1 μ M to 5 mM.
25. The method of treating a red blood cell composition of claim 24, wherein the concentration of the pathogen-inactivating compound in the resulting mixture of step (a) is sufficient to inactivate at least 3log of pathogens, if any, in the red blood cell composition.
26. The method of treating a red blood cell composition of claim 1, wherein the time between step (a) and step (b) is from 1 to 48 hours.
27. The method of treating a red blood cell composition of claim 26, wherein the time between step (a) and step (b) is between 4 and 30 hours.
28. The method of treating a red blood cell composition of claim 27, wherein the treatment inactivates at least 3log of pathogen contaminants in the red blood cell composition, if present.
29. The method of treating a red blood cell composition of claim 1, further comprising the step of reducing the concentration of a pathogen inactivating compound in the mixture.
30. The method of treating a red blood cell composition of claim 29, wherein the steps of reducing the concentration of the quencher in the mixture and reducing the concentration of the pathogen-inactivating compound in the mixture are performed simultaneously.
31. The method of treating a red blood cell composition of claim 1, wherein after step (a), the red blood cells of the resulting mixture have less than 55% antibody binding capacity as compared to the antibody binding capacity value of red blood cells from the same method under the same conditions, but without the use of a base.
32. The method of treating a red blood cell composition of claim 1, wherein at 20 hours after step (a), the red blood cells of the resulting mixture have less than 65% antibody binding capacity as compared to the antibody binding capacity value of red blood cells from the same method under the same conditions, but without the use of a base.
33. The method of treating a red blood cell composition of claim 1, wherein the red blood cells of the resulting mixture have an average antibody binding capacity of less than 50,000.
34. The method of treating a red blood cell composition of claim 33, wherein the red blood cells of the resulting mixture have an average antibody binding capacity of less than 40,000.
35. The method of treating a red blood cell composition of claim 1, wherein the red blood cells of the resulting mixture have an average antibody binding capacity between 25,000 and 70,000.
36. The method of treating a red blood cell composition of claim 35, wherein the red blood cells of the resulting mixture have an average antibody binding capacity between 35,000 and 45,000.
37. The method of treating a red blood cell composition of claim 1, wherein the red blood cells of the resulting mixture have less than 1% hemolysis after step (b).
38. The method of treating a red blood cell composition of claim 37, wherein the red blood cells of the resulting mixture have less than 1% hemolysis at 42 days at 4 ℃ after step (b).
39. The method of treating a red blood cell composition of claim 1, wherein the red blood cells of the resulting mixture have a packed cell volume greater than 50% after step (b).
40. The method of treating a red blood cell composition of claim 39, wherein the red blood cells of the resulting mixture have a packed cell volume greater than 50% at 42 days at 4 ℃ after step (b).
41. The method of treating a red blood cell composition of claim 1, wherein the red blood cells of the resulting mixture have an average red blood cell friability value greater than 140mOsm after 42 days at 4 ℃ after step (b).
42. Use of a red blood cell composition produced by the method of any one of claims 1-41 in the manufacture of a medicament for infusing red blood cells into a subject.
43. A method of reducing dehydration of a red blood cell composition, wherein the composition is a mixture comprising a quencher which is glutathione, 0.5 to 1.5 equivalents of a base, wherein equivalent means equivalent to the molar amount of quencher in the mixture, red blood cells, and a treatment solution or diluent solution; wherein the treatment solution or diluent solution comprises one or more of the following: dextrose, adenine, mannitol, citrate, and citric acid; and wherein the red blood cell composition comprises between 40mM and 100mM chloride ion; the method comprises replacing a solution in a mixture with a fresh additive solution such that the concentration of quencher in the mixture is reduced to less than 10mM, wherein the additive solution comprises one or more of the following components: 10mM to 150mM glucose, 0.5mM to 5mM adenine, 10mM to 150mM mannitol, 5mM to 75mM citrate, 3mM to 75mM phosphate and 50mM to 250mM chloride.
44. The method of reducing dehydration of a red blood cell composition of claim 43 wherein the quencher is glutathione monosodium salt.
45. The method of reducing dehydration of a red blood cell composition of claim 44 wherein the concentration of quencher is reduced to less than 8 mM.
46. The method of reducing dehydration of a red blood cell composition of claim 44 wherein the concentration of quencher is reduced to less than 6 mM.
47. The method for reducing dehydration of a red blood cell composition according to claim 43 wherein the red blood cells of the mixture have less than 1% hemolysis after reducing the concentration of the quencher.
48. The method for reducing dehydration of a red blood cell composition of claim 47 wherein the red blood cells of the mixture have less than 1% hemolysis at 42 days at 4 ℃ after reducing the concentration of the quencher.
49. The method for reducing dehydration of a red blood cell composition according to claim 43 wherein the red blood cells of the mixture have a packed cell volume greater than 50% after reducing the concentration of the quencher.
50. The method for reducing dehydration of a red blood cell composition of claim 49 wherein the red blood cells of the mixture have a packed cell volume greater than 50% at 42 days at 4 ℃ after reducing the concentration of the quencher.
51. The method of reducing dehydration of a red blood cell composition according to claim 43, wherein the red blood cells of the mixture have an average red blood cell friability value greater than 140mOsm after 42 days at 4 ℃ after reducing the concentration of the quencher.
52. A composition comprising red blood cells produced by the method of any one of claims 1-41 or 43-51.
53. A composition comprising red blood cells preparable by the method of any one of claims 1-41 or 43-51.
54. A composition, comprising:
(a) red blood cells preparable by the method of any one of claims 1-41 or 43-51, wherein said red blood cells are covalently bound to an electrophilic group of a pathogen-inactivating compound, said compound being β -alanine, N- (acridin-9-yl), 2- [ bis (2-chloroethyl) amino ] ethyl ester; and
(b) a quencher that is glutathione, wherein the concentration of the quencher is less than 10 mM;
wherein the red blood cells have an average red blood cell friability value greater than 150mOsm after 28 days at 4 ℃.
55. The composition of claim 54, wherein at least 3log of pathogens, if present, are inactivated.
56. The composition of claim 54, wherein the electrophilic group is covalently bound to the cell surface of the red blood cell.
57. The composition of claim 54, wherein the quencher is glutathione monosodium salt.
58. The composition of claim 54, wherein the quencher concentration is less than 8 mM.
59. The composition of claim 54, wherein the quencher concentration is less than 6 mM.
60. The composition of claim 54, wherein the red blood cells have a packed cell volume greater than 55%.
61. The composition of claim 60, wherein the red blood cells have a packed cell volume greater than 60%.
62. The composition of claim 54, wherein the red blood cells have an average antibody binding capacity of less than 50,000.
63. The composition of claim 62, wherein the red blood cells have an average antibody binding capacity of less than 40,000.
64. The composition of claim 54, wherein the red blood cells have an average antibody binding capacity of between 25,000 and 70,000.
65. The composition of claim 64, wherein the red blood cells have an average antibody binding capacity between 35,000 and 45,000.
66. The composition of claim 54, wherein the composition further comprises a fresh additive solution comprising one or more of dextrose, sodium chloride, adenine, guanosine, glucose, citrate, citric acid, phosphate, and mannitol.
67. Use of the red blood cell composition of claim 54 in the manufacture of a medicament for infusing red blood cells into a subject.
68. The method of treating a red blood cell composition of claim 8, wherein the base and the quencher are mixed to form a salt form of the quencher.
69. The method of treating a red blood cell composition of claim 68, wherein the salt form is potassium glutathione or sodium glutathione.
70. The method of treating a red blood cell composition of claim 1, wherein the chloride concentration after step (a) and before step (b) is less than 75 mM.
71. The method of treating a red blood cell composition of claim 1, wherein the composition comprising red blood cells in step (a) has a packed cell volume between 70 and 90%.
72. The method of treating a red blood cell composition of claim 71, wherein the composition comprising red blood cells in step (a) has a packed cell volume of between 75 and 85%.
73. The method of treating a red blood cell composition of claim 1, wherein the composition comprising red blood cells in step (a) has a packed cell volume between 50 and 70%.
74. The method of treating a red blood cell composition of claim 73, wherein the composition comprising red blood cells in step (a) has a packed cell volume of between 55 and 70%.
75. The method of treating a red blood cell composition of claim 1, wherein the composition comprising red blood cells in step (a) has a packed cell volume of between 30 and 50%.
76. The method of treating a red blood cell composition of claim 75, wherein the composition comprising red blood cells in step (a) has a packed cell volume between 35 and 45%.
77. The method of treating a red blood cell composition according to claim 1, wherein the red blood cells in step (a) have been leukoreduced.
78. The method of treating a red blood cell composition of claim 1, wherein the red blood cells in step (a) are not leukoreduced.
79. The composition of claim 54, further comprising one or more of dextrose, sodium chloride, adenine, guanosine, glucose, citrate, citric acid, phosphate and mannitol.
Applications Claiming Priority (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US4366608P | 2008-04-09 | 2008-04-09 | |
| US61/043,666 | 2008-04-09 | ||
| US8703408P | 2008-08-07 | 2008-08-07 | |
| US61/087,034 | 2008-08-07 | ||
| PCT/US2009/040032 WO2009126786A2 (en) | 2008-04-09 | 2009-04-09 | Improved quenching methods for red blood cell pathogen inactivation |
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| Publication Number | Publication Date |
|---|---|
| HK1157140A1 HK1157140A1 (en) | 2012-06-29 |
| HK1157140B true HK1157140B (en) | 2015-08-14 |
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