Quenching methods for red blood cell inactivation process
Methods are provided for improved quenching of undesired side reactions upon treating a red blood cell composition with a pathogen inactivating compound comprising a nucleic acid binding ligand and a functional group which is, or which is capable of forming, an electrophilic group. In some embodiments, the improved methods use a suitably high concentration of quencher that comprises a nucleophilic functional group that is capable of covalently reacting with the electrophilic group, wherein the treatment occurs within a desired pH range to provide sufficient quenching. Preferred quenchers for use in some of the methods include thiols, such as glutathione, which have been suitably neutralized such that addition to a red blood cell composition results in the desired concentration of quencher at a desirable pH range of 6.8 to 8.5.
1. A method of treating a red blood cell composition which contains a red blood cell concentrate in a buffered additive solution, comprising:
(a) mixing a quencher with a base, wherein the quencher comprises a thiol group and an acid group, and is capable of quenching an undesired side reaction of a pathogen-inactivating compound with the red blood cells,
(b) mixing the quencher and base of step (a) with the red blood cell composition,
(c) mixing a pathogen-inactivating compound with the red blood cell composition, wherein the pathogen-inactivating compound contains a nucleic acid binding group and a functional group which is an electrophilic group or is capable of forming an electrophilic group, wherein the electrophilic group is capable of reacting with nucleic acid,
wherein the molar ratio of the quencher to pathogen-inactivating compound is about 10:1 to about 400:1, wherein the quencher is in a final concentration following step (c) of about 2 mM to about 40 mM, and wherein the base is in a molar ratio with the quencher of at least about 1, so that the undesired side reaction is decreased in comparison with the treatment method of a red blood cell composition without mixing of the base with the quencher.
2. The method of claim 1 , wherein the undesired side reaction of the pathogen-inactivating compound with the red blood cells is modification of the surface of the red blood cells by the pathogen-inactivating compound.
3. The method of claim 1 , wherein both the base and the quencher are mixed with the red blood cell composition prior to, at the same time, or no more than about 30 minutes after mixing the pathogen-inactivating compound with the red blood cell composition.
4. The method of claim 3 , wherein both the base and the quencher are mixed with the red blood cell composition prior to mixing the pathogen-inactivating compound with the red blood cell composition.
5. The method of claim 1 , wherein the base is NaOH.
6. The method of claim 1 , wherein the base is a basic buffer.
7. The method of claim 1 , wherein the quencher is in a final concentration following step (c) of about 10 mM to about 30 mM.
8. The method of claim 1 , wherein the resulting mixture comprising the red blood cell composition, the pathogen-inactivating compound, the quencher, and the base has a pH at room temperature of about 6.7.
9. The method of claim 1 , wherein the resulting mixture comprising the red blood cell composition, the pathogen-inactivating compound, the quencher, and the base has a pH at room temperature of about 6.7 or higher.
10. The method of claim 1 , wherein the resulting mixture comprising the red blood cell composition, the pathogen-inactivating compound, the quencher, and the base has a pH at room temperature of about 7.0 to 8.5.
11. The method of claim 1 , wherein the quencher comprises cysteine or a derivative of cysteine.
12. The method of claim 11 , wherein the quencher is glutathione.
13. The method of claim 1 , wherein the functional group is selected from the group consisting of a mustard, a mustard intermediate, and a mustard equivalent.
14. The method of claim 13 , wherein the pathogen-inactivating compound is β-alanine, N-(acridin-9-yl), 2-[bis(2-chloroethyl)amino]ethyl ester.
15. The method of claim 1 , wherein the treatment results in an inactivation of at least 1 log of a pathogen contaminant in the red blood cell composition.
16. The method of claim 1 , further comprising a step of reducing the concentration of the pathogen-inactivating compound in the resulting mixture comprising the red blood cell composition, the pathogen-inactivating compound, the quencher, and the base.
17. The method of claim 1 , wherein the base is in a molar ratio with the quencher of about 1.
18. The method of claim 1 , wherein the level of anti-pathogen inactivating compound antibody binding to the treated red blood cell composition in the resulting mixture which comprises the red blood cell composition, the pathogen-inactivating compound, the quencher, and the base, is reduced by at least about 25% relative to the mixture without the base.
19. The method of claim 18 , wherein the level of anti-pathogen inactivating compound antibody binding to the treated red blood cell composition in the resulting mixture is reduced by at least about 50% relative to the mixture without the base.
20. The method of claim 19 , wherein the level of anti-pathogen inactivating compound antibody binding to the treated red blood cell composition in the resulting mixture is reduced by at least about 75% relative to the mixture without the base.