WO2004050848A2

WO2004050848A2 - Methods for antigen masking of red blood cells resulting in reduced hemolysis

Info

Publication number: WO2004050848A2
Application number: PCT/US2003/038224
Authority: WO
Inventors: Adonis Stassinopoulos; Basha Clark
Original assignee: Cerus Corporation
Priority date: 2002-12-04
Filing date: 2003-12-03
Publication date: 2004-06-17
Also published as: AU2003298827A1; WO2004050029A3; AU2003293237A1; WO2004049914A2; AU2003302499A8; AU2003293237A8; AU2003298827A8; WO2004050029A2; AU2003297614A1; WO2004049914A3; AU2003302499A1; WO2004050897A2; AU2003297614A8; WO2004050848A3; WO2004050897A3

Abstract

Methods are provided for the preparation of an RBC composition which has significantly reduced antigenicity. The methods of preparation of the red cell compositions involve the optimization of reaction conditions for attaching antigen masking compounds to the red cells without significantly affecting the function of the red cells, in particular reducing the hemolysis of the red cells from processing of the cells. The RBC compositions are of particular use for introduction into an individual in cases where the potential for an immune reaction is high, for example in alloimmunized blood recipients or in trauma situations where the possibility of transfusion of a mismatched unit of blood in higher. The RBC compositions of this invention provide a much lower risk of a transfusion associated immune reaction.

Description

METHODS FOR ANTIGEN MASKING OF RED BLOOD CELLS RESULTING IN REDUCED HEMOLYSIS

FIELD OF THE INVENTION

[0001] The present invention relates generally to methods for modifying a red blood cell (RBC) by covalently binding an antigen masking compound, such as polyethylene glycol (PEG), to the surface of the red cell. The PEG modified RBC result in a reduced immune response to RBC antigens on transfusion into an individual and are useful for providing an RBC that either does not need cross matching or can be used for allosensitized individuals.

BACKGROUND

[0002] Blood transfusions are essential in the treatment of patients with anemia, trauma, surgical bleeding and certain inherited disorders. Risks of an immune reaction due to donor red cell antigens in a patient receiving an RBC transfusion include hemolytic transfusion reaction and alloimmunization. Hemolytic transfusion reactions may be due to reaction of a recipient antibody to an antigen on the donor RBC, often resulting from human error in transfusing a mismatched unit of RBC. Elaborate systems of identification and testing are in place to ensure that correctly matched RBC are transfused. However, human performance is a factor in such systems and mistakes are made. A report on transfusion error in New York state indicates ABO incompatible transfusion of 1 in 33,000 units resulting in 3 fatalities [Linden et al., Transfusion 32: 601 (1992)]. Similar error rates have been reported in Great Britain [McClelland et al., BMJ 308:1205 (1994)]. Alloimmunization is of greatest risk to patients who require chronic RBC transfusions and results from the patient forming antibodies to minor group antigens on the RBC, which cause reactions in subsequent transfusions (allosensitization). [0003] Several approaches exist to mask the RBC antigens by attaching long, flexible hydrophilic molecules such as polyethylene glycol (PEG) to the surface of the RBC creating an essentially non-immunogenic RBC [U.S. Patent Numbers 5,908,624, 6,129,912, and 6,312,685, the disclosures of which are hereby incorporated by reference]. The latter patent also discusses improved rheological properties of such modified red cells. The modified red cells have a reduced viscosity at low shear rates, which is beneficial in treating conditions having low blood flow resulting in ischemia. The level of PEG modification required may vary depending on the application. In the case of an RBC that does not need cross matching, a high level of modification may be required in order to mask all antigens. In the case of an allosensitized individual, or for lowering the viscosity, a lesser amount of PEG modification may be sufficient to mask the minor antigens as the blood can be matched for the major antigens.

[0004] While these approaches are promising, the existing methods for binding of an antigen masking compound to the red cells can be detrimental to the function of the red cells. In particular, it is necessary to develop methods for modification of the red cells that do not result in excessive hemolysis of the red cells. If a modified red cell undergoes considerable damage, as indicated by hemolysis, it could not be used in a transfusion due to the excessive iron levels free in solution as well as the toxicity of free hemoglobin. Therefore, methods are needed to prepare antigen masked red cell compositions which can be stored for extended periods without resulting in unacceptable levels of hemolysis.

SUMMARY OF THE INVENTION

[0005] The present invention provides new methods for the modification of RBC with an activated antigen masking compound so that they have significantly reduced immunogenicity and undergo minimal hemolysis, even after extended storage periods. The methods provided reduce the amount of hemolysis during the processing of the red cells, wherein the hemolysis is sufficiently low after processing and remains sufficiently low after extended storage at 4 °C. In some embodiments, the processing of the red cells involves washing the red cells, followed by reaction of the red cells with an activated antigen masking compound, post reaction washing of the red cells, and storage of the red cells in a suitable storage solution. In the various steps an appropriate solution (pre reaction wash solution, reaction solution, post reaction wash solution or storage solution) is added to a red cell solution. In some embodiments, the red cell solution is a red cell concentrate. In preferred embodiments, one or all of the pre reaction wash solution, reaction solution, post reaction wash solution, and storage solution comprise additives such as dextrose or L-camitine. In one embodiment, one or all of these solutions further lack chloride ions. These additives result in reduced hemolysis of the red cells during the process. In a preferred embodiment, all four solutions comprise dextrose and L-carnitine. In a further embodiment, all four solutions lack chloride ions.

[0006] In one embodiment, a red cell composition is mixed with an activated antigen masking compound and a reaction solution and incubated to covalently bind antigen masking compound to the surface of the red cells, wherein the reaction solution comprises dextrose at a concentration of about 50-300 mM, preferably about 75-200 mM, preferably approximately 100 mM. In one embodiment, the reaction solution comprising dextrose further lacks chloride ions. In one embodiment, the reaction solution comprising dextrose further comprises L-carnitine at a concentration of about

2-100 mM, preferably about 2-10 mM, preferably approximately 5 mM. In one embodiment, the reaction solution comprising dextrose and L-carnitine further lacks chloride ions. In one embodiment, a red cell composition is mixed with activated antigen masking compound and a reaction solution and incubated to covalently bind antigen masking compound to the surface of the red cells wherein the reaction solution comprises L-carnitine at a concentration of about 2-100 mM, preferably about

2-10 mM, preferably approximately 5 mM. In one embodiment, the red cell composition is prepared as a red cell concentrate prior to mixing with the reaction solution and activated antigen masking compound. In another embodiment, the reaction mixture is at a hematocrit of about 20-95%, also about 30-95%, also about

40-95%, preferably about 50-95%, preferably about 60-95%, more preferably about

60-80%.

[0007] One embodiment of the invention encompasses a method of preparing a red cell composition comprising a) washing the red cells with a pre reaction wash solution to provide washed red cells; b) mixing the washed red cells with a reaction solution and an activated antigen masking compound to provide a reaction mixture; c) incubating the reaction mixture so that the antigen masking compound covalently binds to the surface of the red cells to provide modified red cells; and d) washing the modified red cells with a post reaction wash solution, wherein at least one of the pre reaction wash solution, reaction solution, and post reaction wash solution comprises dextrose at a concentration of about 50-300 mM, preferably about 75-200 mM, preferably approximately 100 mM. In one embodiment, at least two of the pre reaction wash solution, reaction solution and post reaction wash solution comprise dextrose at a concentration of about 50-300 mM, preferably about 75-200 mM, preferably approximately 100 mM. In one embodiment, the pre reaction wash solution, reaction solution, and post reaction solution all comprise dextrose at a concentration of about 50-300 mM, preferably about 75-200 mM, preferably approximately 100 mM. In one embodiment, at least one of the pre reaction wash solution, reaction solution, and post reaction wash solution further comprises L- carnitine at a concentration of about 2-100 mM, preferably about 2-10 mM, preferably approximately 5 mM. In one embodiment, at least two of the pre reaction wash solution, reaction solution and post reaction wash solution further comprise L- camitine at a concentration of about 2-100 mM, preferably about 2-10 mM, preferably approximately 5 mM. In one embodiment, the pre reaction wash solution, reaction solution, and post reaction solution further comprise L-carnitine at a concentration of about 2-100 mM, preferably about 2-10 mM, preferably approximately 5 mM. In one embodiment, one, at least two, or all of the pre reaction wash solution, reaction solution and post reaction wash solution further lack chloride ions. In one embodiment, step (a) involves washing a red cell concentrate with a volume of pre . reaction wash solution that is greater than or equal to the red cell concentrate volume.

In one embodiment, step (b) involves mixing the reaction solution and an antigen masking compound with a washed red cell concentrate to a hematocrit of about 20-

95%, also about 30-95%, about 40-95%, about 50-95%, preferably about 60-95%, more preferably about 60-80%. In one embodiment, the pre reaction wash solution and reaction solution are at a pH of about 8-10, preferably about 9 and comprise a buffer at a concentration of about 50-350 mM, preferably about 100-200 mM. In one embodiment, the pre reaction solution and the reaction solution are the same. In one embodiment the post reaction wash solution comprises a buffer at a pH of approximately 7. In a preferred embodiment, the pre reaction wash and reaction solution comprises 150 mM CHES pH 9.0, 100 mM dextrose and 5 mM camitine and the post reaction wash comprises 150 mM Na₂HPO , 100 mM dextrose, and 5 mM L- carnitine at a pH of 7.0, preferably wherein all solutions lack chloride ion. DESCRIPTION OF THE DRAWINGS

[0008] Fig. 1 is an exemplary plot of the population maximum fluorescence by flow cytometry for the binding of fluorescent antibody to mPEG modified RBC. [0009] Fig. 2A is an exemplary plot of a standard curve of fluorescence measurement vs. fluorescently labeled activated PEG (FPEG) concentration spiked into red cell ghosts for quantitation of PEG binding to red cells (1.3 billion ghosts per sample). [0010] Fig. 2B is a plot of the FACScan peak (FLl-Height) vs. the number of PEG molecules bound per red cell.

DESCRIPTION OF THE INVENTION

[0011] The present invention generally relates to new methods for attaching polymeric compounds to RBC, wherein the attached polymers mask RBC antigens of the red cells. The invention comprises the resulting polymer modified RBC, wherein the RBC have reduced immunogenicity compared to unmodified RBC. An additional property of the modified red cells is a reduced viscosity at low shear rates. In this instance, the level of antigen masking compound per red cell can be lower, as the viscosity reduction at low shear rates can be done with less antigen masking compound bound than required for antigen masking of the red cells. Such low viscosity red cells can be used to treat ischemic conditions, such as those resulting from stroke, myocardial infarction, sickle cell anemia, and other conditions relating to vascular occlusion (see US Patent 6,312,685). Additional diseases that can be treated include angina, critical limb ischemia, cerebral vasospasm, and subarrachnoid hemorrhage. The modified RBC remain functional and are suitable for in vivo use, e.g. for transfusion. In particular, the modified RBC are not significantly hemolyzed by the methods used to attach polymeric compounds to the RBC, or after extended storage of the resulting RBC.

[0012] The present invention encompasses in vitro or ex vivo methods of treating a composition comprising RBC with a compound comprising a non-immunogenic group having a suitable reactive coupling group, such a compound being referred to as an activated antigen masking compound. The compound selectively masks RBC antigens by covalently binding to the surface of the RBC resulting in an RBC composition having significantly reduced immunogenicity. This reduced immunogenicity would result in a reduced immune response when transfused into an individual having a non-matching blood type. While the ideal reduction in the immunogenicity would provide.a red.ceU that doesji t need to be cross rnatched, it would also be useful in some cases to mask minor antigens without necessarily masking major antigens. Such a red cell could be used in a case where a subject is alloimmunized, i.e. has developed an immune response to minor antigens due to repeated infusions. In this case, a red cell composition could have reduced immunogenicity with respect to the minor antigens and still be cross matched for the major antigens. The immunogenicity can be assessed by reacting the antigen masked red cells with antibodies to the red cell antigens. This can be assessed in vitro, for example using standard blood typing techniques, or in vivo, for example by infusion into an individual of the same species having an incompatible blood type. The details of the activated antigen masking compound and the methods for reacting with the surface of the red cells are optimized to avoid hemolysis during the process and after extended storage of the resulting RBC. The buffers used during the process comprise additives that reduce the level of damage expressed as hemolysis during processing or as delayed hemolysis upon storage. It is expected that this will result in less removal of red cells from circulation upon infusion into an individual. Preferred additives for the reduction in hemolysis are dextrose and L-carnitine. In one embodiment, buffers used during the process lack chloride ions.

[0013] The present invention involves the in vivo use of red cells modified by binding to antigen masking compounds. In vivo use of a material or compound is defined as introduction of the material or compound into a living individual. For example, the transfusion of a blood product into an individual in need of a transfusion would be considered an in vivo use of the blood product. An individual, as defined herein, is a vertebrate, preferably a mammal, including domestic animals, sport animals, and primates, including humans. Ex vivo use of a compound is defined as using a compound for treatment of a biological material outside of a living individual, where the treated biological material is intended for use inside the living individual. Since the activated antigen masking compound is either reacted with the red cells or washed away after processing the red cells, it is considered an ex vivo use of the compounds.

[0014] The compositions included in the present invention are considered to be functional if certain in vitro and in vivo properties are similar to the properties of a control sample that is not treated by the methods of the present invention. In addition, there are certain blood banking standards that must be met by compositions of the present invention. To be suitable for in vivo use, the compositions must also exhibit low levels of Joxicity, as measured for example by cellular assays such as Ames mutagenicity assays and animal studies.

Assessment of antigen masking of red cells. [0015] In one embodiment, the present invention contemplates the antigen masking or immune masking of RBC. RBC comprise several antigenic determinants on their surface that might cause an immune response. For example, the immune system of a recipient of an RBC transfusion may recognize certain antigens on the transfused RBC as foreign and mount an immune response to the RBC. Masking of these antigens involves the modification or hiding of these antigens so that any immune response that would normally be elicited in the recipient is significantly reduced. In one embodiment, the antigens are masked so that they are no longer accessible to or recognized by the immune system of the recipient. Certain compounds may be attached to the RBC surface such that the antigens on the RBC surface are hidden or masked by the compound. In addition, these compounds that can be linked to the RBC surface may have a structure that is not itself recognized by an immune system, i.e. these compounds are non-immunogenic or non-antigenic. By masking antigens in this manner, an immune response elicited in the recipient by the transfused RBC is significantly reduced. RBC antigens are considered to be substantially masked when the treated RBC have significantly reduced reactivity toward antibodies specific for RBC antigens when compared to the reactivity of an untreated RBC toward these antibodies. This reduced immunogenicity can be readily measured using in vitro antibody binding assays or in vitro measurements of the amount of modification of the RBC. Further, the treated red cells can be tested in vivo to assess whether they provide a reduction or elimination of an immune response in a recipient. [0016] In vitro assays include, but are not limited to, ABO reactivity agglutination, measurement of RBC aggregation as a function of antibody added to the composition, reactivity to minor antigens, ELISA assay to measure direct binding of antibody to the antigen masked RBC, and analysis of binding of fluorescent antibody reactive with antigens on the unmodified RBC to assess levels of modification by antigen masking compounds (e.g. Example 5). [0017] As an example of ABO reactivity assessment, a composition containing treated RBC is reacted with serum containing a suitable antibody (e.g. treated type A RBC would be reacted with serum containing anti-A antibodies) and agglutination of the

RBC is observed. This agglutination assay can be used with anti-A, anti-B and anti-D antibodies.and is. described inJExample 1. The_reaction is.repeated with erially diluted aliquots of the antibody containing serum until no agglutination is observed.

Standard blood typing assays used in blood banks involve such an agglutination assay, where typically one drop of antibody solution is mixed with one drop of the red cell solution and observed for agglutination of the red cells. The immunogenicity of minor antigens, including the D antigen, which are generally implicated in alloimmunization, can also be tested in vitro. Rather than using the dilution system, an established system rates the reaction with antisera on a scale of 0, 1+, 24-, 34-, or

44-, 0 being no reaction, 44- being the highest level of agglutination [Walker et al.,

AABB Technical Manual, 10th Ed., pp 528-537 (1990)]. These agglutination assays are typically done in a test tube and are scored by those skilled in the art. Minor antigens that have been implicated in alloimmunization include Jk^a, E, K, Bg, Lu , Pi,

D, Sd^a, Fy^a, M, Yk^a, Ai, Le^a, Kp^a, C, e, and I [Heddle et al., Brit. J. Hemat. 91:1000-5

(1995)]. Another possible assay is an ELISA assay to measure the binding of antibodies to the RBC antigens using an anti-human IgG conjugated to alkaline phosphatase.

[0018] A gel card system using A/B D Monoclonal Grouping Card™ kit (Micro

Typing Systems, Pompano Beach, FL) can be used to look at the reactivity with A, B, and D antibodies (see Example 1). The gel cards contain either A, B, or D antibodies within the gel such that when a red cell is passed through the gel by centrifugation, it will agglutinate with the antibody if it contains an antigen that can bind the antibody.

The agglutinated red cells will remain at the top of the gel while intact red cells pass through to the bottom of the gel, and the cell type can be easily determined by which antibody causes the agglutination. The resulting gels can be assigned a number of either 0, 14-, 24-, 3+, or 44-, where 0 indicates essentially completely intact cells (i.e. no reactions) and 44- indicates complete agglutination. The effect of antigen masking on the antigens can be readily assessed by comparing the gel cards for a modified red cell compared to an unmodified red cell. Ideally, a red cell composition that shows agglutination with a particular antibody will show no reaction with the same antibody after it has been reacted with an antigen masking compound.

[0019] It is also possible to use certain in vivo assays to assess the immune response to a treated RBC relative to an untreated control. In vivo survival studies in animals may be done to assess the immune response, for example by assaying in vivo survival of treated red cells either across species or within a species. For example treated sheep RBC may be assessed for survival in mice. Preferably in vivo survival can be assessed within a model species, such as treated canine RBC transfused into mismatched canines. The mismatched canine RBC would elicit an immune response and survival of treated red cells can be assessed relative to untreated red cells. In such a model, an increase in the survival of treated red cells is most likely the result of the reduction in the immunogenicity of the treated red cell due to the masking of the red cell antigens.

Assessment of the level of modification of the red cells. [0020] Additional techniques may be used to estimate the level of antigen masking with methods of the present invention. One such technique may utilize a detection label on the antigen masking compound, such as radioactive or fluorescent labeled compounds. In such assays, the amount of suitably labeled PEG on the surface of the RBC can be measured directly by isolation and analysis of the RBC membranes (ghosts) or other methods of partitioning measurement for the RBC. Alternatively, the amount of fluorescently labeled PEG (FPEG) on the surface of the RBC can be directly measured using a flow cytometer. The fluorescent signal can be correlated to the relative amount of fluorescent PEG used and compared to a standard curve using, for example, beads containing known amounts of fluorescently labeled molecules. Alternatively, the FPEG results can be cross validated to another quantitative assay. An example of the measurement of the modification density of PEG modified RBC is given in Example 6. Another method of measuring the modification density involves the use of a PEG that incorporates an unnatural amino acid into the coupling group of the compound. For example, the reactive coupling group could contain 6 amino caproic acid such that this group gets attached to the red cell. The modified red cell ghosts can be treated by dissolving in acid to hydrolize proteins to free the 6 amino caproic acid group, which can be quantified by HPLC. The number of 6 amino caproic acid groups per red cell can then be calculated. Methods of the present invention will result in a preferred level of antigen masking compound bound per red cell of about 10⁴-10⁹, also about 10⁵-10⁸, also about 10⁶-10⁸, or about 10⁶-10⁷ molecules of antigen masking compound per red cell. Higher levels of modification are preferred for complete antigen masking while lower levels are adequate for the reduction in viscosity at low shear rates.

Assessment of hemolysis of the red cells. [0021] The hemolysis of a red cell composition can be assessed by comparing the hemoglobin concentration in the supernatant_^as compared to a sample that is_100% lysed. The hematocrit of a red cell composition is measured using a hematocrit centrifuge and reader. To prepare a test sample, the red cell composition is centrifuged at 12000 x g for 2 minutes, and the supernatant removed. The centrifuge step is repeated on the supernatant and 100 μl of the final supernatant is diluted into 1 mL of Drabkin's reagent (Sigma). A 100% lysis sample is prepared as a standard by dilution of 5 μl of the red cell composition into 1 mL of Drabkin's reagent. The absorbance of the test and standard samples is measured at 540 nm and the hemoglobin(Hb) concentration is measured as Hb(g/dL) = A_{5 0} x dilution factor / 6.85. From this, the percent hemolysis is calculated as (100 - hematocrit) x Test Hb (g/dL) / Standard Hb (g/dL). This can be measured at various steps throughout the processing of the red cells to assess the effects of additives on the hemolysis of the red cells. It is most important that hemolysis of the final red cell composition is reduced to acceptable levels. In one embodiment of the invention, the hemolysis observed in the final red cell composition is less than 5%, preferably less than 2%, preferably less than about 0.8%. In one embodiment, the hemolysis measured after storage of the modified red cells at 4 °C for up to 7, 14, 21, 28, 35, or 42 days is less than 2%, preferably less than 0.8%.

Antigen masking compounds. [0022] Activated antigen masking compounds for use in the invention are known in the art. A discussion of the possible activated antigen masking compounds for use in the present invention can be found in US Provisional Patent Application Serial Number 60/338,707, US Patent Application Serial Number 10/310,618, US Provisional Patent Application Serial Numbers 60/431,216, and 60/431,213, the disclosures of which are hereby incorporated by reference. Without intending to be limited to any particular mechanism of action of the present invention, activated antigen masking compounds for covalent binding to RBC will comprise a non- immunogenic group and a coupling group.

[0023] Preferred activated antigen masking compounds comprise PEG and derivatives of PEG attached to a suitable coupling group. Such PEG compounds are also referred to as activated PEG compounds and have the general formula Cp-(OCH₂CH₂) _n-OH wherein n is greater than or equal to 3 and Cp represents a coupling group which reacts with terminal thiol or amine groups on an RBC surface to covalently link the non-immunogenic group to the RBC. In one embodiment, n is about 3-1,000, also about.3-500, .also .about 1_.0_:500, also_. about J 00^500^ Derivatives wherein the end_^ groups are modified include, but are not limited to, PEG ethers (e.g.Cp -(OCH CH₂)_n- OR, such as Cp -(OCH₂CH₂) _n-OCH₃ (methoxy(polyethyleneglycol), mPEG), PEG esters (e.g. Cp-(OCH₂CH₂) _n-OOCR, such as Cp-(OCH₂CH₂) _n-OOC(CH₂)ι₄ CH₃), PEG amides (e.g. Cp-(OCH₂CH₂) _n-OOC(CH₂)₇CONHR), PEG amines (e.g. Cp- (OCH₂CH₂)_n-NH₂,), PEG acids (e.g. Cp-(OCH₂CH₂) _n-OCH₂COOH), PEG aldehydes (e.g. H-(OCH₂CH₂) _n-OCH₂CHO), and electrophilic derivatives such as halogenated PEG (e.g. H-(OCH₂CH ) _n-Br. In these examples, R is an alkyl group, preferably a linear alkyl group such as

-(CH₂)_yCH₃, where y is 0 to 20. The preferred derivatives of the present invention are those of mPEG.

[0024] In one embodiment, the coupling group for linking the non-immunogenic group to the RBCs comprises a reactive group which reacts with terminal thiol or amine groups on the RBC surface. Examples include, but are not limited to, sulphonate esters,' substituted triazines, N-hydroxysuccinimide esters, anhydrides, activated carbonates, substituted phenyl carbonates, oxycarbonylimidazoles, maleimides, aldehydes, glyoxals, carboxylates, vinyl sulphones, epoxides, mustard, mustard equivalents, isocyanates, isothiocyanates, disulphides, acrylates, allyl ethers, silanes, and cyanate esters. Mustards are herein defined as including mono or bis- (haloethyl)amine groups, and mono haloethylsulfide groups. Mustard equivalents are herein defined as groups that react by a mechanism similar to the mustards (i.e. by forming reactive intermediates such as aziridinium or aziridine complexes and sulfur analogs of these complexes). Examples of such mustard equivalents includes aziridine derivatives, mono or bis-(mesylethyl)amine groups, mono mesylethylsulfide groups, mono or bis tosylethylamine groups, and mono tosylethylsulfide groups. Other possible coupling groups are selected from 2,2,2-trifluoroethanesulphonate, pentafluorobenzenesulphonate, fluorosulphonate, 2,4,5-trifluorobenzenesulphonate, 2,4-difluorobenzenesulphonate, 2-chloro-4-fluorobenzenesulphonate, 3-chloro-4- fluorobenzenesulphonate, 4-amino-3-chlorobenzenesulphonate, 4-amino-3- fluorobenzenesulphonate, o-trifluoromethylbenzenesulphonate, m- trifluoromethylbenzenesulphonate, 2-trifluoromethoxybenzenesulphonate, 4- trifluoromethoxybenzenesulphonate, 5-fluoro-2-mefhylbenzenesulphonate, 4,6- dichlorotriazine, 6-chlorotriazine, N-hydroxysuccinimidyl succinate, N- hydroxysuccinimidyl glutarate, N-hydroxysuccinimidyl succinamide, N- hyjdroxysuccinimidylalkanedioicamMes^-hydrrø carboxymethylated polymers, N-hydroxysuccinimidyl esters of amino acids, succinimidylcarbonate, succinate mixed anhydride, succinic anhydride, 2,4,5- trichlorophenol, trichlorophenyl carbonate, nitrophenyl carbonate, 4-nitrophenol, cyanuric chloride, maleimide, N-substituted maleimide, acetaldehyde, propionaldehyde and chemically equivalent sulfur analogs, glyoxal, phenylglyoxal, acrylate, methacrylate, fluoro substituted phenyl esters and their sulfur analogs, fluoro substituted ethyl esters and fluoro substituted ethanethiol esters. In another embodiment, the coupling group is a halogen atom, preferably iodide, bromide or chloride. In some instances the reactivity of the group may be increased through the use of a catalyst. This catalyst may be an enzyme, such as transglutaminase, or a man- made or naturally occurring compound, such as iodide used as a nucleophilic catalyst, used in substoichiometric or stoichiometric amounts.

[0025] Another embodiment of the present invention contemplates branched PEG and branched PEG derivatives in which PEG arms are linked giving multi armed branched molecules. A further embodiment of branched PEG derivatives includes derivatives which can form crosslinks when bound to the red cell surface. Such branched PEG compounds bound to red cells are able to link with intermolecular red cell bound PEG forming crosslinks. Such crosslinks may provide a protective network around the red cell and be more effective at antigen masking the red cells. Generally, the branched PEGs for crosslinking would require at least another reactive electrophilic center and the use of a multivalent nucleophile such as a polyamine or a protein molecule that contains multiple nucleophiles. Reaction of the first electrophilic center of the PEG will result in attachement of the PEG to the RBC, while the other reactive center would be in a position that allows the reaction with one of the valences of the polynucleophile. Since the polynucleophile can react with multiple PEG centers it will generate a crosslinked matrix above the RBC (e.g. see US Patent Number 6,129,912). The molecular weight for the compounds can vary up to approximately 200 kDa or more. Such compounds are difficult to purify as they increase in size such that molecular weights represent an average molecular weight with a distribution in weights around this average. The desired weight ranges refer to an approximate average molecular weight for a given sample. Compounds useful in the present invention have a molecular weight range of about 2-40 kDa, also about 5-40 kDa, about 10-40 kDa, about 15-40 kDa, preferably about 20-40 kDa, more preferably

-about20 30J Da, mostpxeferahly_ahout 20-25J Da_^ The concej trati ιι actiyated_ antigen masking compound that is most effective depends to some extent on the size of the antigen masking compound used. Generally, the larger compounds require lower concentrations than the smaller compounds. Since the antigen masking compounds do not penetrate the red cell membranes, the concentrations are based on the extracellular volume of the samples being reacted. Generally, activated antigen masking compounds can be used over a range of approximately 1-50 mM. For activated antigen masking compounds in the range of about 15-40 kDa, the concentration used may be in the range of about 1-30 mM, also about 1-20 mM, also about 2-15 mM or about 2-10 mM. Antigen masking compounds in the range of about 2-15 kDa can be used at higher concentrations, such as about 5-50 mM, also about 5-30 mM, about 5-25 mM. In one embodiment, the activated PEG is in the range of about 20-30 kDa at a concentration of about 1-20 mM, also about 2-10 mM.

[0026] A preferred embodiment of an activated non-immunogenic compound is 2,2,2- trifluoroethanesulphonylmonomethoxy polyethylene glycol (Tresyl mPEG, or

TmPEG). Other preferred embodiments of an activated non-immunogenic compound are Methoxy(polyethyleneglycol)-succinimidyl propionate (mPEG-SPA-NHS),

Methoxy(polyethyleneglycol)-succinimidyl butanoate (mPEG-SBA-NHS),

Methoxy(polyethyleneglycol)-succinimidyl carbonate (mPEG-SC), N-3-

[Methoxy(polyethyleneglycol)]- øxø-aminopropionate N-hydroxysuccinimide ester

(mPEG-βA-NHS), N-6-[Methoxypoly(ethyleneglycol)]-oxo-aminocaproate N- hydroxysuccinimide ester (mPEG-6AC-ΝHS), N-5-[Methoxypoly(ethyleneglycol)]- øxø-aminovalerate N-hydroxysuccinimide ester (mPEG-5AN-ΝHS), N-4-

[Methoxypoly(ethyleneglycol)] -øxø-aminobutyrate N-hydroxysuccinimide ester

(mPEG-4AB-ΝHS), N-6-[Methoxypoly(ethyleneglycol)]-oxo-aminocaproate pentafluorophenyl ester (mPEG-6AC-PFP), N-6-[Methoxypoly(ethyleneglycol)]-oxø- aminocaproate 2,2,2-trifluoroethyl ester (mPEG-6AC-TFE), N-6-

[Methoxypoly(ethyleneglycol)]-oxo-aminocaproate ethyl ester (mPEG-6AC-OEt), N-

6-[Methoxypoly(ethyleneglycol)]-oxo-aminocaproate pentafluorobenzenethio ester

(mPEG-6AC-PFT), N-6-[Methoxypoly(ethyleneglycol)]-oxo-aminocaproate 2,3,5,6- tetrafluorobenzene thio ester (mPEG-6AC-TFT), N-6-[Methoxypoly(ethyleneglycol)]- oxo-aminocaproate 4-fluorobenzenethio ester (mPEG-6AC-4FTP), N-6- [Methoxypoly(ethyleneglycol)]-oxo-aminocaproate 2,2,2- trifluoroethylfhio ester (mPEG-6AC-TFET), andN-6-[Methoxypoly(ethyleneglycol)]-øxø-aminocaproate ethylthio ester (mPEG-6AC-SEt), the structures of which are as given below,_^ where n_ is greater than or equal to 1, preferably greater than or equal to 3. In one embodiment, n is about 3-1,000, also about 3-500, also about 10-500, also about 100-500.

Methods of reacting activated antigen masking compounds with red cells. [0027] The methods of the present invention provide adequate antigen masking of red cells with sufficiently low levels of hemolysis during processing. The conditions for reaction can be optimized to provide adequate masking of the antigens and adequate function of the resulting red cell composition for use in vivo. The methods can be optimized with respect to the buffers used during processing of the RBC. A thorough discussion of the buffering of the solution for optimal reaction of the activated antigen masking compound with the red cells can be found in US Patent Application Serial Number 10/310,618.

[0028] In one embodiment, a red cell composition is mixed with a suitable reaction solution and activated antigen masking compound. This mixture is incubated so that the activated antigen masking compound covalently binds the surface of the red cells. The incubation is done at a temperature ranging from about 4-40 °C, preferably about 20-25 °C, for at least 30 minutes, typically 30-240 minutes, preferably approximately 60-120 minutes. In one embodiment, it is preferable to wash the red cells with a suitable buffer prior to reacting with the activated antigen masking compounds. In one embodiment, the pre reaction wash solution and reaction solution comprise buffers that provide adequate buffering of the system to Dptimize the reaction of the activated antigen masking compound with the red cell surface. In one embodiment, the buffering is adequately provided by the pre reaction wash solution such that the reaction solution need not be buffered. The buffering solutions of the invention will preferably have a pH in the range of approximately 8-10, preferably approximately 9 and comprise a buffer at a concentration of approximately 50-350 mM, preferably about 100-200 mM. Buffers for use in the present invention include, but are not limited to, [(2-Hydroxy-l,l-bis[hydroxymethyl]ethyl)amino]-l-propanesulfonic acid (TAPS, pKa 8.40), 2-Amino-2-methyl-l,3-propanediol (AMPD, pKa 8.80), N-tris- (Hydroxymethyl)methyl-4-aminobutanesulfonic acid (TABS, pKa 8.90), 3-([l,l- Dimethyl-2-hydroxyethyl]amino)-2-hydroxypropanesulfonic acid (AMPSO, pKa 9.00), N-(2-Hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES, pKa 7.48), 3-(Cyclohexylamino)-2-hydroxy-l-propanesulfonic acid (CAPSO, pKa 9.60), 2-(N- Cyclohexylamino)ethanesulfonic acid (CHES, pKa 9.30), phosphate buffers, and Triethanolamine (pKa 7.8). In one embodiment, the buffers discussed above are used as a wash buffer to prepare the red cells for reaction. When the red cells are adequately washed with these buffers, the reaction solution need not be buffered. For example, the reaction solution could be unbuffered blood bank saline, or an unbuffered dextrose solution that is isotonic or hypertonic. In one embodiment, the reaction solution is an isotonic or hypertonic dextrose solution that lacks chloride ions. In another embodiment, when the red cells are washed with the above- mentioned buffers, the reaction solution can be a buffer with a pH range of about 6- 10, also about 7-9. In one embodiment, the red cells are washed with a solution comprising a buffer having a concentration of about 50-350 mM, preferably about 100-200 mM, and a pH of about 8-10. In one embodiment, the washed red cells are then reacted in a reaction solution comprising blood bank saline. In another embodiment, the washed red cells are reacted in a reaction solution comprising unbuffered dextrose at a concentration that is effectively isotonic or hypertonic, such as about 125-300 mM, preferably about 125-200 mM. In a preferred embodiment, the unbuffered dextrose solution lacks chloride ions. In another embodiment, the pH is maintained by use of a resin. Appropriate buffering conditions for both reaction of activated antigen masking compound and for long term storage may be achieved by addition of a resin material to alter the buffering capacity of the red cell solution. A resin is defined as any solid material that can achieve the change of pH without being

-dissoLvedJutLώe_solutromand-enc.o^^ such as solid minerals. Such a resin could reduce or eliminate any washing requirements in order to achieve a suitable pH for the reaction of the compounds with the red cells. A thorough discussion of such methods can be found in US Provisional

Patent Application Serial Number 60/338,707 and US Patent Application Serial

Number 10/310,618. After reaction with the activated antigen masking compound, the cells can be washed with a buffer that provides suitable conditions for storage of the modified red cells, such as a buffer that restores the pH to physiological value (i.e. approximately pH 7). The post reaction wash will preferably have a pH of about 7-

7.5, preferably about 7 and comprise a buffer at a concentration of approximately 50-

350 mM, preferably about 100-200 mM. In addition to the buffering provided by these solutions, the solutions used in the methods of the present invention are optimized to reduce the amount of hemolysis of the red cells during the processing.

Additives such as dextrose and L-camitine may be included in these solutions in order to minimize hemolysis. In another embodiment, these solutions lack chloride ions.

Any or all of the solutions used in the processing of the red cells comprise dextrose at a concentration of about 50-300 mM, preferably about 75-200 mM, more preferably approximately 100 mM or L-camitine at a concentration of about 2-100 mM, preferably about 2-10 mM, more preferably about 5 mM. In some embodiments, any or all of the solutions lack chloride ions. In some embodiments, any or all of the solutions used will comprise both dextrose and L-camitine. In some embodiments, the solutions comprising both dextrose and L-camitine will also lack chloride ions. In a preferred embodiment, all solutions used in the processing of the red cells will comprise both dextrose and L-camitine. In a further embodiment, all solutions used in the processing will lack chloride ions.

[0029] The methods for reaction of the antigen masking compounds with red cells can also be improved by reacting at higher hematocrit. Reaction of red cells with a 5 kDa mPEG-SPA-NHS at the same extracellular concentration results in the same level of antigen masking of the red cells at 40, 60 or 80% hematocrit as determined by

FACScan analysis of fluorescent labeled antibody binding to the cells. As hematocrit increases, a lesser amount of the mPEG is used with a higher number of red cells. Since level of antigen masking does not change, it is far more cost effective to react at the higher hematocrit. Therefore, it is preferred for this invention to carry out the reaction at a hematocrit of greater than about 50%, also greater than about 60%, or at approximately 60-95%. A d^jleα¹_discjassjorι of the reaction of antigen masking compounds with red cells at high hematocrit can be found in concurrently filed US Provisional Patent Application Serial Number 06/431,215.

In vitro and in vivo function of the modified red cells. [0030] The antigen masked red cell compositions resulting from the methods of the present invention, in order to be useful for in vivo use, must remain sufficiently functional. The compositions of the present invention are assessed for their in vitro and in vivo function. The in vivo function can be assessed by doing in vivo survival studies. Studies can be done to measure the in vivo survival upon infusion to another species, where an unmodified red cell would be readily eliminated by the immune response of the recipient. Such studies could also be done within the same species using a mismatched blood type, for example in a canine model. [0031] In vitro Parameters for red cell suitability are known to those skilled in the art and include, but are not limited to, measurements indicating oxygen transport activity of the RBC (as measured by oxygen affinity), intracellular adenosine 5'-triphosphate (ATP) levels, intracellular 2,3-diphosphogιycerate (2,3-DPG) levels, extracellular potassium levels, reduced glutathione (GSH) levels, hemolysis or vesiculation of the RBC, pH, hematocrit, free hemoglobin levels, osmotic fragility of the RBC, deformability of the RBC by ektacytometry, ion homeostasis (Na⁺, K⁺ and SO ^" fluxes), active cation transport (ouabain sensitive Na⁺ transport, bemetanide sensitive Na⁺, K⁺ transport), dextrose consumption and lactate production. [0032] Methods for determining ATP, 2,3-DPG, dextrose, hemoglobin, hemolysis, glutathione and potassium are available in the art. See for example, Davey et al, Transfusion, 32:525-528 (1992), the disclosure of which is incorporated herein by reference. Methods for determining RBC function are also described in Greenwalt et al, Vox Sang, 58:94-99 (1990); Hogman et al, Vox Sang, 65:271-278 (1993); Beutler et al, Blood, Vol. 59 (1982); and Beutler, RBC Metabolism, 3rd edition, Grune & Stratton, (1984) the disclosures of which are incorporated herein by reference. Extracellular sodium and potassium levels may be measured using a Ciba Coming Model 614 K⁺/Na⁺ Analyzer (Ciba Coming Diagnostics Corp., Medford, MA). The pH can be measured using a Ciba Coming Model 238 Blood Gas Analyzer (Ciba Coming Diagnostics Corp.).

[0033] These measurements are compared to an untreated control RBC composition to determine whether the function of the treated composition has been significantly

-reduced.-Jn-one_emboch^ent,_anJB^ will have extracellular potassium of no more than 3 times and more preferably no more than 2 times the level measured in an untreated control RBC composition 1 day after treatment. In another embodiment, hemolysis of the treated RBC composition is less than about 5% after treatment and after up to 42 days storage at 4 °C. In another embodiment, hemolysis of the treated RBC composition after storage at 4 °C is less than about 3% after 28 days, more preferably less than about 2% after 35 days, more preferably less than or equal to about 0.8% after 35 days, more preferably 42 days. In another embodiment, the treated RBC composition will have intracellular ATP levels that are within about 75%, also about 50%, more preferably about 25%, and more preferably about 10%, of the level of the untreated control composition directly after treatment, preferably within about 50% after 28 days storage at 4 °C, more preferably within about 50% after 42 days storage at 4 °C. In another embodiment, the treated

RBC composition will have GSH levels that are within about 75%, also about 50%, more preferably about 25%, and more preferably about 10%, of the level of the untreated control composition directly after treatment, preferably within about 50% after 28 days storage at 4 °C, more preferably within about 50% after 42 days storage at 4 °C. In another embodiment, the treated RBC composition will have intracellular

2,3-DPG levels that are within about 90%, more preferably about 50%, and more preferably about 25%, of the level of the untreated control composition directly after treatment, preferably after 7 days storage at 4 °C. In some embodiments, the red cell compositions of the invention, upon transfusion into an individual, have an in vivo survival after circulating 24 hours post transfusion of greater than approximately 40%, preferably about 50%, preferably about 60%, more preferably about 75%. In a further embodiment, the in vivo survival of red cells that have been stored up to 14 days, preferably 28 days, preferably 35 days, more preferably 42 days prior to transfusion is greater than approximately 40%, preferably about 50%, preferably about 60%, more preferably about 75% measured at 24 hours post transfusion.

Preferred embodiments of the invention.

[0034] In one embodiment, the red cells are processed by washing with a pre reaction wash solution, then the washed red cells are reacted with activated antigen masking compound with the addition of a reaction solution. In one embodiment at least one of the pre reaction wash solution and the reaction solution comprises dextrose at a concentration of about 50-300 mM, preferably about 75-200 mM, preferably approximately 100 mM. In one embodiment both the pre reaction wash solution and the reaction solution comprise dextrose at a concentration of about 50-300 mM, preferably about 75-200 mM, preferably approximately 100 mM. In one embodiment at least one of the pre reaction wash solution and the reaction solution comprises L- carnitine at a concentration of about 2-100 mM, preferably about 2-10 mM, preferably approximately 5 mM. In one embodiment both the pre reaction wash solution and the reaction solution comprise L-camitine at a concentration of about 2-100 mM, preferably about 2-10 mM, preferably approximately 5 mM. In one embodiment, at least one of the pre reaction wash solution and the reaction solution comprises both dextrose at a concentration of about 50-300 mM, preferably about 75-200 mM, preferably approximately 100 mM and L-carnitine at a concentration of about 2-100 mM, preferably about 2-10 mM, preferably approximately 5 mM. In one embodiment, both the pre reaction wash solution and the reaction solution comprise both dextrose at a concentration of about 50-300 mM, preferably about 75-200 mM, preferably approximately 100 mM and L-camitine at a concentration of about 2-100 mM, preferably about 2-10 mM, preferably approximately 5 mM. In the above embodiments, one or both of the pre reaction wash solution and reaction solutions lacks chloride ions. In one embodiment, the pre reaction wash solution comprises a buffer at a concentration of approximately about 50-350 mM, preferably about 100-

200 mM and a pH of approximately 8-10, preferably about 9. In one embodiment, the pre reaction wash solution comprises a buffer at a concentration of approximately about 50-350 mM, preferably about 100-200 mM and a pH of approximately 8-10, preferably about 9, and the reaction solution comprises dextrose at a concentration of about 50-300 mM, preferably about 125-200 mM, preferably lacking chloride ions. In one embodiment, the reaction solution comprises a buffer at a concentration of approximately 50-350 mM and a pH of approximately 8-10, preferably about 9. In one embodiment, the pre reaction wash solution and the reaction solution are the same.A preferred pre reaction wash solution and reaction solution comprises 150 mM

CHES buffer at a pH of 9, 100 mM dextrose and 5 mM L-carnitine, preferably lacking chloride ions. In one embodiment, the red cells are prepared as a red cell concentrate prior to washing with the pre reaction wash solution. In another embodiment, the reaction solution and activated antigen masking compound are added to a washed red cell concentrate to provide a reaction mixture. In a further embodiment, the reaction mixture is at a hematocrit of about 20-95%, also about 30-95%, also about 40-95%, .preferably_about^0^5S^,_preferably_abouL60^5^,Jtnore preferably_about_60-80L%. In a further embodiment, following the reaction, the modified red cells are washed with a post reaction wash solution that comprises either dextrose at a concentration of about 50-300 mM, preferably about 75-200 mM, preferably approximately 100 mM or L-carnitine at a concentration of about 2-100 mM, preferably about 2-10 mM, preferably approximately 5 mM, or both dextrose and L-camitine. In one embodiment, the post reaction wash solution comprises 150 mM Na₂HPO , 100 mM dextrose and 5 mM L-camitine, preferably lacking chloride ions. [0035] One embodiment includes a method of preparing a modified red blood cell composition comprising washing the red blood cells with a solution comprising a buffer at a concentration of about 50-350 mM and a pH of about 8-10, adding a reaction solution and an activated antigen masking compound to the washed red blood cells to form a reaction mixture, and incubating the reaction mixture so that the antigen masking compound covalently binds to the surface of the red blood cells to provide modified red blood cells, wherein the reaction solution is unbuffered. In a further embodiment, the reaction solution comprises blood bank saline. In another embodiment, the reaction solution comprises dextrose at a concentration of about 125- 200 mM, preferably lacking chloride ions. In one embodiment, the wash solution comprises either dextrose at a concentration of about 50-300 mM, also about 75-200 mM or about 100 mM, or L-camitine at a concentration of about 2-100 mM, also about 2-10 mM or about 5 mM. In a preferred embodiment, the wash solution comprises dextrose at a concentration of about 50-300 mM, also about 75-200 mM or about 100 mM and L-camitine at a concentration of about 2-100 mM, also about 2-10 mM or about 5 mM. In another embodiment, the wash solution lacks chloride ions. [0036] One embodiment of the invention encompasses a method of preparing a red cell composition comprising a) washing the red cells with a pre reaction wash solution to provide washed red cells; b) mixing the washed red cells with a reaction solution and an activated antigen masking compound to provide a reaction mixture; c) incubating the reaction mixture so that the antigen masking compound covalently binds to the surface of the red cells to provide modified red cells; and d) washing the modified red cells with a post reaction wash solution, wherein at least one of the pre reaction wash solution, reaction solution, and post reaction wash solution comprises

L-carnitine at a concentration of about 2-100 mM, preferably about 2-10 mM, preferably approximately 5 mM. In one embodiment, at least two of the pre reaction

_wash-solution,-reaction~solution-and-postreaction_wash-solution-compriseX-carnitine - at a concentration of about 2-100 mM, preferably about 2-10 mM, preferably approximately 5 mM. In one embodiment, the pre reaction wash solution, reaction solution, and post reaction solution comprise L-carnitine at a concentration of about 2-

100 mM, preferably about 2-10 mM, preferably approximately 5 mM. In one embodiment, at least one of the pre reaction wash solution, reaction solution, and post reaction wash solution further comprises dextrose at a concentration of about 50-300 mM, preferably about 75-200 mM, preferably approximately 100 mM. In one embodiment, at least two of the pre reaction wash solution, reaction solution and post reaction wash solution further comprise dextrose at a concentration of about 50-300 mM, preferably about 75-200 mM, preferably approximately 100 mM. In one embodiment, the pre reaction wash solution, reaction solution, and post reaction solution further comprise dextrose at a concentration of about 50-300 mM, preferably about 75-200 mM, preferably approximately 100 mM. In one embodiment, one, at least two, or all of the pre reaction wash solution, reaction solution and post reaction wash solution further lack chloride ions. In one embodiment, step (a) involves washing a red cell concentrate with a volume of pre reaction wash solution that is greater than or equal to the red cell concentrate volume. In one embodiment, step (b) involves mixing the reaction solution and an antigen masking compound with a washed red cell concentrate to a hematocrit of about 20-95%, also about 30-95%, about 40-95%, about 50-95%, preferably about 60-95%, more preferably about 60-

80%. In one embodiment, the pre reaction wash solution and reaction solution are at a pH of about 8-10, preferably about 9 and comprise a buffer at a concentration of about

50-350 mM, preferably about 100-200 mM. In one embodiment, the pre reaction solution and the reaction solution are the same. In one embodiment the post reaction wash solution comprises a buffer at a pH of approximately 7.

[0037] In further embodiments, the activated antigen masking compound has a molecular weight of approximately 15-40 kDa, preferably about 15-30 kDa, preferably about 20-30 kDa, more preferably about 20-25 kDa. In a further embodiment, the activated antigen masking compound is an activated PEG. In a further embodiment, the activated PEG is a methoxy(polyethyleneglycol) (mPEG) with the formula Cp - (OCH₂CH₂)_n-OCH₃, where Cp represents an activated coupling group and n is about 3-1,000, also about 3-500, also about 10-500, also about 100-500. In a further embodiment, the activated mPEG is selected from the group consisting of mPEG- SBA-ftTHS_rιιιP^G_JSBA=I.fflS,.^^ mPEG-5AN-NHS, mPEG- 4AB-NHS, mPEG-6AC-PFP, mPEG-6AC-TFE, mPEG- 6AC-OEt, mPEG-6AC-PFT, mPEG-6AC-TFT, mPEG-6AC-4FTP, mPEG-6AC- TFET, and mPEG-6AC-SEt. In a preferred embodiment, the activated mPEG is selected from the group consisting of mPEG-6AC-NHS, mPEG-5AN-NHS, and mPEG-4AB-NHS. In a more preferred embodiment, the activated mPEG is mPEG- 6AC-NHS. In one embodiment, the incubation of red cells with the antigen masking compound is at room temperature (approximately 20-25 °C). In one embodiment, the incubation is done at approximately 4 °C. In another embodiment, the incubation is for approximately 30 - 120 minutes, preferably for approximately 1 hour. [0038] One embodiment of the invention encompasses a method of preparing a modified red cell composition comprising a) providing i) a red cell concentrate, ii) an mPEG compound having the formula Cp -(OCH₂CH₂) _n-OCH₃, where Cp represents an activated coupling group and n is about 3-1,000, also about 3-500, also about 10- 500, also about 100-500, iii) a reaction solution comprising dextrose at a concentration of about 50-300 mM, and iv) a wash solution comprising dextrose at a concentration of about 50-300 mM; b) washing the red cell concentrate with the reaction solution to provide a washed red cell concentrate c) mixing the washed red cell concentrate with the reaction solution and the mPEG compound; d) incubating the red cell composition with the reaction solution and the mPEG compound such that the mPEG covalently binds to the surface of the red cells providing a modified red cell solution; and e) washing the modified red cell solution with the wash solution. In one embodiment, In a further embodiment, the activated mPEG is selected from the group consisting of mPEG-SPA-NHS, mPEG-SBA-NHS, mPEG-SC, mPEG-βA-NHS, mPEG-6AC-NHS, mPEG-5AN-NHS, mPEG- 4AB-NHS, mPEG-6AC-PFP, mPEG- 6AC-TFE, mPEG-6AC-OEt, mPEG-6AC-PFT, mPEG-6AC-TFT, mPEG-6AC-4FTP, mPEG-6AC-TFET, and mPEG-6AC-SEt. In a preferred embodiment, the activated mPEG is selected from the group consisting of mPEG-6AC-NHS, mPEG-5AV-NHS, and mPEG-4AB-NHS. In a more preferred embodiment, the activated mPEG is mPEG-6AC-NHS. In one embodiment, the incubation of red cells with the mPEG compound is at a hematocrit of about 20-95%, also about 30-95%, about 40-95%, about 50-95%, preferably about 60-95%, more preferably about 60-80%. In one embodiment, the incubation is at room temperature (approximately 20-25 °C). In one embodiment, the incubation is done at approximately 4 °C. In another embodiment,

hour. In a further embodiment, the reaction solution comprises a buffer selected from the group consisting of TAPS, AMPD, TABS, AMPSO, HEPES, CAPSO, and CHES. In a further embodiment, the reaction solution comprises a buffer at approximately 150 mM and a pH of approximately 9. In a preferred embodiment, the reaction solution comprises approximately 150 mM CHES at pH 9. In one embodiment, the reaction solution further comprises L-camitine. In one embodiment, the reaction solution further lacks chloride ions. , In a preferred embodiment, the reaction solution comprises 150 mM CHES, 100 mM dextrose, and 5 mM L-carnitine at a pH of 9, preferably lacking chloride ions. In one embodiment, the wash solution comprises a buffer at approximately 150 mM and a pH of approximately 7. In one embodiment, the wash solution comprises phosphate. In one embodiment, the wash solution further comprises L-carnitine. In one embodiment, the wash solution further lacks chloride ions. In a preferred embodiment, the wash solution comprises 150 mM disodium phosphate, 100 mM dextrose, and 5 mM L-carnitine, preferably lacking chloride ions. In a preferred embodiment, the post reaction wash involves adding a volume of the wash solution equal to that of the reaction mixture directly to the reaction mixture. This solution is centrifuged and the supernatant removed to give a red cell concentrate, which can be washed again with wash solution. In another embodiment, following the post reaction wash of the red cells, they are stored in a suitable storage solution at a hematocrit of approximately 40-60%. In a further embodiment, the suitable storage solution is either Adsol™ (comprising 154 mM NaCl, 2.0 mM adenine, 41.2 mM mannitol, and 111.0 mM dextrose, Baxter Healthcare, EL), or Erythrosol™ (Erythrosol consists of 94 mL part A (25.0 mM sodium citrate, 16.0 mM disodium phosphate, 4.4 mM monosodium phosphate, 1.5 mM adenine, 39.9 mM mannitol), and 20 mL part B (8% dextrose), Baxter Healthcare, IL). In another embodiment, the post reaction wash solution is the same as the storage solution. Other red cell storage solutions include Nutricel^® (70 mM NaCl, 2.2 mM adenine, 61 mM dextrose, 2 mM sodium citrate, 23 mM Na₂HP0₄, 2.2 mM citric acid, Miles, IN), Optisol^® (150 mM NaCl, 2.2 mM adenine, 45.4 mM dextrose, 45.4 mM mannitol, Terumo) and SAGM (150 mM NaCl, 1.6 mM adenine, 50 mM dextrose, 29 mM mannitol). In another embodiment, the above methods are performed using an automated system that provides the appropriate concentration of compound at the desired reaction hematocrit. In one embodiment, the invention comprises a

-comμosi onxifj odifleiljed-cells comprising red cells that have been prepared by the_ methods discussed above. In another embodiment, the invention comprises the use of such red cell compositions for transfusion using techniques known in the art. An additional embodiment of the present invention is a medicament comprising RBC prepared by the methods discussed above. Another embodiment contemplates an

RBC processing system comprising compositions or medicaments as described above and a suitable container for storing the RBC composition wherein the RBC composition is suitable for delivery to an individual. In a preferred embodiment, the container is a blood bag. .,

EXPERIMENTAL

EXAMPLE 1

Determination of agglutination reaction of RBC of the present invention.

[0039] The process of antigen masking of red cells is carried out under appropriate conditions on CPDA-1 collected RBC. PEG derivatives are commercially available (e.g. Shearwater Polymers Huntsville, AL). Agglutination reactions of the treated RBC are assayed by standard techniques as described in Walker et al., AABB Technical Manual, 10th Ed., pp. 528-537 (1990). The agglutination reaction is assessed on serially diluted samples. The dilution level at which agglutination no longer is observed is recorded for treated RBC compared to untreated RBC. This assay is carried out using type A RBC and anti-A antibody or antiserum or type B RBC and anti-B antibody or antiserum. The processing of the RBC with respect to antigen masking can be optimized in part based on this assay. [0040] Similar assays can be done using Rh positive RBC and anti-D antiserum. In this assay, the agglutination will be scored as described in the AABB technical manual. The treated RBC will be compared to an untreated control sample to assess ability of the process to mask the D antigen.

[0041] Similarly, a non-immunogenic red cell composition can be assayed using an A/B D Monoclonal Grouping Card™ kit (Micro Typing Systems, Pompario Beach, FL). The desired red cell sample at a hematocrit of approximately 40 % is diluted to approximately 4 % with MTS Diluent 2 Plus (typically, 50 μl of red cells are diluted with 0.5 mL of diluent). A 10-12.5 μl aliquot of the red cell sample is added to an Ηti=A/B/ ~rrucrotabeτ- τ ie^^ centrifuged using the MTS centrifuge. The Gel Card is then observed and scored for agglutination. Agglutination is graded as 0, 14-, 24-, 34-, or 44-. This range has 0 indicating no reaction with the red cells, all cells pelleting at the bottom of the microtube and 44- indicating complete agglutination with a layer of cells at the top of the gel). There may be cases where a mixed field results, i.e. there are some cells at both the top and bottom of the gel.

EXAMPLE 2 Assessment in vitro of RBC function after processing of RBC.

[0042] The intracellular adenosine-5'-triphosphate (ATP), intracellular 2,3- diphosphoglyceric acid (2,3-DPG), extracellular potassium, extracellular and intracellular pH, and hemolysis levels are readily assessed following processing of the RBC with compounds and methods of the present invention. The results are compared to untreated control samples to assess whether the treated RBC are suitable for their intended use, such as transfusion. Intracellular ATP and 2,3-DPG are measured using a Sigma ATP Kit or 2,3-DPG kit respectively (Sigma, St. Louis, Mo.). The ATP kit was used following Sigma procedure No. 366-UN hereby incorporated by reference. Extracellular potassium levels can be measured using a Ciba Coming 614 K⁺/Νa⁺ Analyzer (Ciba Coming Diagnostics Corp., Medfield, Ma.). The extracellular pH can be measured by centrifuging the cells at 4 °C for 15 minutes at 12,000 x g and removing the supernatant. The supernatant pH is measured on a standard pH meter at room temperature (e.g. Beckman, Epoxy Calomel electrode). For the intracellular pH, the remaining pellet in a centrifuge tube is capped and stored at approximately -80 °C for at least 2 hours, then lysed by adding deionized water. The lysed sample is well mixed and the pH of the solution is measured either at room temperature using a standard pH meter or at 37 °C using a Ciba-Coming model 238 blood gas analyzer. EXAMPLE 3 Evaluation of the oxygen affinity of the processed RBC.

[O-043JJEollowing-me_processmg present invention, oxygen affinity of the RBC samples is measured with a Hemox analyzer. The Hemox analyzer is pre-equilibrated at 37 °C. Fifty μL of the RBC sample is mixed with 3.97 mL Hemox buffer solution (TCS Scientific Corp., New Hope, PA), containing 20 μL of 20% Bovine Serum Albumin (TCS Scientific Corp.) and 10 μL anti-foaming reagent (TCS Scientific Corp.) before transferring into the Hemox Analyzer cuvette. After the diluted sample is drawn into the cuvette, the temperature of the mixture is equilibrated with stirring for 8 minutes at 37 °C. Subsequently, the diluted sample is fully oxygenated by exposure to air for 8 minutes. The instrument is calibrated for the partial pressure reading and the degree of hemoglobin saturation for each sample. The log ratio of the solution absorption at 560 to the absorption at 570 nm is recorded on the Y-axis while the partial pressure of oxygen (pθ₂) obtained from a Clark electrode is recorded on the X-axis. The X-axis is calibrated by assigning values of 0 and the maximum calculated pθ₂ for the day to readings obtained from 100% nitrogen and 100% air. The Y-axis is calibrated by assigning values of 0 and 1 to readings obtained from hemoglobin equilibrated under nitrogen or oxygen, respectively. For each sample an oxygen affinity curve is obtained by lowering the pθ₂ through the introduction of nitrogen to the space above the liquid sample and measuring the percent of oxygen saturation of hemoglobin. The numerical data is converted to a graph of the oxygen affinity curve through the use of the computer program Kaleidagraph 3.0.5 (Synergy Software, Reading, PA) and the P5₀ is determined from the half point of the curve. Measurements can be made on treated samples and compared to measurements of untreated control samples.

EXAMPLE 4 Evaluation of the osmotic fragility of the processed RBC.

[0044] The osmotic fragility of samples is measured for RBC processed with compounds and methods of the present invention and compared to untreated control samples. Reagent is prepared at 0.1, 0.2, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.75, and 0.9 % PBS (1.0% PBS is 9g NaCl, 1.365g Na₂HPO₄, and 0.186g NaH₂PO₄ to a final volume of 1 liter in water). A 10 μL aliquot of RBC sample is added to 1.0 mL of each of these solutions, mixed gently and incubated at room temperature for 30

the_sample is mixed gently and centrifuged for 2 minutes at 2,000 x g. A spectrophotometer is zeroed with water and the absorption of the supernatant of the sample is measured at 540 nm. The % lysis is calculated using the following formula, in which the 0.9% PBS sample is considered background lysis and the 0.1% PBS sample is considered to be 100% lysis.

[0045] % lysis = (A₅₄₀ - 0.9% A₅₄₀) ÷ (0.1% A₅₄₀% - 0.9% A₅₄₀) x 100

The % lysis is plotted as a function of the %PBS and the plots are compared for treated RBC and untreated control RBC.

EXAMPLE 5 Flow Cytometry analysis of RBC to assess levels of PEG modification.

[0046] A unit of ABO-typed whole blood (Sacramento Blood Center, CA) is leukofiltered according to standard blood banking methods. The RBC are washed with a buffer comprising 150 mM CHES at a pH of 9.0 to eliminate plasma proteins and adjust the pH of the extracellular domain to the desired value for the reaction. A solution of activated mPEG (5 kDa) is prepared in the CHES buffer and an aliquot of the RBC suspension is added to this solution resulting in a final concentration of mPEG of 22 mM in the extracellular volume at a hematocrit of 40%. The solution is mixed by gentle vortexing and inversion and incubated for 1 hour at room temperature. Following this room temperature incubation, the solution is washed three times with blood bank saline (BBS, 154 mM NaCl, Baxter Healthcare) to remove any excess mPEG and any other reaction side products. Following this wash, an approximately equal volume of Adsol is added. The resulting RBC suspension is stored at 4°C.

[0047] The modified cells are analyzed for their ability to bind fluorescently labeled antibody with a flow cytometry method using a FACScan. An aliquot of cells is centrifuged and the supernatant removed. A 50 μL portion of RBC (approximately lxlO⁶ cells) is incubated at room temperature for 1 hour with 5μL of an appropriate stock antibody solution (i.e. antibody would bind non mPEG modified RBC, e.g. anti- A FITC conjugate BRIC-145, anti-B FITC conjugate BGRL1, or anti-D FITC conjugate BRAD-3 depending on the blood type, International Blood Group Reference Laboratory, UK). The cells are subsequently washed with BBS to remove the excess of the antibody and are analyzed by flow cytometry for bound fluorescent antibodies. The level of bound fluorescent antibodies is compared to either non mPEG modified cells (positive control) or cells which are not incubated with FITC antibody (negative control). The relative degree of PEG modification is estimated based on the ratio of the population maximum fluorescence (test article - negative control) / (positive control - negative control). This is represented as A2/A1 in Figure 1. This calculation can be reported as a percent binding of antibody relative to a positive control.

EXAMPLE 6 Measurement of modification density for PEG Modified RBC

[0048] Leukofiltered RBC (approximately 60% hematocrit) containing a suitable additive solution such as Erythrosol are centrifuged to an 80-95% hematocrit (red cell concentrate), washed twice with buffer (e.g. pH 9.0 CHES, 150 mM CHES, 50 mM NaCl) and subsequently diluted to a hematocrit of 40% into a CHES buffer solution containing mPEG-SPA-NHS (or other activated PEG) at an appropriate concentration. In addition, the activated PEG is a mixture of the activated mPEG plus FITC labeled activated PEG (FPEG) with the same coupling group, which bears a fluorescent label on the end opposite the coupling group. Alternatively, the activated mPEG is modified with other detectable labels, such as a radioactive isotope. A 50:50 mixture of mPEG-SPA-NHS to FPEG-SPA-NHS is used for this experiment. The reaction is allowed to proceed for 2 hours at room temperature (RT) and the cells are subsequently washed to remove the reaction side products and any fluorescent label that is not attached to the red cells. RBC concentrate (200 μL) is subsequently used to make ghost membranes through controlled lysis with chilled hypotonic lysis buffer (1600μL,7.5 mM sodium phosphate, lmM NaEDTA, pH 7.5). The resulting ghosts are isolated through centrifugation (14000 x g; 2 min) and washed a total of 4 times with chilled lysis buffer and then suspended in a 250μL volume of the same buffer. SDS is added to the suspension to a final concentration of 1% SDS in order to achieve complete dissolution of the membranes. The resulting solution is further diluted 5 fold in lysis buffer and then analyzed for fluorescent label content (λ_exc=490nm, λ_emm=525nm). The amount of fluorescent label is quantitated versus a standard curve prepared by adding specific amounts of FPEG in the dissolved ghost membranes in a lysis buffer containing SDS, prepared as per the reacted samples above. The fluorescence reading is plotted against the known concentration of FPEG added.to he_^ghQs.tjmernbrane-preparation and this curve is used to calculate he FFjBG concentration corresponding to a fluorescence reading in the ghosts that have been reacted with the activated FPEG. Based on this calculated concentration of FPEG on the ghosts (or the concentration of another suitable label) and the number of cells used to prepare the ghosts, the amount of FPEG per cell (membrane modification density) can be calculated for a given experiment (Figure 2A). From the FPEG:mPEG ratio, the number of total PEG molecules per cell can be calculated. An aliquot of the red cells that are modified with the FPEG can also be analyzed by FACScan (counting a set number of red cells). The calculated PEG molecules per cell can be plotted against the FACScan peak value (FL1 -Height) and this curve can be used on new samples to calculate the amount of binding directly from the FACScan reading of the modified red cells (Figure 2B).

[0049] As a an example of the calculations involved, ghosts were prepared at a level of 1.3 x 10⁹ cells and dosed with known concentrations of FPEG and the fluorescence measured to generate the line from Figure 2A. Ghosts were prepared from 5 kDa mPEG-SPA-NHS / FPEG-SPA-NHS modified red cells (reacted as described above, only using pH 8.0 HEPES, 150 mM HEPES, 50 mM NaCl, 75 mM glucose) and the modification density was measured at each concentration of PEG used in the reaction.

The 13.2 mM PEG sample gave a fluorescence reading of 1495, which from Figure

2A was calculated to be 9.8 μM. As the actual samples were only 50% FPEG, the total PEG for this sample was 19.6 μM. Based on the known cell number in the sample (1 mL volume), the total number of moles of PEG per cell was calculated as

19.6 μM x 10^"6 (mole/μmole) x 10^"3 (L/mL). The moles of PEG per cell was then

1.96 x 10^"8 moles / 1.3 x 10⁹ cells. Using Avogadros number, the total amount of

PEG per cell was calculated to be 9 x 10⁶.

[0050] An additional method of use of the FPEG approach for the measurement of

RBC PEG modification is achieved through the use of flow cytometry analysis of the pegylated RBC using a FACScan device. The RBC are directly analyzed for fluorescence intensity through a commercial device. The number of PEG molecules attached to the RBC surface is proportional to the percent of active FPEG in the active

PEG. The FACScan fluorescent signal intensity is proportional to the PEG content. A standard curve of PEG modification done either through the method above or by comparison to beads containing known amounts of fluorescent molecules on them can be used to quantify fluorescent label amounts. Beads used in a FACScan device are j.ommexciaUy_ay ilab e_and^an.be_prepared to custom specifications (Bangs_.

Laboratories, Fisher, IN).

[0051] An alternative method for the quantitation of the PEG molecules is the use of radioactively labeled activated mPEG (labeled with covalently attached ³H, ¹⁴C or other appropriate radioactive atom). The RBC are washed after the end of the PEG modification procedure and then the washed RBC are lysed, decolorized and the radioactivity content is measured through liquid scintillation. The extent of PEG modification is calculated using the specific activity of the radiolabeled activated mPEG.

[0052] Another method involves the use of an mPEG that contains an unnatural amino acid in the coupling group, such as mPEG-6AC-NHS (6-amino caproic acid is an unnatural amino acid). The reaction of this with red cells will deposit a number of the unnatural amino acids on the surface of the red cells that corresponds to the number of mPEG molecules on the surface of the red cells. The RBC are lysed after

PEG modification along with control RBC that are unmodified. Ghosts are prepared from both populations for a known number of cells, the samples are treated to release free amino acids, and the amino acid content is measured for both preparations through the use of an HPLC assay (Sartore et al., Applied Biochemistry and

Biotechnology 31:213, 1991). For example, with mPEG-6AC-NHS, the 6AC content will be compared to the number of natural amino acids. Since the control sample will give you the number of natural amino acids per red cell, the ratio between 6AC and the natural amino acids can be used to quantify the amount of 6AC per red cell, which gives the amount of mPEG per red cell. Alternatively, the number of 6AC can be determined for a known number of red cells and the mPEG per red cell can be calculated directly.

[0053] The modification density for an antigen masking compound of a certain size can be determined as a function of the concentration used and correlated with agglutination assays or antibody binding assays to estimate the level of modification density necessary to get adequate coverage of the red cell antigens. EXAMPLE 7

The effect of pH on the extent of PEG modification of RBC.

[0054] A unit of B-t- whole blood (Sacramento Blood Center, CA) was leukofiltered according to standard blood banking methods. The RBC were centrifuged at 4 °C at 4100 x g for 6 minutes and the plasma was removed to provide a red cell concentrate. The red cell concentrate was divided into four samples and washed with four different buffers and reacted with a 50:50 mixture of 5 kDa mPEG-SPA-NHS and SPA FPEG in the same buffer. The buffers used were PBS pH 7.0 (150 mM Na₂HPO₄, 50 mM NaCl), HEPES pH 8.0 (150 mM HEPES, 50 mM NaCl), CHES pH 9.0 (150 mM CHES, 50 mM NaCl), and CAPSO pH 10.0 (150 mM CHES, 50 mM NaCl). These particular buffers were selected to have good buffering capacity at the desired pH ranges. The red cells were washed twice with lx volume of the buffer. A solution of mPEG-SPA-NHS / SPA FPEG was prepared in each buffer and added to the blood to give a final concentration of the PEG mixture of approximately 22 mM in the extracellular volume at a hematocrit of 40%. The reaction was allowed to proceed for 2 hours at RT in each of the four buffers. The pH was monitored at 30 minute intervals during the reaction and the amount of PEG bound to the red cells was assessed by measurement of the fluorescence associated with the red cells by FACScan analysis (Figure 5). The fluorescent intensity peak value for the samples were pH 9 = 4032, pH 10 = 3786, pH 8 = 3220 and pH 7 = 1928. The results indicate that a buffer pH in the range of 8-10 is preferable with the pH 9 buffer giving the greatest amount of PEG modification of the cells. A similar experiment was done using the same buffers with the inclusion of 75 mM dextrose in all buffers. The PEG modification with the pH 10 buffer was reduced with dextrose while the others were not changed significantly. The fluorescent peak values for this study were pH 9 = 1155, pH 8 = 890, pH 10 = 784, pH 7 = 573. Note that the peak values are not comparable from one experiment to another as the extent of PEG modification depends on the red cells. EXAMPLE 8

Comparison of hemolysis levels for PEG modification of red cells with and without dextrose.

[0055] A unit of A4- whole blood (Sacramento Blood Center, CA) was leukofiltered according to standard blood banking methods. The RBC (4 x 10 mL samples) were centrifuged at 4 °C at 4100 x g for 6 minutes and the plasma was removed to provide a red cell concentrate (RCC). Red cell concentrates were washed twice with 150 mM CHES pH 9 containing either 100 mM dextrose or 50 mM NaCl, or both 50 mM NaCl and 100 mM dextrose (wash volume approximately equal to RCC volume). A sample of 5 kDa mPEG-SPA-NHS was prepared by dissolving in each of the wash solutions. These were added to the red cell concentrate washed with the same buffer to give a 22 mM mPEG-SPA-NHS concentration in the extracellular volume at a hematocrit of approximately 40%. In addition, a sample of mPEGOH was dissolved in the 100 mM dextrose buffer and added to a 100 mM dextrose washed red cell concentrate as a control (22 mM, 40% hematocrit). The samples were gently mixed and incubated at room temperature for one hour. Following the incubation, the red cells are washed with an equal volume of 150 mM Na₂HP0 at a pH of 7, containing either 50 mM NaCl or 100 mM dextrose, or both 50 mM NaCl and 100 mM dextrose (the pH 7 wash components corresponded to the reaction solutions). These are then centrifuged at 4 °C at 4100 x g for 6 minutes and the supernatant is discarded. This wash is repeated and the final red cell pellet is suspended in an approximately equal volume of Adsol. Each sample is assayed using the FACScan assay described in Example 5, using an anti-A FITC conjugate. The level of hemolysis was also measured for each sample after Adsol addition. The results are given in Table 1. Although the sample without glucose showed better coverage of antigens, all samples showed adequate blocking of anti-A antibody binding (0% antibody binding indicates complete blocking while 100% antibody binding indicates no effect). These results show that dextrose added to the buffers, with or without NaCl, provides less hemolysis during the processing, where dextrose without NaCl is preferred. The control sample suggests that the hemolysis is due mainly to the reaction of the mPEG with the red cells. Table 1. Anti-A antibody binding and hemolysis results for red cells modified with 5 kDa mPEG-SPA-NHS using buffers with and without dextrose.

EXAMPLE 9 Comparison of hemolysis levels for PEG modification of red cells with dextrose at various steps in the processing.

[0056] This example compares the effects of having dextrose in either the pre reaction wash solution, the reaction solution, or both. A unit of AH- whole blood (Sacramento Blood Center, CA) was leukofiltered according to standard blood banking methods. The RBC were centrifuged at 4 °C at 4100 x g for 6 minutes and the plasma was removed to provide a red cell concentrate (RCC). Red cell concentrates were washed twice with 150 mM CHES pH 9 containing either 100 mM dextrose or 50 mM NaCl (wash volume approximately equal to RCC volume). A sample of 5 kDa mPEG-SPA- NHS was prepared by dissolving the solid in 150 mM CHES pH 9 containing 50 mM NaCl, or CHES pH 9 containing 100 mM dextrose. These were added to the red cell concentrate washed with the same buffer, to give a 22 mM mPEG-SPA-NHS concentration in the extracellular volume at a hematocrit of approximately 40%. An mPEGOH control sample was prepared as well (150 mM CHES pH 9 with 50 mM NaCl wash and reaction, 22 mM mPEGOH, 40% hematocrit). The samples were gently mixed and incubated at room temperature for one hour. Following the incubation, all samples are washed with an equal volume of 150 mM Na₂HPO₄ pH 7 with 75 mM dextrose. These are then centrifuged at 4 °C at 4100 x g for 6 minutes and the supernatant is discarded. This wash is repeated and the final red cell pellet is suspended in an approximately equal volume of Adsol. Each sample is assayed using the FACScan assay described in Example 5, using an anti-A FITC conjugate. The level of hemolysis was also measured jfor each sample._. The results are given in Table 2. The results indicate that dextrose in either the pre reaction wash or in the reaction solution results in less hemolysis, with dextrose in both solutions giving the lowest level of hemolysis. Samples all resulted in the same amount of anti-A antibody binding.

Table 2. Anti-A antibody binding and hemolysis results for red cells modified with 5 kDa mPEG-SPA-NHS using buffers with dextrose in the pre reaction wash vs. reaction steps.

[0057] Another experiment was done as above to look at the effect of dextrose in the post reaction wash solution. Samples were processed with 150 mM CHES pH 9 and 100 mM dextrose in the pre reaction wash and reaction solution, then 150 mM Na₂HPO₄ pH 7, 50 mM NaCl with or without 100 mM dextrose in the post reaction wash, or with 150 mM CHES pH 9 and 50 mM NaCl in the pre reaction wash and reaction, and 150 mM Na₂HPO₄ pH 7, 50 mM NaCl with or without 100 mM dextrose in the post reaction wash. The mPEGOH control was done with 150 mM CHES pH 9 and 50 mM NaCl in the pre reaction wash and mock reaction, and 150 mM Na₂HPO pH 7, 50 mM NaCl with 100 mM dextrose as the post reaction wash. The results for anti-A antibody binding and hemolysis are given in Table 3. The results indicate that dextrose in the post reaction wash reduces hemolysis considerably when there is no dextrose in any other part of the process. It also shows slight improvement when dextrose is used in both the pre reaction wash and reaction steps. The antibody results show adequate masking of antigens, although the samples without dextrose in the reaction step showed slightly better masking of antigens. Table 3. Anti-A antibody binding and hemolysis results for red cells modified with 5 kDa mPEG-SPA-NHS using buffers with or without 100 mM dextrose and 50 mM NaCl.

EXAMPLE 10 Comparison of hemolysis with dextrose at 100 mM vs. 200 mM.

[0058] A unit of AH- whole blood was processed as per Example 9 with reaction of a 5kDa mPEG-SPA-NHS at 22 mM and 40% hematocrit. A control mPEGOH sample is processed with 100 mM dextrose in the CHES and phosphate buffers. One rnPEG- SPA-NHS sample is processed with 150 mM CHES pH 9, 50 mM NaCl (pre reaction wash and reaction) and 150 mM Na₂HPO pH 7, 75 mM dextrose (post reaction wash). One sample was processed with 150 mM CHES pH 9, 100 mM dextrose (pre reaction wash and reaction) and 150 mM Na₂HPO H 7, 100 mM dextrose (post reaction wash). One sample was processed with 150 mM CHES pH 9, 200 mM dextrose (pre reaction wash and reaction) and 150 mM Na₂HPO₄ pH 7, 200 mM dextrose (post reaction wash). The final red cell pellet is suspended in an approximately equal volume of Adsol. Each sample is assayed using the FACScan assay described in Example 5, using an anti-A FITC conjugate. The level of hemolysis was also measured for each sample. The results are given in Table 4. The results indicate that dextrose at 100 mM results in lower hemolysis than dextrose at 200 mM, although 200 mM is still preferable to no dextrose in the pre reaction and reaction. Table 4. Anti-A antibody binding and hemolysis results for red cells modified with 5 kDa mPEG-SPA-NHS using buffers with 100 vs. 200 mM dextrose.

EXAMPLE 11 Hemolysis of PEG modification of red cells with 5 kDa or 20 kDa mPEG-6AC-MHS with dextrose present.

[0059] A unit of A+ whole blood was processed as per Example 9 with reaction of a 5kDa (22 mM) or 20 kDa (5.5 mM) mPEG-6AC-NHS at 40% hematocrit. A control mPEGOH (5 and 20 kDa) sample is processed as well. All samples are processed with 150 mM CHES, 100 mM dextrose pH 9 (pre reaction wash and reaction solution) and 150 mM Na₂HPO , 100 mM dextrose pH 7 (post reaction wash). The final red cell pellet is suspended in an approximately equal volume of Erythrosol. Each sample is assayed using the FACScan assay described in Example 5, using an anti-A FITC conjugate. The level of hemolysis was also measured for each sample. The results for antibody binding and hemolysis are given in Table 5.

Table 5. Anti-A antibody binding and hemolysis for red cells modified with 5 kDa or 20 kDa mPEG-6AC-NHS with 100 mM dextrose in all buffers.

EXAMPLE 12 The effect of L-carnitine on hemolysis for reaction of 5 kDa mPEG-SPA-NHS with red cells.

[0060] A unit of A+ whole blood was processed as per Example 9 with reaction of a 5kDa mPEG-SPA-NHS at 22 mM and 40% hematocrit. Reaction buffer contained 150 mM CHES pH 9, 100 mM dextrose. Post reaction wash solution contained 150 mM Na₂HPO pH 7, 100 mM dextrose. These buffers were prepared with and without 5 mM L-carnitine and reactions were done with L-camitine in the reaction buffer only, both pre reaction wash and reaction buffer, in the post reaction wash only, in all three buffers, or in none of them. mPEGOH control was processed without L- carnitine in any buffer. The final red cell pellet is suspended in an approximately equal volume of Erythrosol. Each sample is assayed using the FACScan assay described in Example 5, using an anti-A FITC conjugate. The level of hemolysis was also measured for each sample. The results for antibody binding and hemolysis are given in Table 6. The L-carnitine reduced hemolysis in all samples, with the addition of L-carnitine to all buffers having the greatest reduction in hemolysis. Table 6. Anti-A antibody binding and hemolysis for red cells modified with 5 kDa mPEG-SPA-NHS with buffers including dextrose with L-camitine present as indicated.

EXAMPLE 13 Hemolysis of red cells reacted with 5 kDa or 20 kDa with dextrose in all buffers with and without L-carnitine.

[0061] A unit of A+ whole blood was processed as per Example 9 with reaction of a 5kDa mPEG-SPA-NHS at 22 mM and 40% hematocrit. Reaction buffer contained 150 mM CHES pH 9, 100 mM dextrose with and without 5 mM L-carnitine and was used for the pre reaction wash of the red cells. Post reaction wash solution contained 150 mM Na₂HPO₄ pH 7, 100 mM dextrose with and without 5 mM L-carnitine. Control mPEGOH samples (5 kDa) were prepared using buffers with and without L- carnitine. The final red cell pellet is suspended in an approximately equal volume of Erythrol. Each sample is assayed using the FACScan assay described in Example 5, using an anti-A FITC conjugate. The level of hemolysis was also measured for each sample. The results are given in Table 7. Table 7. Anti-A antibody binding and hemolysis for red cells modified with 5 kDa mPEG-SPA-NHS with dextrose with or without L-camitine in all steps.

EXAMPLE 14 Reaction of a full RBC unit with 20 kDa mPEG-6AC-NHS.

[0062] A unit of A+ whole blood was leukofiltered according to standard blood banking methods. The RBC, contained in a blood bag, were centrifuged at 4 °C for 6 minutes at 4100 x g and the plasma was removed. The red cell concentrate (approximately 80% hematocrit) was washed with approximately an equal volume of 150 mM CHES pH 9, 100 mM dextrose, 5 mM L-carnitine (approximately 200 mL red cell concentrate and 200 mL buffer) and centrifuged as above, and the supernatant was removed. The wash procedure was repeated. A 10.7 g sample of 20 kDa mPEG- 6AC-NHS was dissolved in 67 mL of 150 mM CHES pH 9, 100 mM dextrose, 5 mM L-carnitine and added to the approximately 200 mL washed red cell concentrate to give approximately 5 mM mPEG-6AC-NHS in the extracellular volume at a hematocrit of approximately 60%. The reaction mixture was mixed by grasping each end-θf .the, blood bag and using a figure 8 motion approximately 30 times and incubated at room temperature for approximately 1 hour. Following incubation 200 mL of 150 mM Na₂HPO₄ pH 7, 100 mM dextrose, 5 mM L-camitine was added to the approximately 267 mL RBC sample. This was mixed using the figure 8 technique and centrifuged at 4 °C for 6 minutes at 4100 x g. The supernatant was removed and the red cell concentrate was washed again with 200 mL of the phosphate buffer. The wash buffer was removed and the final red cell concentrate was suspended in

Erythrosol (Erythrosol is added as 94 mL part A and 20 mL part B(8% dextrose)), and stored at 4 °C. The amount of anti-A antibody binding was assessed as per Example

6, agglutination tested as per Example 1 initially and after 21 and 42 days storage at 4

°C. The anti-A antibody binding was 0% at all points and the gel cards showed no reaction as well. The hemolysis, ATP levels, potassium levels, glutathione levels

(GSH), and both intracellular and extracellular pH, were measured initially and after storage at 4 °C for 2, 7, 14, 21, 28, 35, and 42 days. These were compared to control samples, either red cells stored by standard methods (4 °C control prepared directly with Erythrosol) or red cells that are processed as above without the mPEG-6AC-NHS

(wash control) and red cells that are processed with 20 kDa mPEG-OH. The in vitro function results are found in Tables 8A-H. The results show that a full unit can be modified to adequately mask antigens and provide suitable values for hemolysis, potassium, glutathione, ATP, and both intracellular and extracellular pH.

Table 8A Day 0 in vitro measurements of red cell function for a full unit modified with 20 kDa mPEG-6AC-NHS.

Table 8B Day 2 in vitro measurements of red cell function for a full unit modified with 20 kDa mPEG-6AC-NHS .

Table 8C Day 7 in vitro measurements of red cell function for a full unit modified with 20 kDa mPEG-6AC-NHS.

Table-8D— Day-14-wι^ttrø-measuremente-Qf-red-Gell-function-fΘr-a-^full-unit-modified with 20 kDa mPEG-6AC-NHS.

Table 8E Day 21 in vitro measurements of red cell function for a full unit modified with 20 kDa mPEG-6AC-NHS.

- data not measured.

Table 8F Day 28 in vitro measurements of red cell function for a full unit modified with 20 kDa mPEG-6AC-NHS.

- data not measured.

Table 8G Day 35 in vitro measurements of red cell function for a full unit modified with 20 kDa mPEG-6AC-NHS.

Table 8H Day 42 in vitro measurements of red cell function for a full unit modified with 20 kDa mPEG-6AC-NHS.

EXAMPLE 15 Survival of mPEG modified human red cells infused into mice.

[0063] In order to assess the immunogenicity of an mPEG modified red cell, it can be infused into another species and assessed for survival in a cross species model. Survival of the red cells suggests that it is not reactive with the immune system of the other species. In order to assess the survival, the red cells are labeled with PKH-26 (using PKH-26-GL kit from Sigma), a fluorescently labeled protein that tags the red cells so they can be measured by FACScan analysis. RBC are centrifuged for 2 minutes at 790 x g. The packed red cells (2-3 mL) are resuspended with Diluent C (from Sigma kit) to about 10 mL. In another tube, 40 μl of PKH-26 dye solution is diluted into 10 mL of Diluent C. The two solutions are mixed by inverting in a 50 mL tube approximately 60 times in a minute. Inactivated fetal calf serum is added to a total volume of 50 mL, and this is centrifuged for 5 minutes at 790 x g. The supernatant is removed and 50 mL of PBS (Ca and Mg free, Gibco) is added. The sample is centrifuged for 5 minutes at 790 x g. This PBS wash is repeated for a total of four washes. The treated cells are transferred to a tube for FACScan analysis. The PKH-26 labeled red cells can then be mPEG modified and assessed for the effectiveness of the mPEG modification as described in the examples above. The PKH-26 labeled red cells (either mPEG modified or unmodified controls) can then be infused into mice through a tail vein injection. Blood samples can be removed over time and assayed by FACScan to see how many labeled red cells survive. Another possible method includes the reaction of the RBC with carboxyfluorescein diacetate succinimidyl ester (CFSE), which enters the cells and reacts with proteins in the cell.

Ihis auses iιe ceUsJo_heJιighlyJlu^

EXAMPLE 16 Synthesis of mPEG-6AC-NHS, mPEG-5AV-NHS and mPEG-4AB-NHS esters.

[0064] N-6-[Methoxypoly(ethyleneglycol)]-øxσ-aminocaproic acid 20 kDa (mPEG- 6AC):

[0065] 6-Aminocaproic acid (840 mg, 6.40 mmol) and NaHCO₃ (538 mg, 6.40 mmol) were dissolved in a mixture of H₂0 (160 mL) and ethanol (50 mL). Methoxypoly(ethyleneglycol) succinimidyl carbonate (n = 453, 64.2 g, 3.20 mmol, prepared from the Methoxypoly(ethyleneglycol)-OH (Shearwater Polymers, Huntsville, AL) by known methods) was added and the mixture was stirred at room temperature for 3 hours. HC1 (I N) was added until the solution reached pH 5. The resulting solution was extracted with CH₂C1₂ (3 x 150 mL) and the combined organic extracts were dried (Na₂S0 ), filtered and concentrated under vacuum to give mPEG- 6AC (60.11 g, 2.99 mmol, 93%) as a white solid.

[0066] 1H NMR (200 MHz, OMSO-d₆): δ_H 7.14-7.29 (m, 1 H), 4.07 (t, J = 4.44, 2 H), 3.60-3.30 (m, 1810 H), 3.25 (s, 3 H), 2.89-2.99 (m, 2 H), 2.20 (t, / = 7.32, 2 H), 1.10- 1.60 (m, 6 H). IR; nujol, cm^"1,1721, 1643, 1099.

N-6- [Methoxypoly(ethyleneglycol)] -øxø-aminocaproate N-hydroxysuccinimide ester 20 kDa (mPEG-6AC-ΝHS):

mPEG-δAC mPEG-6AC-NHS

[0067] N-6-[methoxypoly(ethyleneglycol)]-oxo-aminocaproic acid (n = 453, 55.16 g, 2.74 mmol) was dissolved in CH C1₂ (200 mL) and cooled in an ice bath under an atmosphere of argon. N-Hydroxysuccinimide (631 mg, 5.48 mmol) and dicyclohexylcarbodiimide (DCC,1.13 g, 5.48 mmol, in CH₂C1₂ (5 mL)) were added and the resulting mixture was stirred at room temperature for 17 hours. The reaction mixture was filtered to remove the crystals which had formed and the filtrate was concentrated under vacuum. The resulting white solid was washed with ether (600 mL), filtered and covered with isopropanol (800 mL). The suspension was warmed to 50 °C until the solid completely dissolved after which the solution was cooled in an ice bath for 2 hours. The suspension was filtered, and the solid redissolved in CH₂C1₂ (160 mL). Ether (1600 mL) was added and the resulting white precipitate was collected by filtration and dried under vacuum to give mPEG-6AC-ΝHS (53.2 g, 2.63 mmol, 96%) as a white solid.

[0068] 1H NMR (200 MHz, DMSO- ): δ_H 7.09-7.29 (m, 1 H), 3.98-4.09 (m, 1 H), 3.11-3.93 (m, 1810 H), 3.25 (s, 3 H), 2.91-3.04 (m, 2 H), 2.82 (s, 4 H), 2.65 (t, J = 7.59, 2 H), 1.51-1.67 (m, 2 H), 1.26-1.49 (m, 4 H). ER; nujol, cm^-1, 1813, 1783, 1740, 1713, 1148, 1109, 1061.

[0069] N-5-[Methoxypoly(ethyleneglycol)]-øxø-aminovaleric acid 5 kDa (mPEG- 5AN):

[0070] 5-Aminovaleric acid (91 mg, 0.778 mmol) and ΝaHCO₃ (65 mg, 0.778 mmol) were dissolved in a mixture of H₂0 (14 mL) and ethanol (6 mL). Methoxypoly(ethyleneglycol) succinimidyl carbonate (n = 114, 2.0 g, 0.39 mmol) was added and the mixture was stirred at room temperature for 2 hours. HC1 (1 N) was added until the solution reached pH 3. The resulting solution was extracted with

-CH₂Cl₂-(3-X^0-mL)-and he-combined-organic-extracts-were.dried-(Na₂SO ),-filtered- and concentrated under vacuum to give mPEG-5-AN acid (1.66 g, 83%) as a white solid.

[0071] 1H ΝMR (200 MHz, DMSO-<£₆): δ_H 7.14-7.29 (m, 1 H), 4.05 (m, 2 H), 3.55 (s,

454 H), 3.25 (s, 3 H), 2.95 (q, J = 5.94 Hz, 2 H), 2.20 (t, /= 7.32, 2 H), 1.25-1.6 (m,

4H). IR; nujol,

1103, 1099.

[0072] N-5-[Methoxypoly(ethyleneglycol)]-øxø-aminovalerate N-hydroxysuccinimide ester(mPEG-5AN-ΝHS):

[0073] N-5-[methoxypoly(ethyleneglycol)]-oxo-aminovaleric acid (n = 114, 1.66 g, 0.323 mmol) was dissolved in CH₂C1₂ (8.5 mL) and cooled in an ice bath under an atmosphere of argon. N-Hydroxysuccinimide (74 mg, 0.65 mmol) was added, and DCC (133 mg, 0.65 mmol, in CH₂CI₂ (0.5 mL) was added. The resulting mixture was stirred at room temperature for 17 hours. The reaction mixture was filtered to remove the crystals which had formed and the filtrate was concentrated under vacuum. The resulting white solid was recrystallized with isopropanol (10 mL). The solid was redissolved in CH₂C1₂ (3 mL). Ether (50 mL) was added and the resulting white precipitate was collected by filtration and dried under vacuum to give mPEG-5AN- ΝHS (1.23 g, 0.24 mmol, 72%) as a white solid.

[0074] 1H ΝMR (200 MHz, DMSO-d₆): δ_H 7.09-7.29 (m, 1 H), 3.98-4.09 (m, 1 H), 3.55 (s, 454 H), 3.25 (s, 3 H), 3.0 (q, J = 5.78, 2H), 2.83 (s, 4 H), 2.70 (t, / = 6.31 Hz, 2 H), 1.42-1.80 (m, 4 H). IR; nujol, cm^"1, 1813, 1783, 1740, 1713, 1148, 1109, 1061.

[0075] N-4-[Methoxypoly(ethyleneglycol)]-0X0-aminobutyric acid 5 kDa (mPEG- 4AB):

[0076] 4-Aminobutyric acid (80 mg, 0.778 mmol) and NaHCO₃ (65 mg, 0.778 mmol) were dissolved in a mixture of H₂O (14 mL) and ethanol (6 mL). Methoxypoly(ethyleneglycol) succinimidyl carbonate (n = 114, 2.0 g, 0.39 mmol) was added and the mixture was stirred at room temperature for 2 hours. HC1 (1 N) was added until the solution reached pH 3. The resulting solution was extracted with CH₂C1₂ (3 x 50 mL) and the combined organic extracts were dried (Na₂SO ), filtered and concentrated under vacuum to give mPEG-4-AB acid (1.77 g, 88%) as a white solid.

[0077] 1H NMR (200 MHz, DMSO- ): δ_H 7.14-7.29 (m, 1 H), 4.05 (t, / = 4.44, 2 H), 3.55 (s, 454 H), 3.25 (s, 3 H), 3.0 (q, J = 6.31 Hz, 2 H), 2.22 (t, J = 7.4, 2 H), 1.62 (pent, J = 7.4 Hz, 2H). IR; nujol, cm^_1,1716, 1103, 1099. [0078] N-4-[Methoxypoly(ethyleneglycol)]-øxø-aminobutyrate N- hydroxysuccinimide ester 5 kDa (mPEG-4AB-ΝHS):

mPEG-4AB mPEG-4AB-NHS

[0079] N-4-[methoxypoιy(ethyleneglycol)]-oxo-aminobutyric acid (n = 114, 1.77 g, 0.345 mmol) was dissolved in CH C1₂ (8.2 mL) and cooled in an ice bath under an atmosphere of argon. N-Hydroxysuccinimide (79 mg, 0.69 mmol) was added, and DCC (142 mg, 0.69 mmol, in CH₂C1₂ (0.5 mL) was added. The resulting mixture was stirred at room temperature for 17 hours. The reaction mixture was filtered to remove the crystals which had formed and the filtrate was concentrated under vacuum. The resulting white solid was recrystallized with isopropanol (15 mL). The solid was redissolved in CH₂C1₂ (5 mL). Ether (100 mL) was added and the resulting white precipitate was collected by filtration and dried under vacuum to give mPEG-4AB- ΝHS (1.35 g, 0.26 mmol, 75%) as a white solid. [0080] 1H NMR (200 MHz, DMSO- ): δ_H 7.09-7.29 (m, 1 H), 3.98-4.09 (m, 1 H),

3.55 (s, 454 H), 3.25 (s, 3 H), 2.98 (q, J = 5.85, 2H), 2.83 (s, 4 H), 2.68 (t, / = 7.59, 2

H), 1.52-1.72 (m, 2H). IR; nujol, cm^"1, 1813, 1783, 1740, 1713, 1148, 1109, 1061.

-[-008L]The-abo-V-e-reactions-can~be-done-starting^with-^PEGn-place-of-me-nιPEG-to- make the succinimidyl carbonates, so that the succinimidyl carbonate will be at both ends of the compound (di-succinimidyl carbonate PEG), and subsequently the øxø- amino acid N-hydroxysuccinimide ester will be at both ends of the compound (di-

6AC-ΝHS-PEG, see Example 18).

EXAMPLE 17

Synthesis of various mPEG-6AC esters and thio esters.

[0082] The following procedures can be followed to make a variety of active mPEG- 6AC derivatives. As per Example 1, the mPEG-6AC could also be prepared as di- 6AC-PEG by making the di-succinimidyl carbonate PEG from PEG instead of mPEG. The resulting compounds would have the øxø-6-aminocaproic acid esters and thiols at both ends. Procedure A:

[0083] N-6-[methoxypoly(ethyleneglycol)]-oxo-aminocaproic acid (e.g. 1 g, 49.7 μmol) was dissolved in CH₂C1₂ (10 mL). l-[3-(dimethylamino)propyl]-3- ethylcarbodimide (EDCI, 1.5 eq., Aldrich), and 4-(dimethylamino)pyridine (DMAP, 0.1 eq., Aldrich) were mixed with the alcohol (10 eq.) or thiol (10 eq.), and stirred at room temperature under an atmosphere of nitrogen until Thin Layer Chromatography (TLC, reverse phase, C8, CH₃OH/H2θ 4/1, v/v) indicated complete conversion to a less polar product. Ether (100 mL) was added and the resulting suspension was cooled to 0 °C for 1 hour. The suspension was filtered and the white solid recrystallized from isopropanol (20 mL). The solid was redissolved in CH₂C1₂ (10 mL) and ether (100 mL) was added. The resulting solution was cooled to 0 °C for 1 hour, after which the white precipitate was collected by filtration and dried under vacuum. Procedure B:

[0084] N-6-[methoxypoly(ethyleneglycol)]-oxo-aminocaproic acid (e.g. 1 g, 49.7 μmol) was dissolved in CH₃CΝ (10 mL). Benzotriazole-1-yl-oxy-tris-pyrrolidino- phosphonium hexafluorophosphate (PyBOP, 1 eq., Calbiochem, San Diego, CA), and 1-hydroxybenzotriazole (HOBT, 1 eq., Aldrich), were mixed with triethylamine

(Et₃N, 2 eq., Aldrich) and the alcohol (5 eq.) or thiol (5 eq.), and stirred at room temperature under an atmosphere of nitrogen until TLC (reverse phase, C8,

(100 mL) was added and the resulting suspension was cooled to 0 °C for 1 hour. The suspension was filtered and the white solid recrystallized from isopropanol (20 mL). The solid was redissolved in CH₂C1 (10 mL) and ether (100 mL) was added. The resulting solution was cooled to 0 °C for 1 hour, after which the white precipitate was collected by filtration and dried under vacuum. Procedure C:

[0085] N-6-[methoxypoly(ethyleneglycol)]-oxo-aminocaproic acid (1 g, 49.7 μmol) was dissolved in CH₃CN (10 mL). 2-(lH-Benzotriazole-l-yl)-l, 1,3,3- tetramethyluronium hexafluorophosphate (HBTU, 1 eq., Calbiochem, San Diego, CA) and Et₃N (2 eq.) were mixed with the alcohol (5 eq.) or thiol (5 eq.), and stirred at room temperature under an atmosphere of nitrogen until TLC (reverse phase, C8, CH₃OH/H₂O 4/1, v/v) indicated complete conversion to a less polar product. Ether (100 mL) was added and the resulting suspension was cooled to 0 °C for 1 hour. The suspension was filtered and the white solid recrystallized from isopropanol (20 mL). The solid was redissolved in CH C1₂ (10 mL) and ether (100 mL) was added. The resulting solution was cooled to 0 °C for 1 hour, after which the white precipitate was collected by filtration and dried under vacuum.

[0086] N-6-[Methoxypoly(ethyleneglycol)]-øxø-aminocaproate pentafluorophenyl ester 20 kDa (mPEG-6AC-PFP):

[0087] Prepared using procedure C (n = 453, 1 g, 49.7 μmol). Obtained 953 mg, 47.0 μmol, 87% as a white solid.

1H NMR (200 MHz, OMSO-d₆): δ_H 7.17-7.30 (m, 1 H), 3.99-4.10 (m, 2 H), 3.11-3.93 (m, 1810 H), 3.25 (s, 3 H), 2.89-3.04 (m, 2 H), 2.80 (m, 2 H), 1.57-1.79 (m, 2 H), 1.26-1.51 (m, 4 H). IR; nujol, cm^"1, 1788, 1719, 1674, 1105, 1061. [0088] N-6- [Methoxypoly(ethyleneglycol)] -øxø-aminocaproate 2,2,2-trifluoroethyl ester 20 kDa (mPEG-6AC-TFE):

mPEG-6AC mPEG-6AC-TFE

[0089] Prepared using procedure A (n = 453, 1 g, 49.7 μmol). Obtained 957 mg, 47.4 μmol, 82% as a white solid.

1H NMR (200 MHz, DMSO- ): δ_H 7.19-7.31 (m, 1 H), 4.65-4.84 (m, 2 H), 3.97-4.10 (m, 2 H), 3.18-3.95 (m, 1813 H), 2.87-3.06 (m, 2 H), 2.37-2.48 (m, 2 H), 1.14-1.62 (m, 6 H). IR; nujol, cm^"1, 1756, 1717, 1641, 1148, 1109, 1057.

[0090] N-6-[Methoxypoly(ethyleneglycol)] -øxø-aminocaproate ethyl ester 20 kDa (mPEG-6AC-OEt):

mPEG-βAC mPEG-6AC-OEt

[0091] Prepared using procedure A (n = 453, 1 g, 49.7 μmol). Obtained 940 mg, 46.7 μmol, 79% as a white solid.

1H NMR (200 MHz, DMSO- ₆): δ_H 7.05-7.26 (m, 1 H), 4.01-4.19 (m, 2 H), 3.11-4.01 (m, 1813 H), 2.92-3.04 (m, 2 H), 2.20-2.36 (m, 2 H), 1.10-1.65 (m, 9 H),. IR; nujol, cm^"1, 1720, 1141, 1111, 1060.

[0092] N-6-[Methoxypoly(ethyleneglycol)] -øxø-aminocaproate pentafluorobenzenethio ester 20 kDa (mPEG-6AC-PFT):

[0093] Prepared using procedure C (n = 453, 1 g, 49.7 μmol). Obtained 0.84 g, 41.4 μmol, 89% as a white solid. ^JH NMR (200 MHz, DMSO- ₆): δ_H 7.13-7.25 (m, 1 H), 3.99-4.12 (m, 2 H), 3.08-3.95 (m, 1813 H), 2.79-3.07 (m, 4 H), 1.19-1.71 (m, 6 H). IR; nujol, cm^"1, 1719, 1640, 1145, 1111, 1061.

[0094] N-6-[Methoxypoly(ethyleneglycol)]-øxø-aminocaproate 2,3,5,6- tetrafluorobenzene thio ester 20 kDa (mPEG-6AC-TFT):

mPEG-6AC mPEG-6AC-TFT

[0095] Prepared using procedure C (n = 453, 1 g, 49.7 μmol). Obtained 1.10 g, 54.2 μmol, 98% as a white solid. 1H NMR (200 MHz, DMSO- ): δ_H 8.06-8.31 (m, 1 H), 7.13-7.25 (m, 1 H), 3.99-4.11 (m, 2 H), 3.08-3.95 (m, 1810 H), 3.26 (s, 3 H), 2.77- 3.07 (m, 4 H), 1.53-1.75 (m, 2 H), 1.22-1.53 (m, 4 H). IR; nujol, cm^"1, 1720, 1633, 1148, 1110, 1061.

[0096] N-6-[Methoxypoly(ethyleneglycol)]-øxø-aminocaproate 4-fluorobenzenethio ester 20 kDa (mPEG-6AC-4FTP):

mPEG-6AC mPEG-6AC-4FTP

[0097] Prepared using procedure B (n = 453, 1 g, 49.7 μmol). Obtained 0.94 g, 54.2 μmol, 88% as a white solid. 1H NMR (200 MHz, DMSO-d₆): δ_H 7.25-7.59 (m, 3 H), 3.97-4.11 (m, 2 H), 3.07-3.96 (m, 1813 H), 2.90-3.03 (m, 2 H), 2.64-2.77 (m, 2 H), 1.53-1.76 (m, 2 H), 1.25-1.49 (m, 4 H). IR; nujol, cm^"1, 1710, 16.76, 1633, 1147,

1108, 1061.

[O098] V_^6 Methoxypol 4e±ylenegLyx^ trifluoroethylthio ester 20 kDa (mPEG-6AC-TFET):

mPEG-6AC mPEG-6AC-TFET

[0099] Prepared using procedure B (n = 453, 1 g, 49.7 μmol). Obtained 1.04 g, 51.4 μmol, 86 % as a white solid. 1H NMR (200 MHz, DMSO-d₆): δ_H 7.07-7.25 (m, 1 H), 3.99-4.10 (m, 2 H), 3.09-3.94 (m, 1813 H), 2.89-3.00 (m, 2 H), 2.62-2.77 (m, 2 H), 1.51-1.74 (m, 2 H), 1.19-1.48 (m, 4 H). IR; nujol, cm^"1, 1722, 1142, 1108, 1062.

[00100] N-6-[Methoxypoly(ethyleneglycol)] -øxø-aminocaproate ethylthio ester 20 kDa (mPEG-6AC-SEt):

mPEG-6AC mPEG-6AC-SEt

[00101] Prepared using procedure A (n = 453, 1 g, 49.7 μmol). Obtained 0.86 g, 42.6 μmol, 85% as a white solid. 1H NMR (200 MHz, DMSO- ₆): δ_H 7.03-7.30 (m, 1 H), 3.98-4.11 (m, 2 H), 3.04-3.97 (m, 1813 H), 2.78-3.03 (m, 4 H), 1.06-1.78 (m, 9 H). IR; nujol, cm^"1, 1785, 1718, 1646, 1148, 1109, 1061.

EXAMPLE 18 Synthesis of di-6AC-NHS-PEG

[00102] The following procedure can be used to make a variety of sizes of activated branched PEG compounds based on the size of the starting material. [00103] Poly(ethyleneglycol)- ,ω-di-(succinimidyl carbonate) (SC-PEG-SC)

[00104] Poly(ethyleneglycol) (n=453, 20 kDa, 20 g, 1.00 mmol, Fluka) was dissolved in a mixture of CH₂C1₂ (30 mL) and CH₃CN (30 mL) under an atmosphere of argon by stirring at room temperature for 5 minutes. Pyridine (2.02 mL, 25 mmol) and DSC (2.05 g, 8 mmol) were added and the reaction mixture was stirred under argon for 20 hours to give a light brown colored solution. The solution was filtered and the filtrate concentrated under vacuum. Ether (200 mL) was added to the residue followed by stirring to give an off white solid which was isolated by filtration. The solid was re-dissolved in ethyl acetate (200 mL) at 50 °C to give a cloudy brown solution which was subsequently filtered. The clear filtrate was triturated with ether (200 mL) and cooled in an ice bath for 2 hours. The resulting white solid was isolated by filtration to give SC-PEG-SC (19.29 g, 0.952 mmol, 95%). 1H NMR (200 MHz, OMSO-d₆): δ_H 4.42-4.50 (m, 4 H), 3.30-3.78 (m, 1808 H), 2.81 (s, 8 H). IR; nujol, cm^"1, 1811, 1788, 1742, 1716, 1641, 1101.

[00105] Poly(ethyleneglycol)- ,ω-di-øxø-aminocaproic acid (6AC-PEG-6AC).

6-aminocaproic acid

[00106] 6-Aminocaproic acid (468 mg, 3.57 mmol) and NaHCO₃ (300 mg, 3.57 mmol) were dissolved in a mixture of H₂0 (45 mL) and ethanol (15 mL). Poly(ethyleneglycol)-α,ω-di-(succinimidyl carbonate) (18.1 g, 0.893 mmol) was added and the mixture was stirred at room temperature for 2.5 hours. HC1 (1 N) was added until the solution reached pH 4. The resulting solution was extracted with CH C1₂ (3 x 50 mL) and the combined organic extracts were dried (Na₂S0₄), filtered and concentrated under vacuum to give poly(ethyleneglycol)-α,ω-di-øxø- aminocaproic acid (17.78 g, 0.876 mmol, 98%) as a white solid.

[00107] 1H NMR (200 MHz, DMSO-d₆): δ_H 7.17-7.25 (m, 2 H), 4.05-4.10 (m, 4

H ₇^40^ 57-(-m₇4-808-H 2-95-( 4 ^^

IR; nujol, cm^_1,1719, 1648, 1099

[00108] Poly(ethyleneglycol)-α,ω-di-øxø-aminocaproate N-hydroxysuccinimide ester (ΝHS-6AC-PEG-6AC-ΝHS).

[00109] Poly(ethyleneglycol)-α,ω-di-øxσ-aminocaproic acid (17.01 g, 0.838 mmol) was dissolved in CH C1₂ (70 mL) and cooled in an ice bath under an atmosphere of argon. N-Hydroxysuccinimide (386 mg, 3.35 mmol) and DCC (691mg, 3.35 mmol, in CH₂C1₂ (5 mL)) were added and the resulting mixture was stirred at room temperature for 18 hours. The reaction mixture was filtered to remove the white solid which had formed and the filtrate was concentrated under vacuum. The resulting white solid was washed with ether (200 mL), filtered and covered with isopropanol (250 mL). The suspension was warmed to 50 °C until the solid completely dissolved after which the solution was cooled in an ice bath for 2 hours. The suspension was filtered, and the solid re-dissolved in CH₂C1₂ (50 mL). Ether (500 mL) was added and the resulting white precipitate was collected by filtration and dried under vacuum to give ΝHS- 6AC-PEG-6AC-ΝHS (16.48 g, 0.804 mmol, 96%) as a white solid. 1H NMR (200 MHz, DMSO- ): δ_H 7.15-7.24 (m, 2 H), 4.00-4.09 (m, 4 H), 3.30-3.58 (m, 1808 H), 2.90-2.98 (m, 4 H), 2.82 (s, 8 H), 2.65 (t, 4 H), 1.51-1.70 (m, 4 H), 1.30- 1.49 (m, 8 H). IR; nujol, cm^"1, 1812, 1783, 1740, 1713, 1641, 1148, 1107.

EXAMPLE 19 Synthesis of 4-Arm-PEG-6AC-NHS

[00110] The following procedure can be used to make a variety of sizes of activated branched PEG compounds based on the size of the starting material. Further, the corresponding 3 arm compound can be made by starting with the 3 arm PEG (available from Shearwater).

Pentaerythritoxy poly(ethylene glycol) Succinimidyl Carbonate (4-Arm-PEG- sn

[00111] Disuccinimidyl carbonate (DSC, 1.54 g, 6 mmol, Aldrich) was added with stirring to pentaerythritol poly(ethylene glycol) (n=114, 20 kDa, 10 g, 0.5 mmol (2 mmol OH groups), Shearwater) in dichloromethane (20 mL) and acetonitrile (40 mL). Pyridine (1 mL) was added. The mixture was stirred at ambient temperature for 4 hours. The reaction solution was filtered, and concentrated in vacuo to a minimal volume. The residue was dissolved in ethyl acetate (90 mL) with heating. Ether (90 mL) was added to this solution, and the resulting solution was cooled with an ice bath. A white precipitate was collected under reduced pressure, and dried under vacuum at room temperature overnight, 10.2 g.

[00112] 1H NMR (200 MHz, OMSO-d₆): δ_H 4.5 (m, 8H), 3.60-3.30 (m, 1824 H), 2.82 (s, 16H). IR; nujol, cm^"1, 1811, 1789, 1742, 1717, 1633, 1099.

[00113] Pentaerythritoxy poly(ethylene glycol) øxø-aminocaproic acid 20 kD (4- Arm-PEG-6AC):

δ-aminocaproic acid

[00114] 6-Aminocaρroic acid (500 mg, 3.91 mmol, Aldrich) and NaHCO₃ (330 mg, 3.91 mmol, EM Science) were dissolved in H₂O (60 mL). Pantaerythritoxy poly(ethylene glycol) succinimidyl carbonate (10 g, 0.49 mmol (1.95 mmol NHS ester groups)) from above was added and the mixture was stirred at room temperature for 2 hours. H O (200 mL) was added to the reaction solution. HC1 (1 N) was added until the solution reached pH 5. The resulting solution was extracted with CH₂C1₂ (3 x 50 mL) and the combined organic extracts were dried (Na₂SO₄), filtered and concentrated

.und r-vacuum θ pve-Pentaer^trιritQ^

(9.47 g, 0.46 mmol, 95%) as a white solid.

[00115] 1H NMR (200 MHz, OMSO-d₆) δ_H 4.07 (t, J = 4.44, 8 H), 3.60-3.30 (m,

1832 H), 2.20 (t, = 7.32, 8 H), 1.10-1.60 (m, 24 H). IR; nujol, cm^"1,1721, 1643,

1099.

[00116] Succinimidyl Pentaerythritoxy poly(ethylene glycol) øxø-aminocaproate (4-Arm-PEG-6AC NHS):

[00117] Pentaerythritoxy poly(ethylene glycol) øxø-aminocaproic acid (9 g, 0.5 mmol (2 mmol carboxylic acid groups), from previous preparation) was dissolved in CH₂C1₂ (50 mL) and cooled in an ice bath under an atmosphere of argon. N- Hydroxysuccinimide (402 mg, 3 mmol, Aldrich) and dicyclohexylcarbodiimide (DCC) (0.721 g, 3 mmol, Aldrich), in CH₂C1₂ (5 mL)) were added and the resulting mixture was stirred at room temperature for 17 hours. The reaction mixture was filtered to remove the crystals which had formed and the filtrate was concentrated under vacuum. The resulting white solid was washed with ether (100 mL), recrystallized in IPA (105 mL), and the solid redissolved in CH₂θ₂ (25 mL). Ether (150 mL) was added and the resulting white precipitate was collected by filtration and dried under vacuum to give Succinimidyl Pentaerythritoxy poly(ethylene glycol) øxø- aminocaproate (8.72 g, 0.42 mmol, 95%) as a white solid.

[00118] ^XH ΝMR (200 MHz, DMSO-rf₆): δ_H 7.09-7.29 (m, 4 H), 4.01-4.1 (m, 8 H), 3.55 (s, 1824 H), 2.91-3.04 (m, 8 H), 2.82 (s, 16 H), 2.65 (t, J = 7.59, 8 H), 1.51-1.67 (m, 8 H), 1.26-1.49 (m, 16 H). IR; nujol, cm^"1, 1813, 1783, 1740, 1713, 1148, 1109, 1061. EXAMPLE 20

Modification of RBC using methods known in the art.

[00119] Published methods for the attachment of activated PEG .compounds were assessed. In one case, a 5 kDa activated PEG was used with a 30 mM triethanolamine (TE) buffer at pH8.6 with 150 mM NaCl [Blackall et al., Blood 97(2):551-556 (2001)]. The red cells were washed twice with 10 mM PBS pH 7 and the reaction was prepared at 9.6 mM combined PEG-SPA-NHS and FPEG-SPA-NHS at a hematocrit of 10% using the TE buffer. Alternatively, either PEG-SPA-NHS or cyanuric chloride PEG were used at 9.6 mM without the FPEG. The reaction was done for 1 hour at room temperature and the cells were washed twice with the PBS and suspended in PBS for analysis. This was compared to using a method of the invention where the red cells are washed twice with 150 mM CHES pH 9, 50 mM NaCl and reacted in the same buffer and washed with phosphate buffer pH 7 (150 mM Na₂HPO , 50 mM NaCl) using 11 mM 5 kDa combined mPEG-SPA-NHS and FPEG-SPA-NHS or 22 mM of the mPEG-SPA-NHS. The pH during the reaction step for the TE buffer protocol went from 8 down to 7.8 for the cyanuric chloride PEG and from 7.6 down to 7.5 for the SPA PEG. For the studies done without FPEG, the anti-A antibody binding was >98% for the TE protocol while it was approximately 9.6% for the CHES protocol. For the FPEG studies, the fluorescent measurement was 4255 for the CHES method and only 1459 for the TE method.

[00120] In another study, 5 kDa cyanuric chloride activated PEG (Sigma) was used at a 5 mM concentration in 50 mM K₂HPO₄, 105 mM NaCl pH 8.0 buffer at a 40% hematocrit [Bradley et al., Transfusion 41:1225 (2001)] to modify a type B+ red cell sample. This was prepared from whole blood without washing and was reacted for 30 minutes at room temperature and then washed three times with PBS (10 mM Na₂HP0₄, 105 mM NaCl pH 7.0). These were compared to 11 mM and 5.5 mM 10 kDa mPEG-SPA-NHS with CHES pH 9 as detailed above. The gel card agglutination showed no reaction with the CHES reacted samples while the phosphate buffered reaction showed some agglutination with anti-B (grade 2+) and a mixed field with anti-D antibody. The anti-B antibody binding by FACScan showed no binding with the 11 mM sample in CHES and 11.7 % binding with the 5.5 mM sample in CHES while the phosphate buffered sample showed 63% binding of the antibody. These examples indicate considerable improvement in modification of the red cells with buffers of the invention compared to known methods.

EXAMPLE 21 Antigen masking of red cells reacted with mPEG-6AC, 5AN or 4AB-ΝHS and mPEG-6AC-PFP.

[00121] A unit of ABO matched whole blood (Sacramento Blood Center, CA) was leukofiltered according to standard blood banking methods. The RBC were centrifuged at 4 °C at 4100 x g for 6 minutes and the plasma was removed to give a red cell concentrate. The red cell concentrate was washed with an equal volume of reaction buffer, centrifuged as above, the supernatant removed and the wash repeated. The washed red cell concentrate was reacted with an activated mPEG by dissolving the mPEG in reaction buffer and adding it to the red cell concentrate to a hematocrit of approximately 40% and the desired mPEG concentration in the extracellular volume. The reaction buffer was either 150 mM CHES pH 9 with 100 mM dextrose and 5 mM L-camitine (CHES-GC) or 150 mM CHES pH 9 with 50 mM NaCl (CHES-Na). The reaction mixture was incubated for 2 hours at room temperature and an equal volume of wash solution was added, the samples centrifuged as above, the supernatant removed and the wash repeated. The wash solution was either 150 mM Na₂HPO₄ pH 7, 50 mM NaCl (PBS), or 150 mM Na₂HPO₄ pH 7, 100 mM dextrose, 5 mM L-carnitine (PB-GC). The final red cell solution is stored in an approximately equal volume of Erythrosol and assayed for anti-A antibody binding as per Example 5. The results are found in Table 9. All compounds showed effective masking of the red cell antigens.

Table 9 Modification of red cells with various activated mPEGs as measured by anti-type antibody binding.

EXAMPLE 22

Antigen masking of red cells reacted with 4-Arm-PEG-6AC-NHS.

400-122] — A-unit-of-A— w-hole-blood_was-p pare.d^s Ed_cellxoτιc£πtι^e_pex Example 13. The red cell concentrate was washed with an equal volume of CHES GC pH 9.0 (150 mM CHES, 100 mM glucose, 5 mM L-camitine), incubated for 5 minutes at room temperature, then centrifuged at 4100 x g for 6 minutes at room temperature and the supernatant removed. This wash was repeated, resulting in a washed red cell concentrate of approximately 80% hematocrit. An aliquot of this was put into separate tubes labeled control and 1-4 (see Table 10). Samples of PEG were weighed into additional tubes similarly labeled (see Table 10). A 0.5 mL of CHES GC was added to each PEG sample. These were sonicated and vortexed to dissolve the PEGs and then added to the appropriate tube containing 0.5 mL of washed red cell concentrate according to Table 10. The samples were mixed with gentle vortexing and incubated static for 1 hour at room temperature. Following incubation, 0.5 mL of PBGC pH 7.0 (150 mM phosphate, 100 mM glucose, 5 mM L-camitine) was added to each sample with mixing. After 5 minutes at room temperature, the samples were centrifuged at 8,000 x g for 2 minutes at room temperature and the supernatant discarded. This wash was repeated and a 50-100 mL aliquot of the concentrate was removed for FACScan analysis. For all samples, the inhibition of anti-A or anti-D antibody binding was assessed by FACScan per Example 5, and the agglutination assay with gel cards per Example 1. For the gel card assay, the 5 mM mPEG-6AC- NHS control and the 2.5 mM 4-Arm-PEG-6AC-NHS samples showed no agglutination. The 1.25 mM 4-Arm-PEG-6AC-NHS sample showed no agglutination with anti-A and a mixed field for anti-D. The 0.5 mM 4-Arm-PEG-6AC-NHS showed complete agglutination for both anti-A and anti-D. The FACScan results are indicated in Table 10. The results indicate some efficacy for the 4-Arm-PEG-6AC- NHS, but this was not as effective as the single arm mPEG-6AC-NHS. Table 10 Modification of red cells with 4-Arm-PEG-6AC-NHS as measured by anti-type antibody binding.

* concentration of PEG based on extracellular volume.

Claims

We claim:

1. A method of preparing a modified red blood cell composition comprising a) washing the red blood cells with a pre reaction wash solution to provide washed red blood cells; b). mixing the washed red blood cells with a reaction solution and an activated antigen masking compound to provide a reaction mixture; c) incubating the reaction mixture so that the antigen masking compound covalently binds to the surface of the red blood cells to provide modified red blood cells; and d) washing the modified red blood cells with a post reaction wash solution, wherein at least one of the pre reaction wash solution, reaction solution, and post reaction wash solution comprises dextrose at a concentration of about 50-300 mM.

2. The method of claim 1, wherein at least two of the pre reaction wash solution, reaction solution, and post reaction wash solution comprises dextrose at a concentration of about 50-300 mM.

3. The method of claim 1, wherein the pre reaction wash solution, reaction solution, and post reaction wash solution all comprise dextrose at a concentration of about 50-300 mM.

4. The method of claim 1, wherein the at least one of the pre reaction wash solution, reaction solution, and post reaction wash solution comprising dextrose further comprises L-carnitine at a concentration of about 2-100 mM.

5. The method of claim 2, wherein the at least two of the pre reaction wash solution, reaction solution, and post reaction wash solution comprising dextrose further comprises L-camitine at a concentration of about 2-100 mM.

6. The method of claim 3, wherein the pre reaction wash solution, reaction solution, and post reaction wash solution all further comprise L-camitine at a concentration of about 2-100 mM.

7. The method of claim 4, wherein the at least one of the pre reaction wash solution, reaction solution, and post reaction wash solution comprising dextrose lacks chloride ions.

8. The method of claim 5, wherein the at least two of the pre reaction wash solution, reaction solution, and post reaction wash solution comprising dextrose lacks chloride ions.

9. The method of claim 6, wherein the pre reaction wash solution, reaction solution, and post reaction wash solution all lack chloride ions.

10. The method of claim 7, wherein the at least one of the pre reaction wash solution, reaction solution, and post reaction wash solution comprising dextrose and L-camitine lacks chloride ions.

solution, reaction solution, and post reaction wash solution comprising dextrose and L-camitine lacks chloride ions.

12. The method of claim 9, wherein the pre reaction wash solution, reaction solution, and post reaction wash solution all lack chloride ions.

13. A method of preparing a modified red blood cell composition comprising a) washing the red blood cells with a pre reaction wash solution to provide washed red blood cells; b) mixing the washed red blood cells with a reaction solution and an activated antigen masking compound to provide a reaction mixture; c) incubating the reaction mixture so that the antigen masking compound covalently binds to the surface of the red blood cells to provide modified red blood cells; and d) washing the modified red blood cells with a post reaction wash solution, wherein at least one of the pre reaction wash solution, reaction solution, and post reaction wash solution comprises L- carnitine at a concentration of about 2-100 mM.

14. The method of claim 13, wherein at least two of the pre reaction wash solution, reaction solution, and post reaction wash solution comprises L- carnitine at a concentration of about 2-100 mM.

15. The method of claim 14, wherein the pre reaction wash solution, reaction solution, and post reaction Wash solution all comprise L-carnitine at a concentration of about 2-100 mM.

16. A method of preparing a modified red blood cell composition comprising washing the red blood cells with a solution comprising a buffer at a concentration of about 50-350 mM and a pH of about 8-10, adding a reaction solution and an activated antigen masking compound to the washed red blood cells to form a reaction mixture, and incubating the reaction mixture so that the antigen masking compound covalently binds to the surface of the red blood cells to provide modified red blood cells, wherein the reaction solution is unbuffered.

17. The method of claim 16, wherein the reaction solution comprises blood bank saline.

18. The method of claim 16, wherein the reaction solution comprises dextrose at a concentration of about 125-200 mM.

19. The method of claim 18, wherein the reaction solution lacks chloride ions.