CA2136698A1 - Resin compositions and methods for their use - Google Patents

Abstract

Novel resin-antigen compositions for removing undesired antibodies from blood-derived compositions containing the antibodies and coagulation factors, such that the coagulation factors are substantially retained in the composition and methods for making such novel resin-antigen compositions are provided. In particular, the undesired antibodies may be blood group antibodies. Blood-derived compositions in which undesired antibodies have been removed and desired coagulation factors are substantially retained are also provided.

Description

NOVEL RESIN COMPOSITIONS AND METHODS
FOR THEIR USE

This invention relates to novel resin compositions useful for the removal of undesired antibodies from blood-derived compositions, which compositions contain both undesired antibodies and desired coagulation factors. The novel resin compositions effect removal of the antibodies in such a manner that the 10 desired coagulation factors are substantially retained in the resulting compositions. More particularly, the invention is directed to contacting the novel resin compositions with a blood-derived composition cont~ining specific antibodies, including blood group antibodies, and coagulation factors where the resin compositions include covalently bound antigens specific for the antibodies5 to be removed from the blood-derived composition, the resin being selected such that its use does not also result in the removal of the desired coagulation factors from the blood-derived composition.

BACKGROUND OF THE INVENTION
The term "blood group" is used to identify any one of the many types into which a person's blood may be classified, based on the presence or absence of certain inherited antigens on the surface of the red blood cells. Blood of one group contains or may contain antibodies in the serum that react against the cells 25 of other groups. These antibodies are referred to herein as "blood group antibodies" and are also referred to in the art as isoagglutinins.

There are more than thirty blood group systems, one of the most important of which is the ABO system. This system is based on the presence or 3 o absence of carbohydrate antigens A and B on the cell surface of red blood cells.
Blood of groups A and B contain antigens A and B, respectively. Group AB
contains both antigens, and group O contains neither.

'_ The major blood group antibodies are anti-A and anti-B antibodies, which are mainly of the IgM and IgG isotypes. Blood of group A contains antibodies to antigen B. Blood of group B contains antibodies to antigen A. Blood of group AB has neither antibody, and blood of group O has both. A person whose 5 blood contains either (or both) of the anti-A and anti-B antibodies cannot receive a transfusion of blood cont~ining the corresponding antigens.

Specifically, when blood group antibodies are mixed with blood of an incompatible group, the antibodies coat the red blood cells of the incompatible 0 group and cause agglutination (clumping or sticking together) thereof.
Incompatible blood group antibodies can fix complement, cause transfusion reactions and induce hemolysis which is the destruction of red blood cells.
Hemolysis can lead to anemia and other complications.

In order to avoid immunohemolysis and transfusion reactions caused by blood group antibodies in donor plasma which is incompatible with the blood group type of the blood transfusion recipient, the donor plasma and the blood group type of the recipient must be cross-matched or typed and screened.

2 o In an effort to avoid the necessity of cross-matching and to create "universal" blood-derived compositions (compositions, such as plasma, that can be ~tlmini.~tered without regard for the blood group of donor and recipient), methods have been developed for removing blood group antibodies from blood-derived compositions. Typically, artificial antigens specific for the blood group 2 5 antibody which is to be removed are attached to a support, such as a resin, which is then used to remove such antibodies. Examples of such artificial antigens can be found in U.S. Patents Nos. 4,362,720, 4,308,376, 4,238,473, 4,195,174 and 4,137,401.

Commercially available resins, such as Synsorb (available from the 5 Alberta Research Council) and Chromosorb (available from Manville), when used with appl-opliate antigens, are able to remove blood group antibodies from blood-derived compositions. However, because some commercially available resins non-specifically adsorb coagulation factors, removal of blood group antibodies from blood-derived compositions with such resins also results in the o undesired removal of an unacceptably high level of coagulation factors from such compositions.

Coagulation factors, also known as blood clotting factors, are substances present in blood that undergo a series of chemical reactions which lead to the 15 conversion of the blood from a liquid to a solid state. Coagulation factors include Factor V, Factor VIII, Factor IX and Factor XI. Since these factors work sequentially (in a series of reactions termed a "cascade"), lack of sufficiently high level of any one of these factors in the blood results in the inability of the blood to clot. Hence, the removal of any coagulation factors 2 o from blood-derived compositions for use in hllm~n~ or ~nim~l~ is not desired and is dangerous. As a result, a need has arisen to develop a method of removing blood group antibodies from blood-derived compositions so that said compositions may be "universal, " without removing coagulation factors from said compositions so that blood clotting ability is not impaired.
Another problem with the commercially available resins cullelllly used to remove blood group antibodies from blood-derived compositions, is that such '- 2136698 resins are only capable of completely removing blood group antibodies from such compositions at a volume ratio of 30 ml of composition per 1 ml of resin. This required ratio results in the inefficient removal of blood group antibodies.
Hence, a need has also arisen to develop a more efficient method of removing 5 blood group antibodies from blood-derived compositions without removing coagulation factors therefrom.

It is therefore an object of this invention to provide resin-antigen compositions capable of and methods for efficiently removing undesired 0 antibodies from blood-derived compositions, without substantially removing coagulation factors from the compositions. These compositions and methods provide blood-derived compositions which are effectively free of undesired antibody, yet have substantially retained their coagulation factors.

This invention is directed to novel resin-antigen compositions useful in methods for removing undesired antibodies, including blood group antibodies, from blood-derived compositions without substantially removing coagulation 2 o factors from said blood-derived compositions so as to avoid risks inherent in ~lmini~tering compositions with tlimini~hed levels of blood clotting factors.
Methods for making the novel resin-antigen compositions are also provided. The methods of using the novel resin-antigen compositions of the invention include contacting blood-derived compositions which contain blood group antibodies and 2 5 one or more coagulation factors with a resin which includes antigens capable of binding (and therefore removing) the blood group antibodies. The resin is selected such that its use does not result in removal of the coagulation factors ~ 2136~g8 from the composition. This invention is further directed to blood-derived compositions which, having once contained both blood group or other undesired antibodies and coagulation factors, are effectively specific antibody-free but have substantially retained their coagulation factors.

213669~

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term "blood-derived compositions" includes whole blood, blood plasma, blood plasma fractions, blood plasma precipitate (e.g., 5 cryoprecipitate, ethanol precipitate or polyethylene glycol precipitate), supernatant (e.g., cryosupernatant, ethanol supernatant or polyethylene glycol supernatant) or other compositions derived from human or animal blood and characterized by the presence of coagulation factors and blood groups or other undesired antibodies. Blood-derived compositions also include purified 0 coagulation factor concentrates (e.g., Factor VIII concentrate, Factor IX
concentrate, Fibrinogen concentrate) prepared by any of various methods including ion exchange, affinity, gel permeation, and/or hydrophobic chromatography or by dirr~l~lllial precipitation.

This invention relates to methods for removing undesired antibodies, including blood group antibodies, from blood-derived compositions Cont~ining both blood group antibodies and coagulation factors wherein said resulting specific antibody-free blood-derived compositions retain a high percentage of their coagulation factors. This invention is also directed to specific antibody-free 2 o compositions cont~ining coagulation factors. In the methods of this invention, a blood-derived composition which contains both undesired antibodies and coagulation factors is put into contact (e.g., by chromatography or by batch adsorption) with a resin-antigen composition capable of removing the undesired antibodies without removing coagulation factors th~l~rlo-l-. Such resin is covalently bonded to, for example, blood group or type-specific (e.g., type-A, type-B or type-D) antigens, such as synthetic oligosaccharide antigens or protein antigens to form a resin-antigen composition. The blood group or type-specific 21366`98 -antigens on the resins bind to the blood group or other specific antibodies in the blood-derived composition, while allowing the remainder of the blood-derived composition to pass through the resin intact.

The resin-antigen combinations of this invention are capable of removing blood group antibodies from blood-derived compositions without substantially removing coagulation factors thelerlolll. The inventors have discovered that theretention of coagulation factors is apparently effected by the resin selected, substitution level (i.e. micromoles of antigen per gram of resin), and the matrix o of the resin. Examples of commercially available resins and the corresponding matrix for each are shown in Table I below.

213fi698 Table I
Resin Resin Description Method of /Manufacturer/Supplier Functionalization Chromosorb P crystalline macroporous aminosilylation silica/Celite Corp.
Antab fastflow unknown Sepharose/Pharmacia Eupergit N,N' -methylene ethylen~ minP (EDA) bis(acrylamide) opening of epoxide crosslinked groups methacrylamide with oxirane contAining monomer/Rohm-Pharma/Fluka Toyopearl-NH2 copolymer of glycidyl commercial methacrylate, pentaerythritol dimethacrylate and polyethylene glycol plus amine cont~ining monomer/Toso-Haas/Supelco Sephacryl N,N' -methylene epoxidation with bis(acrylamide) H202/AcOH, 1-5 hours, crosslinked allyl EDA treatment dextran/Pharmacia Poros polystyrene/divinylbenz commercial ene copolymer/Perseptive Biosystems 21~6~8 SynsorbsTM (available from the Alberta Research Council) are immunoadsorbent compositions produced by: aminosilylation of Chromosorb P;
conversion of hapten methyl ester first to hydrazide then to a reactive acyl azide;
reaction of acyl azide with amine cont~ining Chromosorb P; and acetylation of 5 rem~inin~ unreacted amines with acetic anhydride in methanol.

Toyopearl resins (m~nllf~ red by Toso-Haas and supplied by Supelco) are provided in a variety of formats. The one pr~fell~d for use in the present invention contains an amine cont~ining monomer so further activation of the o matrix is not required.

Commercially available resins currently used in the removal of blood group antibodies are unable to remove blood group antibodies from blood-derived compositions without also removing an unacceptably high level of 5 coagulation factors thelerlolll. However, we have discovered that, for example, the commercially available Toyopearl resin, which has a polymethacrylic matrix (also called backbone), may be covalently bonded to an ~propliate antigen at an appropriate substitution level to remove blood group antibodies while retaining over 85 % of the coagulation factors. Further, the Toyopearl resin-antigen 2 o composition of the present invention may be used to more efficiently remove blood group antibodies from plasma at a volume of 120 ml plasma per 1 ml of resin.

Covalent bonding of antigens to resins (also referred to herein as 2 5 haptenation) was generally accomplished by methods similar to those described in the art. See, for example, Immobilized A~finity Ligand Techniques by G. T.
Hermanson, A. Krishna Mallia, and P. K. Smith, Academic Press 1992, where `-- 213fi69~

many of the supports, methods for their functionalization and haptenation are described. We exemplify the methods for haptenation l1tili7ing carbohydrate antigens, but the methods are applicable to other types of antigens as well.

The oligosaccharide antigens we employ may optionally contain an 8- ~
methoxycarbonyloctyl aglycon. This organic spacer keeps the carbohydrate antigen sufficient distance from the matrix such that the matrix will not hlLelre-e in the carbohydrate/protein binding. The terminus of the aglycon is a carboxylicacid ester. This functionality can be easily converted to a hydrazide by treatment 0 with hydrazine, then to a reactive acyl azide as has been previously described for the preparation of Synsorb. Alternatively the ester can be hydrolyzed to its acid and this material activated l~tili7ing known methods described, for example, foractivation of amino acids utilized in peptide synthesizers and the like. Preferred methods for preparing the resin-antigen compositions of the present invention are disuccinimidyl carbonate activation method and the water soluble carbodiimide activation method. Other well known methods for activation use carboxylic acids such as carbonyl diimidazole and/or 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinolone (EEDQ) may be used.

2 o Additionally, the carboxylic acid ester of an antigen may typically be reacted with large stoichiometric excesses of ethylene~ min~ or other diamino cont~inin~ material to provide an amine-cont~ining hapten suitable for coupling to matrices functionalized with carboxylic acid moieties. In these instances, the matrix is activated by the methods described above and then treated with the amine-cont~ining hapten. The amine-cont~ining haptens may also be reacted directly with oxirane(epoxide) cont~ining matrices. Hydroxyl-cont~ining matrices, particularly those with sugar polymer backbones may be treated with ~ 2136~98 epichlorohydrin and like materials to provide for epoxy-activated matrices.
Epoxy-activated matrices, such as the commercially available EupergitTM may be treated with ethylen~ min~ to provide for an ~min~tecl surface ready for reaction with an activated carboxyl cont~ining hapten. The epoxy activated 5 matrix may be opened to a diol then oxidized with periodate or similar oxidant to give a matrix cont~ining formyl groups. These materials bind amine groups, particularly lysine groups found in peptides, to give Schiff's bases that are not easily hydrolyzed. The Schiff's base can be reduced with borohydride cont~ining reducing agents to provide non hydrolyzable secondary amines.

The ability of the carbohydrate or other antigen bound to the matrix to bind antibody depends on several factors. The surface area able to be functionalized and the density of available reactive moieties will determine thelevel of hapten incorporation required for op~ l binding of antibody to the 5 resin-antigen composition of the present invention. Generally, not all available sites are reacted with hapten so that excess reactive sites may be blocked afterhaptenation. In the case of carboxylic acid activated carbohydrate antigens reacted with amine cont~ining matrices this is accomplished by acetylation in methanol using acetic anhydride.
The available surface area of a matrix, particularly those matrices employed in affinity techniques depends on the porosity of the matrix, both the size and the number, of the pores. Most of the binding of antigen to its target will then depend on the ability of the target to diffuse into the pores of the 2 5 matrix. If the pores of the matrix are small and the target is a large protein, then any hapten contained in these pores will not be able to bind the target. Target proteins that have more than one binding site, particularly antibodies, benefit 213fi~98 from high hapten densities where greater avidity of binding is achieved because both(IgG) or several(IgM) arms of the antibody bind hapten siml-lt~n~ously.

Hapten densities of 1 ~Lmole/g are utilized in Synsorbs. For example, 5 higher hapten densities have not been found to give better capacity of these resins to bind antibody. This may be due to the saturation of the available surface areas of the matrix and/or the non-uniformity of the porosity in the Chromosorb P
matrices. Organic matrices such as ToyopearlTM and EupergitTM may have larger surface area per gram, more ~miro~ porosity and a higher density of functional 0 groups available for haptenation, allowing incorporations even above 10 ~moles/g which yield better capacity to bind antibody per unit weight of resin-antigen composition. Their limitations are reflected in their greater deformity under conditions of flow resulting in poorer flow rates for certain applications.

The pf~felled levels of hapten incorporation for the resin-antigen compositions of the present invention are 1-10 llmoles per gram of material.
The most preferred levels of incorporation are about 5-10 ~umoles per gram.

Higher capacity resins are desirable since they require less resin and 2 0 result in less non-specific adsorption of proteins. The non-specific adsorption of proteins can be the result of hydrophobic interaction or electrostatic interactions that occur between the protein and the matrix. The composition of the matrix has much to do with this interaction and the use of less resin minimi7.~s these interactions.
We also found that preadsorption of resin-antigen compositions with human serum albumin (HSA) could elimin~te depletion of coagulation factors ~r 2136~98 without affecting the resin's ability to bind specific antibodies. For example, Synsorbs with blood group antigens A and B bound thereto were incubated for 30 minutes with 5 % HSA, then used to adsorb plasma. Results showed that pre-incubation with HSA resulted in depletion of specific blood group antibodies and5 100% recovery of coagulation Factor XI.

In a pl~r~lled embodiment, the resin-antigen composition is Toyopearl haptenated at an incorporation level of 1-10 ~umoles/g with synthetic blood group antigen A(A-trisaccharide) and/or synthetic blood group antigen B (B-tri-10 saccharide). Incorporation at 5 ~moles/g is most preferred.

Tmmllnoaffinity techniques known to those of skill in the art, such aschromatography and batch adsorption, may be used to remove blood group antibodies from blood-derived compositions without substantially removing 5 coagulation factors thel~rlolll. Where chromatography is used, a resin-antigencombination capable of removing blood group antibodies without substantially removing coagulation factors is equilibrated in a buffer, such as PBS or saline,with a pH in the range of about 5.5-9.5. The preferred pH range is 6.4-7.8.
The equilibration temperature is about 4-45C, with the pl~r~ d temperature 2 o being room temperature. A blood-derived composition cont~ining blood group antibodies and coagulation factors is then run through the resin column with a contact time in the range of about 1-60 minutes, with 4-10 minutes being the preferred contact time.

2 5 Another immunoaffinity procedure which may be used to remove blood group antibodies from blood-derived compositions without substantially removing coagulation factors therefrom, is batch adsorption. To utilize batch adsorption, a resin-antigen composition capable of removing blood group antibodies from blood-derived compositions without substantially removing coagulation factors thel~rlolll is added to a composition cont~inin~ blood groupantibodies and coagulation factors in a suitable container, mixed at a temperature 5 of 4 to 45C, with the p~felled temperature being ambient, for a period of at least 1 hour, with a pler~lled period being 4 hours, the resin sedimented by normal gravity or centrifugation and then the unbound composition is removed.

Blood-derived compositions which may be used include blood products, lo blood plasma, blood plasma precipitate (e.g., cryoprecipitate, ethanol precipitate or polyethylene glycol precipitate), supernatant (e.g., cryosupernatant, ethanolsupernatant or polyethylene glycol supernatant), or any other composition derived from blood and characterized by the presence of one or more coagulation factors and blood group or other undesired antibodies.
In order to determine whether blood group antibodies have been removed from a blood-derived composition, it is n.oces~ry to determine the blood group antibody titer within the composition after contacting the composition with a resin-antigen combination of the invention. This may be performed by direct 2 o blood group antibody test (DAT) or indirect Coombs test (ICT). In order to determine the amount of recovery of coagulation factors in the blood-derived compositions treated by the methods of this invention, the treated blood-derivedcompositions may be assayed for activities of the coagulation factor or factors of interest, e.g., Factors V, VIII, IX and XI, by detern ining the degree of 2 5 correction in the clotting time of a plasma deficient in the particular factor or factors in the presence of activated partial thromboplastin (APTT) reagent, or by other assay methods known in the art. In order to evaluate the protein content ~ 21366~8 and distribution of the blood-derived compositions treated by the methods of this invention, protein content may be measured by Biuret reagent and protein distribution may be measured by SDS-polyacrylamide gel electrophoresis.

F,x~nlples Fxample 1: Covalent Attachment of Blood Group B Antigen to Toyopearl Resin A slurry of AF-Amino-650 M Toyopearl resin in aqueous 0.02% sodium azide (Toso-Haas, available through Bioseparation Specialists, Philadelphia, PA)o was used as the starting material. Weight equivalent of dry resin was 6 g for every 25 ml of slurry. A volume of 250 ml AF-Amino-650M Toyopearl resin weighing 261.6 g was washed in a column with 1 1 RO water.

A 204 mg amount of B-Trisaccharide acid (BTS acid), which has the formula: a Gal (1~3) ~ Gal (1~2)-O-(CH2)8-COOH
I (1~2) aFuc and 213 mg di(N-succinimidyl)-carbonate (DSC) were added to a flask with a magnetic stirring bar and 25 ml anhydrous DMF added. The mixture was stirred 2 0 for 3 hours or until the reaction had stopped, as determined by thin layer chromatography (TLC), which was done on samples taken every hour.

After the h~ptenaLion reaction was complete, the BTS acid mixture was added to the resin slurry in a three-neck flask with stirring. This mixture was 25 stirred slowly for 21 hours.

i~ 213fi6~8 Samples of the mixture were removed and assayed for incorporation using a phenol-sulfuric acid assay (PSA). The results of the PSA showed hapten incorporation of 9.46 ~lmoles/g resin.

The haptenated resin was transferred to a Buchner funnel (using #1 or #2 Whatman filters) and washed with 1000 ml RO water. After washing, the resin (weighing 258.0 g) was stored in 250 ml 0.02% sodium azide solution until used.

0 In an alLelllalive method, AF-amino 650M Toyopearl resin was shaken tohomogeneity and 26 ml (approximately 27 g) was transferred to a column and washed with 100 ml of RO water then 100 ml of 0.1 N MES(N-morpholineethanesulfonic acid) pH 4.75. The resin was transferred to a round bottom flask with a minimllm of MES buffer. B trisaccharide, in its acid form, 21 mg was dissolved in 5 ml of MES buffer then treated with 753 mg of EDC(l-ethyl-3(3'-dimethylaminopropyl)carbodiimide), stirred for 5 minutes and added to the Toyopearl resin. The mixture was stirred for 3.5 hours then filtered and washed with RO water, NaCl solution and again with RO water.

2 o An aliquot was taken, dried and the phenol sulfuric acid assay performed to determine hapten loading. The hapten incorporation was 1.46 ,umoles per g.

In another alternative approach 0.1 N NaPO4 buffer, pH 7.4, can be used and sulfo-NHS can be added to the carbodiimide treated hapten. In this approach 2 5 a sulfo-NHS ester is generated which is more stable to hydrolysis than thecarbodiimide and very reactive with amine cont~inin~ supports.

'-2136~98 Example 2: Plepald~ion of Ethylene~ mine, Termin~t-od A Trisaccharide One gram of A trisaccharide methyl ester was added to 50 ml of reagent ethylene~ min~. The reaction was heated at 50C for 24-48 hours under a blanket of nitrogen. The reaction was checked for completion by thin layer 5 chromatography (tlc). The excess ethylen~ minP was removed under reduced pressure on a rotary evaporator and the residue pumped under high vacuum for 16 hrs. The residue was taken up in water and applied to a column of reversed phase silica. Several column volumes of water were applied to the column and then 25 % aqueous methanol used to elute the desired ethylene ~ min~
10 terminated A hapten. The solvent was removed under reduced pressure and the residue dried for 24 hours under high vacuum. The product can be dissolved in a small quantity of water and lyophilized.

Example 3: Reaction of Ethylene~ mine Termin~ted A Trisaccharide with 5 Eupergit The ethylen~ min~, termin~tçcl A trisaccharide was dissolved either in water or 0.1 M sodium phosphate buffer at a concentration of 0.34 mg/ml. One hundred milligrams of EupergitTM resin was slurried in 1 to 3 ml of water or 2 0 buffer to which was added the amount of ethylene(li~min~ tel " ~ A
trisaccharide in excess of the desired level of hapten incorporation. The materials were incubated at room temperature for 2 days then filtered and water washed. The level of hapten incorporation was measured by phenol sulfuric acid assay.

Example 4: Reaction of EDA Opened Eupergit with DSC Activated A
Trisaccharide A 5 g sample of Eupergir was suspended in 50 ml of ACS grade ethylene ~ minP and incubated for 2 hours at 40C. The Eupergit was centrifuged, the ethylen~ min~ dçc~ntçcl off and the Eupergit taken up in water and washed thoroughly to pH 8. Four 100 mg samples of Eupergit were prepared in 3, 3, 2 and 1 ml of water. A solution of 1.81 mg of A trisaccharide acid dissolved in 5.3 ml of dry DMF was treated with 2 mg of DSC (di-N-0 succinimidyl carbonate) at room temperature (r.t.) for 3 hours then 0.1, 0.2, 1 and 2 ml of this solution added respectively to the Eupergit samples in 3, 3, 2 and 1 ml of water. These samples were mixed for 2 days at r.t. on a hematology mixer. The samples were filtered and washed thoroughly with RO water.
Hapten incorporations were measured by phenol-sulfuric acid assay and were respectively; 0.5, 0.69, 2.5 and 4 ~lmoles of ATS per g of resin.

In the above procedure amino-ToyopearlTM can be substituted for Eupergit 2 o Example 5: Removal of Anti-A and Anti-B Blood Group Antibodies From Plasma Usin~ Chromatography Resins cont~ining covalently attached blood group A antigens and blood group B antigens were used to remove blood group antibodies from plasma.
Resins cont~ining blood group A antigens are identified by the suffix "A".
2 5 Resins cont~ining blood group B antigens are identified by the suffix "B " .

The resins were first equilibrated in phosphate buffered saline, pH 7.2 (PBS). Either 1 ml or 500 ,ul of human O plasma was then added to 50 mg of -each resin and the samples were incubated for 2 hours at room temperature with constant mixing. The samples were then centrifuged and the supernatant assayed for antibody by hemagglutination. This procedure was repeated. The results are shown in Table II below. As shown in Table II, all of the resins removed blood 5 group antibodies, although certain resins had substantially higher capacities than other resins for such removal.
Table II

Resin Hapten 1st 2nd 3rd 4th incorp. Adsorp- Adsorp- Adsorp- Adsorp-,umol/g tion tion tion tion Plasma 1024 1024 1024 1024 control Synsorb A 1.17 4 32 128 128 Antab A unknown 4 64 256 128 Eupergit-C 8.8 32 32 128 128 A
Toyopearl 8.4 16 N.D.* N.D.* N.D.*

Biosynsorb 0.88 16 N.D.* N.D.* N.D.*
A

Biosynsorb 0.88A 64 N.D.* N.D.* N.D.*
A/B 0.98B
Poros 8.6 16 N.D.* N.D.* N.D.*

1 0 *Not done Similarly, Toyopearl resins prepared as described above were used to adsorb human plasma and direct agglutination titers of Anti-A and Anti-B

~ 213fi~8 antibody were determined using the methods described above. The results are presented in Table III below.

Table III
Direct Agglutination Titer of Adsorbed Antisera Anti-A Anti-B
Control 64/64 64l64 Toyopearl B (7.87 ~mole/g) 64/64 l/l 10 Example 6: Retention of Coagulation Factors in Plasma Using Various Resins Various resins were used in chromatography columns and assessed for their binding of coagulation factors in the plasma. Input plasma and pooled eluted unbound plasma were assayed for the activities of Factors VIII and XI by 5 d~tellllhlillg the degree of correction in the clotting time of plasma deficient in each of those particular coagulation factors in the presence of activated partial thromboplastin (APTT) reagent, using standard methods known to those of skill in the art.

2 o Table IV
Percent Recovery Resin Factor Factor VIII XI

Eupergit B 100% 100%
Toyopearl B 88% 88%
Sepharose B 100% 96%

~ 2136698 Example 7: Removal of Anti-B Blood Group Antibodies and Retention of Coagulation Factors in Plasma Using Toyopearl Resin and Batch Adsorption One ml of Toyopearl B prepared as described above was used to remove antibodies from 130 ml of human blood plasma. The titer of anti-B blood group 5 antibody was measured by DAT. The anti-B blood group antibody was reduced from a starting titer of 24 to an lln(letect~ble titer 2. Greater than 90% of each of coagulation factors V, VII, IX and XI was retained in the plasma.

In another experiment carried out as above using Toyopearl B, the anti-B
0 antibody was reduced from 25 to 22 and recovery of coagulation factors was 86%.

Although the invention herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely5 illustrative of various aspects of the invention. For example, the resin-antigen compositions of the present invention could be used to remove any undesired antibody while not substantially removing coagulation factors. Thus, it is to beunderstood that numerous modifications may be made in the illustrative embodiments, and other arrangements may be devised without departing from 2 o the spirit and scope of the invention.

Claims (5)

1. A method for removing undesired antibodies from a blood-derived composition which contains said antibodies and one or more coagulation factors, comprising the steps of contacting said composition with a resin-antigen combination capable of removing said antibodies without substantially removing coagulation factors therefrom, such that the treated composition is effectively free of the undesired antibodies but retains an average of at least 85 % of its coagulation factors.

2. The method of Claim 1 wherein said blood-derived composition is selected from the group consisting of blood products, blood plasma, blood plasma precipitate, supernatant and other compositions derived from blood and characterized by the presence of coagulation factors and undesired antibodies.

3. The method of Claim 1 wherein said antibodies are blood group antibodies.

4. The method of Claim 1 wherein the means of contacting the blood-derived composition with the resin-antigen combination is chromatography or batch adsorption.

5. The method of Claim 1 wherein the resin does not substantially adsorb coagulation factors. 6. The method of Claim 1 wherein said antibodies are anti-A and anti-B blood group antibodies.
7. The method of Claim 1 wherein the coagulation factors are selected from the group consisting of Factor V, Factor VIII, Factor IX and Factor XI.
8. A specific antibody-free blood-derived composition which has retained an average of at least 85 % of its coagulation factors, the blood-derived composition having once contained specific antibodies and coagulation factors and thereafter put in contact with a resin-antigen combination capable of removing said specific antibodies without substantially removing said coagulation factors.
9. The composition of Claim 8 wherein said antibodies are blood group antibodies.
10. The composition of Claim 8 wherein said blood-derived composition is selected from the group consisting of blood products, blood plasma, blood plasma precipitate and supernatant.
11. The composition of Claim 8 wherein said antibodies are anti-A or anti-B blood group antibodies.
12. The composition of Claim 8 wherein the coagulation factors are selected from the group consisting of Factor V, Factor VIII, Factor IX and Factor XI.