Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
2136598
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.
2136698
'_
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
2136698
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
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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.
15 SUMMARY OF THE INVENTION
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~
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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
- 12-
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
2136698
- 14-
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
- 16 -
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.
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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
-
2136698
- 19 -
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
- 20 -
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.
