CA2026717C

CA2026717C - Absorbent for the removal of biomacromolecules such as ldl and endotoxins from whole blood in extracorporeal circuits

Info

Publication number: CA2026717C
Application number: CA002026717A
Authority: CA
Inventors: Veit Otto
Original assignee: Fresenius SE and Co KGaA
Current assignee: Fresenius Medical Care Deutschland GmbH
Priority date: 1989-10-03
Filing date: 1990-10-02
Publication date: 2000-01-25
Anticipated expiration: 2010-10-02
Also published as: DE3932971C2; EP0424698A1; ATE146700T1; ES2098237T3; EP0424698B1; CA2026717A1; JPH03254756A; DE3932971A1; DE59010619D1; JP3100974B2

Abstract

A material for the elimination of biomacromolecules, in particular LDL and ignotoxins from a whole blood circuit, comprising an absorbent, comprising a porous carrier material of a homo-, co-, or terpolymerizate of acrylic acid and/or methacrylic acid with a particle range of between 50 and 250 mmol and an exclusion level of at least 5 x 10 5 daltons, as well as organic ligands which are covalently bound to the carrier material via a spacer, wherein the carrier material has a spherical shape and suitably, the spacer contains no double bonds.

Description

RV3 011090 1 LUDR3.~
FIELD OF THE INVENTION
Absorbents for Removal of Toxins from Whole Blood.
BACKGROUND OF THE INVENTION
It is well accepted in the art that it is preferable to undertake the cleansing of whole blood rather than blood plasma in extracorporeal circuits, since such a procedure requires substantially less equipment and also reduces the requirement of supervision by additional professionally trained personnel, who are not always available in every hospital. In the cleansing of whole blood, it is not necessary to expand substantial efforts in the prior removal of cells ( i.e., leuco-, erthyro- and thrombocytes), for example in the filter, which also requires constant supervision.
Heretofore, the cleaning of whole blood ex-vivo, utilized absorbents of activated charcoal or activated charcoal bearing certain coatings, such as those provided by solutions of polyacrylic acid or polyacrylic acid and polyethylene imine (see USSR SU
732,207).
Such activated charcoal absorbents, because of their grounding in activated charcoal, however suffer from the disadvantage of reduced mechanical stability in particular at high pressures, as well as a low level of selectivity, with respect to the biomacromolecules to be removed.
As a consequence of the foregoing, considerable experimentation was undertaken to replace absorbents based on charcoal (as well as, for other reasons, those based on other inorganic materials) by, for example, modified natural or synthetic polymers which had a higher mechanical stability and a higher level of selectivity for the elimination of certain body generated, in particular pathogenic biomacromolecules, in body fluids such as blood, plasma, or serum.

,2026717 RV3 011090 2 LUDR3.0-041 By use of the process of suspension polymerization, certain porous homopolymers as well as co- or terpolymers of vinyl containing monomers, for example, acrylic acid received particular attention, as the carrier materials.
Such acrylic acid polymers are presently available in commerce, for example, "TSK-Gel Toyopearl~, manufactured by Toyo Soda Kogyo Co., Ltd, Japan and Toso Haas, Philadelphia, PA, USA) and Fractogel~ TSK (manufactured by Merck GmbH, Darmstadt, Federal Republic of Germany). Such substances are designated as hard gels which, in a chemical sense, are substantially identical, and, due to the presence of OH groups, are also hydrophilic.
These materials not only in their original form, but especially after modification (activation) by reaction first with an oxirane containing compound, for example, epichlorhydrin and the subsequent reaction with ammonia, an amino, or carboxyl containing compound, or with cyanuric chloride, may be used as carrier materials (see for example J. Chromatogr. 239, 747-754 (1982)) and Toya Soda Kenkyuhokoku, 25 (2), 81-88 (1981)). As further examples of such modified products which could be used as absorbents or carrier materials in absorbents, there may be mentioned those which are activated with glutaraldehyde and then reduced, for example, with sodium borohydride (see Shin-jikkenkagaku-koza, ed. S. Ishii, Maruzen, Tokyo, 141 (1978)).
Such activated carriers offer the possibility of having a specific operating mode.
By the use organic bridging members of different chemical structure and length (generally known as spacers) one may introduce specific covalent organic ligands.
Thus, one can produce so-called specifically tailored absorbents of higher selectivity with respect to the biomacromolecules which one wishes to remove from the system.
Thus, in EP-A 83 112 042, certain absorbents are suggested as particularly suitable for the selective removal of VLDL (lipoprotein of very low density) and/or LDL
(lipoprotein of low density) from body fluids such as blood or plasma in an extracorporeal circuit. Such materials utilize as carrier material Toyopearl~
TSK of the type HW 75, 65, 60 as well as 55 (having a grain size of approximately 50 to 100 ~,m . 20 2677 RV3 011090 3 LUDR3.0-041 (see in comparison Examples 1 and 2 to 4 of the EP application), however having different exclusion limits, to which, after reaction with epichlorhydrin a ligand, such as heparin or chondroitin-polysulfate is covalently bound.
In DE-OS 36 17 672, there are named a substantial number of porous adsorbents including those which are suitable for the elimination of pathogenic biopolymers from aqueous fluids such as body fluids, for example, blood, plasma or serum, which comprise an organic solid phase as the carrier material which, via a covalently bound bridge member (spacer), which may be a mercapto-, amino-, and/or carboxyl group containing mono-, oligo-, or polymer and is covalently bonded to a ligand, suitably a polycarboxy acid or a derivative thereof which can be converted into the free acid form. The carrier material is pretreated with a coupling agent suitably an epoxy compound such as a diglycidal compound which can subsequently be reacted (derivatized) with an amino compound such as ammonia to form the bridge member. The thus modified carrier material is then further reacted, for example with a polycarboxy acid or a derivative thereof which is further activated with a carbamino acid ester, for example, N-ethoxy-carbonyl-2-ethoxy-1,2-dihydro-quinoline, to form the ligand.
Many other materials may also be used as ligands. However, especially preferred are polymerizates or copolymerizates of acrylic acid and as the carrier material, the commercially available Fractogel~ may be mentioned.
It is disclosed in GIT Fachz. Lab. 27, 380-389 (1983) that all types of Fractogel~ TSK, that is to say, types HW 40, 50, 55 and 65 comprise spherical and entirely porous particles having a grain size for the subtype S in the range of 25-40~m and for the subtype F, in the range of 32-63~.m. There is an exception in type HW 40 in the form of subtype C, which comprises a grain~size range of 50-100~,m.
From Figure 4 of the above-identified publication, it is clear that type HW 40 exists in a size range of only 102 to 104 daltons. Thus, as a result of this relatively small pore diameter, it is unusable for the separation of larger biomacromolecules, for example, LDL which has a molecular weight greater than 106.

RV3 011090 4 LUDR3.0-041 The term "exclusion limit" is understood to be the minimum molecular weight of a molecule which, in gel permeation chromatography cannot (anymore) enter into the pores of the absorbent.
It is further to be deduced from the foregoing publication, see in particular page 385, first column, last paragraph, that utilizing absorbents of a smaller particle size, leads to a substantial improvement in the efficiency of separation between the biopolymers to be eliminated (at constant selectivity). For this reason, in such chromatography where high demands are made on the absorptive properties, in particular the separation efficiency, the finest possible, that is to say "super fine"
material, with a grain size in the range of 20 to 50~m is used.
A spacer and a ligand, for example, a polycarboxy acid, suitably a polyacrylic acid, or a Polymyxin, for example Polymyxin B, may be used in conjunction with known carrier materials formed from homo-, co-, or terpolymers of acrylic acid or methacrylic acid, such as for example, the above identified commercially available materials to provide substrates which can be utilized for the removal of biomacromolecules, for example, LDL and endotoxins from blood plasma. This is particularly so when the carrier material is chosen from the point of view of porosity, that is to say, the exclusion limit wherein, with respect to efficiency of separation, the smallest particle size is sought.
When utilized in an extracorporeal circuit with whole blood on the other hand, particles of a grain size of at least 50um should be utilized. This requirement is based on the fact that the largest blood cell particles present in whole blood have a diameter of approximately 20~m, so that the sieve which holds back the absorbed material must have a pore width of at least 40~.m, in order to let the blood cells through. It has been found that utilizing packed columns of absorbent particles of a size of 50~cm, there is sufficient room between these particles to permit the blood cells to pass through.

RV3 011090 5 LUDR3.0-041 Unexpectedly however, it has been found that the foregoing carrier materials having a covalently bound spacer and a ligand covalently bound thereto, for example, a polycar-bonic acid such as polyacrylic acid or Polymyxin B, having a particle size greater than 50~.m ostensibly utilizable with whole blood, when utilized for example, for the separation of LDL and endotoxins which require an exclusion limit of at least 5 x 105 daltons (measured with lypoproteins), are not suitable for the removal of such biomacro-molecules because an entirely undesired thrombocyte aggregation then occurs.
This phenomenon of blood cell incompatibility is shown in Figure 1 (dashed surfaces) when what one would consider as suitable absorbent for the removal of LDL and endotoxin from full blood (that is to say, one with a particle size greater than um and a cut-off barrier of approximately 5x105 daltons measured with lipoproteins) was tested and found unsuitable. Here, a carrier material, i.e., the 15 commercially available Toyopearl~ (HW 75 SC) was used which has a spacer and a ligand in accordance with Example 1 hereof (this material is, as stated above, is made of copolymer of glycidylmethacrylate, erythrodimethacyrlate and ethylene glycol.
However, while this material has the appropriate particle size and exclusion limit, it is not spherical but rather exists in the form of irregularly formed aggregates.
SUMMARY OF THE INVENTION
The purpose of the present invention is to provide a absorbent material which may be used in an extracorporeal circuit with whole blood which has, suitably, a high selectivity with respect to removal of biomacro molecules in particular, LDL
and endotoxin while having an acceptable compatibility with blood cells.
This task is achieved in accordance with the absorbents comprising a porous carrier material of a homo-, co-, or terpolymerizate of acrylic acid and/or methacrylic acid with a particle range of between 50 and 250 mmol and an exclusion level of at least 5 x 105 daltons, as well as organic ligands which are covalently bound to the carrier material via a spacer, wherein the carrier material has a spherical shape.

.202677 RV3 011090 6 LUDR3.0-041 A further problem to be solved by this invention is the preparation of an absorbent, in particular for the removal of endotoxins from whole blood which gives rise to the lowest possible level of retention for thrombocytes. This is solved by an absorbent wherein the spacer contains no double bonds.
It has been found that in order to formulate absorbents, which are in large measure suitable for the removal of endotoxins from whole blood that it is desirable to provide the carrier material with a relatively long covalently bound spacer having, for example, 13 atoms. It has also been shown that the presence of double bonds, which occur in the spacer as a result of a requirement of production, gives rise to an undesired retention level of thrombocytes (see Fgure 2). An example of a spacer containing two double bonds is the 13 atom spacer which is introduced in the reaction of OH containing carrier materials such as Fractogel~ and Toyopearl~
with ethylenediamine and glutaraldehyde. Surprisingly, it has been found that the hydration of double bonds, for example with sodium borohydride in the conventional manner, gives rise to an absorbent which retains much lower amounts of thrombocytes (see Figure 2, right).
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a block plot showing recovery of blood cells of different types for spherical and non-spherical carriers.
Figure 2 is a block plot showing recovery of blood platelets using different types of spacers.
Figure 3 is a comparative pressure/flow diagram for adsorption in whole blood and saline.
Figure 4 (a) and (b) are electron micrographs of non spherical and spherical carrier material.

2p2s717 RV3 011090 7 LUDR3.0-041 Figure 5 is a block diagram showing the influence of spacers of different length on the LDL adsorption capacity.
Figure 6 a block diagram showing the recovery of blood cells of the three different types.
Figure 7 shows the structure of a 13 atom spacer beofre and after hydration.
The invention may be further illustrated by the following examples.

RV3 011090 8 LUDR3.0-041 In this example, there will be described the formation of an absorbent suitable for the removal of LDL from whole blood wherein, as well as in Example 2, there is utilized as the starting carrier material Toyopearl~, under the internal characterization, HW 70 SC (56H), manufactured by Toyo Haas Company, Philadelphia, PA, U. S. A., under the this material was spherical (see Fgure 4b), has a size range of between 70 and 150um. This product is a copolymer of ethylene glycol, glycidylmethacrylate and erythritol-dimethacrylate.
The absorbent utilized for the following comparison tests for blood cell compatibility comprises, as the carrier material, the known commercial product Toyopearl~ 76 SC, which may be distinguished from the foregoing described product in that it is not spherical. Furthermore, it demonstrated (as a result of particle aggregation) an irregular condition (see Figure 4a) at the same particle size.
The foregoing carrier material is amino derivatized in the usual manner, namely as follows:
a) epichlorhydrin followed by ammonia, whereby a 5 atom spacer is introduced; b) with epichlorhydrin and ethylenediamine, whereby an 8 atom spacer is introduced;
c) with 1,4-butandiol-diglycidoxyether and then ammonia, whereby a 13 atom spacer is introduced.
The preparation of the amino derivatized carrier (b) has been carried out as follows:
The Toyopearl~ gel was washed on a glass filter (G-1 ) with distilled water and dried in vacuo at 60°C. Fifty grams of the dried gel, 300 ml of 1 N-NaOH, and 11 ml of epichlorhydrin were placed in a 500 ml-separable flask and stirred for 3 hours at 30°, filtered by a glass filter (G4) and washed with cold distilled water. This gel was placed in a 500 ml-separable flask with 63 ml of ethylenediamine, 35 mol distilled water and stirred for 1.5 hours at 80°C. After the reaction, the mixture was filed by RV3 011090 9 LUDR3.0-041 a glass filter (G4), the gel was washed with acetone several times to remove unreacted ethylenediamine and dried in vacuo at 60°C overnight.
For the introduction of polyacrylic acid as a ligand 200 mg of polyacrylic acid were dissolved in 12 ml at 0.15 M aqueous sodium chloride to which were added ~mg of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline as activating agent.
After 30 minutes, the above described amino derivatized carrier material was added to the polyacrylic acid solution. The mixture was permitted to react at room temperature for 12 hours. The composite absorbent on the spherical carrier material was subjected to a battery of tests:
A1 Whole Blood Measurament/S~ecific Binding Caaacitv These were carried out with an absorbent in accordance with Example 1 for the determination of the LDL binding capacity obtained from fresh blood drawn from healthy volunteers.

2 ml. of the absorbent of Example 1 were charged to a 10 m. diameter column.
ml. of fresh blood treated with ACDA (citrate anticoagulant) (1:1 ) and pumped through the absorber at the rate of 1 ml. per minute. The blood cells were measured 20 before and after this treatment.
The absorbent produced in accordance with Example 2 however was utilized for the measurements of the endotoxin binding capacity in physiological aqueous sodium chloride (standard: E.coli 055:85). For an absorbent produced in accordance with Example 1 (a), an LDL binding capacity of 8 mg/ml. of absorbent was given. In vitro tests with human blood which were carried out to determine the effect of spacer lengths in the absorbents according to Examples 1 a, 1 b and 1 c, with respect to LDL
capacity, are shown in Figure 5.
Blood Cell Compatibility An absorbent was produced in accordance with Example 1, however, utilizing the non-spherical absorbent (Toyopearl~ (HW 75 SC). Tests were carried out with 20 ml. of blood/2 ml. of absorbent. The results are illustrated in Figure 1.
They show .2026717 RV3 011090 10 LUDR3.0-041 a substantial superiority of the absorbent in the spherical form over the non-spherical form, with respect to compatibility with leuco- and thrombocytes.
Toxicological Experiments The following toxicological experiments were carried out with respect to the LDL absorbent:
1. Physicochemical tests (USP XXI) 2. Systematic Acceptability in the Mouse (USP XXI) 3. Intracutaneous Test with Rabbits (USP XXI) 4. Sensitization Test with Guinea Pigs (DECD Guideline No. 406) 5. Hemolysis Test with Rabbits 6. Agar Overlay Test (Compare Evaluation of Hemodialysis Membranes, US
Depart. Health and Welfare, 1977, Publ. No. NIH-77/1234).

7. Ames Test, DECD Guideline No. 471 ).
None of these tests showed the presence of any toxic effect. The absorptive material is therefore biologically compatible.
Pressure Stability 400 ml. of absorber produced in accordance with Example 1 was packed into an absorption housing of 48 mm. diameter.
Whole blood was pumped over 15 minutes through the absorber at a plurality of rates. The pressure was measured at the filter input. The pressure was found to be constant over 15 minutes. The pressure flow diagram is shown in Figure 3.
The stability of pressure, as well as the linearity of the pressure flow diagram, shows that the absorber is stable at these pressures.
Utilizing the same housing and similarly utilizing 400 m. of an absorbent produced in accordance with Example 1, a particle count was carried out in accordance with the provisions of DIN Schedule 58 362 (determination of particulate impurity levels).

20267t7.
RV3 011090 11 LUDR3.0-041 Size of Particles (gym) 25-50 51-100 Over 100 after ~assaae of 10 liters Number of Particles 31 2 0 All of these experiments were carried out with sterilized absorbent which had been previously autoclaved at 121 °C for 30 minutes.
E~-The spherical carrier material described in Example 1 was treated, in a manner well known in the art, with epichlorhydrin and subsequently with ethylenediamine or with 1,4-butandiol-diglycidoxyether, followed by ethylenediamine to produce an activated carrier material.
In this procedure, 40 grams of carrier material were dissolved in 125 ml. of a 1 molar aqueous sodium hydroxide solution with 8.25 ml. of epichlorhydrin or with 1,4-butandiol-diglycidoxyether, followed by 47 ml. of ethylenediamine.
The thus activated carrier material was incubated for 12 hours with a 5%
aqueous solution of glutardialdehyde. These procedures introduced into the carrier material - a 13 atom spacer and a 19 atom spacer respectively, each containing two double bonds. The structure of the 13 atom spacer (before and after hydration) is shown in Figure 7, wherein the term "POL." shows a polymyxin ligand bound thereto.
Thereafter, Polymyxin B was covalently bound to the ligand in the conventional manner in which the coupling reaction was carried out in 250 ml. of a 10 mM
solution of magnesium chloride containing 0.6 g. of Polymyxin B sulfate to yield absorbent (I).
A portion of the absorbent with the 13 atom spacer was hydrated in the usual manner with 5% aqueous sodium borohydride to hydrate the double bonds of the spacer to yield absorbent (ll).

.202677 RV3 011090 12 LUDR3.0-041 The retention of thrombocytes was examined, utilizing 20 ml. of blood/2 ml.
of absorbent with (I) (with unreduced spacer), as well as (II) (with reduced spacer) for the 13 atom spacer as well as the 19 atom spacer. The surprising result, as 5 shown in Figure 2, was that (II) showed the retention of a substantially smaller number of thrombocytes.

.202677 RV3 011090 13 LUDR3.0-041 In accordance with the detailed description set forth immediately below, spherical carrier material based upon methacrylic acid terpolymer was amino derivatized in accordance with Example 1, an 8 atom spacer introduced and bonded to the polyacrylic acid in a covalent manner as described in Example 1, whereby an absorbent was obtained which proved itself to be useful in the separation of LDL from whole blood.
The starting carrier material was a terpolymer of methacrylamide, N-methylene-bis-methylacrylamide and allylgylcidylether, having a size distribution of spherical particles in the region of 50 to 200 ~.m and an exclusion limit of > 5 x 105 daltons.
These particles have a macroporous structure comprising channels, openings and cavitations. The pore volume (determined by the mercury method) was 1.74 cm3/g.
and the mean pore diameter was 35 nm. The specific BET-surface (isothermal nitrogen absorption) was 183 m2/g.
Toxicological determinations (USP XXI) showed that the carrier material and the absorbent produced therefrom are toxicologically harmless. It was also shown further to be biologically compatible.
The method utilized in Example 1 showed the LDL binding capacity of the absorbent from whole blood to be 3.6 mg./ml.
This value is less than that obtained for the measurements of the material produced in accordance with Example 1 (a) which can be explained by the fact that the absorbent of the present Example 3 has a smaller exclusion limit than the absorbent of Example 1.
The blood cell compatibility of the absorbent is carried out in accordance with procedures of Example 1. The results are shown in Figure 6. The absorbent thus illustrates an unexpectedly low level of retention for leucocytes, erythrocytes and thrombocytes.

Claims

1. An absorbent material for the elimination of biomacromolecules, from a whole blood circuit, comprising a porous carrier material of a homo-, co- or terpolymerizate of acrylic acid and/or methacrylic acid with a particle grain size range of between 50 and 250µm and an exclusion limit of at least 5 x daltons, as well as organic ligands which are covalently bound to the carrier material via a spacer, characterized in that the carrier material has a spherical shape.

2. An absorbent material for the elimination of LDL from a whole blood circuit, comprising a porous carrier material of a homo-, co-, or terpolymerizate of acrylic acid and/or methacrylic acid with a particle grain size range of between 50 and 250µm and an exclusion limit of at least 5 x 10 5 daltons, as well as organic ligands which are covalently bound to the carrier material via a spacer, characterized in that the carrier material has a spherical shape.

3. An absorbent material for the elimination of endotoxins from a whole blood circuit, a porous carrier material of a homo-, co-, or terpolymerizate of acrylic acid and/or methacrylic acid with a particle grain size range of between 50 and 250µm and an exclusion limit of at least 5 x 10 5 daltons, as well as organic ligands which are covalently bound to the carrier material via a spacer, characterized in that the carrier material has a spherical shape.

4. An absorbent in accordance with any one of claims 1, 2 or 3, characterized in that the spacer contains no double bonds.

5. An absorbent in accordance with any one of claims 1-4, characterized in that the carrier material has a particle size of between 50 and 150µm and an exclusion limit of at least 10 5 daltons.

6. An absorbent in accordance with any one of claims 1-4, characterized in that the carrier material has a particle size of between 50 and 150µm.