NO905496L - BIOLOGICAL COMPATIBLE, SUBSTANCE-SPECIFIC REAGENTS FOR TREATMENT OF PHYSIOLOGICAL LIQUIDS. - Google Patents

Description

Technical area

The present invention relates to the production and use of biologically compatible water-insoluble reagents

refers to the removal of substances from physiological fluids, including specific binding ligands and biocompatibility agents immobilized on the surface of a carrier substrate.

The background of the invention

The selective removal of harmful substances from blood, blood plasma or other physiological fluids can provide important therapeutic benefits. High levels of circulating lipids, especially cholesterol, which is carried in low-density lipoprotein (LDL), are e.g. a cause of atherosclerosis. When it is not stopped, atherosclerosis can lead to serious diseases in the circulatory system, leading to stroke and/or biocardial infarction. Reduction of plasma LDL levels is believed to substantially reduce the incidence of such events in high-risk patients (A.M. Gotto, et al Circulation, 69, 1065A-1090A (1984)). Selective removal of LDL should be an effective method to reduce atherosclerosis. Similarly, the ability to intervene in the development of arthritis by removing circulating rheumatoid factor, immune-related diseases by removing antibodies, or cancer by removing malignant cells is sought after in medical science. Currently, selective dilution plasma apheresis, which involves passing 2-4 liters of blood or plasma through an extracorporeal device containing the selective absorption agent, represents an attractive therapy.

Clinical application of such necessary treatment modalities brings the physiological fluid into contact with foreign surfaces. In the case of blood and blood plasma, one of the resulting sequelae is activation of the complement cascade which can lead to life-threatening anaphylaxis. When practicing such treatment modalities, blood or plasma is anticoagulated using citrate and/or heparin. Citrate is often used as much for its ability to inhibit the complement cascade as for its ability to block coagulation. The reliance on citrate to inhibit the complement cascade can be risky because citrate is toxic at doses very close to the dose used as an anticoagulant. Citrate is typically used in a dose where mild toxicity is observed (numbness and tingling in the lips). The toxicity of citrate is due to calcium gelation which disrupts normal nerve conduction. The applicability of extracorporeal devices containing highly selective adsorbent materials is therefore not only dependent on the specificity and affinity of the material, but also its biological compatibility with body tissues and biochemical systems.

Stoffel and Demant (Proe. Nat. Acad. Sei. USA, 78, 611-615 (1981)) describe the use and effectiveness of immunoadsorption chromatography for removing low density lipoprotein from plasma. Using polyclonal anti-swine or LDL antibodies covalently bound to Sepharose CL-4B, pigs were treated with an extracorporeal device containing a plasma separator and the immunoadsorbant interspersed in series in an arterial-venous parallel circuit. The plasma LDL concentration was found to decrease progressively with the duration of the treatment and the increase of the flow volume.

Koren et al. (Biochemica et Biophysica Acta. 8 7 6, 202-207 (1986)) describes the use of a mouse monoclonal anti-human LDL antibody coupled to an agarose support to remove LDL from plasma samples. No apheresis was performed on any organisms.

Parker et al. (Proe. Nat. Acad. Sei. USA, 8^, 777-781 (1986)) treated patients by LDL apheresis using sheep polyclonal anti-LDL antibodies attached to Sepharose. Substantial reductions in LDL levels were achieved with this therapy.

European Patent Application No. 83112042.3, published June 6, 1984, describes the use of immobilized heparin or dextran sulfate to remove LDL from plasma. The adsorbent used comprised a water-insoluble porous hard gel, to which a binding ligand was attached. The ligands were attached through reaction with epoxy groups introduced into polymer gels by reaction with epichlorohydrin or a polyoxyran compound, or in the case of inorganic gels, with epoxy groups introduced by reaction with epoxysilane coupling agents. Gels with surface hydroxyl groups were therefore preferred. Although many selective binding ligands were described, only heparin and dextran sulfate (adsorbents that remove many plasma proteins) were exemplified as LDL removal systems.

Japanese patent application JP60087854 describes a blood-purifying adsorbent for removing malignant substances or cells from blood or plasma. The adsorbent comprises a carrier such as glass, charcoal, cellulose triacetate,

and various polymers to which a negatively charged compound and an organic ligand are attached. However, the binding requires the use of strong electrophilic reagents that cannot be used in simultaneous or subsequent reactions that include complex biological binding ligands.

European patent application EP-103184A describes biospecific polymers carrying immobilized biological agents.

The biocompatible polymer support material is usually a hydrogel or other wettable polymer to which a biological agent is covalently bound. No biocompatible agents have been disclosed which would generally provide protection against adverse reactions in vivo.

Japanese patent application J602239425 describes the use of solid carriers to which are bound monoclonal antiapo-lipoprotein B antibodies, to remove LDL from human plasma. No biocompatibility agents were used to promote protection against adverse reactions with tissues or systems in vivo.

Japanese patent application JP59200655 describes the use of an adsorbent consisting of an insoluble carrier, preferably hydrophilic, with a bound organic compound containing a negatively charged group. The immobilization of the binding ligand takes place through the negatively charged group. The use of the adsorbent to remove malignant cells is discussed.

A clear need exists today for an adsorbent incorporating specific binding ligands and biocompatibility agents to remove harmful substances from physiological fluids. Such agents should extend both high selectivity and protection against activation of the complement system or blood coagulation. Increased biocompatibility of the reagent would allow for a reduction in the amounts of anti-coagulants administered. The activity of the complex biological ligands is extremely sensitive to the immobilization technique. It is also required that a method of preparing such reagents be mild enough to avoid denaturation, and yet flexible enough to allow simultaneous or sequential immobilization of binding ligands and biocompatibility agents.

Summary of the invention

A method has been invented for the immobilization of both specific binding ligands and biological compatibility agents on many different carrier substrates. The newly produced reagents remove substances from physiological fluids, while adverse reactions such as complement activation are minimised. A specific aspect of the present invention is a water-insoluble reagent for removing a substance from physiological fluids comprising (a) a carrier substrate, and immobilized on the surface of the carrier substrate, (b) a binding ligand specific to the substance, and (c) a biocompatibility agent comprising a organic molecule with a nucleophilic part and a negatively charged part separated by a spacer arm, where the biocompatibility agent is immobilized on the surface through reaction with the nucleophilic part. Another aspect of the present invention involves a method of preparing such a water-insoluble reagent for removing a substance from physiological fluids, comprising the step of contacting and reacting a carrier substrate with an aqueous solution of a binding ligand specific to the substance, and a biocompatibility agent comprising an organic molecule with a nucleophilic moiety and a negatively charged moiety separated by a spacer arm. This method can include bringing the carrier substrate into contact with and reacting with the aqueous solutions of binding ligand and biological compatibility agent both simultaneously and sequentially. Still another aspect of the present invention includes a method for treating a physiological fluid to remove a substance, comprising contacting the fluid with a water-insoluble reagent as described above. As a final aspect, the present invention provides an apparatus for the extracorporeal treatment of physiological fluids to remove a specific substance, including methods for removing fresh blood from a mammal, methods for separating plasma from the fresh blood, methods for treating the plasma, including a chamber containing a water-insoluble reagent as described above which will react with and remove the substance from the plasma, and methods for recombining the substantially substance-free plasma with the rest of the fresh blood and for returning the recombined fresh blood to the mammal.

In the case of removal of LDL from blood plasma, preferred reagents and methods will include an activated, neutral polymer carrier substrate, a polyclonal anti-LDL antibody, and a biocompatibility agent, wherein the immobilization is carried out in two steps at a pH of from approximately 4 to 10, controlled by a non-nucleophilic buffer. Preferably, due to ease of preparation and/or efficiency of use, the reagent is formed by immobilizing monoclonal anti-LDL antibody and sulfamic acid on tresyl-Sepharose in a one-step simultaneous reaction at approximate pH

8 in potassium phosphate buffer.

Brief description of the figures

Figure 1 is a bar graph representing LDL and HDL plasma concentrations before and after treatment for two experiments using human plasma in vitro.

Detailed description of the invention

Production of materials

The present invention provides a versatile method for synthesizing new biologically compatible reagents for the removal of specific substances from physiological fluids through the immobilization of specific binding ligands and biological compatibility agents on substrate carriers under mild conditions, which preserve the activity of sensitive biological ligands. Within the framework of the present condition, a number of designations are used. As used herein, a "support substrate" refers to a solid support matrix "activated" by well-known coupling chemistry to introduce reactive surface moieties susceptible to nucleophilic attack and displacements by binding ligands or biocompatibility agents to be immobilized on the support substrate. "Binding ligand" refers to any substance, or group of substances, that has specific binding affinity for the substance to be removed from the physiological fluid and excludes other substances. The binding ligand can consist of a polyclonal or monoclonal antibody in the form of antiserum or ascites fluid, an IgG fraction or in the form of an active fragment of such materials. Other specific binding proteins, e.g. lectins, antigens or protein A can be used. As used herein, "biocompatibility moiety" refers to a substance that makes the carrier substrate more biocompatible to complement activation. The term "nucleophilic moiety" refers to chemical groups, e.g. NH^, SH or OH, which contains an unoccupied pair of electrons and usually attacks another molecule in a place where the atomic nucleus is poorly protected by outer electrons. As used herein, the term "negatively charged moiety" refers to chemical groups, e.g. OSO^, RSO^, RSC^, C00 , which contain a net negative charge. The nucleophilic part and the negatively charged part are contained within the same molecular entity which forms a "spacer arm" that separates the two functional parts.

As used herein, the term "neutral polymer" refers to a water-insoluble material, either natural or synthetic, which consists of repeating subunits and which can be formed into many different possible configurations such as flat sheets, fibers, beads, porous beads, membranes and foams.

"Non-nucleophilic buffer" refers to any water-buffering substance that does not contain a nucleophilic moiety such as R-OH, R-SH, R-NH^, R2NH. "Polyclonal antibody" refer-

refers to any collection of antibodies against a substance, where the collection of antibodies against a substance arises from many different antibody-producing cells. "Monoclonal antibody" refers to any antibody produced from a single antibody-producing cell line.

Carrier substrate

The aqueous soluble reagents according to the present invention involve the immobilization of binding ligands and biological compatibility agents on activated solid support matrices (carrier substrates), using such a mild coupling chemistry that sensitive biological ligands and biological compatibility agents can be reacted simultaneously. The immobilization process is shown schematically in reaction equation 1,

which shows the possibility of either sequential or simultaneous reaction of the binding ligand (L) and the biocompatibility agent (BA). Activation of various solid support matrices is achieved by using a suitable chemistry for the material to introduce a chemical moiety (ACT) susceptible to nucleophilic attack by chemical groups in the binding ligand molecules and the biocompatibility agent.

Many different materials are suitable as solid support materials for the present invention. These materials generally have the following characteristics: (1) sufficient surface area to effectively capture and remove the target substance, either due to the presence of pores or availability in a finely divided state, (2) physiological fluid is allowed to flow over or through the material when enclosed in an extracorporeal treatment apparatus, (3) chemical surface groups that can be activated following reaction with a binding ligand and biological compatibility agent, (4) stable in the biological environment. Materials that meet these criteria include synthetic polymers, natural polymers, silicon oxide, alumina, and zirconia. The physical geometry of the solid support matrix may be in the form of a particle, bead, fiber, hollow fiber, thin sheet, foam, or any other form that allows fluid flow over or through the material. Table I shows a list of various commercially available carrier substrates that can be used to practice the present invention. The list is representative of the other materials and activation chemistries that satisfy the above four criteria will be known to the person skilled in the art.

Activation of support matrix materials to produce suitable support substrates with surface groups susceptible to nucleophilic attack is performed using well-known chemical techniques and process reagents. See Scouten, W.H., Affinity Chromatography: Bioselective Adsorption on Inert Matrices, Wiley&Sons, New York, (1981), incorporated herein by reference. In the case of synthetic and natural polymers, activating reagents may include cyanogen halides, epichlorohydrins, polyoxiranes, periodates, polyethylene imines, or glutaraldehyde. When the most preferred reagent for removing LDL from blood plasma, Sepharose^4B (Pharmacia Fine Chemicals, Uppsala, Sweden; agarose gel beads) containing surface hydroxyl groups, is reacted with tresyl chloride (2,2,2-trifluoroethanesulfonyl chloride) to produce a surface -sulfonate esters (tresyl-activated Sepharose<®>4B, Pharmacia Fine Chemical) as depicted schematically in reaction equation 2.

agarose-tresyl chloride "tresyl-activated" agarose

Such sulfonate esters have been reported to undergo nucleophilic displacement of amines, thiols, hydroxyl, and imidazole groups to produce stable chemical bonds that immobilize various binding ligands (Nilsson, K. and Mosbach, K., Biochem Biophys. Res. Comm., 102, 449-457 (1981)

and Methods in Enzymology, 104, 56-69 (1984).

In the case of inorganic support matrix material including silica, alumina or zirconia, activation can be achieved by reaction with an epoxy silane such as gamma-glycidoxypropyltrimethoxysilane, an aminosilane such as gamma-aminopropyltriethoxysilane, or polyethyleneimine glutaraldehyde.

Binding ligand

Many different binding ligands can be used in the present invention depending on the specific substance to be removed from the physiological fluid. Binding ligands possessing high specificity allow the selective removal of the substance with the least overall impact on the composition of the physiological fluid being treated. A representative list of binding ligands, conjugated substance to be removed, and disease to be treated is provided in Table II.

It should be understood that the binding ligands according to the present invention are not limited to those listed in Table II, which is intended to be illustrative.

These binding ligands can be used alone or in mixtures if appropriate.

In the case of removal of LDL from blood plasma, highly specific monoclonal antibodies are most preferred as binding ligands. An anti-LDL antibody was prepared using a standard monoclonal antibody immunization and fusion protocol (Vernon T. Oi and Leonard A. Herzenberg. Selected Methods in Cellular Immunology, Eds.

B. MØishell and S. Shiigi, 1980, pp. 351-372) Balb/CJ mice were immunized with purified human LDL with and without complete Freund's adjuvant. The mice were injected with a booster

at regular intervals and blood drawn at various times for several weeks after the immunizations. Serum samples were tested by ELISA to determine the amount of anti-LDL antibodies present in the immunized mice relative to the non-immunized Balb/CJ.

Several months after the first immunization, the mice were re-injected with antigen and the first fusion was performed. A spleen cell suspension from a treated mouse was fused with SP2/O myeloma cells, mixed with an irradiated spleen feeder cell suspension and spread. The clones were fed at various intervals and observed for growth.

Clones were selected and analyzed to determine the amount of IgG produced, and each clone was isotyped. Clones were tested regularly to monitor IgG production. At a later time, cell supernatants from selected clones were tested in an LDL ELISA to determine specific activity of each selected clone. The most productive clones were selected and deep frozen in liquid nitrogen. Several of the better clones were subcloned and analyzed to ensure that a monoclonal anti-LDL was produced and to try to increase IgG production and LDL specificity. All the clones that produced specific anti-LDL monoclonal antibody were further analyzed with respect to other factors, such as stability, antibody production and affinity, which would provide selection of the best clones for use in the present invention.

Biocompatibility agent

A number of bifunctional chemical entities comprising both a nucleophilic moiety, for reaction with the carrier substrate, and a negatively charged moiety separated by a spacer arm, are considered biocompatibility agents according to the present invention. The negatively charged moiety attaches to the surface after the reaction of the nucleophilic moiety with the support substrate to immobilize the biocompatibility agent.

Many combinations of nucleophilic moiety and negatively charged moiety are contemplated by the present invention. Nucleophilic moieties including e.g. RNH2, RHS, ROH or R2NH can be used. Negatively charged moieties including OSO^, RSO^, RSC>2, CC>2 are suitable. A representative list of biocompatibility agents is shown in Table III.

It appears from Table III that a number of bifunctional molecules with separate nucleophilic and negatively charged parts can act as biological compatibility agents. It is understood that the molecular structure into which such moieties are incorporated serves as a spacer arm to separate the moieties and allow independent function, i.e., reaction with carrier substrate and protection against adverse biological interactions. In itself, the spacer is not critical and the person skilled in the art can select different molecular structures as spacers into which the functional parts are to be incorporated.

Water-soluble reagent

Binding ligands and biocompatibility agents are immobilized on the surface of the carrier substrate by means of nucleophilic displacement reactions involving a nucleophilic part of the binding ligand or biocompatibility agent, and the activated part on the carrier substrate. In the case of protein binding ligands such as antibodies, the large number of amino groups within the antibody serve as nucleophilic moieties for a suitable reaction. The process according to the present invention is shown schematically in the reaction equation

ing 1.

Simultaneous binding of a binding ligand and a biological compatibility agent to the carrier substrate: In this process, the water-soluble reagent is prepared by adding the carrier substrate to a reaction solution containing the biological compatibility agent and the binding ligand, or vice versa. The carrier substrate, such as those listed in Table I, can be added in the form of a dry material, or moistened by means of a suitable buffer. The reaction solution contains binding ligand in a concentration from approximately 10 mg/ml to 100 mg/ml, and biocompatibility agent in a concentration from approximately 0.01 M to 5 M. The reaction solution can also be buffered with any non-nucleophilic buffer , such as phosphate, at a buffer concentration of from approximately 0.01 M to 5 M. The pH range of the reaction solution may vary from approximately 4 to 10. After combining the reaction solution with the carrier substrate, the resulting mixture is stirred for 30 minutes or longer, up to several days if necessary, at any temperature that does not freeze the mixture or deform the support substrate, typically 4°C to 45°C. After coupling, the reaction solution is separated from the water-soluble reagent and rinsed to remove unreacted biocompatibility agent and binding ligand. The resulting derivatized support substrate comprises the water-insoluble reagent.

Stepwise binding of a binding ligand and a biological compatibility agent to the carrier substrate: In this process, the water-insoluble reagent is prepared first by reacting the carrier substrate with a binding ligand and then adding a biological compatibility agent to the reaction solution in a second step. This is the preferred method in cases where the binding ligand and the biological compatibility agent are not compatible in the same solution, or if the reaction conditions for immobilizing the binding ligand and biological compatibility agent are significantly different. The ranges for the reaction parameters in stepwise reactions are the same as for the simultaneous reactions described above.

EXAMPLE 1

Preparation of Anti-LDL water-insoluble reagent using simultaneous binding of anti-LDL monoclonal antibody CLL1 (binding ligand) and sulfamic acid (biocompatibility agent): Tresyl-Sepharose (carrier substrate; 35 g) was sterilized by washing with acetone and filtered through a sterile sintered glass funnel. The acetone-washed beads were then transferred to a sterile rotary flask (as an aid in transferring the beads, it is recommended that the beads be moistened lightly with acetone). To the rotary container was added 105 ml of the reaction mixture containing monoclonal antibody CLL1 (3.0 mg/ml), sulfamic acid (0.25 M) and potassium phosphate (0.25 M) at pH 8.0. The mixture was gently stirred overnight (16 hours) at ambient temperature. After stirring overnight, the mixture was filtered using a sintered glass funnel and the combined sorbent was washed with 1 L of citric acid (pH 3.0), followed by 1 L of buffered saline (pH 7.1). The filtrate was collected for treatment and reuse. The sterile anti-LDL sorbent was transferred to a sterile container where merthiolate had been added (final concentration 0.1%) as a sterilizing preservative until further use.

EXAMPLE 2

Preparation of anti-LDL water-insoluble reagent using stepwise binding of anti-LDL monoclonal antibody CLL1 (binding ligand) and sulfamic acid (biologically unifying ligand 1): Tresyl-sepharose (carrier substrate; 0.5 g) was sterilized by washing with acetone and filtering through a sterile sintered glass funnel. The acetone-washed beads were then transferred to a sterile centrifuge tube (as an aid to transferring the beads, it is recommended that the beads be moistened lightly with acetone). To the centrifuge tube was added 5 ml of the reaction solution containing monoclonal antibody CLL1 (3.0 mg/ml), and sodium bicarbonate (0.10 M) at pH 8.0. The mixture was stirred gently for 4 hours at ambient temperature. The antibody solution was separated from the carrier substrate by centrifugation at 1000 x g for 10 min and decanting the unreacted antibody Sulfamic acid (0.20 M) in sodium carbonate buffer (0.05 M), pH 9.0, was then added and the mixture was stirred overnight (16 h) at ambient ice temperature. After stirring overnight, the mixture was filtered using a sintered glass funnel and the anti-LDL water-insoluble reagent was washed with 1 L of citric acid (pH 3.0), followed by 1 L of buffered saline (pH 7.1). The decanted supernatant and filtrate were collected for processing and reuse. The sterile anti-LDL water-insoluble reagent mixture was transferred to a sterile container to which merthiolate (final concentration 0.1%) had been added as a sterilizing preservative until further use.

Application

The water-insoluble reagent described above can be used for the selective removal of substances from physiological fluids which are bound by means of the binding ligand. The water-insoluble reagent may be contained in a container that allows the flow of the physiological fluid through the water-insoluble reagent. If the physiological fluid is blood or blood plasma, the water-insoluble reagent can be used to remove a substance associated with a disease or a disease process from the blood or plasma. This may be the case in a number of diseases where it is known or assumed that the removal of the disease-associated substance would be beneficial. These diseases include auto-immune diseases, metabolic disorders and others. A more complete list of diseases that may be amenable to this therapy, the agent(s) to be removed, and the possible ligands that may accomplish this are shown in Table II. The water-insoluble reagent is listed in Table I, one or more of the binding ligands is listed in Table II, and one or more of the biocompatibility agents are listed in Table III.

To illustrate the application of the present invention, a therapeutic purpose will contain a volume or quantity of a reagent as described above in a container that will allow the flow of blood or plasma. Anticoagulated blood or plasma from a patient is pumped through the container containing the water-insoluble reagent and then returned to the patient. The blood or plasma that is treated in this way will thus be unaffected, with the exception that the unwanted substance bound by means of the binding ligand in the water-insoluble reagent has been removed. There are two parameters that can be used for evaluating the performance or applicability of the system: 1) the effectiveness of the binding ligand, and; 2) the effectiveness of the biocompatible agent.

The effectiveness of the binding ligand can be determined by measuring the amount of a substance in the physiological fluid before and after passing through the water-insoluble reagent. The amount of material removed is a measure of the performance of the reagent. The analysis system for measuring the amount of substance removed is of course dependent on the substance. To further illustrate how this can be carried out, in the case of LDL, plasma that is anticoagulated is soaked over an anti-LDL water-insoluble reagent. Samples of the plasma, before and after soaking over the combined sorbent, are collected and analyzed. Plasma samples are then analyzed for total cholesterol and HDL. The LDL levels are easily calculated from the values for HDL and total cholesterol, because the total cholesterol level i

plasma is assumed to be the sum of HDL and LDL.

The determinations of total cholesterol are performed using standard clinical laboratory procedures. This method is a quantitative enzymatic reaction using cholesterol oxidase followed by chemical saponification of the cholesterol esters. The by-product of cholesterol oxidase ^H2^2^ is coupled with peroxidase and a cholerimetric substrate. This reaction leads to a colored end product which can be measured using a spectrophotometer (Stein, E. A., In, Textbook of Clinical Chemistry, Tietz, N. W., Ed.,

WB Saunders, Philadelphia, 1986, pp. 879-886, 1818, and 1829).

The HDL determinations are carried out by first precipitating LDL, using a precipitating agent, followed by analysis of the supernatant via the above-described method for determining total cholesterol.

The effectiveness of the biocompatibility agent can be determined by measuring the amount of complement activation in the plasma as a result of the action of the combined sorbent. Impairment of complement activation compared to the untreated polymer vehicle demonstrates the performance of the biocompatibility agent. Activation of the complement cascade by means of a foreign substance takes place via the activation of an alternative reaction pathway. The components of the complement cascade that are quantitatively determined to determine the amount of activation are C, and • C^a(or ) is determined by a known RIA technique (see Chenweth, D.E. and Hugli, T.E., J. Biol. Chem 250. 8293 (1975 ), incorporated herein by reference) in which antibodies specific to human C3, a (or Cv Id a) are allowed to react with a heparin-anticoagulated plasma sample exposed to the water-insoluble reagent. The use of other anticoagulation agents such as citrate can lead to a significant change in complement activation. The degree of binding of the specific antibody to ^/-i3a (or C^g) is determined by means of a radioisotope probe. The amount of Cj0 a (or Cc ba) in a plasma sample can then be calculated from the measured amount of radioactivity. The amounts of C^g and C^a are summed to give a value representative of the total in vitro complement activation of the tested material.

The water-insoluble reagent can be contained in variously designed containers depending on the physical form of the solid support material or the application. The design of the apparatus used is not critical in this connection, any apparatus which retains the water-insoluble reagent and allows the physiological fluid to flow through can be used.

Using the techniques described above, the effectiveness of the water-insoluble reagent in removing LDL from plasma can be demonstrated by measuring the LDL levels before and after soaking a plasma sample over the water-insoluble reagent. Using the water-insoluble reagent according to example 1, comprising anti-LDL monoclonal antibody CLL1 (binding ligand) and sulfamic acid (biocompatibility agent) immobilized on tresyl sepharose, the detection of LDL binding to the immunosorbent and removal of LDL from plasma is illustrated by the two experiments shown in Figure 1. The values for LDL-cholesterol (filled bars) and HDL-cholesterol (shaded bars; total cholesterol is the sum of HDL and LDL) before and after plasma treatment with the immunosorbent are shown. The immunosorbent effectively removes LDL from plasma whether a plasma with a high cholesterol content (> 300 mg/dl) or a plasma with a low cholesterol content (155 mg/dl) is used. It should also be noted that HDL levels are unaffected by treatment with the LDL immunosorbent, confirming the specificity of the water-insoluble reagents.

The effectiveness of the biocompatibility agent in suppressing complement activation in the plasma, compared to the untreated polymer vehicle, is shown in Table IV.

As previously indicated, this total complement activation is the sum of C^a and C and is expressed as jjg/ml in the test plasma. Treatment with sulfanilic acid and sulfamic acid leads to the use of a biocompatibility agent from Table III, and a significant reduction in the complement activation of this material is observed. Treatment with tris (tris(hydroxymethyl)aminomethane, Sigma Chemical Company)) or water hydrolyzed tresyl sepharose does not incorporate the negatively charged moiety into the surface and does not provide the protection obtained from the use of the biocompatibility agent.

A combination sorbent prepared with a binding ligand and a biocompatibility agent provides both binding properties and biocompatibility properties to the water-insoluble reagent. The water-insoluble reagent prepared using anti-LDL antibody CLL1 and sulfamic acid (Example 1) particularly shows the combined effect; the ability of the biocompatibility agent to weaken complement, and the ability of the binding ligand to bind and remove LDL. The results in Table V show the amount of bound LDL

in mg per ml of water-insoluble reagent, as well as the associated complement activation.

Example 1

Use of anti-LDL combined sorbent to remove LDL from plasma;

The anti-LDL reagent, as described above (1.5 ml) was packed into a 1.0 x 10 cm column containing a fritted disc at the bottom to retain the water-insoluble reagent material. The packed regent was washed with several column volumes of phosphate buffered saline at pH 7.4 to remove the merthiolate. Citric acid-treated plasma (9.0 mL) was then recirculated through the column for 1 hour. Samples of plasma before and after recirculation were analyzed to determine the amount of LDL or other constituents removed from the plasma. The removal of LDL from plasma is illustrated in Figure 1. Quantitative determination of the lipoprotein levels (HDL and LDL) in plasma is achieved by measuring the cholesterol content in the respective lipoprotein. The LDL cholesterol values (solid bars) and HDL cholesterol values (shaded bars; total cholesterol is the sum of HDL and LDL) before and after treatment of the plasma with anti-LDL water-insoluble reagent are shown. In the experiments depicted in the figure, two plasma samples were soaked over the reagent. It should be noted that the reagent removes LDL efficiently both when a plasma with a high cholesterol level (> 300 mg/dl) or a plasma with a low cholesterol level (155 mg/dl) is used. Table VI shows the effect of the plasma recycling over the water-soluble reagent, on the other components before and after recycling. No significant changes were observed in the levels of the other plasma constituents.

The above detailed description and the specific examples indicate preferred embodiments of the present invention, and are intended to be illustrative. A person skilled in the art can ascertain from the present description

about the essential characteristics of the present invention, and carry out various changes and modifications of the invention to adapt it to different application areas and conditions, without deviating from the scope of the invention.

Claims (22)

1. Water-soluble reagent for removing a substance from physiological fluids, characterized in that it comprises (a) a carrier substrate, and immobilized on the surface of the carrier substrate, (b) a binding ligand specific to the substance, and (c) a biocompatibility agent comprising an organic molecule having a nucleophilic moiety and a negatively charged moiety separated by a spacer arm, wherein the biocompatibility agent is immobilized on the surface through reaction with the nucleophilic moiety. 2. Insoluble reagent according to claim 1, characterized in that the carrier substrate is a neutral polymer. 3. Water-soluble reagent according to claim 2, characterized in that the neutral polymer is selected from the group consisting of Affigel 10, Affigel 15, tresyl-sepharose, CNBr-sepharose, Reacti-gel (CDI activated agarose/dextran), Eupergit C, epoxy-activated sepharose . PEI-glutaraldehyde-activated nylon, PEI-glutaraldehyde-activated silica, glutaraldehyde-activated agarose, isocyanate-activated agarose, maleimide-activated agarose, and chloromethyl polystyrene. 4. Water-soluble reagent according to claim 3, characterized in that the neutral polymer is tresyl sepharose. 5. Water-soluble reagent according to claim 1, characterized in that the binding ligand is selected from the group consisting of a monoclonal antibody, a polyclonal antibody, hormone receptor, lectin, antigen and vitamin binding protein. 6. Water-soluble reagent according to claim 5, characterized in that the binding ligand is a monoclonal antibody specific to the substance to be removed from a physiological fluid. 7. Water-soluble reagent according to claim 6, characterized in that the monoclonal antibody is specific to low-density lipoprotein. 8. Water-soluble reagent according to claim 1, characterized in that the biological compatibility agent comprises an organic molecule with the negatively charged part selected from the group consisting of OSO^ , RSO^ , RSO~, and CO0~. 9. Water-soluble reagent according to claim 1, characterized in that the biological compatibility agent comprises an organic molecule in which the nucleophilic part. is selected from the group consisting of NH^ , SH and OH. 10. Water-soluble reagent according to claim 1, characterized in that the biological compatibility agent is selected from the group consisting of sulfamic acid, taurine, sulfanilic acid, glycine, aspartate, cysteine, isethionic acid, hydroxybenzenesulfonic acid, glucose sulfate, lactic acid, glucuronic acid, gluconic acid, 2-mercaptoethanesulfonic acid, mercaptoacetic acid, thiosalicylic acid, thiolactic acid. 11. Water-soluble reagent according to claim 10, characterized in that the biological compatibility agent is sulfamic acid. 12. Method for producing a water-insoluble reagent for removing a substance from physiological fluids, characterized in that a carrier substrate is brought into contact with, and reacted with, an aqueous solution of a binding ligand specific to the substance, and a biological compatibility agent comprising a organic molecule with a nucleophilic part and a negatively charged part separated by a spacer arm. 13. Method according to claim 12, characterized in that the concentration of the binding ligand is from approximately 10 mg/ml to 100 mg/ml, and that the concentration of the biocompatibility agent is from approximately 0.01 M to 5.0 M. 14. Method according to claim 13, characterized in that the pH is controlled using a non-nucleophilic buffer with a pH in the range from approximately 4 to 10. 15. Method for producing a water-insoluble reagent for removing a substance from physiological fluids, characterized by the steps; (a) a carrier substrate is contacted with and reacted with an aqueous solution of a drug-specific binding ligand, and (b) the product of step (a) is contacted with and reacted with an aqueous solution of a biocompatibility agent comprising an organic molecule with a nucleophilic moiety and a negatively charged moiety separated by a spacer arm, to form the water-insoluble reagent. 16. Method according to claim 15, characterized in that the concentration of the binding ligand is from approximately 10 mg/ml to 100 mg/ml, and that the concentration of the biocompatibility agent is from approximately 0.01 M to 5.0 M. 17. Method according to claim 15, characterized in that the pH is controlled using a non-nucleophilic buffer with a pH in the range from approximately 4 to 10. 18. Method for treating a physiological fluid to remove a substance, characterized in that the liquid is brought into contact with a water-insoluble reagent according to claim 1. 19. Method according to claim 18, characterized in that the physiological fluid is mammalian plasma. 20. Method according to claim 18, characterized in that the substance is low-density lipoproteins. 21. Apparatus for removing a specific substance from physiological fluids, characterized in that it comprises a means for removing fresh blood from a mammal, a means for separating plasma from fresh blood, a means for treating the plasma, including a chamber containing a water-insoluble reagent according to claim 1, which will react with and removing the substance from the plasma, and means for recombining the plasma with the remainder of the fresh blood and for returning the recombined blood to the mammal. 22. Apparatus according to claim 21, characterized in that the water-insoluble reagent comprises tresyl-sepharose containing a monoclonal antibody against low-density lipoprotein and sulfamic acid, immobilized on the surface of the tresyl-sepharose.