WO2000041718A9 - Methode et dispositif permettant d'extraire selectivement des anticorps xenoreactifs du sang, du serum ou du plasma - Google Patents

Methode et dispositif permettant d'extraire selectivement des anticorps xenoreactifs du sang, du serum ou du plasma

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Publication number
WO2000041718A9
WO2000041718A9 PCT/US2000/000410 US0000410W WO0041718A9 WO 2000041718 A9 WO2000041718 A9 WO 2000041718A9 US 0000410 W US0000410 W US 0000410W WO 0041718 A9 WO0041718 A9 WO 0041718A9
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WO
WIPO (PCT)
Prior art keywords
membrane
blood
plasma
ligand
serum
Prior art date
Application number
PCT/US2000/000410
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English (en)
Other versions
WO2000041718A1 (fr
Inventor
Sujatha Karoor
Lee Henderson
Jeanette Molina
Original Assignee
Baxter Int
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Filing date
Publication date
Application filed by Baxter Int filed Critical Baxter Int
Priority to AU24087/00A priority Critical patent/AU2408700A/en
Publication of WO2000041718A1 publication Critical patent/WO2000041718A1/fr
Publication of WO2000041718A9 publication Critical patent/WO2000041718A9/fr

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/34—Filtering material out of the blood by passing it through a membrane, i.e. hemofiltration or diafiltration
    • A61M1/3472—Filtering material out of the blood by passing it through a membrane, i.e. hemofiltration or diafiltration with treatment of the filtrate
    • A61M1/3486—Biological, chemical treatment, e.g. chemical precipitation; treatment by absorbents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/34—Filtering material out of the blood by passing it through a membrane, i.e. hemofiltration or diafiltration
    • A61M1/3472—Filtering material out of the blood by passing it through a membrane, i.e. hemofiltration or diafiltration with treatment of the filtrate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/34—Filtering material out of the blood by passing it through a membrane, i.e. hemofiltration or diafiltration
    • A61M1/3472—Filtering material out of the blood by passing it through a membrane, i.e. hemofiltration or diafiltration with treatment of the filtrate
    • A61M1/3475—Filtering material out of the blood by passing it through a membrane, i.e. hemofiltration or diafiltration with treatment of the filtrate with filtrate treatment agent in the same enclosure as the membrane
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
    • B01J20/28014—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their form
    • B01J20/28033—Membrane, sheet, cloth, pad, lamellar or mat
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3202—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
    • B01J20/3204—Inorganic carriers, supports or substrates
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3202—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
    • B01J20/3206—Organic carriers, supports or substrates
    • B01J20/3208—Polymeric carriers, supports or substrates
    • B01J20/321—Polymeric carriers, supports or substrates consisting of a polymer obtained by reactions involving only carbon to carbon unsaturated bonds
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3202—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
    • B01J20/3206—Organic carriers, supports or substrates
    • B01J20/3208—Polymeric carriers, supports or substrates
    • B01J20/3212—Polymeric carriers, supports or substrates consisting of a polymer obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3214—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the method for obtaining this coating or impregnating
    • B01J20/3217—Resulting in a chemical bond between the coating or impregnating layer and the carrier, support or substrate, e.g. a covalent bond
    • B01J20/3219—Resulting in a chemical bond between the coating or impregnating layer and the carrier, support or substrate, e.g. a covalent bond involving a particular spacer or linking group, e.g. for attaching an active group
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3268—Macromolecular compounds
    • B01J20/3272—Polymers obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds
    • B01J20/3274—Proteins, nucleic acids, polysaccharides, antibodies or antigens

Definitions

  • This invention relates in general to a system for removing xenoreactive antibodies from blood, serum or plasma, and in particular to an apparatus containing an immunoadsorption membrane and a ligand immobilized thereto which is capable of binding with xenoreactive antibodies such as IgM, and a method of removing xenoreactive antibodies from blood, serum or plasma by passing these fluids through an immunoaffinity membrane having a ligand capable of binding xenoreactive antibodies covalently linked to its surface, collecting the blood fluids from which the xenoreactive antibodies have been removed, and reintroducing them back into a patient, who will be less likely to suffer a hyperacute rejection when receiving a transplanted organ from a donor animal because of the removal of the xenoreactive antibodies.
  • Hyperacute rejection is thought to occur through an antibody-mediated complement activation, such as described in Dalmasso et al., Amer. J. Pathol . 140:1157-1166 (1992), and is characterized by conditions such as thrombosis, hemorrhage and edema which almost invariably lead to a decline in graft function and irreversible rejection of the grafted tissues.
  • hyperacute rejection Studies on the course of hyperacute rejection have indicated that the process appears to be initiated by binding of xenoreactive natural antibodies to certain carbohydrate structures present on the endothelial cells of the graft, which leads to the activation of the complement cascade. See Platt et al., Transplantation 50:817-822 (1990). In its normal course, hyperacute rejection ultimately results in additional reactions of the host's complement defense system after initiation by xenoreactive natural antibodies, and causes rapid decline and destruction of the graft.
  • the xenoreactive natural antibodies involved in this process generally belong to the IgG, IgM and IgA classes of antibodies. See Parker et al., J. Immunol . 153:3791- 3803 (1994) .
  • xenoreactive IgM xenoreactive IgM
  • X- IgM xenoreactive IgM
  • Gal Gal epitopes
  • xenograft survival can be prolonged by techniques such as peritransplant depletion of antibodies via plasmapheresis, organ perfusion, and removal of all of the IgG, IgM, IgA antibodies.
  • plasmapheresis removes all of the immunoglobulin, not just the X-IgM antibody, and removes plasma proteins such as clotting factors and other proteins that are important during the peritransplant period.
  • organ perfusion With regard to organ perfusion, this procedure is cumbersome o perform, requiring sacrifice of a donor animal to obtain the target organ. Furthermore, organ perfusion causes loss of recipient blood volume as well as activation of complement and coagulation cascades as the perfused organ is rejected. Finally, methods such as organ perfusion involve complex and cumbersome equipment which greatly increases the cost and inconvenience involved in such procedures.
  • the present invention provides a method of selectively removing xenoreactive antibodies from blood, serum or plasma comprising the steps of immobilizing a ligand capable of binding the specific xenoreactive antibodies that bind the Gal epitope to the surface of an immunoaffinity membrane, and then passing the patient's blood or serum through this membrane so that these specific xenoreactive antibodies will be removed.
  • the present invention utilizes a non-compressible immunoaffinity membrane and a ligand which specifically binds the xenoreactive antibodies responsible for hyperacute rejection of grafts containing the antigenic Gal epitope so that other important blood proteins, such as clotting factors, are allowed to pass through and are retained in the treated blood fluids.
  • FIGS. 1A-1C are graphic representations of the effect of flow rate and other parameters on the selective removal of xenoreactive antibodies using the method of the present invention.
  • FIGS. 2A-2C are graphic representations showing the selective removal of xenoreactive antibodies using the recycle mode of the method of the present invention.
  • FIGS. 3A-3E are graphic representations of the removal of antibodies using the method of the present invention.
  • FIGS. 4A-4C are graphic representations of the removal of antibodies using the method of the present invention.
  • FIG. 5 is a schematic view of an embodiment of a mode of operation using an immunoaffinity membrane in accordance with the present invention.
  • FIGS. 6A-6C are graphic representations of the removal of antibodies using the method of the present invention.
  • a method and apparatus which allows for the selective removal of the xenoreactive antibodies specific to the antigenic Gal epitope from blood, serum or other blood fluids so that when the treated blood fluids are reintroduced, the patient will be less likely to undergo a hyperacute reaction following the transplantation of donor tissues or organs from a non-human animal whose cells express the Gal epitope.
  • the present invention comprises a method of immunoadsorption using a ligand bound to an immunoaffinity membrane which specifically targets only the xenoreactive antibodies ("X-Ab” or "anti-Gal antibodies”) which bind to the Gala(1,3) Gal residues (or "Gal epitopes").
  • selective removal of the X-Ab's from plasma or whole blood is carried out through immobilization of a ligand to all accessible surfaces of a microporous membrane contained in a suitable device, and by passing whole plasma or whole blood through this device. It is also preferred that the ligand immobilized to this membrane will be one which will bind the X-Ab's yet allow other important blood proteins, such as clotting factors, to pass through unabated. Accordingly, ligands such as Gal ⁇ l-3Gal ⁇ l- 4GlcNAc or Gal ⁇ l-3Gal ⁇ l-4GluNAc are particularly preferred in accordance with the present invention.
  • the microporous membrane of the present invention can comprise any configuration suitable for membrane plasmapheresis, and these suitable forms include hollow fibers, flat sheets, a stack of flat plates, spiral wound, or any other form wherein the membrane could carry out immunoadsorption in accordance with the present invention.
  • further steps can be carried out to increase or maximize the amount of membrane surface area that will be placed in contact with the blood fluids of the patient so as to increase the percentage of xenoreactive antibody removal, as will be described more fully below.
  • the material of the microporous membrane of the invention is preferably any organic polymer or inorganic material to which a suitable ligand can be attached, including nylon, polysulfone, cellulose triacetate, regenerated cellulose (cuprophane) or ethylene vinylalcohol copolymer (EVAL) .
  • suitable ligand including nylon, polysulfone, cellulose triacetate, regenerated cellulose (cuprophane) or ethylene vinylalcohol copolymer (EVAL) .
  • Other examples of the immunoaffinity membranes suitable for use in the present invention include microporous membranes such as those disclosed in U.S. Patent Nos. 5,310,688 and 5,683,916, incorporated herein by reference.
  • the pore size of the membrane should be suitable for membrane plasmapheresis, and it is generally preferred that the pore size be from about 0.1 to 0.75 ⁇ m.
  • the wall thickness of the microfiltration fibers used in the microporous membrane of the invention is preferably from about 50 ⁇ m to about 500 ⁇ m so that the ligand can be immobilized not only to the surface but also in the walls of the pores, which ensures that the surface area will be very high. It is also preferred that the membrane should exhibit minimal non-specific binding to plasma, serum or blood components other than the xenoreactive antibodies removed by the ligands of the invention. In accordance with the present invention, as explained further below, the membrane will be part of a conduit through which whole blood or whole plasma can be processed.
  • a method of selective removal of X-Ab from plasma or whole blood wherein a suitable ligand is immobilized to the surface of a dialysis or ultrafiltration membrane contained in a device through which plasma or whole blood may be passed.
  • the membrane may be in any suitable form, including hollow fiber or flat sheet, or any other configuration commonly used in dialysis or ultrafiltration.
  • the use of dialysis or ultrafiltration fibers is particularly preferred in cases where the amount of antibody to be removed is small.
  • the membrane surface area used for adsorption is small for these fibers, they are less practical to use when one has to remove ligate in very large (e.g., gram or more) quantities.
  • the dialysis membrane can be any organic polymer or inorganic material to which a suitable ligand can be attached, for example nylon, polysulfone, cellulose triacetate, cuprophane, cellulose acetate or ethylene vinylalcohol copoly er (EVAL) , and the membrane will employed as part of a conduit through which plasma or whole blood can be processed.
  • EVAL ethylene vinylalcohol copoly er
  • the pore size is preferably one that is suitable for dialysis, or for ultrafiltration or a non-porous surface will also be suitable. Generally, pore sizes in Angstroms will be suitable, and dry wall thicknesses of the dialysis fiber are preferably about 5-10 ⁇ m, or more.
  • the ligand is preferably attached to the surface of the membrane, and thus both the inside and outside membrane surfaces can be used for the immobilization of the ligand.
  • immunoaffinity adsorption of xenoreactive antibodies in accordance with the present invention is carried out in an apparatus that will allow the entire process to be performed continuously with a single piece of equipment, and which will avoid the use of a separate plasma separation step.
  • the method of the present invention is thus advantageous over prior methods such as those which employ a two-step procedure of plasma separation from blood followed by adsorption of xenoreactive IgM. See, e.g., Kessler, Blood Purif. 11:150-157 (1993) .
  • the use of a conventional sepharose bead column requires a plasmapheresis device for the separation of plasma from other components of blood.
  • a bead column is then used to adsorb the xenoreactive antibodies.
  • the membranes of the present invention can be activated by any suitable method which will allow a suitable ligand to be attached to the membrane surface, including those processes which generate amine groups on the membrane surface for the attachment of the ligand.
  • amine groups are generated on the membrane material by direct chemical modification of the surface, or indirectly by coating the surface with a suitable activating material, such as polyethyleneimine (PEI) , polyvinylamine (PVA) or polyallylamine (PAA) , or any other chemical material generally known to be used for such purposes.
  • PEI polyethyleneimine
  • PVA polyvinylamine
  • PAA polyallylamine
  • one mode of attachment of the ligand to the surface comprises generating amine groups on the membrane material by direct chemical modification of the surface or indirectly by coating with polyethyleneimine (PEI) , polyvinylamine (PVA) or polyallylamine (PAA) . These methods generate amine groups for attachment of the ligand.
  • PEI polyethyleneimine
  • PVA polyvinylamine
  • PAA polyallylamine
  • the ligand which is preferably Gal ⁇ l-3Gal ⁇ l-4GlcNAc with 6 carbon spacers and an end carboxyl group, is preferably linked to an end amine group of the membrane by suitable chemistry, such as by (1-ethyl- 3-(3-dimethylaminopropyl) carbodiimide (EDC) chemistry.
  • EDC (1-ethyl- 3-(3-dimethylaminopropyl) carbodiimide
  • the amine-containing microporous membrane is washed thoroughly with a coupling buffer such as 0.2M (2-(N- morpholino(ethanesulfonic acid) (MES) , pH 4.0 to 6.0.
  • MES 2-(N- morpholino(ethanesulfonic acid)
  • the ligand along with sulfo-N hydroxysuccinimide (sulfo-NHS) and EDC (l-ethyl-3-(3-dimethylaminopropyl) carbodiimide, is dissolved in the coupling buffer.
  • the ratio of ligand to sulfo-NHS to EDC is preferably about 1.0:0.45:4.5.
  • This solution is then pumped through a module containing the microporous membrane at low flow rates in one of two different modes for approximately 16 hours. In a flow-through mode, the coupling buffer is pumped through the lumen and shell side of the device separately.
  • the coupling buffer is pumped only through the lumen side, and the other end of the lumen is closed so that all the coupling buffer passes through the membrane and into the shell side.
  • PBS phosphate buffered saline
  • the pH is restored to neutral by 0.5M NaCl plus PBS.
  • the module is then preferably stored in 0.02% sodium azide.
  • Addition of sulfo-NHS increases the efficiency of amide bond formation by creating an active sulfo- NHS ester intermediate which is less prone to hydrolysis than the EDC ester precursor, thus giving greater yields in the reaction with amine-containing substrate. Similar methods can be used when a hydrazide end group is used instead of an amine.
  • these membranes can also be activated by a number of different methods which involve generating amine groups on the surface for attachment of the ligand.
  • amine groups may be generated on the membrane material by direct chemical modification of the surface, or indirectly by coating with PEI, PVA or PAA.
  • an activated matrix including using a compound such as tosyl chloride, ethyleneglycol diglycidyl ether (EDGE) , carbonyl- diimidizol, iodoacetic acid or bromoacetic acid to activate the cellulose matrix followed by a reaction with diamine or a polyamine spacer such as PVA, PAA or PEI.
  • This modified surface can then be reacted with the N-hydroxysuccinimide (NHS) ester of Gal to achieve activation.
  • the cellulose membrane is treated with NaOH to disrupt hydrogen bonding of the surface sugar residues, which makes the hydroxyl groups more available for reaction.
  • This treatment is preferably followed with a reaction with the NHS ester of Gal to obtain the activated membrane.
  • Still another suitable mode of activation is via the sodium metaperiodate oxidation of the cellulose membrane to produce aldehyde functional groups. Condensation with diamine or any suitable polyamine spacers is preferably followed by a reaction with the NHS ester of Gal to give an activated membrane.
  • sodium metaperiodate can be used to oxidize hydroxyl compounds to form aldehyde groups suitable for reductive amination coupling.
  • a solution containing sodium 0.2 M sodium metaperiodate can be pumped through both the lumen side and the shell side of a module containing the membrane of the present invention at a flow rate of about 3 l/min for an hour.
  • This step will produce the aldehyde groups which can be used to couple to amine groups.
  • the membrane fibers are then washed extensively with deionized water.
  • polyethyleneimine at a concentration of 50 mg/ml is dissolved in 0.1 borax buffer (at pH 9.2) and 0.1 sodium cyanoborohydride, and is then pumped through both the shell and lumen side of a membrane module for a period of about 16 hours.
  • the terminal end of the EDGE is then reacted with ligands containing amine groups.
  • the module is then rinsed with deionized (milli Q) water, and a 2% PEI solution is pumped through both the lumen and shell side of the membrane in the module for a period of about 3 hours at a flow rate of 3 to 5 ml/min.
  • the module containing the membrane is then washed extensively with deionized water so as to become ready for carbohydrate immobilization.
  • the ligand to be immobilized to the dialysis membrane which will be capable of binding to a xenoreactive antibodies present in blood or serum will comprise a carbohydrate such as the Gal epitope, e.g., Gal ⁇ l-3Gal ⁇ l-4GlcNAc or Gal ⁇ l- 3Gal ⁇ l-4GluNAc, with 6 carbon spacers and an end carboxyl group.
  • the ligand will be linked to an amine end group of the membrane via an appropriate chemical moiety such as 1-ethyl- 3-(3-dimethylaminopropyl carbodiimide) (EDC) .
  • the amine-containing membrane is washed thoroughly with a coupling buffer such as O.IM 2-N-morpholino ethanesulfonic acid (MES) at pH 4.0 to 6.0. Then the ligand, along with sulfo- NHS and EDC, is dissolved in the coupling buffer.
  • the ratio of ligand to sulfo-NHS to EDC is preferably about 1.0:0.45:4.5.
  • this solution is then pumped through the tube side of a module containing the membrane at a flow rate of about 3 to 6 ml/min for around 16 hours.
  • the coupling buffer is pumped through the lumen and shell side of the device or module separately.
  • PBS phosphate buffered saline
  • the module can then be stored in 0.02% sodium azide.
  • Addition of sulfo-NHS during the immobilization of carbohydrate is preferred because it increases the efficiency of amide bond formation by creating an active sulfo-NHS ester intermediate. The intermediate is less prone to hydrolysis than the EDC ester precursor, thus giving greater yields in the reaction with amine-containing substrate.
  • Immobilization of the carbohydrate ligand can be carried out if necessary without the addition of sulfo-NHS, and a similar method can be used when a hydrazine end group is used instead of an
  • a method for extracorporeally treating the blood of a patient so that the xenoreactive antibodies will be removed, after which the blood devoid of antibodies can be returned to the patient.
  • this method comprises the steps of attaching a ligand to an immunoaffinity membrane as described above, and passing the blood or plasma of a patient through a module that contains the ligand-containing membrane.
  • extracorporeal treatment is preferably carried out by passing blood through a module containing the microporous membrane, and in this mode, blood flows through the lumen of the hollow fiber, the outlet of the shell compartment is open, and the blood side is pressurized.
  • the present method and apparatus can thus be utilized to remove xenoreactive antibodies from a patient ' s own blood in preparation for a xenotransplantation procedure, or, if necessary, can be used on donated blood which is intended to be transfused into a human patient prior to xenotransplantation.
  • the process of the present invention ideally proceeds in a series of steps as follows: (1) Loading plasma or whole blood:
  • the membrane is preferably devoid of any specifically bound xenoreactive antibody.
  • Whole blood or plasma is then passed through the ligand-containing microporous membrane which is situated in an appropriate compartment in a module which can be set up to receive blood or other blood fluid directly from a patient.
  • a module which can be set up to receive blood or other blood fluid directly from a patient.
  • plasma flows across the membrane. Accordingly, whole blood or plasma passes through the blood compartment of the module, while plasma filters through the wall of the membrane and comes in contact with the ligand which is covalently linked to the membrane.
  • xenoreactive antibodies will bind to the ligand.
  • the filtrate and outlet samples can be combined and cycled through the module repeatedly.
  • the functionalized membrane surface should have the property that it minimally binds plasma or blood components nonspecifically.
  • the xenoreactive antibody that is specifically bound to the membrane can be removed by any suitable means, such as by lowering the pH to 3.0 using O.IM glycine.
  • the module containing the microporous membrane can be regenerated and made ready for the next cycle via introduction of a suitable physiological buffer solution.
  • a method is also provided for extracorporeally treating the blood of a patient using the dialysis or ultrafiltration membrane described above.
  • extracorporeal treatment is preferably carried out by passing blood through a module containing the dialysis or ultrafiltration membrane, and in this mode, blood flows through the lumen of the hollow fiber, the outlet of the shell compartment is closed. As blood flows through the module, the X-Ab comes in contact with the ligand where it is specifically adsorbed. The blood that has been depleted of X-Ab can then be returned to the patient.
  • the process of the present invention ideally proceeds in a series of steps as follows: (1) Loading plasma or whole blood:
  • the membrane is preferably devoid of any specifically bound xenoreactive antibody.
  • Feed solution whole blood or plasma
  • ligand which is covalently linked to the membrane.
  • Xenoreactive antibodies bind to the ligand as blood flows through the tube side of the device.
  • the plasma or blood that is depleted of xenoreactive antibodies can be collected at the end of the run.
  • the system can be operated such that all of the X-Ab can be adsorbed with one device, in which case there is no need for wash, elution, and regeneration cycles. However, when desired, these cycles preferably are carried out as follows: (2 ) Washing :
  • the washing cycle proceeds from the tube side only.
  • the functionalized membrane surface should have the property that it minimally binds plasma or blood components nonspecifically.
  • the xenoreactive antibody that is specifically bound to the membrane can be removed by any suitable means, such as by lowering the pH to 3.0 using 0.1 M glycine and 0.5 M salt.
  • the membranes suitable for use in the invention can be made of a wide variety of materials, and these materials can be arranged in a variety of suitable configurations.
  • a wide range of polymers which can be activated for covalent attachment of the ligand can be used in the present invention.
  • polymeric materials which covalently attach the ligand to the membrane include such materials as dextran, hydroxyethyl cellulose, polyhydrazide, polyallylamine (PAA) , polyaldehyde, polyethyleneimine (PEI) and polyvinylamine (PVA) .
  • these polymers can be modified so as to increase the number of activatable groups by coating them with dextran or hydroxyethyl cellulose.
  • the ligand end group can be modified so as to be able to use new activation chemistries.
  • the primary ligands preferred for use in the invention will be Gal ⁇ l-3Gal ⁇ l-4GlcNAc with 6 carbon spacers and an end carboxyl group, and Gal ⁇ l- 3Gal ⁇ l-4GluNAc with 6 carbon spacers and an end carboxyl group, with possible end groups including an amine, a hydrazine, biotin, avidin, hydroxyl, or aldehyde.
  • additional ligands which can be immobilized to the affinity membranes of the present invention and which bind with xenoreactive antibodies can also be employed in the present invention.
  • the affinity membrane can be further configured in any suitable manner in order to increase or maximize the surface area that will be available to remove xenoreactive antibodies, and the ligand and xenoantibody can be brought into contact with each other in a great number of ways, as would be recognized by one skilled in this art.
  • the length of the device was constant (8.0 cm, effective length 5.5 cm) and the number of fibers was varied from module-to-module as shown in the table.
  • the fibers were potted into devices, followed by surface modification to alter the chemical characteristics of the membrane.
  • the surface modification of the devices was accomplished by coating with PEI as follows:
  • the terminal free, unbound end of the EDGE molecules was then reacted with the amine containing polymer PEI.
  • the module was rinsed with deionized (milli Q) water.
  • a 2% PEI solution at pH 14 (temp.
  • 60-70°C was recirculated through both the lumen and shell side of the membrane in the module for a period of 3 hours at a flow rate of 3 to 5 ml/min.
  • the module containing the membrane was washed extensively with deionized water and was then ready for carbohydrate immobilization.
  • PBS phosphate buffered saline
  • the module was then stored in 0.02% sodium azide. All the devices (except CF-12) had carbohydrate attached to the inside surface (lumen) and also the outside surface. In any one experiment, only the lumen or the inside surface area was utilized, but not both.
  • FIG. 1A The effect of feed flow rate on the removal of xenoreactive antibody from serum using device CF-13 is illustrated in Figure 1A.
  • the same device was used for all the runs.
  • the experiment was carried out in a single pass mode, i.e., the total 50ml volume of feed serum was passed through the device once and collected.
  • the feed concentration was kept constant at 0.034 mg/mL for all the runs.
  • flow rate ranged from 1 to 120 mL/min.
  • the serum was pumped through the blood side (lumen side) of the device at the constant specified flow rate.
  • the blood exiting from the device was pooled.
  • Xeno- IgM analysis was performed on the serum before and after passing through the device.
  • the level of xeno-IgM in the pooled sample was measured by ELISA (see Parker W. et al., J. Immunol. 153:3791-3803 (1994)), using commercially prepared Gal ⁇ l-3Gal ⁇ l-4GlcNAc, attached to BSA
  • Gal-BSA (Gal-BSA) .
  • a 30 ⁇ g/mL solution of Gal-BSA was added to an ELISA plate and incubated for one hour. The plate was then washed four times with IX PBS. After that, the plate was blocked with a 2% solution of BSA for one hour, then washed four times with IX PBS, and finally incubated with the pooled samples diluted 1:40 in 2%
  • the fraction removal is equal to (1-Relative concentration) times 100.
  • Relative concentration is feed concentration divided by the outlet concentration.
  • This experiment was carried out for 190 minutes, and the blood flow rate was set at 15 ml/min throughout the experiment. During this experiment, the reservoir blood was sampled for concentration measurement at specified time intervals. The X-IgM concentration in the blood decreased to 72% of its original value after about 50 minutes of run time with no regeneration of the module.
  • the capacity (defined as the mass removed by the membrane divided by the membrane lumen surface area) of the module CF-9 was higher by a factor of 2.8 times (3.66 X 10 "2 mg/cm 2 ) when compared to module CF-7 (1.3 X l ⁇ "2 mg/cm 2) . This capacity does not necessarily represent the saturation capacity, since the mass removed may depend upon the final reservoir concentration.
  • module CF-14 was 0.054 mg/c _> whi.ch i.s hi.gher when compared to module CF-7 by a factor of 4.
  • Modules CF-13, CF-18 and CF-17 were used previously in other experiments, but module CF-16 was a new module with a lower capacity.
  • An overall removal of 82% of X-IgM and 53% of X-IgG was ultimately achieved.
  • pass 1 35 % removal of X-IgM was obtained, but virtually no X-IgG was removed.
  • pass 2 76% removal of X-IgM was obtained accompanied by 55% of removal of the remaining X-IgG.
  • This closed loop system was used to permit time dependent accumulation of complement derived anaphylatoxins C3a and C5a.
  • Plasma samples 450 ⁇ l were removed at regular time intervals (i.e., 15 min, 30 min, 60 min, 120 min and 180 min) and added to 50 ml of 0.2M disodium ethylenediamine tetracetate (EDTA) to prevent additional complement activation during storage.
  • EDTA disodium ethylenediamine tetracetate
  • Plasma samples were also used simultaneously.
  • 10 ml of plasma was pumped through the circuit without the module at 1.0 ml/min at 37 °C.
  • Plasma samples 450 ⁇ l were also removed at regular time intervals (i.e., 15 min, 30 min, 60 min, 120 min and 180 min) and added to 50 ml of 0.2M disodium ethylenediamine tetracetate (EDTA) to prevent additional complement activation during subsequent storage.
  • EDTA disodium ethylenediamine tetracetate
  • xymosan was added at a concentration of 10 mg/ml to 10 ml of plasma, and plasma samples (450 ⁇ l) were once again removed at regular time intervals (i.e., 15 min, 30 min, 60 min, 120 min and 180 min) and added to 50 ml of 0.2M disodium ethylenediamine tetracetate (EDTA) to prevent additional complement activation during storage.
  • EDTA disodium ethylenediamine tetracetate
  • modules containing microfiltration membranes prepared in accordance with the present invention were performed with modules containing microfiltration membranes prepared in accordance with the present invention in order to address various issues, including (1) capacities as a function of coating with PEI or PVA, (2) binding specificity of the ligand, (3) reproducibility of results, (4) effect of fiber base material when coated with PEI, (5) effect of capacity when the membrane was coated with PAA, (6) adsorptive performance changes when contacting whole blood, and (7) comparison between crossflow and starlings flow using whole blood.
  • Table 4 gives the modules used and the operating condition for the adsorption breakthrough experiments.
  • Either Nylon or polysulfone fibers were coated with PEI or PVA before making the hollow fiber devices.
  • the fibers were washed extensively with deionized water. The fibers were then dried and stored for future carbohydrate immobilization.
  • a similar procedure was carried out for coating with PVA or PAA, substituting PEI for PVA or PAA in the above described procedure, except that a 1% solution PAA was used, instead of the 2% employed for PEI.
  • the concentration of PVA was similar to that of PEI at 2%.
  • the PEI fibers were used to make devices modules-9 and modules-12 , PVA fibers were used to make modules-ll. modules-14 and modules-18 and PAA fibers used when making modules 3-27 and 3-30.
  • the coupling buffer was pumped through only the lumen side and one end of the lumen was closed. This forces all the coupling buffer to pass through the membrane into the shell side.
  • the module was then stored in a 0.02% sodium azide solution.
  • the carbohydrate was immobilized using the above procedure, except for devices 3-27 and 3-30, where no sulfo-NHS was " used during the immobilization procedure.
  • Figure 3A compares the breakthrough curve performance of the modules coated with either PVA or PEI respectively, followed by the ligand immobilization procedure discussed above.
  • Samples of the filtrate serum were collected and analyzed for X-IgM concentration
  • the relative concentration (C/Co) of x-IgM is plotted as a function of mass throughput CnQft, where C is the concentration of the filtrate, CQ is the concentration in the feed, Qf is the filtrate flowrate, and t is the time during which filtrate exits the downstream side of the membrane.
  • C the concentration of the filtrate
  • CQ the concentration in the feed
  • Qf the filtrate flowrate
  • t is the time during which filtrate exits the downstream side of the membrane.
  • the capacity of the module was obtained by dividing the area above the breakthrough curve by the membrane volume (Charcosset et al., Biotech. and Bioencr' ⁇ . 48:415-427 (1995)). A capacity of 14.2mg/ml mem. vol. was obtained (Fig. 3A) .
  • a capacity of 17.4mg/ml mem. vol. was obtained (Fig. 3A) when 300ml of serum (feed x-IgM concentration 0.026mg/ml) was passed through the module at a flow rate of 1.0 ml/min.
  • fibers coated with PVA provided about 25% higher capacity than fibers coated with PEI.
  • the x- IgM concentration of the filtrate reached its maximum value (feed concentration) after 0.06mg of x-IgM was loaded, indicating that the x-IgM was not significantly retained by the membrane.
  • the ligand when present, bound specifically to the x-IgM.
  • Figure 3D shows the effect of membrane base material on the shape of the breakthrough curve.
  • the polysulfone fiber give a very broad breakthrough curve when compared to the nylon for the same coating material.
  • the membrane volumes for these membranes are very similar.
  • Figure 3E shows the effect of coating the microfiltration fiber with 1% PAA instead of PEI or PVA.
  • 4.7 mg of X-IgM was adsorbed with a module having a membrane volume of 0.125ml(37mg/ml) .
  • the residence time was 7.5 sec in this experiment.
  • the capacity of the device for xenoreactive IgM doubled as the coating polymer was changed.
  • FIG 4A data are plotted from an experiment in which 45ml of whole blood was passed through the blood compartment at a flow rate of 13.1 ml/min. The shell side was open and plasma was collected at a rate of 0.5 ml/min. The concentration of X-IgM in the blood was 0.096 mg/ml and the blood hematocrit was 39.
  • Figure 4C shows the comparison between the two modes of operation in terms of the concentration in the blood reservoir divided by its initial value.
  • concentration of X-IgM in the reservoir drops to less than 10% of its initial value after approximately 50 min in crossflow mode as compared to about 100 min in the starlings flow mode.
  • crossflow mode is more effective than starlings flow mode under the given operating conditions.
  • Nylon fiber with PEI and carbohydrate Three different modules with membrane volume of 0.125ml for were fabricated: 1) Nylon fiber with PEI and carbohydrate; 2) Nylon fiber with PEI; and 3) Unmodified Nylon base fiber.
  • Plasma was used as negative control and xymosan was used as positive control.
  • a volume of 10ml of plasma was circulated from a reservoir at 37 °C through the nylon fiber modules 1 to 3 at a flow rate of lml/min from the tube side to the shell side of the device at 37 °C (deadend mode). The outlet from the shell was returned to the plasma pool. This closed loop system was used to permit time dependent accumulation of complement derived anaphylatoxins C3a and C5a.
  • Plasma samples (450 ⁇ l) were removed at regular time intervals (15 min, 30 min, 60 min, 120 min, 180 min) and added to 50ml of 0.2M disodium ethylenediamine tetracetate (EDTA) to prevent additional complement activation during storage. Appropriate control samples were also used simultaneously.
  • EDTA disodium ethylenediamine tetracetate
  • 10 ml of plasma was pumped through the circuit without the module at 1.0 ml/min at 37°C.
  • Plasma samples (450 ⁇ l) were removed at regular time intervals (15 min, 30 min, 60 min, 120 min, 180 min) and added to 50 ⁇ l of 0.2M disodium EDTA to prevent additional complement activation during subsequent storage.
  • xymosan was added at a concentration of 10 mg/ml to lOml of plasma.
  • Plasma samples 450 ⁇ l were removed at regular time intervals (15 min, 30 min, 60 min, 120 min, 180 min) and added to 50ml of 0.2M disodium EDTA to prevent additional complement activation during storage.
  • a clinical apparatus was set up in order to test the adsorption of xenoreactive IgM and xenoreactive IgG in non-immunosuppressed baboons in vivo using a nylon microfiltration membrane apparatus of the present invention.
  • prototype baboon- scale modules was used.
  • a schematic diagram of the equipment setup which incorporated device is illustrated in FIG. 5.
  • the device 10 consisted of 600 PAA-modified nylon fibers of 10.2 cm length.
  • the total membrane volume in the device was approximately 10 mL.
  • the devices were modified or coated with PAA and the carbohydrate was immobilized onto them overnight (as described below) .
  • the devices were then steam-sterilized in an autoclave and stored in a sterile bag until needed.
  • the nylon hollow fibers were obtained from Akzo Nobel.
  • the nylon membrane was then coated with polyallylamine to increase the number of free amino groups on the membrane as described in example 2.
  • the attachment of carbohydrate is similar to that carried out in example 2 except that the carbohydrate concentration was lower and the unreacted amine groups were capped.
  • a brief description of the immobilization procedure is given here.
  • the ligand is Gal ⁇ l-3Gal ⁇ l-4GlcNAc with a six-atom carbon spacer and carboxy terminus. It is linked to the membrane through free amino groups by (l-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) ) chemistry.
  • the amine-containing membrane is washed thoroughly with the coupling buffer, which is 0.2 M (2-(N- morpholino (ethanesulfonic acid))) (MES) , at pH 5.5. Then the carbohydrate ligand along with EDC is dissolved in the MES coupling buffer. The ratio of ligand to EDC is 1.0:4.5 by weight.
  • the carbohydrate concentration for coupling was 10 mg/mL. Using a peristaltic pump, 50 ml of the carbohydrate solution was pumped through the device in recirculation mode overnight. For each module, approximately 250 mL of PBS + 0.5 M NaCl solution was flushed from the shell inlet to the shell outlet. Solution was collected in a waste container.
  • Acetic anhydride solution was prepared: 30 mL of acetic anhydride was mixed with 300 mL of 0.2 M sodium acetate solution for each module. Lines and module were emptied. All air was purged from the module using the acetic anhydride solution, first from the shell inlet to the shell outlet then from the lumen inlet to the lumen outlet. The module was flushed for 30 minutes with the acetic anhydride solution from lumen inlet to shell outlet in recirculation mode.
  • modules were emptied of their solution, which was returned to the recirculation pool, and 30 mL of acetic anhydride was added. The new solution was mixed well. Air was purged again from the module and the modules were flushed another 30 minutes in recirculation mode. Then modules were flushed with IL each of PBS + 0.5 M NaCl, 0.1 M Glycine + 0.5 M NaCl, PBS, and sterile water in the following manner. A volume of 250 mL was flushed from shell inlet to shell outlet, 250 mL from lumen inlet to lumen outlet, and 500 mL from lumen inlet to shell outlet. The device was then sterilized. Physical and chemical properties of the carbohydrate immobilized affinity membrane are summarized in Table 6.
  • the experimental arrangement 10 for the baboon trials is shown in Figure 5, it includes the blood pump (BM11A) , the arterial and venous tubing set and the device.
  • the device was operated in a crossflow mode. Blood was pumped into the inlet of the device from the right femoral artery, where Feed-In samples were taken. Blood plasma was passed across the pores of the hollow fibers, while the cellular components did not cross the wall of the membrane.
  • the targeted antibodies (i.e., the xenoantibodies) in the plasma came into contact with the carbohydrate antigen, that had previously been immobilized on the internal surface of the pores.
  • the treated plasma devoid of xenoantibodies
  • the blood recombined with the cellular component of the blood at the venous chamber, and the blood re-entered the animal at the left cephalic vein.
  • the Feed-Out samples were taken just before the blood reentered the animal as shown in the figure.
  • the baboons' heart rate, blood pressure, breathing and anesthesia were monitored during the perfusion. The experiment were carried out for about 190 min. After the experiment was complete, the module was washed with saline. A single device was used to remove the xenoantibodies from the baboon and the device was not reused.
  • the concentration of xenoreactive IgG and IgM decreased as a function of time. Nearly complete removal of both xeno-IgM and xeno-IgG occurred in each of the trials. Xeno- IgM removal after 60 minutes was approximately 85% for all three trials. Xeno-IgG removal after 60 minutes was almost 100% for Trials 1 and 2 and 85% for Trial 3.
  • the xeno-IgG feed concentrations were very small, too small to be measured by even low-concentration standards at equal dilution. Therefore the final concentration of xeno-IgG was near zero, showing the modules can remove even very low concentrations of xenoreactive antibody. Overall, the modules were highly successful at removing xenoreactive antibody in a short period of time from baboons. Similar results were obtained when the Blood In samples were plotted as a function of time.

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Abstract

Cette invention concerne une méthode et un dispositif permettant d'extraire sélectivement des anticorps xénoréactifs du sang, du sérum ou du plasma. Sur une membrane à immunoaffinité, on immobilise un ligand capable de se lier à un anticorps xénoréactif présent dans le sang, le sérum ou le plasma. En traversant la membrane à immunoaffinité renfermant le ligand, le sang, le sérum ou le plasma abandonnent les anticorps xénoréactifs avant d'être réintroduits dans l'organisme. Grâce à cette invention, il est possible d'extraire uniquement les anticorps xénoréactifs qui sont liés au rejet hyperaigu observable lorsque des organes ou des tissus de certains animaux non humains entrent en contact avec du sang ou des liquides sanguins, notamment pendant une xénotransplantation. Cette invention offre l'avantage de pouvoir être mise en oeuvre simplement et efficacement avec un seul matériel. Elle convient tout particulièrement bien pour réduire ou éliminer les risques de rejets hyperaigus et la destruction rapide de tissus greffés normalement observables après une xénotransplantation.
PCT/US2000/000410 1999-01-11 2000-01-10 Methode et dispositif permettant d'extraire selectivement des anticorps xenoreactifs du sang, du serum ou du plasma WO2000041718A1 (fr)

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