WO1990004416A1 - Extraction specifique de lipo-proteines de faible densite contenues dans le sang - Google Patents

Extraction specifique de lipo-proteines de faible densite contenues dans le sang Download PDF

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Publication number
WO1990004416A1
WO1990004416A1 PCT/US1989/004716 US8904716W WO9004416A1 WO 1990004416 A1 WO1990004416 A1 WO 1990004416A1 US 8904716 W US8904716 W US 8904716W WO 9004416 A1 WO9004416 A1 WO 9004416A1
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beads
ldl
agarose
composition
agarose beads
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PCT/US1989/004716
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English (en)
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Richard E. Ostlund, Jr.
Gustav Schonfeld
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Invitron Corporation
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Publication of WO1990004416A1 publication Critical patent/WO1990004416A1/fr

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    • 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
    • A61M1/36—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits
    • A61M1/3679—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits by absorption
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • 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
    • A61M2202/00—Special media to be introduced, removed or treated
    • A61M2202/04—Liquids
    • A61M2202/0413—Blood
    • A61M2202/0456—Lipoprotein
    • A61M2202/046—Low-density lipoprotein

Definitions

  • the invention relates to clinical practices for regulating blood components. Specifically, it concerns methods and materials useful in the lowering of cholesterol levels in plasma through the removal of LDL from whole blood.
  • Extracorporeal treatment of blood and plasma for various therapeutic purposes has been known -for some time.
  • Losgen, H., et al, Biomat, Med Dev, Art Orq (1978) 6/151-173 describes the use of large agarose beads of 1-10 m diameters prepared from ' Sepharose R.
  • the beads were approximately 4% agarose and used to remove various components from plasma by passage of whole blood over the beads.
  • the beads could be activated with cyanogen bromide to immobilize proteins as specific re- actants as well.
  • adsorbent materials encapsulated with a high concentration agarose to obtain spheres of 1 mm or more to remove materials from the bloodstream.
  • Such supports have been used both nonspecifically, and in con- nection with specific agents such as albumin for the removal of bilirubin and UDP-glucuronyl transferase to effect the glucuronidation of toxins by passage of blood over the solid support.
  • the invention herein concerns the removal of • apolipoprotein B (apoB) from plasma. Removal of apoB is of interest because apoB is the only protein component of low density lipoprotein (LDL) and a major component of very low density lipoprotein (VLDL) . LDL is the principal.
  • U.S. Patents 4,656,261 and 4,654,420 which describe a water insoluble hard cellulose gel chemically sulfated to obtain a polymer which adsorbs LDL and VLDL from body fluids. The resulting support is disclosed as appropriate only for plasmapheresis.
  • U.S. Patent 4,637,994 discloses a water insoluble porous hard gel, to which is bound a sulfated moiety such as dextran sulfate for use in removing LDL or VLDL from body fluids. The disclosure distinguishes the hard gels included therein from "soft" gels, such as agarose.
  • Monoclonal antibodies reactive with specified epitopes on apolipoprotein B have also been prepared (EPO Application 0257778, published 20 July 1987).
  • the invention satisfies the need for a rapid and efficient way to treat blood extracorporeally to diminish its capacity to support the presence of cholesterol.
  • the method is based on the direct perfusion of whole blood through an adsorbent which selectively removes LDL from the plasma. The process is completed by returning the LDL-depleted blood to the patient.
  • the invention is directed to a method to treat whole blood which comprises passing the blood over a solid phase consisting essentially of 0.5-3% agarose beads, preferably 2% agarose beads, of relatively small dimensions (60-500 micron diameter, preferably 200-300 micron diameter) which have been cross- linked and derivatized to a ligand specifically reactive with LDL or VLDL.
  • the ligand is typically an antibody or a polyanion which is specific for apolipoprotein B- containing moieties, such as LDL or VLDL, the best known example of which is dextran sulfate.
  • This method can be used directly in conjunction with the bloodstream of the patient by placing the adsorbent in an extracorporeal shunt through which blood is withdrawn and passed and then returned to the patient.
  • the invention also relates to the 0.5-3% (preferably 2%) agarose 60-500 micron (preferably 200-300 micron) beads derivatized to ligands, which constitute the affinity support.
  • the invention in another aspect, relates to a method to selectively remove LDL from plasma which method comprises passing whole blood through a support consisting essentially of 0.5-3%, preferably 2%, agarose beads.of 60- 500 micron, preferably 200-300 micron, diameter to which is derivatized a monoclonal antibody preparation cross- reactive with a particular monoclonal preparation secreted by the cell line described below.
  • the monoclonal prepara ⁇ tion is reactive with the N-terminal portion of apolipoprotein B, and is designated C3D1.
  • the invention also includes monoclonal antibodies cross-reactive with C3D1 and cell lines capable of secreting them.
  • the invention is directed to a method to treat whole blood which comprises passing the blood over a solid phase consisting es ⁇ sentially of 0.5-3%, preferably 2%, agarose beads of 60- 500 micron, preferably 200-300 micron, diameter which have been cross-linked and derivatized to a polyanion specific for LDL and VLDL, preferably through an amino functionality.
  • This support too, can be used directly with the bloodstream of the patient and is useful to remove LDL or VLDL from blood or plasma.
  • the invention is directed to these derivatized supports.
  • the invention is also directed to a column sterilization method.
  • Figure 1 shows a map of the apoB protein along with the binding sites for various monoclonal antibody preparations.
  • Figure 2 shows a diagram of a hemoperfusion ap ⁇ paratus which includes the immunosupport of the invention.
  • Figure 3 shows the binding of anti-LDL to sup ⁇ port beads as a function of their agarose concentration.
  • Figure 4 shows LDL binding capacity of the various agarose concentration beads prepared as for Figure 3.
  • the columns may employ antibodies or their derivatives, wherein these derivatives include, for example, F(ab')_ or Fab' or Fab fragments.
  • antibodies or derivatives thereof immunoreactive with LDL refers both to intact antibodies and to specifically immunoreactive fragments.
  • Immunoglobulins or their immunoreactive derivatives which react with the same epitope are "cross-reactive". Cross- reactivity among such immunoreactive molecules can be determined by the ability of one such immunoglobulin to compete with the other for binding to the antigen.
  • Immunoglobulins which are cross-reactive will block each other's -binding to the antigen; those not cross-reactive, and which bind to different epitopes on the antigen will not.
  • the apoB protein,' of 4536 amino acids has a number of identified epitopes which are specific to particular monoclonal preparations described below. Alternate monoclonal preparations may be obtained which cross-react with the same epitopes as, for example, C3D1 which represents the most useful monoclonal for the purpose of LDL removal when bound to agarose.
  • antibodies specific for LDL or its major" component are conjugated to the agarose beads.
  • polyclonal antisera raised against purified LDL or apolipoprotein B, and previously purified by affinity chromatography using LDL conjugated supports are conveniently used.
  • These antibody preparations are raised in suitable mammals, such as goats, sheep, or mice and are purified from the immune serum using a technique suf- ficiently specific to obtain antibodies of the required specificity. Either the purified whole antibodies or the immunoreactive fragments are used.
  • a monoclonal preparation can be obtained by immunization of a subject mammal with LDL or apolipoprotein B and fusion of the antibody-secreting cells with myelomas or otherwise immortalizing them to obtain cell lines capable of secret ⁇ ing anti-LDL antibodies.
  • the immortalized cell lines are screened for desired antibody secretion using standard ELISA or other immunoassay techniques as a preliminary screen.
  • selection of the ideal candidate will be either by assaying the supernatants after their conjuga ⁇ tion to the agarose support of the invention and/or by a competitive immunoassay for cross-reactivity with the monoclonal antibody disclosed herein, designated C3D1.
  • Standard methods are used to conjugate the monoclonal or polyclonal preparations to the support.
  • the derivatization should result in 2-10 mg/ml gel for monoclonal preparations cross-reactive with C3D.I or for polyclonal preparations.
  • the agarose supports of the invention may be conjugated, preferably using the functionality of an amino group, to an LDL/VLDL-specific polyanion, such as dextran sulfate.
  • LDL/VLDL-specific polyanion such as dextran sulfate.
  • the polyanion specifically adsorbs LDL and VLDL from the blood.
  • Such polyanions are typically polysulfated products of various saccharides or alcohols.
  • a particuarly preferred polyanion is dextran sulfate.
  • the polysulfated forms of other monosaccharides, oliqosaccharides or polysaccharides can also be -used, as well as those of polyhydroxy compounds such as glucuronic or ascorbic acids, or polyhydric alcohols such as glycerol.
  • Polysulfation of the alcohol groups of these compounds also provide suitable polyanions.
  • Other apolipoprotein B-specific ligands include the sulfation products of starch, chitin, pectin, chondroitin, and ' the like.
  • the preferred polyanion, dextran sulfate is available in a range of molecular weights and a range in percentage sulfation.
  • the dextran sulfate should have a molecular weight of 2,000-10,000, preferably around 5,000, and should have a sulfur content of 15-20%, preferably around 17%.
  • Dextran sulfate is commercially available and can be made by reaction of a polysaccharide produced by Leuconostoc mesentieroides with, for example, chlorosulfonic acid.
  • the polyanionic sulfate ligand is preferably attached to the agarose supports of the invention by mediation of an amino group linkage supplied to the ligand as described below.
  • conjugation of the ligand to the agarose supports of the invention for example using alternate methods such as direct reaction using cyanogen bromide, epichlorohydrins, a polyoxirane compound such as bisepoxide or triazine halide, all as described by Tani et al in U.S. Patent 4,637,994, fails to supply adequate derivatization of agarose with ligand to make the support an effective adsorbent of VLDL, LDL or apoB.
  • the most effective method for attachment of the polyanionic sulfate affinity ligand is mediation by an amino group.
  • Two major, approaches for effecting this mediation are herein described; both •involve derivatization of the polyanionic sulfates so as to provide amino groups.
  • the polyanionic sulfate is activated first with cyanogen bromide, and then derivatized to a diamirioalkane such as diaminohexane.
  • the polyanionic sulfate is first activated with a diglycidyl ether and then reacted with a diaminoalkane.
  • the resulting polyanionic sulfates containing amino groups can then be directly bound to the agarose beads using a number of cross-linking agents including, for example, cyanogen bromide and glutaraldehyde, as exemplified below.
  • the resulting polyanionic sulfate derivatized agarose should have the ligand immobilized at a concentra ⁇ tion in a range of 0.5-20 mg of the ligand/ml column volume, preferably 3 mg/ l. Below this range of effective concentration, the binding of LDL to the support is inadequate for effective removal of LDL from serum.
  • the derivatized supports using either antibody preparations or polyanionic sulfate as affinity ligands are then suitable not only for plasmapheresis techniques, but also for hemoperfusion using whole blood. Of course, the derivatized supports can also be used for standard Ln vitro chromatographic techniques for adsorption and assay of LDL, VLDL, or other apolipoprotein B containing moieties.
  • hemoperfusion is meant the passage of whole blood through a solid support to obtain a product blood which is different in composition from the blood initially passed through the adsorbent.
  • the process can be conducted in several ways. For example, a portion of blood can be removed as a batch, treated with anti ⁇ coagulant, passed through the immunoadsorbent, and then used for whatever purpose it is intended. If, however, -li ⁇
  • the hemoperfusion is intended for therapeutic purposes in an individual patient, this is generally conducted as a continuous process where the blood is recycled into the patient after passage through the appropriate support.
  • a typical, but of course nonlimiting, arrangement is shown in Figure 2.
  • blood is removed from the patient and treated with an anticoagulant shown in the figure as heparin.
  • Alternative anticoagulants can also be used, and are preferred, including citrates such as citrate phosphate dextrose (CPD) or the commonly used preparation anticoagulant citrate dextrose (ACD) .
  • CPD citrate phosphate dextrose
  • ACD preparation anticoagulant citrate dextrose
  • the blood containing the anticoagulant is then pumped through a drip chamber to prevent bubble formation, and then through a column containing the adsorbent and back through an additional drip chamber to the patient.
  • a variety of designs for the hemoperfusion system can be used, as well as a variety of configurations with regard to the column containing the adsorbent.
  • the invention herein is directed to an improvement in hemoperfusion to remove LDL from blood which comprises the use of 60-500 micron, preferably 200-300 micron, 0.5-3%, preferably 2%, agarose beads as a support.
  • 60-500 micron preferably 200-300 micron, 0.5-3%, preferably 2%
  • agarose beads as a support.
  • Particularly preferred are diameters of 212-300 microns; they have been sieved in water from 50-100 mesh agarose. Chromatographic size (60-140 micron) and hemoperfusion size (300-450 microns) are less desirable.
  • the 212-300 micron (200-300 micron) beads showed accept ⁇ able flow rates.
  • beads were prepared at various agarose concentrations of 1%, 2%, 4%, 6% and 8% agarose, 50-100 wet mesh (Bio-Rad, Richmond, California) and cross- linked and desulfated as described by Kristiansen, T. , et al, Meth Enzymol (1974) _3_4:331-341 and Porath, J. , et al, J Chromat (1971) 6_0:167-177, respectively. The beads were then passed through a 300 micron hand sieve, and retained on a 212 micron sieve.
  • the beads were washed 70 times with water on the smaller sieve.
  • the beads were activated using cyanogen bromide as described by March, S.C., et al. Anal Biochem (1974) 6):149-152.
  • CNBr activated beads showed excellent flow rates.
  • the activated gel cake was added to 2-4 times its volume of 0.2 M sodium bicarbonate, pH 9 containing the desired antibody or derivative thereof and rotated for two hours at room temperature. Unreacted sites were blocked by rotating the gel overnight at 4°C with 0.2 M glycine, pH 8 containing 0.15 M sodium chloride.
  • Figure 3 shows the effect of bead agarose concentration on the capacity to be derivatized with anti ⁇ body.
  • the antibody preparations specific for LDL were raised in goats, harvested by plasmapheresis and purified by affinity chromatography on LDL agarose as described by Semenkovich, C.F., et al, J Lab Clin Med (1985) 106:42-47. Fragments were prepared by digestion with papain to produce Fab (Mage, M.G., Meth Enzymol (1980) 70:142-150) or with pepsin to produce F(ab')_ as described by Hudson, L., et al. Practical Immunology (1976) Oxford: Blackwell Scientific Publications, pp. 186-188.
  • the ability of the resulting derivatized beads to bind LDL was tested using an in vitro test tube assay 5 for adsorption.
  • the beads to be tested are suspended at 10% volume/volume in water and 100-200 micro- liter of the suspension are pipetted into 1.5 ml conical plastic microfuge tubes.
  • the tubes are centrifuged for 5 seconds, the supernatant fluid removed under suction, and* 10 a 40-80 microliter sample of EDTA-bovine serum albumin buffer (0.15 M NaCl, 50 mM tris, 1 mM EDTA, 2 mg/ml BSA, pH 7.4) containing 2.5 mg 125I-labeled LDL protein/ml were added.
  • the tubes are then vortexed and reset in the microfuge and then rotated end over end for two hours at
  • the binding' was 64% completed in 15 minutes and completed in 2 hours.
  • the beads are then washed 6 times with EDTA/BSA buffer and eluted 3 times for two hours with 0.5 ml 1 M
  • the beads could be activated using glutaraldehyde according to the procedure of Cambiaso, C.L., et al, Immunochem (1975) 1 ⁇ :273-278.
  • the activated beads are washed 5 times in 0.1 M sodium bicarbonate, pH 8.5 and reacted with 6 volumes of antibody in 0.1 M sodium bicarbonate, pH 8.5 for two hours at room temperature. Unreacted groups are blocked as described above. All gel beads are washed with 1 M acetic acid with three cycles of alternating acidic and basic washes of 0.5 M NaCl containing 0.1 M sodium acetate, pH 4.5 and 0.5 M NaCl containing 0.1 M sodium bicarbonate, pH 8.3, and with water.
  • the 2% beads were markedly inferior to higher percentage agarose beads — 2% beads adsorbed about 3 mg goat IgG per ml gel; 4% and higher beads adsorbed about 6 mg/ml or approximately twice as much.
  • the effect of the amount of antibody on the columns was also studied, and it was found that after 5 mg of antibody was bound per ml beads, there was no further improvement in the capacity of the immunoadsorbent to bind LDL. This result was obtained both for 2% and 4% beads.
  • the amount of Fab for example, reacted with the activated beads was increased in the range of 0-32 mg/ml, the amount of Fab bound increased monotonically to about 15 mg/ml.
  • the number of mg of LDL capable of binding per ml of beads leveled off at about 2 mg/ml for 4% and at about 4 mg/ml for 2 percent agarose beads after, in each case, about 5 mg/ml had been bound.
  • antibody in the amount of about 3-15 mg per ml gel could be bound using various methods including the CNBr and glutaraldehyde methods described above, as well as methods involving trichloro- triazine and adsorption methods. .Considerable variation resulted in the resulting ability of the beads to bind LDL; clearly the best results were achieved for CNBr or glutaraldehyde.
  • the amount of LDL bound to the immunosorbent is expressed as mg LDL protein/ml gel volume, as determined by the micro test tube assay.
  • a Ab is affinity-purified anti-human LDL raised in goats.
  • Agarose beads were derivatized with diaminohexane using the CNBr technique and then exposed to 2.5% glutaraldehyde, followed by anti-LDL IgG, as described in Methods.
  • c Protein A-Sepharose 4B-C1 beads (2 mg protein A/ml gel) were rotated overnight with affinity-purified anti- LDL antibody. After washing twice with EDTA-BSA buffer, the gels were assayed for 125I-LDL binding in the usual manner, using 1 M acetic acid as eluting agent.
  • Affigel-10 Bio-Rad, Richmond, CA, U.S.A.
  • a 10- atom spacer arm separates the active ester from the agarose bead.
  • Fab antibody fragments were bound to gel beads in 0.1 M sodium bicarbonate, pH 8, for 4 h at 4°C. e Prepared as in Finlay, T,H., et al. Anal Biochem
  • the invention also includes agarose bead sup- - ports derivatized to dextran sulfate through the mediation of amino group linkages as described herein, and to methods of plasmapheresis and hemoperfusion using these ⁇ supports.
  • agarose bead sup- - ports derivatized to dextran sulfate through the mediation of amino group linkages as described herein, and to methods of plasmapheresis and hemoperfusion using these ⁇ supports.
  • Detailed examples of the preparation of dextran sulfate derivatized supports is exemplified below.
  • the immunosorbent can be reused if properly sterilized. Since autoclavin ' g destroys the activity, alternative sterilization methods must be used, and a variety of such methods are suggested in the above-referenced paper.
  • a particularly preferred method employs a mixture of phosphoric acid in alcohol at a slightly elevated temperature.
  • An illustrative mixture containing 0.34% phosphoric acid and 80% ethanol at 37°C is capable of sterilizing the immunoadsorbents for reuse.
  • An improved sterilant 60-80%, preferably 70% ethanol containing 60-120 mM, preferably 90 mM phosphate adjusted to pH 2.5-4.0, preferably 3.0 and employed at elevated temperatures, e.g., 37 C for 4-24 hours, prefer ⁇ ably 8-16 hours, has been developed.
  • Example 1 Reduction of LDL Levels in Dogs Agarose beads conjugated to goat anti-LDL Ig were prepared as described in Ostlund, R. (supra). The beads were washed with 1% Liquinox ' to reduce leakage of nonspecifically adsorbed antibody and the sorbent was shown to contain 3.24 mg antibody/ml. The beads were packed to within 1 cm of the top of a cylindrical 300 ml polycarbonate hemoperfusion canister (7.1 cm diameter x
  • Blank columns were prepared in the same manner and treated with CNBr and glycine without addition of antibody. The columns were disinfected by perfusing with 1.5 liters 1 M acetic acid and stored until use.
  • Two mongrels weighing 20 kg were perfused once per week for four weeks with blank columns and once per week for four weeks with antibody columns.
  • the animals were placed in a sling and sedated with acepromazine.
  • the hemoperfusion used an arrangement similar to that shown in Figure 2, containing a Gambro AK-10 blood monitor and standard adult hemodialysis tubing. Pressure monitors were connected before and after the column. The tubing was primed and acetic acid washed out of the column in a -19-
  • ACD-A anticoagulant citrate dexcrose solution A
  • ACD-saline ACD-saline
  • Sixteen gauge catheters were placed in each external jugular vein and flushed with ACD-saline. The left catheter was used to draw blood and was connected to a three-way stopcock into which was infused ACD-A at a rate 1/13 that of blood flow. Venous return to the animal was not connected until blood cells appeared in the venous drip chamber, so as to avoid providing saline to the animal.
  • Blood was pumped at 15 ml/min for one hour and flowed through the column in an upward direction, then washed from the column in a downward direction for an ad ⁇ ditional 20-30 min.
  • Samples for assay were taken from the animal before infusions of saline and after the procedure was terminated. Samples were taken from the blood lines before and after the column after 40 minutes of blood withdrawal. ' Biochemical and blood cell values were cor ⁇ rected for dilution based on the red cell concentration of the sample. The columns could be disinfected for storage after washing with saline by perfusion with 1.5 1 1 M acetic acid at room temperatures.
  • the blood was pumped easily through the columns; at a flow rate of 15 ml/min, the trans-column pressure was 15 mm Hg and at a flow rate of 30 ml/min, it was 25 mm Hg.
  • the arterial and venous blood pressures were 35 and 50 mm Hg, respectively, at,a 15 ml/min blood flow, showing that resistance to blood flow was due to catheters and tubing rather than the column. No clotting was observed in the columns or tubing for the most part, although small clots, not of troublesome dimensions, are sometimes observed after extensive perfusion.
  • Data for Dogs 1 and 2 represent 4 and 3 procedures, respectively. Data are corrected for red blood cell count and presented as mean + SEM.
  • the average ApoB reduction was 81% across the column and 33.5% in the animal before and after the procedure.
  • the dog is not a precise model for the human, as the major lipoprotein of the dog is high-density lipoprotein, and thus this procedure would not be expected to decrease, the level of cholesterol in the plasma of dogs, per se.
  • C3D1 When tested in standard im unoassays, C3D1 was of average binding capacity to LDL, while B1B3 showed the highest affinity. These results were not the same as those obtained in the alternative assay described above, wherein LDL was provided to microfuge tubes containing the antibody derivatized to agarose beads.
  • the immunosorbents were prepared as described above using CNBr-derivatized cross-linked 212-300 micron beads of varying agarose concentration. All of the antibodies bound significantly to the beads. In most cases, the level of binding was similar across all agarose percentages and comparable to that of goat polyclonal antibody.
  • Literature values for immunoadsorbents are approximately 3-5 mg cholesterol/ml. Similar results were obtained when derivatization was effected using glutaraldehyde.
  • Example 3 Production of C3D1 Cell culture containing C3D1 was deposited at the American Type Culture Collection on , 1988, with accession no. ' The C3D1 cell line was expanded in mice by injecting with the cell culture and recovery of the ascites fluid.
  • the C3D1 cell line can also be proliferated in vitro.
  • C3D1 cells from a single frozen vial are recovered into a T flask which is subsequently expanded to multiple new T flasks. Cells from these flasks axe used to inoculate a 500 ml spinner vessel, and these cells progressively expanded to 3-1 and 14-1 spinner vessels, respectively.
  • a 100-1 perfusion reactor is then inoculated with cells from 2X14-1 spinner vessels and grown in standard media supplemented with 5% fetal bovine serum. Approximately 250 1 of conditioned medium are produced and the IgG concentrated from the medium using LDL affinity columns. -23-
  • A.l In one method, 2 g CNBr was added to 12 ml water at 4°C and stirred. Dextran sulfate, 2g in 2 ml water, was added and the pH was raised to 11.0 with sodium hydroxide and maintained for 20 minutes. After lowering the. pH to 9.0, the cyanogen bromide-activated dextran sulfate was added to 18 ml of 2.8 M diaminohexane, pH 9, and rotated for 24 hours at room temperature. The result ⁇ ing aminohexyl-dextran sulfate was separated from unreacted CNBr and diaminohexane by dialysis against water in 1 kd MW cutoff dialysis tubing. The product contained 0.7 moles amino group per mole sulfate group.
  • the prepared support was then tested for capacity to adsorb LDL in the microfuge tube assay described above.
  • the results, as a function of agarose bead concentration, are shown in Table 3, for beads derivatized using the aminohexyl dextran sulfate prepared as in A.1.

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Abstract

Un procédé particulièrement efficace permet de réduire le taux de cholestérol chez des patients par traitement du sang entier avec des perles d'agarose conjuguées avec des anticorps anti-lipo-protéines de faible densité ou avec un sulfate polyanionique, spécialement lorsque les perles sont conjuguées au moyen d'une fonction amine. Dans des modes préférentiels de réalisation, les perles contiennent entre 0,5 et 3 % d'agarose, de préférence 2 % d'agarose, et ont des diamètres compris entre 200 et 300 microns. Des anticorps préférentiels peuvent former des réactions croisées avec des anticorps C3D1.
PCT/US1989/004716 1988-10-25 1989-10-23 Extraction specifique de lipo-proteines de faible densite contenues dans le sang WO1990004416A1 (fr)

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5187010A (en) * 1990-11-27 1993-02-16 W. R. Grace & Co.-Conn. Membrane having high affinity for low density lipoprotein-cholesterol from whole blood
US5236644A (en) * 1990-11-27 1993-08-17 W. R. Grace & Co.-Conn. Process of making membrane for removal of low density lipoprotein-cholesterol from whole blood
US5364793A (en) * 1990-11-30 1994-11-15 Monoclonetics International, Inc. Methods for the diagnosis of peripheral nerve damage
US5496637A (en) * 1990-11-27 1996-03-05 W. R. Grace & Co.-Conn. High efficiency removal of low density lipoprotein-cholesterol from whole blood
EP0710135A4 (fr) * 1993-07-23 1997-05-21 Meir Strahilevitz Dispositifs de traitment extracorporel bases sur l'adsorption par affinite
US5844097A (en) * 1990-11-30 1998-12-01 Monoclonetics International, Inc. Methods for the diagnosis of peripheral nerve damage
US6127339A (en) * 1995-06-21 2000-10-03 Asahi Kasei Kogyo Kabushiki Kaisha Peptide for binding thereto a low density lipoprotein
WO2002050550A2 (fr) * 2000-12-21 2002-06-27 Henogen S.A. Fractions de ldl oxydees
EP1029928A3 (fr) * 1999-01-27 2002-09-18 Matsushita Electric Industrial Co., Ltd. Procede de determination du cholesterol et capteur utilisable pour sa mise en oeuvre
WO2022034025A1 (fr) * 2020-08-14 2022-02-17 Idris Oncology B.V. Procédé d'application d'un revêtement comprenant de l'acide hyaluronique sur la surface d'un dispositif d'échantillonnage médical, et dispositif d'échantillonnage médical pour la capture de cellules tumorales circulantes fournies avec le revêtement
CN117654450A (zh) * 2023-12-15 2024-03-08 武汉睿奇生物工程有限公司 一种血液中脂蛋白的吸附材料及其制备方法和应用

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DE4300412A1 (de) * 1993-01-09 1994-07-14 Behringwerke Ag Hydrophobe Adsorbentien und ihre Verwendung zur Absorption von Lipoproteinen

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Cited By (14)

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US5496637A (en) * 1990-11-27 1996-03-05 W. R. Grace & Co.-Conn. High efficiency removal of low density lipoprotein-cholesterol from whole blood
US5236644A (en) * 1990-11-27 1993-08-17 W. R. Grace & Co.-Conn. Process of making membrane for removal of low density lipoprotein-cholesterol from whole blood
US5187010A (en) * 1990-11-27 1993-02-16 W. R. Grace & Co.-Conn. Membrane having high affinity for low density lipoprotein-cholesterol from whole blood
US5844097A (en) * 1990-11-30 1998-12-01 Monoclonetics International, Inc. Methods for the diagnosis of peripheral nerve damage
US5583201A (en) * 1990-11-30 1996-12-10 Monoclonetics International, Inc. Methods for diagnosis of peripheral nerve damage
US5364793A (en) * 1990-11-30 1994-11-15 Monoclonetics International, Inc. Methods for the diagnosis of peripheral nerve damage
EP0710135A4 (fr) * 1993-07-23 1997-05-21 Meir Strahilevitz Dispositifs de traitment extracorporel bases sur l'adsorption par affinite
US6127339A (en) * 1995-06-21 2000-10-03 Asahi Kasei Kogyo Kabushiki Kaisha Peptide for binding thereto a low density lipoprotein
EP1029928A3 (fr) * 1999-01-27 2002-09-18 Matsushita Electric Industrial Co., Ltd. Procede de determination du cholesterol et capteur utilisable pour sa mise en oeuvre
US6762062B2 (en) 1999-01-27 2004-07-13 Matsushita Electric Industrial Co., Ltd. Method of determining cholesterol and sensor applicable to the same
WO2002050550A2 (fr) * 2000-12-21 2002-06-27 Henogen S.A. Fractions de ldl oxydees
WO2002050550A3 (fr) * 2000-12-21 2004-03-04 Henogen S A Fractions de ldl oxydees
WO2022034025A1 (fr) * 2020-08-14 2022-02-17 Idris Oncology B.V. Procédé d'application d'un revêtement comprenant de l'acide hyaluronique sur la surface d'un dispositif d'échantillonnage médical, et dispositif d'échantillonnage médical pour la capture de cellules tumorales circulantes fournies avec le revêtement
CN117654450A (zh) * 2023-12-15 2024-03-08 武汉睿奇生物工程有限公司 一种血液中脂蛋白的吸附材料及其制备方法和应用

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