Selective removal of apolipoprotein B-containing serum lipoproteins from blood plasma (extracorporeal blood filtration/monospecific anti-low density lipoprotein-Sepharose/plasma low density lipoprotein immunoadsorption/ kinetics of low density lipoprotein replenishment/low density lipoprotein adsorption by heparin-Sepharose 4B)

Studies were undertaken to determine the applicability and effectiveness of immunoadsorption chromatography on anti-low density lipoprotein (LDL) columns as a plasma-cholesterol-lowering procedure. Mass production and isolation of monospecific antibodies against swine LDL (p = 1.006-1.063 g/ml) was carried out by immunization of sheep with swine LDL and selective antibody adsorption from their antisera by chromatography on LDL-Sepharose. The isolated LDL antibodies were then covalently linked to Sepharose CL-4B. Pig plasma LDL was effectively removed in vitro by the anti-LDL-Sepharose beads. In vivo studies were performed in pigs with (i) a plasma-separator membrane permeable to solutes below M, 2,000,000 or (ii) a blood centrifuge interposed into an arteriovenous shunt to separate the corpuscular elements of blood from the plasma. In either case, the plasma was passed through the anti-LDL-Sepharose column and recirculated into the venous part of the shunt. Plasma cholesterol levels were reduced 70-80% by this procedure and rebounded 3-4 days after the extracorporeal immunoadsorption procedure. This continuous plasma separation-immunoadsorption procedure may have broad applicability for the elimination of any plasma component with antigenic properties. Familial hypercholesterolemia (type II hyperbetalipoproteinemia) (1) is characterized by increased plasma cholesterol concentrations and increased plasma low density lipoprotein (LDL) levels. It has been established that this form of hypercholesterolemia is due to a reduced fractional catabolic rate and to a 2to 3-fold overproduction of LDL (1-3); as a result, there is premature development of atherosclerosis. Three independent studies have been reported (4-6) that were designed to reduce the plasma cholesterol concentrations in patients with familial hypercholesterolemia by nonsurgical methods: plasmapheresis techniques (4, 6) and extracorporeal removal of LDL by means of heparin-agarose beads in blood venous packs (5). These approaches have produced transient reductions in plasma LDL levels. We report a procedure that results in specific removal ofapolipoprotein B (apo B)-containing lipoproteins (VLDL and LDL) by immunoadsorption. This study was undertaken to evaluate (i) the extent to which plasma LDL can be removed in vitro by an anti-LDL-Sepharose immunoadsorbent and (ii) whether and to what extent LDL (and VLDL) can be removed by continuous extracorporeal immunoadsorption in swine. Monospecific LDL antibodies were prepared from the serum of sheep immunized against pig LDL and were covalently linked to Sepharose. When swine plasma was passed through a column ofanti-LDLSepharose, LDL was absorbed from the plasma. In vivo studies in pigs were performed by shunting blood from the arterial branch between the common carotid artery and internal jugular vein, separating the blood into a cell concentrate and a plasma fraction by means of a selective plasma-separator membrane technique or a continuous-flow blood cell-separator centrifuge. The plasma fraction was passed over an anti-LDL-Sepharose column, and the LDL-depleted plasma was recycled into the cell concentrate and returned to the animal through the venous branch of the shunt. A comparison of continuous immunoadsorption and continuous heparin-Sepharose adsorption of LDL was also performed. MATERIALS AND METHODS Animals. Swine from a local farm and Hanford minipigs (provided by Nattermann & Cie, Koln, Federal Republic of Germany) were used for in vivo studies. Pig blood was obtained from a local slaughterhouse. Isolation of Low Density Lipoprotein. Plasma from heparinor EDTA-treated pig blood (1 mg/ml) was separated by centrifugation, and the LDL was isolated by ultracentrifugation in a 60 Ti rotor of a Beckman L5-65B centrifuge (7, 8). For the first centrifugation, a density of 1.006 g/ml was achieved with KBr. The supernatant was removed, and the density of the infranatant was adjusted to 1.063 g/ml with KBr. After ultracentrifugation, the upernatant containing LDL with density 1.006-1.063 g/ ml was collected, sedimented at density 1.006, washed once at density 1.063, and used immediately for coupling to Sepharose for immunization of sheep. LDL values given represent the total weight, with protein content given in parentheses. Preparation of LDL-Sepharose CL-4B. Sepharose CL-4B (30 ml) was washed with water and suspended in 90 ml of icecold 1M Na2CO3. CNBr (6 g in 3 ml of acetonitrile) was added with stirring, and the activated Sepharose beads were collected on a fritted-glass funnel after 2 min. The Sepharose cake was washed with 5 vol of ice-cold 0.2 M bicarbonate buffer (pH 9.5) and 5 vol of 0.5 M bicarbonate buffer (pH 8.5) and was resuspended immediately in a solution of 300 mg of LDL (60 mg of apo B) in 60 ml of 0.5 M bicarbonate buffer (pH 8.5). The incubation was continued for 20 hr at 4°C, the suspension was centrifuged, the supernatant was decanted, and the sediment was resuspended in 30 ml of phosphate-buffered saline/2M glycine for 12 hr at room temperature. Finally, the LDL-Sepharose beads were thoroughly washed with 20 vol of phosphate-buffered saline. Between 5.6 and 9.5 mg of LDL (1-2 mg of apo B per ml ofwet Sepharose beads) was covalently linked, as determined from the unbound protein and the cholesterol in the supernatant and from the cholesterol content in chloroform/ methanol extracts of aliquots of LDL-Sepharose 4B. Immunization and Isolation of Sheep Anti-Pig LDL. Sheep were immunized by three subcutaneous injections at intervals Abbreviations: LDL, low density lipoprotein; VLDL, very low density lipoprotein; HDL, high density lipoprotein; apo B, apolipoprotein B of