Reduced leaching of a ligand

Abstract

A column for removal of a component from a fluid is disclosed. The column has a compartment with a cross sectional area. The compartment contains beads having a diameter. A ligand selected to bind to the component is coupled to the beads. The cross-sectional area and bead diameter are selected to maintain a flow velocity of the fluid within the compartment below a first threshold, thereby reducing leaching of the ligand into the fluid. Also described herein is an adsorbent comprising a ligand that is attached to a substrate by an amine bond, wherein the ligand is resistant to dissociation from the substrate.

What is claimed is: 
     
         1 . A method of preparing an adsorbent, comprising the steps of:
 activating a substrate by exposing the substrate to an activation solution comprising an inorganic salt;   forming a bond between the activated substrate and a capture ligand by exposing the substrate to a ligand solution comprising the capture ligand, wherein the ligand solution has a pH in the range of 8.5-10; and   strengthening the bond by exposing the bond to a coupling solution comprising sodium cyanoborohydride, wherein the coupling solution has a pH in the range of 8.5-10 so that the bond forms a secondary amine linkage while avoiding thioether and ether linkages.   
     
     
         2 . The method of  claim 1 , wherein:
 the substrate comprises agarose; and   the inorganic salt comprises sodium metaperiodate.   
     
     
         3 . The method of  claim 1 , wherein:
 the capture ligand comprising a portion of Tumor Necrosis Factor alpha (TNF-alpha).   
     
     
         4 . The method of  claim 3 , wherein:
 the portion of TNF-alpha comprises a single-chain TNF-alpha trimer.   
     
     
         5 . The method of  claim 4 , wherein:
 the substrate comprises a plurality of beads; and   the ligand solution comprises approximately 1 milligram of single-chain TNF-alpha trimer per 1 milliliter of beads when a bed volume of the ligand solution is added to a bed volume of beads.   
     
     
         6 . The method of  claim 1 , further comprising the steps of:
 aspirating the ligand solution and the coupling solution, after the step of strengthening the bond, from the substrate;   quenching the substrate, after the step of aspirating the ligand solution and the coupling solution, by exposing the aspirated substrate to a quench solution comprising ethanolamine;   aspirating the quench solution, after the step of quenching, from the substrate thereby producing an aspirated and quenched substrate; and   adding a phosphate-buffered saline (PBS) solution having a pH in the range of 7.1-7.3, to the aspirated and quenched substrate.   
     
     
         7 . The method of  claim 1 , further comprising the steps of:
 aspirating the ligand solution and the coupling solution, after the step of strengthening the bond, from the substrate; and   adding a phosphate-buffered saline (PBS) solution having a pH in the range of 7.1-7.3, after the step of aspirating the ligand solution and without quenching the substrate, to the aspirated substrate.   
     
     
         8 . An adsorbent, comprising:
 a substrate; and   a capture ligand;   wherein the adsorbent is prepared by the steps of:
 activating the substrate by exposing the substrate to an activation solution comprising an inorganic salt; 
 forming a bond between the activated substrate and the capture ligand by exposing the substrate to a ligand solution comprising the capture ligand, wherein the ligand solution has a pH in the range of 8.5-10; and 
 strengthening the bond by exposing the bond to a coupling solution comprising sodium cyanoborohydride, wherein the coupling solution has a pH in the range of 8.5-10 so that the bond forms a secondary amine linkage while avoiding thioether and ether linkages.