Novel separation matrix

Abstract

The present invention relates to a separation matrix comprised of ligands coupled to the surfaces of a porous support, such as one or more porous particles, wherein the ligands provide at least one chemical gradient within the support. In the most advantageous embodiment, the chemical gradient is a ligand density gradient. The invention also relates to a method of providing a separation matrix comprising ligands coupled to the surfaces of a porous support, in which method at least one ligand density gradient is provided by solvent-controlled diffusion of at least one reagent into the porous support.

1 . A separation matrix comprising a porous support; and ligands coupled to the surfaces of said porous support, wherein the ligands provide at least one chemical gradient in the support.  
   
   
       2 . The separation matrix of  claim 1 , wherein the support comprises porous particles and the ligand gradient(s) extend between the center and the exterior surface of each porous particle.  
   
   
       3 . The separation matrix of  claim 1 , wherein at least one gradient is a ligand density gradient formed by a changing density of ligands on the support.  
   
   
       4 . The separation matrix of  claim 3 , wherein two or more chemical gradients are present in the support and at least one gradient is a ligand density gradient.  
   
   
       5 . The separation matrix of  claim 3 , wherein in the ligand density gradient(s), the ligand concentration increases towards the center of the support.  
   
   
       6 . The separation matrix of  claim 3 , wherein in the ligand density gradient(s), the ligand concentration decreases towards the center of the support.  
   
   
       7 . The separation matrix of  claim 1 , which matrix is a chromatography matrix comprised of a plurality of essentially spherical particles, wherein each particle presents one or more gradient(s) perpendicular to the direction of the liquid flow applied in chromatography.  
   
   
       8 . The separation matrix of  claim 1 , wherein at least one gradient is the result of varying pKa values of functional groups of the ligands present on the support.  
   
   
       9 . The separation matrix of  claim 1 , wherein at least one gradient is the result of a varying net charge of the ligands present on the support.  
   
   
       10 . The separation matrix of  claim 1 , wherein at least one gradient is a continuous and smooth gradient.  
   
   
       11 . The separation matrix of  claim 1 , wherein the ligands present on the support provide at least two different functionalities.  
   
   
       12 . The separation matrix of  claim 11 , wherein said functionalities are selected from the group consisting of cation exchange ligands, anion exchange ligands, hydrophobic interaction chromatography (HIC) ligands, reversed phase chromatography (RPC) ligands, immobilised metal chelating ligands (IMAC), thiophilic ligands, and affinity ligands.  
   
   
       13 . The separation matrix of  claim 11 , wherein said at least two different functionalities are present on the same ligand.  
   
   
       14 . The separation matrix of  claim 11 , wherein the ligands present zwitterionic functionalities.  
   
   
       15 . The separation matrix of  claim 11 , wherein said at least two different functionalities are present on different ligand kinds, and each such ligand kind provides a separate chemical gradient within the support.  
   
   
       16 . A chromatography column packed with a separation matrix comprised of a porous support; and ligands coupled to the surfaces of said porous support, wherein the ligands provide at least one chemical gradient within the support.  
   
   
       17 . The chromatography column of  claim 16 , wherein the support comprises essentially spherical porous particles and the ligands provide at least one chemical gradient between the center and the exterior surface of each porous particle.  
   
   
       18 . The chromatography column of  claim 16 , wherein at least one gradient is a ligand density gradient formed by a changing density of ligands on the support.  
   
   
       19 . (canceled)  
   
   
       20 . In a method of providing a separation matrix containing ligands coupled to the surfaces of a porous support, the improvement comprises providing at least one ligand density gradient by solvent-controlled diffusion of at least one reagent into the porous support.  
   
   
       21 . The method of  claim 20 , wherein the solvent-controlled diffusion is obtained by contacting a first solvent comprising said reagent(s) with the support, in the pores of which a second solvent is present, said first and second solvents presenting different solubilities.  
   
   
       22 . The method of  claim 21 , wherein the first solvent is aqueous and the second solvent is organic.  
   
   
       23 . The method of  claim 21 , wherein the first solvent is organic and the second solvent is aqueous.  
   
   
       24 . The method of  claim 20 , wherein the diffusion rate is controlled by adjusting one or more conditions selected from the group consisting of temperature; air flow; solvent properties; and concentration and/or nature of functionalities.  
   
   
       25 . The method of  claim 24 , wherein the diffusion of reagent(s) is assisted by providing an essentially continuous air flow through the reaction mixture during the reaction.  
   
   
       26 . The method of  claim 20 , wherein a ligand density gradient that decreases towards the center of the support is provided by diffusion-controlled addition to the porous support of a reagent that comprises at least one functionality.  
   
   
       27 . The method of  claim 26 , wherein either the reagent or groups present on the surface of the porous support have been activated before the reaction.  
   
   
       28 . The method of  claim 20 , wherein the support presents activated groups.  
   
   
       29 . The method of  claim 28 , wherein a ligand density gradient that increases towards the center of the support is provided by diffusion-controlled addition to the porous support of a first reagent, which comprises deactivating groups, to deactivate in a controlled fashion some of the surface groups of the support, and subsequent addition of another reagent, which comprises at least one functionality, to couple said at least one functionality to the surface groups that have not been deactivated.  
   
   
       30 . The method of  claim 20 , wherein the reagent comprises two different functionalities in a predetermined ratio to provide to different ligands in the separation matrix.  
   
   
       31 . The method of  claim 20 , wherein the porous support comprises essentially spherical particles.  
   
   
       32 . A method of preparing a separation matrix that comprises ligands coupled to the surfaces of a porous support, which method comprises the steps of: 
 (a) providing activatable groups on the surface of a porous support;    (b) activating said groups with an activation agent;    (c) reacting groups activated according to step b) with a compound which comprises at least one functionality;    wherein control of the reactivity in step (c) results in at least one chemical gradient within the support.    
   
   
       33 . The method of  claim 32 , wherein the reactivity is controlled by the concentration of the compound that comprises the functionalities in step (c).  
   
   
       34 . The method of  claim 32 , wherein the activatable groups of step (a) are carbon-carbon double bonds.  
   
   
       35 . The method of  claim 32 , further comprising providing the activatable groups present at the surface of at least one porous particle in a step preceding step (a).  
   
   
       36 . The method of  claim 35 , wherein the step preceding step (a) comprises allylating hydroxyl groups present on the surface of a porous support.  
   
   
       37 . The method of  claim 35 , wherein steps (a)-(c) are replaced by a single step of reacting an activated ligand with the activatable groups present on the surface of the support.  
   
   
       38 . The method of  claim 32 , wherein the activation agent used in step (b) is a halogen.  
   
   
       39 . The method of  claim 32 , wherein at least one chemical gradient is a ligand density gradient.  
   
   
       40 . The method of  claim 32 , wherein at least one chemical gradient is a continuous and smooth gradient.  
   
   
       41 . The method of  claim 32 , wherein least two different functionalities are provided in step (c).  
   
   
       42 . The method of  claim 41 , wherein said at least two different functionalities are provided by one compound.  
   
   
       43 . The matrix of  claim 41 , wherein said at least two different functionalities are provided by different compounds.  
   
   
       44 . The method of  claim 32 , wherein two or more chemical gradients are provided in the support, one of which is a ligand density gradient.  
   
   
       45 . The method of  claim 32 , wherein the support comprises porous particles and at least one gradient extends between the center and the exterior surface of each porous particle.  
   
   
       46 . A separation matrix prepared by the method of  claim 20 .  
   
   
       47 . A process of liquid chromatography, comprising contacting a liquid including at least one target molecule with a separation matrix, said separation matrix comprises ligands coupled to the surfaces of a porous support; and adsorbing the target molecule(s) to the matrix, wherein the ligands provide at least one chemical gradient within the support.  
   
   
       48 . The process of  claim 47 , wherein the liquid is applied in a flow direction which is perpendicular to at least one chemical gradient within the support.  
   
   
       49 . The process of  claim 47 , further comprising a step of eluting the adsorbed target molecule from the matrix by contacting the matrix with an eluent.  
   
   
       50 . The process of  claim 47 , wherein at least one chemical gradient is a ligand density gradient.  
   
   
       51 . The process of  claim 47 , wherein the support comprises essentially spherical porous particles and at least one chemical gradient extends between the center and the exterior surface of each porous particle.  
   
   
       52 . The process of  claim 49 , wherein the functional groups of the ligands are zwitterions and the elution is performed at a pH different from that during the adsorption.  
   
   
       53 . (canceled)  
   
   
       54 . The method of  claim 45 , wherein the porous particles are spherical particles.  
   
   
       55 . A separation matrix prepared by the method of  claim 32.