Composite chromatographic sorbent of mineral oxide beads with hydroxyapatite-filled pores

Abstract

A new adsorbent of a porous mineral oxide material with apatite crystals, preferably hydroxyapatite crystals, in the pores of the mineral oxide material is disclosed. The adsorbent is useful for protein and nucleic acid separations

1 - 12 . (canceled)  
   
   
       13 . A chromatography column, comprising: 
 (a) a tubular member having an inlet end and an outlet end;    (b) first and second porous members disposed within said tubular member; and    (c) a composite chromatography sorbent comprising: 
 porous mineral oxide beads that have a pore volume which exceeds about 10% of the bead volume and an average pore diameter of at least about 500 Å, wherein the pores of the beads contain apatite crystals which have been formed within the pores of the beads by solutions that are allowed to penetrate the pores, and wherein said composite chromatography sorbent is packed within said tubular member between said first and second porous members.  
   
   
   
       14 . The chromatography column of  claim 13 , wherein the column volume is between about 50 liters and about 5000 liters.  
   
   
       15 . The chromatography column of  claim 13 , further comprising: 
 means for flowing a liquid sample upward through said composite chromatography sorbent.    
   
   
       16 . The chromatography column of  claim 15 , further comprising: 
 a series of stages between said inlet end and said outlet end.    
   
   
       17 . A chromatographic separation method comprising: 
 contacting a solution comprising biomolecules with a composite chromatography sorbent,    wherein said composite chromatography sorbent comprises porous mineral oxide beads that have a pore volume which exceeds about 10% of the bead volume and an average pore diameter of at least about 500 Å, wherein the pores of the beads contain apatite crystals, wherein the solution permeates the pores of the mineral oxide beads, and wherein some biomolecules in the solution are bound to the apatite crystals and other, different biomolecules remain in the solution.    
   
   
       18 . A chromatographic separation method comprising: 
 a first flowing solution comprising biomolecules through a chromatography column comprising: 
 (a) a tubular member having an inlet end and an outlet end;  
 (b) first and second porous members disposed within said tubular member; and  
 (c) a composite chromatography sorbent comprising porous mineral oxide beads that have a pore volume which exceeds about 10% of the bead volume and an average pore diameter of at least about 500 Å, wherein the pores of the beads contain apatite crystals, and wherein said composite chromatography is packed within said tubular member between said first and second porous members, wherein the solution permeates the pores of the mineral oxide beads, and wherein some biomolecules in the solution are bound to the apatite crystals and other, different biomolecules remain in the solution.  
   
   
   
       19 . The method of  claim 18 , further comprising: 
 a second flowing solution through the chromatography column to elute the biomolecules bound to the apatite crystals.    
   
   
       20 . The method of  claim 18 , wherein the biomolecules are selected from the group consisting of polypeptides, nucleic acids, antibodies, and glyco-iso-forms.  
   
   
       21 . The method of  claim 18 , wherein the composite chromatography sorbent is formed by a method comprising: 
 (i) providing porous mineral oxide beads that have a pore volume which exceeds about 10% of the bead volume and an average pore diameter of at least about 500 Å;    (ii) contacting the porous mineral oxide beads with a maximum of one pore volume of a solution of either (A) calcium chloride or (B) potassium or sodium phosphate so that it permeates the pores of the beads;    (iii) drying the beads from (ii);    (iv) contacting the dried beads with a maximum of one pore volume of a solution of the other of either (A) calcium chloride or (B) potassium or sodium phosphate so that it permeates the pores of the beads, thereby forming calcium phosphate in the pores;    (v) washing the beads from (iv) with water to eliminate excess calcium or phosphate ions;    (vi) contacting the washed beads from (v) with a solution of sodium hydroxide;    (vii) washing the beads from (vi) with water; and    (viii) contacting the washed beads from (vii) with a solution of disodium phosphate to form hydroxyapatite crystals in the pores of the beads.    
   
   
       22 . The method according to  claim 21 , wherein the beads are washed with a phosphoric acid solution before (ii).  
   
   
       23 . A chromatographic separation method comprising: 
 a first flowing solution comprising biomolecules through a chromatography column comprising: 
 (a) a tubular member having an inlet end and an outlet end;  
 (b) first and second porous members disposed within said tubular member;  
 (c) a composite chromatography sorbent comprising porous mineral oxide beads that have a pore volume which exceeds about 10% of the bead volume and an average pore diameter of at least about 500 Å, wherein the pores of the beads contain apatite crystals, and wherein said composite chromatography is packed within said tubular member between said first and second porous members; and  
 (d) means for flowing a liquid sample upward through said composite chromatography sorbent,  
 wherein the solution permeates the pores of the mineral oxide beads, and wherein some biomolecules in the solution are bound to the apatite crystals and other, different biomolecules remain in the solution.  
   
   
   
       24 . The method of  claim 23 , further comprising: 
 a second flowing solution through the chromatography column to elute the biomolecules bound to the apatite crystals.    
   
   
       25 . The method of  claim 23 , wherein the biomolecules are selected from the group consisting of polypeptides, nucleic acids, antibodies, and glyco-iso-forms.  
   
   
       26 . The method of  claim 23 , wherein the composite chromatography sorbent is formed by a method comprising: 
 (i) providing porous mineral oxide beads that have a pore volume which exceeds about 10% of the bead volume and an average pore diameter of at least about 500 Å;    (ii) contacting the porous mineral oxide beads with a maximum of one pore volume of a solution of either (A) calcium chloride or (B) potassium or sodium phosphate so that it permeates the pores of the beads;    (iii) drying the beads from (ii);    (iv) contacting the dried beads with a maximum of one pore volume of a solution of the other of either (A) calcium chloride or (B) potassium or sodium phosphate so that it permeates the pores of the beads, thereby forming calcium phosphate in the pores;    (v) washing the beads from (iv) with water to eliminate excess calcium or phosphate ions;    (vi) contacting the washed beads from (v) with a solution of sodium hydroxide;    (vii) washing the beads from (vi) with water; and    (viii) contacting the washed beads from (vii) with a solution of disodium phosphate to form hydroxyapatite crystals in the pores of the beads.    
   
   
       27 . The method according to  claim 26 , wherein the beads are washed with a phosphoric acid solution before (ii).  
   
   
       28 - 29 . (canceled)  
   
   
       30 . The chromatography column according to  claim 13 , wherein the apatite crystals are hydroxyapatite crystals.  
   
   
       31 . The chromatography column according to  claim 13 , wherein the mineral oxide is zirconia.  
   
   
       32 . The chromatography column according to  claim 13 , wherein the apatite crystals are hydroxyapatite crystals and the mineral oxide is zirconia.  
   
   
       33 . The separation method of  claim 17 , wherein the apatite crystals are hydroxyapatite crystals.  
   
   
       34 . The separation method of  claim 17 , wherein mineral oxide is zirconia.  
   
   
       35 . The separation method of  claim 17 , wherein the apatite crystals are hydroxyapatite crystals and the mineral oxide is zirconia.  
   
   
       36 . The method of  claim 18 , wherein the apatite crystals are hydroxyapatite crystals.  
   
   
       37 . The method of  claim 18 , wherein mineral oxide is zirconia.  
   
   
       38 . The method of  claim 18 , wherein the apatite crystals are hydroxyapatite crystals and the mineral oxide is zirconia.  
   
   
       39 . The method of  claim 23 , wherein the apatite crystals are hydroxyapatite crystals.  
   
   
       40 . The method of  claim 23 , wherein the mineral oxide is zirconia.  
   
   
       41 . The method of  claim 23 , wherein the apatite crystals are hydroxyapatite crystals and the mineral oxide is zirconia.  
   
   
       42 . The chromatography column of  claim 13 , wherein the mineral oxide is selected from the group consisting of alumina, titania, hafnia, silica, zirconia and mixtures thereof.  
   
   
       43 . The chromatography column of  claim 13 , wherein the mineral oxide comprises zirconia.  
   
   
       44 . The chromatography column of  claim 13 , wherein the mineral oxide comprises silica.  
   
   
       45 . The chromatography column of  claim 13 , wherein the beads are coated with a layer of a hydrophilic polymer.  
   
   
       46 . The chromatography column of  claim 13 , wherein the apatite crystals comprise: 
 (a) calcium ions; and    (b) a metal ion or a metalloid ion.