Free solution ligand interaction molecular separation method

Abstract

The present invention is directed to novel methods for enhancing the ability to separate a species of interest from other different species present in a free solution mixture thereof which takes advantage of interactions that occur between soluble, small molecular weight ligands and the species of interest. The small molecular weight ligands employed in the present invention function to interact with a species of interest in a mixture of different species through either affinity, hydrophobic and/or ionic interactions, thereby altering the molecular weight, hydrodynamic volume and/or isoelectric point of the species of interest and rendering it separable from other component(s) in the mixture.

What is claimed is:  
     
         1 . A method for separating a first species of interest from a second species of interest in a free solution mixture comprising said first and second species of interest, said first and second species of interest being different, said method comprising: 
 contacting said free solution mixture with a soluble small molecular weight ligand that interacts with said first species of interest, wherein the molecular weight of said ligand is smaller than the molecular weight of said first species of interest and wherein said interaction forms a first species of interest/ligand complex which is capable of being separated from said second species of interest in free solution; and    separating said first species of interest/ligand complex from said second species of interest.    
     
     
         2 . The method according to  claim 1 , wherein said first species of interest is a biological entity selected from the group consisting of a protein, a polypeptide, an amino acid, a colloid, an endotoxin, a virus and a nucleic acid.  
     
     
         3 . The method according to  claim 1 , wherein said first species of interest is a protein.  
     
     
         4 . The method according to  claim 3 , wherein said protein is recombinantly produced.  
     
     
         5 . The method according to  claim 1 , wherein said ligand is selected from the group consisting of a protein, a peptide, an antibody and a nucleic acid.  
     
     
         6 . The method according to  claim 5 , wherein said ligand is Protein A.  
     
     
         7 . The method according to  claim 1 , wherein said ligand is a multi-ionic molecule.  
     
     
         8 . The method according to  claim 1 , wherein said ligand hydrophobically interacts with said first species of interest to form said first species of interest/ligand complex.  
     
     
         9 . The method according to  claim 1 , wherein said first and second species of interest have about the same hydrodynamic volume and said first species of interest/ligand complex and said second species of interest have different hydrodynamic volumes.  
     
     
         10 . The method according to  claim 1 , wherein said first species of interest/ligand complex and said second species of interest have about the same hydrodynamic volume.  
     
     
         11 . The method according to  claim 1 , wherein said first species of interest and said first species of interest/ligand complex have about the same isoelectric point.  
     
     
         12 . The method according to  claim 1 , wherein said first and second species of interest have about the same isoelectric point and said first species of interest/ligand complex and said second species of interest have different isoelectric points.  
     
     
         13 . The method according to  claim 1 , wherein said first species of interest/ligand complex and said second species of interest have about the same isoelectric point.  
     
     
         14 . The method according to  claim 1  further comprising the step of altering the pH or ionic strength of said free solution mixture, said step of altering resulting in an increase in the difference in the hydrodynamic volume between said first species of interest/ligand complex and said second species of interest.  
     
     
         15 . The method according to  claim 1 , wherein said step of separating is accomplished by size exclusion chromatography.  
     
     
         16 . The method according to  claim 15 , wherein said size exclusion chromatography is performed at an ionic strength of less than about 100 mM.  
     
     
         17 . The method according to  claim 1 , wherein said step of separating is accomplished by membrane filtration.  
     
     
         18 . The method according to  claim 17 , wherein said membrane filtration is tangential flow filtration.  
     
     
         19 . The method according to  claim 18 , wherein said tangential flow filtration is high performance tangential flow filtration.  
     
     
         20 . The method according to  claim 17 , wherein said membrane filtration is ultrafiltration, reverse osmosis or microfiltration.  
     
     
         21 . The method according to  claim 1  further comprising the step of dissociating said first species of interest/ligand complex into its individual components, wherein dissociating occurs after said step of separating.  
     
     
         22 . The method according to  claim 1 , wherein more than one ligand interacts with said first species of interest.  
     
     
         23 . The method according to  claim 1 , wherein said ligand does not interact with said second species of interest.  
     
     
         24 . The method according to  claim 1 , wherein said first species of interest is an impurity.