Antibody purification via affinity chromatography

Abstract

Embodiments herein provide methods of purifying monoclonal and polyclonal anti-bodies (e.g., immunoglobulins) from biological fluids, such as cell lysates, cell supernatant and ascites fluids, using small molecule affinity chromatography. Various embodiments disclose a class of small molecules that selectively bind a nucleotide binding site that is inherent to all immunoglobulins, and in various embodiments, methods are disclosed that use one of these small molecules as a capture molecule in small molecule affinity chromatography. In some embodiments, the small molecule may be an indole, and in particular embodiments, the small molecule may be indole-3-butyric acid.

What is claimed is: 
     
         1 . A method of binding an immunoglobulin, comprising:
 providing a small molecule comprising an aromatic ring, a fused aromatic ring or an indole, that specifically binds a nucleotide binding site on an immunoglobulin;   contacting a composition containing an immunoglobulin with the small molecule under conditions suitable for binding of the immunoglobulin to the small molecule; and   separating the small molecule from the composition, wherein the immunoglobulin remains bound to the small molecule.   
     
     
         2 . The method of  claim 1 , wherein the small molecule has a monovalent Kd of less than about 150 μM for the nucleotide binding site. 
     
     
         3 . The method of  claim 1 , wherein the immunoglobulin comprises a light chain and a heavy chain, and wherein the nucleotide binding site comprises:
 a first amino acid in an amino acid sequence of the light chain, wherein the first amino acid is a phenylalanine or a tyrosine;   a second amino acid in the amino acid sequence of the light chain, wherein the second amino acid is a tyrosine;   a third amino acid in an amino acid sequence of the heavy chain, wherein the third amino acid is a tyrosine; and   a fourth amino acid in the amino acid sequence of the heavy chain, wherein the fourth amino acid is a tryptophan.   
     
     
         4 . The method of  claim 3 , wherein:
 the first amino acid corresponds to phenylalanine 35 of SEQ. ID NO: 1 or tyrosine 36 of SEQ. ID. NO: 2;   the second amino acid corresponds to tyrosine 86 of SEQ. ID NO: 1 or tyrosine 87 of SEQ. ID. NO: 2;   the third amino acid corresponds to tyrosine 95 of SEQ. ID NO: 3 or tyrosine 95 of SEQ. ID. NO: 4; and   the fourth amino acid corresponds to tryptophan 111 of SEQ. ID NO: 3 or tryptophan 110 of SEQ. ID. NO: 4.   
     
     
         5 . The method of  claim 3 , wherein the small molecule has a monovalent Kd of less than about 150 μM for a nucleotide binding site defined as:
 A. phenylalanine 35 of SEQ. ID NO: 1;
 tyrosine 86 of SEQ. ID NO: 1; 
 tyrosine 95 of SEQ. ID NO: 3; and 
 tryptophan 111 of SEQ. ID NO: 3; or 
 
 B. tyrosine 36 of SEQ. ID. NO: 2;
 tyrosine 87 of SEQ. ID. NO: 2; 
 tyrosine 95 of SEQ. ID. NO: 4; and 
 tryptophan 110 of SEQ. ID. NO: 4. 
 
 
     
     
         6 . The method of  claim 1 , wherein the small molecule comprises an indole. 
     
     
         7 . The method of  claim 6 , wherein the indole is indole-3-butyric acid. 
     
     
         8 . The method of  claim 1 , wherein the small molecule is coupled to a solid support, and wherein separating the small molecule from the composition comprises separating the solid support from the composition. 
     
     
         9 . The method of  claim 1 , further comprising causing the small molecule to release the immunoglobulin. 
     
     
         10 . The method of  claim 9 , wherein the solid support comprises a resin, a bead, a particle, a membrane, a capillary, a microarray, a surface, or a multiple well plate. 
     
     
         11 . The method of  claim 10 , wherein the solid support comprises a resin, and wherein the resin is packed in a chromatography column. 
     
     
         12 . The method of  claim 9 , wherein the solid support comprises a resin, and wherein the resin is utilized as a slurry. 
     
     
         13 . The method of  claim 11 , wherein the small molecule is coupled to the resin via an amide bond. 
     
     
         14 . The method of  claim 8 , wherein contacting the composition containing an immunoglobulin with the small molecule comprises contacting the solid support with a mobile phase comprising the composition. 
     
     
         15 . The method of  claim 11 , wherein contacting the composition containing an immunoglobulin with the small molecule comprises applying a mobile phase comprising the composition to the chromatography column. 
     
     
         16 . The method of  claim 8 , wherein separating the small molecule from the composition comprises washing the solid support. 
     
     
         17 . The method of  claim 15 , wherein separating the small molecule from the composition comprises applying an elution buffer to the chromatography column. 
     
     
         18 . The method of  claim 1 , wherein the composition comprises ascites fluid, cell supernatant, or cell lysate. 
     
     
         19 . The method of  claim 1  wherein the immunoglobulin is a monoclonal antibody, a polyclonal antibody, or a mixture thereof. 
     
     
         20 . A method of purifying an antibody, comprising:
 providing a small molecule comprising an aromatic ring, a fused aromatic ring or an indole that specifically binds a nucleotide binding site on an immunoglobulin, wherein the small molecule is coupled to a resin;   contacting the small molecule with a mobile phase comprising the antibody under conditions suitable for binding of the antibody to the small molecule;   washing the resin to remove the mobile phase, wherein the antibody remains bound to the small molecule; and   applying an elution buffer to the resin, thereby eluting the purified antibody from the resin.   
     
     
         21 . The method of  claim 20 , wherein the nucleotide binding site comprises a first amino acid, a second amino acid, a third amino acid, and a fourth amino acid, wherein the first amino acid comprises a tyrosine or a phenylalanine or a tyrosine on an immunoglobulin light chain, wherein the second amino acid comprises a tyrosine on an immunoglobulin light chain, wherein he third amino acid comprises a tyrosine on an immunoglobulin heavy chain, and wherein the fourth amino acid comprises a tryptophan on an immunoglobulin heavy chain. 
     
     
         22 . The method of  claim 20 , wherein the small molecule comprises an indole. 
     
     
         23 . The method of  claim 22 , wherein the indole is indole-3-butyric acid. 
     
     
         24 . A method of identifying a specific binding partner for an immunoglobulin, comprising determining whether a candidate molecule will specifically bind to a nucleotide binding site on the immunoglobulin, wherein the immunoglobulin comprises a light chain and a heavy chain, and wherein the nucleotide binding site comprises:
 a first amino acid in an amino acid sequence of the light chain, wherein the first amino acid is a phenylalanine or a tyrosine;   a second amino acid in the amino acid sequence of the light chain, wherein the second amino acid is a tyrosine;   a third amino acid in an amino acid sequence of the heavy chain, wherein the third amino acid is a tyrosine; and   a fourth amino acid in the amino acid sequence of the heavy chain, wherein the fourth amino acid is a tryptophan.   
     
     
         25 . The method of  claim 24 , wherein:
 the first amino acid corresponds to phenylalanine 35 of SEQ. ID NO: 1 or tyrosine 36 of SEQ. ID. NO: 2;   the second amino acid corresponds to tyrosine 86 of SEQ. ID NO: 1 or tyrosine 87 of SEQ. ID. NO: 2;   the third amino acid corresponds to tyrosine 95 of SEQ. ID NO: 3 or tyrosine 95 of SEQ. ID. NO: 4; and   the fourth amino acid corresponds to tryptophan 111 of SEQ. ID NO: 3 or tryptophan 110 of SEQ. ID. NO: 4.   
     
     
         26 . The method of  claim 24 , wherein specific binding comprises having a Kd of less than about 150 μM for the nucleotide binding site.