Purification of immunoglobulins using affinity chromatography and peptide ligands

Abstract

An immunoglobulin binding peptide having the general formula, from amino terminus to carboxy terminus, of Z-R 1 —R 2 —R 3 —R 4 —R 5 —R 6 —X, is described, wherein: R 1 is H or Y; R 2 is a hydrophobic, preferentially aromatic, amino acid (for example W, F, Y, V); R 3 is a positively charged or aromatic amino acid (for example R, H, F, W); R 4 is a hydrophobic or positively charged amino acid (for example G, Y, R, K, L); R 5 is a positively charged or aromatic amino acid (for example W, F, R, H, Y); R 6 a random amino acid but preferably hydrophobic or negatively charged (for example V, W, L, D, H); X is present or absent and when present is a linking group; and Z is present or absent and when present is a capping group bonded to the N terminus of R 1 ; and wherein the amino acids of said peptide are in D form, L form, or a combination thereof. Methods of using such peptides for the purification of Immunoglobulins are also described

1 . An immunoglobulin binding peptide having the general formula, from amino terminus to carboxy terminus, of
   Z-R 1 —R 2 —R 3 —R 4 —R 5 —R 6 —X   
       wherein:
 R 1  is H or Y; 
 R 2  is W, F, Y or V; 
 R 3  is R, H, F, or W; 
 R 4  is G, Y, R, K or L; 
 R 5  is W, F, R, H or Y; 
 R 6  is V, W, L, D or H; 
 X is present or absent and when present is a linking group; and 
 Z is present or absent and when present is a capping group bonded to the N terminus of R 1 ; 
 and wherein the amino acids of said peptide are in D form, L form, or a combination thereof. 
 
     
     
         2 . The peptide of  claim 1  selected from the group consisting of:
 (a) the peptide HWRGWV (SEQ ID NO: 4),   (b) the peptide HYFKFD (SEQ ID NO: 11),   (c) the peptide HFRRHL (SEQ ID NO: 21),   (d) the peptide HVHYYW (SEQ ID NO: 12),   (e) the peptide YYWLHH (SEQ ID NO: 8),   (f) peptides of (a), (b), (c), (d) or (e) having a linking group bonded to the C terminus thereof; and   (g) peptides of (a), (b), (c), (d), (e) or (f) having a capping group bonded to the N terminus thereof.   
     
     
         3 . The peptide of  claim 1 , wherein said capping group is selected from the group consisting of: R 10 CO—, R 10 —O—CO—, R 10 —PO—, R 10 —SO 2 —, and arylalkyl-;
 wherein R 10  is selected from the group consisting of H, alkyl, alkenyl, alkynyl, aryl, and arylalkyl.   
     
     
         4 . The peptide of  claim 1 , wherein said capping group is selected from the group consisting of: acetyl, benzoyl, formyl, trifluoroacetyl, benzyloxycarbonyl, tert-butyloxycarbonyl, biphenylylisopropyloxycarbonyl, triphenylmethyl, o-nitrobenzenesulfenyl, and diphenylphosphinyl. 
     
     
         5 . The peptide of  claim 1 , wherein said linking group is selected from the group consisting of monomers and polymers. 
     
     
         6 . The peptide of  claim 1 , wherein said linking group is a chain of from 1 to 10 amino acids. 
     
     
         7 . The peptide of  claim 1 , wherein said linking group is selected from the group consisting of polyethylene glycol, polypropylene glycol, polyesters, polypeptides, polyethers, and polysaccharides. 
     
     
         8 . The peptide of  claim 1  coupled to a detectable group. 
     
     
         9 . The peptide of  claim 8 , wherein said detectable group is selected from the group consisting of fluorescent groups, radioactive groups, and enzymatic groups. 
     
     
         10 . A solid support having an immunoglobulin binding peptide according to  claim 1  immobilized thereon. 
     
     
         11 . The solid support of  claim 10 , wherein said solid support is a bead, particle, membrane, semi-permeable membrane, capillary, microarray or multiple well plate. 
     
     
         12 . The solid support of  claim 10 , wherein said solid support comprises inorganic material, organic material or combinations thereof. 
     
     
         13 . The solid support of  claim 10 , wherein said solid support comprises an inorganic material. 
     
     
         14 . The solid support of  claim 10 , wherein said solid support comprises an inorganic material selected from the group consisting of glass, alumina, silica, silicon, zirconia, magnetite, semiconductors, and combinations thereof. 
     
     
         15 . The solid support of  claim 10 , wherein said solid support comprises an organic material 
     
     
         16 . The solid support of  claim 10 , wherein said solid support comprises an organic material selected from the group consisting of polysaccharides including agarose, dextran, cellulose, chitosan, polyacrylamide, polyacrylate, polystyrene, polyvinyl alcohol, and combinations thereof. 
     
     
         17 . The solid support of  claim 10 , wherein said solid support is a polymer of an acrylate. 
     
     
         18 . The solid support of  claim 17 , wherein said polymer is a methacrylate polymer. 
     
     
         19 . The solid support of  claim 17 , wherein the methacrylate polymer is hydroxylated polymethacrylate amino resin. 
     
     
         20 . A method of binding an immunoglobulin, comprising:
 (a) providing a binding peptide according to  claim 1 ;   (b) contacting a composition containing an immunoglobulin to said binding peptide; and then   (c) separating said binding peptide from said composition, with said immunoglobulin bound to said binding peptide.   
     
     
         21 . The method of  claim 20 , wherein said peptide is coupled to a solid support, and wherein said separating step is carried out by separating said solid support from said composition. 
     
     
         22 . The method of  claim 21 , further comprising the step of separating said immunoglobulin from said solid support. 
     
     
         23 . The method of  claim 20 , wherein said solid support is a bead, particle, membrane, semi-permeable membranes, capillary, microarray or multiple well plate. 
     
     
         24 . The method of  claim 20 , wherein said contacting step (b) is carried out in solution. 
     
     
         25 . The method of  claim 24 , wherein said separating step (c) is carried out by fluorescence-activated cell sorting, membrane filtration, or mass spectrometry. 
     
     
         26 . The method of  claim 20 , wherein said composition comprises blood or blood plasma. 
     
     
         27 . The method of  claim 20 , wherein said composition comprises plasma fractions. 
     
     
         28 . The method of  claim 20 , wherein said composition comprises ascites fluid, milk or colostrum. 
     
     
         29 . The method of  claim 20 , wherein said composition comprises an aqueous cell culture containing said immunoglobulin. 
     
     
         30 . The method of  claim 20  wherein said immunoglobulin is selected from the group consisting of monoclonal antibodies and polyclonal antibodies. 
     
     
         31 . The method of  claim 20 , wherein said immunoglobulin is mammalian immunoglobulin. 
     
     
         32 . The method of  claim 20 , wherein said immunoglobulin is selected from the group consisting of human, goat, bovine, mouse, rat, rabbit, monkey, horse, and sheep immunoglobulin. 
     
     
         33 . The method of  claim 20 , wherein said immunoglobulin is selected from the group consisting of IgG, IgM, IgA, IgD and IgE. 
     
     
         34 . The method of  claim 20 , wherein said immunoglobulin is a human IgG. 
     
     
         35 . The method of  claim 20 , wherein said immunoglobulin is a humanized IgG. 
     
     
         36 . A method of identifying small peptide ligands with high affinity for immunoglobulins of interest, said method comprising the steps of:
 (a) screening a library of peptides, wherein the peptides are attached to a solid support, and wherein the peptides consist essentially of from 5 to 7 amino acid residues;   (b) contacting a labeled immunoglobulin or labeled fragment thereof to said peptides; and then   (c) determining the identity of the peptides bound to the said labeled immunoglobulin or fragment thereof.   
     
     
         37 . The method of  claim 36 , wherein said determining step is carried out by sequencing. 
     
     
         38 . The method of  claim 36 , wherein said label is selected from the group consisting of enzyme labels, fluorescent labels, and radiolabels. 
     
     
         39 . The method of  claim 36 , wherein said immunoglobulin is an IgG. 
     
     
         40 . The method of  claim 36 , wherein said immunoglobulin is a human IgG. 
     
     
         41 . The method of  claim 36 , wherein said immunoglobulin is a humanized IgG. 
     
     
         42 . The method of  claim 36 , wherein said immunoglobulin is an IgG Fc fragment. 
     
     
         43 . The method of  claim 36 , wherein said immunoglobulin is a human IgG Fc fragment. 
     
     
         44 . The method of  claim 36 , wherein said immunoglobulin is a human IgM, IgA, IgD or IgE Fc fragment. 
     
     
         45 . The method of  claim 36 , wherein said immunoglobulin is IgG. 
     
     
         46 . The method of  claim 45 , wherein said IgG is human IgG. 
     
     
         47 . The method of  claim 36 , wherein said immunoglobulin is IgM, IgA, IgD or IgE.