Ligands for antibody and fc-fusion protein purification by affinity chromotography iv

Abstract

The present invention relates to the use, for affinity purification of an antibody or an fragment of an antibody, of a ligand-substituted matrix comprising a support material and at least one ligand covalently bonded to the support material, the ligand being represented by formula (I) L-(Sp) v -Ar 1 -Am-Ar 2   (I) wherein L, SP, Ar 1 , AM, Ar 2 and v are defined herein.

1 .- 17 . (canceled) 
     
     
         18 . A method of affinity purification of an antibody or a fragment of an antibody, of comprising:
 contacting the antibody or the fragment of the antibody with a ligand-substituted matrix according to  claim 23 .   
     
     
         19 . The ligand-substituted matrix according to  claim 23 , wherein
 Ar 1  is selected from benzimidazole, 2,3-dihydrobenzimidazole, pyridine and 4-triazolylbenzene;   wherein   Ar 1  is connected to Sp or L via a chemical bond attached to the 1- or 2-position of benzimidazole, the 1-position of 2,3-dihydrobenzimidazole, the 2-position of pyridine or to the 1-position of the triazole part of 4-triazolylbenzene;   wherein   Ar 1  is connected to Am via a chemical bond attached to the 5- or 6-position of benzimidazole, the 5- or 6-position of 2,3-dihydrobenzimidazole, to the 5-position of pyridine or to the 4-position of the benzene part of 4-triazolylbenzene; and   wherein   Ar 1  is either not further substituted or attached to at least one substituent selected from: methyl, ethyl, methoxy=S and=O; and combinations thereof;   Ar 2  is a 5-membered mononuclear aromatic ring selected from 1,2,4-thiadiazole, 1,3,4-thiadiazole, and thiazole; which is unsubstituted, or via a chemical bond attached to at least one substituent selected from: methyl, ethyl, methoxy, ethylthio, and cyclopropyl; a 5- or 6-membered mononuclear aromatic ring selected from pyridine, furan, isoxazole, oxazole, isothiazole, thiazole; and combinations thereof.   
     
     
         20 . The ligand-substituted matrix according to  claim 23 ,
 wherein the support material comprises a material selected from carbohydrates or crosslinked carbohydrates, preferably agarose, cellulose, dextran, starch, alginate and carrageenan, Sepharose, Sephadex; synthetic polymers, preferably polystyrene, styrene-divinylbenzene copolymers, polyacrylates, PEG-Polycacrylate copolymers polymethacrylates, polyvinyl alcohol, polyamides and perfluorocarbons; inorganic materials, preferably glass, silica and metal oxides; and composite materials.   
     
     
         21 . The method according to  claim 18 , wherein the antibody is an IgG type antibody, or an Fc fusion protein. 
     
     
         22 . The method according to  claim 18 , wherein the antibody fragment is a Fc fragment or domain or the antibody belongs to the IgG antibody class, more preferably to human, IgG or to polyclonal or monoclonal IgG of human origin, in particular to IgG 1 , IgG 2  IgG 3  and IgG 4 . 
     
     
         23 . A ligand-substituted matrix, comprising:
 a support material and   at least one ligand covalently bonded to the support material, and binding to the Fc part of the antibody or the antibody fragment, the ligand being represented by formula (I)
   L-(Sp) v —AR 1 —Am—Ar 2    (I)
 
   wherein   L is the linking point on the support material to which the ligand is attached;   Sp is a spacer group;   v is 0 or 1;   Am is an amide group —NR 1 —C(O)—, and wherein either NR 1  is attached to Ar 1  and —C(O)— is attached to Ar 2 , or —C(O)— is attached to Ar 1  and NR 1  is attached to Ar 2 ; and
 R 1  is hydrogen or methyl; and most preferably hydrogen 
   Ar 1  is a 5- or 6-membered mononuclear aromatic ring or partially saturated aromatic ring selected from: imidazole, benzene, pyridine, 2,3-dihydro-1H-imidazole, and triazole, which is connected to Sp or L via a chemical bond and which is optionally furthermore   (a) attached to benzene via a chemical bond; or   (b) fused to benzene as part of a multinuclear ring system   wherein   Ar 1  is directly connected to Am via a chemical bond present on the 5- or 6-membered aromatic ring or partially saturated aromatic ring constituting Ar 1 , or indirectly via a chemical bond which is either present at the benzene ring attached to Ar 1 , or on the benzene ring fused to Ar 1 ; and   wherein   Ar 1  is either not further substituted or attached to at least one substituent selected from: methyl, ethyl, propyl, methoxy, ethoxy, difluoromethyl, trifluoromethyl, trifluoromethoxy, difluoromethoxy; cyclopropyl, hydroxymethyl, —NO 2 , ═O, ═S; and combinations thereof;   Ar 2  is a 5-membered mononuclear aromatic ring containing at least one atom selected from N, S, O, preferably from N, S, which is unsubstituted, or via a chemical bond attached to at least one substituent selected from: methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, difluoromethyl, trifluoromethyl, trifluoromethoxy, difluoromethoxy, cyclopropyl, hydroxymethyl, t-butyl, butyl, sec-butyl, fluro, chloro, carbamoyl, ethylthio, methylthio, preferably methyl, ethyl, methoxy, ethylthio, and cyclopropyl; a 5- or 6-membered mononuclear aromatic ring; and combinations thereof; and wherein Ar 2  optionally, further to the substituents to which it may be attached via a chemical bond as cited above, is fused to benzene or thiazole as part of a multinuclear ring system.   
     
     
         24 . A method of synthesis of a ligand-substituted matrix according to  claim 23  wherein a ligand according to formula (I)
   L-(Sp) v —Ar 1 —Am—Ar 2    (I)
 
 is attached to the support material. 
 
     
     
         25 . A method for affinity purification, preferably affinity chromatography, of a protein, comprising:
 contacting a protein to be purified with a ligand-substituted matrix according to  claim 23 .   
     
     
         26 . The method of  claim 25  wherein the protein is an antibody or an Fc fusion protein. 
     
     
         27 . A ligand as shown in formula (II), binding to the Fc part of an antibody or a fragment of an antibody,
   (SpP) v —Ar 1 —Am—Ar 2    (II)
   wherein   SpP is a spacer group;   v is 0 or 1;   Am is an amide group —NR 1 —C(O)—, and wherein either NR 1  is attached to Ar 1  and —C(O)— is attached to Ar 2 , or —C(O)— is attached to Ar 1  and NR 1  is attached to Ar 2 ; and
 R 1  is hydrogen or methyl; and most preferably hydrogen, 
   Ar 1  is a 5- or 6-membered mononuclear aromatic ring or partially saturated aromatic ring selected from: imidazole, benzene, pyridine, 2,3-dihydro-1H-imidazole, and triazole, which is connected to SpP via a chemical bond and which is optionally furthermore   (a) attached to benzene via a chemical bond; or   (b) fused to benzene as part of a multinuclear ring system   wherein   Ar 1  is directly connected to Am via a chemical bond present on the 5- or 6-membered aromatic ring or partially saturated aromatic ring constituting Ar 1 , or indirectly via a chemical bond which is either present at the benzene ring attached to Ar 1 , or on the benzene ring fused to Ar 1 ; and   wherein   Ar 1  is either not further substituted or attached to at least one substituent selected from: methyl, ethyl, propyl, methoxy, ethoxy, difluoromethyl, trifluoromethyl, trifluoromethoxy, difluoromethoxy; cyclopropyl, hydroxymethyl, —NO 2 , ═O, ═S; and combinations thereof;   Ar 2  is a 5-membered mononuclear aromatic ring containing at least one atom selected from N, S, O, preferably from N, S, which is unsubstituted, or via a chemical bond attached to at least one substituent selected from: methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, difluoromethyl, trifluoromethyl, trifluoromethoxy, difluoromethoxy, cyclopropyl, hydroxymethyl, t-butyl, i-butyl, sec-butyl, fluro, chloro, carbamoyl, ethylthio, methylthio, preferably methyl, ethyl, methoxy, ethylthio, and cyclopropyl; a 5- or 6-membered mononuclear aromatic ring; and combinations thereof; and wherein Ar 2  optionally, further to the substituents to which it may be attached via a chemical bond as cited above, is fused to benzene or thiazole as part of a multinuclear ring system.   
     
     
         28 . A ligand having the structural element depicted in formula (III), binding to the Fc part of an antibody or a fragment of an antibody,
   Ar 1 —Am—Ar 2    (III)
   wherein   Am is an amide group —NR 1 —C(O)—, and wherein either NR 1  is attached to Ar 1  and —C(O)— is attached to Ar 2 , or —C(O)— is attached to Ar 1  and NR 1  is attached to Ar 2 ; and
 R 1  is hydrogen or methyl; and most preferably hydrogen 
   Ar 1  is a 5- or 6-membered mononuclear aromatic ring or partially saturated aromatic ring selected from: imidazole, benzene, pyridine, 2,3-dihydro-1H-imidazole, and triazole, which is optionally furthermore   (a) attached to benzene via a chemical bond; or   (b) fused to benzene as part of a multinuclear ring system   wherein   Ar 1  is directly connected to Am via a chemical bond present on the 5- or 6-membered aromatic ring or partially saturated aromatic ring constituting Ar 1 , or indirectly via a chemical bond which is either present at the benzene ring attached to Ar 1 , or on the benzene ring fused to Ar 1 ; and   wherein   Ar 1  is either not further substituted or attached to at least one substituent selected from: methyl, ethyl, propyl, methoxy, ethoxy, difluoromethyl, trifluoromethyl, trifluoromethoxy, difluoromethoxy; cyclopropyl, hydroxymethyl, —NO 2 , ═O, ═S; and combinations thereof; Ar 2  is a 5-membered mononuclear aromatic ring containing at least one atom selected from N, S, O, preferably from N, S, which is unsubstituted, or via a chemical bond attached to at least one substituent selected from: methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, difluoromethyl, trifluoromethyl, trifluoromethoxy, difluoromethoxy, cyclopropyl, hydroxymethyl, t-butyl, i-butyl, sec-butyl, fluro, chloro, carbamoyl, ethylthio, methylthio, preferably methyl, ethyl, methoxy, ethylthio, and cyclopropyl; a 5- or 6-membered mononuclear aromatic ring; and combinations thereof; and wherein Ar 2  optionally, further to the substituents to which it may be attached via a chemical bond as cited above, is fused to benzene or thiazole as part of a multinuclear ring system.   
     
     
         29 . A method for affinity purification of an antibody or a fragment of an antibody, preferably after attaching the ligand to an appropriate matrix, comprising:
 contacting the antibody or the fragment of the antibody with the ligand according to  claim 27 .   
     
     
         30 . The ligand-substituted matrix according to  claim 23 , wherein the matrix furthermore comprises at least one antibody or a fragment of an antibody, bonded to the ligand via the Fc part. 
     
     
         31 . A method for affinity purification of an antibody or a fragment of an antibody, comprising: contacting the antibody or the fragment of the antibody with the ligand according to  claim 28 . 
     
     
         32 . The method according to  claim 31 , wherein the ligand is attached to an appropriate matrix prior to contacting the antibody or the fragment of the antibody.