Novel affinity chromatography media for removal of anti-a and/or anti-b antibodies

Abstract

Embodiments described herein relate to novel chromatography media for removing anti-A and/or anti-B antibodies from a sample, as well as methods of using the same. The media described herein have several advantages over previously described media including, acid and alkaline stability.

What is claimed is: 
     
         1 . A media for removing anti-A antibodies from a sample, the media comprising a solid support with a blood group A antigen ligand attached thereto, wherein the ligand is attached to the solid support at a ligand loading of at least 0.8 mg/ml of solid support, and wherein the media is stable under acid and/or alkaline conditions. 
     
     
         2 . A media for removing anti-B antibodies from a sample, the media comprising a solid support with a blood group B antigen ligand attached thereto, wherein the ligand is attached to the solid support at a ligand loading of at least 0.8 mg/ml of solid support, and wherein the media is stable under acid and/or alkaline conditions. 
     
     
         3 . The media according to  claim 1 , wherein the ligand loading is at least 1 mg/ml of solid support or at least 1.5 mg/ml of solid support or at least 1.65 mg/ml of solid support. 
     
     
         4 . The media according to  claim 2 , wherein the ligand loading is at least 1 mg/ml of solid support or at least 1.2 mg/ml of solid support. 
     
     
         5 . A media for removing anti-A and anti-B antibodies from a sample, the media comprising: (a) a solid support with both blood group A antigen ligand and blood group B antigen ligand attached thereto, each at a ligand loading of at least 0.8 mg/ml of solid support; or (b) a mixture of two solid supports, one comprising blood group A antigen ligand thereto and another comprising blood group B antigen ligand, each media having ligand loading of at least 0.8 mg/ml of solid support, wherein the media are stable under acid and/or alkaline conditions. 
     
     
         6 . The media of  claims 1 , wherein the solid support comprises a polymer selected from the group consisting of polyvinylether, polyvinylalcohol, polymethacrylate, polyacrylate, polystyrene, polyacrylamide, polymethacrylamide and polycarbonate. 
     
     
         7 . The media of  claims 2 , wherein the solid support comprises a polymer selected from the group consisting of polyvinylether, polyvinylalcohol, polymethacrylate, polyacrylate, polystyrene, polyacrylamide, polymethacrylamide and polycarbonate. 
     
     
         8 . The media of  claim 1 , wherein the solid support is a polyvinyl ether based bead. 
     
     
         9 . The media of  claim 2 , wherein the solid support is a polyvinyl ether based bead. 
     
     
         10 . The media of  claim 1 , wherein the blood group A antigen ligand has the following structure: 
       
         
           
           
               
               
           
         
       
     
     
         11 . The media of  claim 2 , wherein the blood group B antigen ligand has the following structure: 
       
         
           
           
               
               
           
         
       
     
     
         12 . The media of  claims 1 , wherein the blood group A and/or B antigen ligands are attached via reductive amination chemistry or pAA tentacle chemistry to a solid support. 
     
     
         13 . The media of  claims 2 , wherein the blood group A and/or B antigen ligands are attached via reductive amination chemistry or pAA tentacle chemistry to a solid support. 
     
     
         14 . The media of  claims 1 , wherein the media is packed in a device. 
     
     
         15 . The media of  claims 2 , wherein the media is packed in a device. 
     
     
         16 . The media of  claim 14 , wherein the device is a chromatography column. 
     
     
         17 . The media of  claim 15 , wherein the device is a chromatography column. 
     
     
         18 . A method of removing anti-A antibodies from a sample, the method comprising the steps of:
 (a) providing a sample comprising a known amount of anti-A antibodies;   (b) incubating the sample with the media of  claim 1  for the media to bind anti-A antibodies;   (c) recovering portion of the sample which is not bound to the media; and   (d) measuring amount of anti-A antibodies in the portion of the sample in (c), wherein the amount of anti-A antibodies in (d) is at least 50% less than the amount of anti-A antibodies in the sample in (a).   
     
     
         19 . A method of removing anti-B antibodies from a sample, the method comprising the steps of:
 (a) providing a sample comprising a known amount of anti-B antibodies;   (b) incubating the sample with the media of  claim 2  for the media to bind blood group B antibodies;   (c) recovering portion of the sample which is not bound to the media; and   (d) measuring amount of anti-B antibodies in the portion of the sample in (c), wherein the amount of anti-B antibodies in (d) is at least 50% less than the amount of anti-B antibodies in the sample in (a).   
     
     
         20 . A method of sanitizing an affinity chromatography column containing a chromatography media suitable for removing anti-A antibodies or anti-B antibodies from a sample after use, while maintaining the ability of the media to remove anti-A antibodies or anti-B antibodies, respectively, from a sample, wherein the method comprises contacting the affinity chromatography column with a solution comprising phosphoric acid, acetic acid and benzyl alcohol for at least three hours. 
     
     
         21 . The method of  claim 20 , wherein maintaining the ability to remove anti-A antibodies or anti-B antibodies comprises the ability of media to remove at least 50% of anti-A or anti-B antibodies from a sample. 
     
     
         22 . A method of purifying a monoclonal anti-A IgM antibody from a clarified cell culture feed, the method comprising the steps of:
 (a) providing a clarified cell culture feed containing a monoclonal anti-A IgM antibody;   (b) incubating the feed with the media of  claim 1  to facilitate the binding of anti-A IgM antibody to the media;   (c) washing the media with an aqueous buffer having a pH ranging from 3.5 to 9.0;   (d) eluting the anti-A IgM antibody from the media using a buffer having a pH ranging from 2.0 to 3.0, thereby to obtain an eluate; and   (e) recovering the purified anti-A IgM antibody in the eluate.   
     
     
         23 . A method of purifying a monoclonal anti-B IgM antibody from a clarified cell culture feed, the method comprising the steps of:
 (a) providing a clarified cell culture feed containing a monoclonal anti-B IgM antibody;   (b) incubating the feed with the media of  claim 2  to facilitate the binding of anti-B IgM antibody to the media;   (c) washing the media with an aqueous buffer having a pH ranging from 3.5 to 9.0;   (d) eluting the anti-B IgM antibody from the media using a buffer having a pH ranging from 2.0 to 3.0, thereby to obtain an eluate; and   (e) recovering the purified anti-B IgM antibody in the eluate.   
     
     
         24 . The method of  claim 22 , wherein the media are packed in a chromatography column. 
     
     
         25 . The method of  claim 23 , wherein the media are packed in a chromatography column. 
     
     
         26 . The method of  claim 23 , wherein the clarified cell culture feed is flowed through the column.