NOVEL AFFINITY CHROMATOGRAPHY MEDIA FOR REMOVAL OF ANTI-A AND/OR ANTI-B ANTIBODIES
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.
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.