Use of cation-exchange chromatography in the flow-through mode to enrich post-translational modifications

Abstract

The present invention relates to improved methods in the separation recombinant polypeptides with post-translational modifications from complex mixtures through the use of a cation exchange medium.

What is claimed is: 
     
         1 . A method for enriching the level of post-translational modification of recombinant polypeptides, comprising:
 contacting a composition which comprises an initial population of recombinant polypeptides having different levels of post-translational modification with a cation exchange chromatography (CEX) medium operated in a flow-through mode;   wherein recombinant polypeptides that do not bind the CEX medium are separated from recombinant polypeptides that bind the CEX medium, and wherein the recombinant polypeptides that do not bind the CEX medium comprise a higher level of post-translational modification compared to the bound recombinant polypeptides.   
     
     
         2 . A method for enriching the level of post-translational modification of recombinant polypeptides, comprising:
 a) contacting a composition which comprises an initial population of recombinant polypeptides having different levels of post-translational modification with a cation exchange chromatography (CEX) medium operated in a flow-through mode; and   b) separating recombinant polypeptides that do not bind the CEX medium from recombinant polypeptides that bind the CEX medium;
 wherein the recombinant polypeptides that do not bind the CEX medium that are recovered comprise a higher level of post-translational modification compared to the bound recombinant polypeptides. 
   
     
     
         3 . A method for enriching the level of post-translational modification of recombinant polypeptides, comprising:
 a) contacting a composition which comprises an initial population of recombinant polypeptides having different levels of post-translational modification with a cation exchange chromatography (CEX) medium operated in a flow-through mode;   b) separating recombinant polypeptides that do not bind the CEX medium from recombinant polypeptides that bind the CEX medium; and   c) recovering the recombinant polypeptides that do not bind the CEX medium;
 wherein the recombinant polypeptides that do not bind the CEX medium comprise a higher level of post-translational modification compared to the bound recombinant polypeptides. 
   
     
     
         4 . A method for enriching the level of post-translational modification of recombinant polypeptides, comprising:
 a) providing a composition which comprises an initial population of recombinant polypeptides having different levels of post-translational modification;   b) contacting the composition with a cation exchange chromatography (CEX) medium operated in a flow-through mode;   c) separating recombinant polypeptides that do not bind the CEX medium from recombinant polypeptides that bind the CEX medium; and   d) recovering the recombinant polypeptides that do not bind the CEX medium;
 wherein the recombinant polypeptides that do not bind the CEX medium comprise a higher level of post-translational modification compared to the bound recombinant polypeptides. 
   
     
     
         5 . The method of  claim 1  or  2 , further comprising recovering the recombinant polypeptides that do not bind the CEX medium. 
     
     
         6 . The method of any one of  claims 1 - 5 , wherein the post-translational modification is sialylation or gamma-carboxyglutamate (Gla) formation. 
     
     
         7 . The method of  claim 6 , wherein the post-translational modification is sialylation. 
     
     
         8 . The method of  claim 7 , wherein the total sialic acid content of the recombinant polypeptides that do not bind the CEX medium is between about 0.5 and about 6 moles of sialic acid per mole of protein higher than that of the initial population of recombinant polypeptides. 
     
     
         9 . The method of  claim 8 , wherein the total sialic acid content of the recombinant polypeptides that do not bind the CEX medium is between about 0.5 and about 4 moles of sialic acid per mole of protein higher than that of the initial population of recombinant polypeptides. 
     
     
         10 . The method of  claim 7 , wherein the total sialic acid content of the recombinant polypeptides that do not bind the CEX medium is between about 5% and about 100% higher than that of the initial population of recombinant polypeptides. 
     
     
         11 . The method of  claim 10 , wherein the total sialic acid content of the recombinant polypeptides that do not bind the CEX medium is between about 5% and about 40% higher than that of the initial population of recombinant polypeptides. 
     
     
         12 . The method of any one of  claims 7 - 11 , wherein the total sialic acid content of the recombinant polypeptides that do not bind the CEX medium is between about 12 and about 20 moles of sialic acid per mole of protein. 
     
     
         13 . The method of  claim 12 , wherein the total sialic acid content of the recombinant polypeptides that do not bind the CEX medium is between about 14 and about 17 moles of sialic acid per mole of protein. 
     
     
         14 . The method of any one of  claims 7 - 10 , wherein the total sialic acid content of the initial population of recombinant polypeptides is about 10 to about 14 moles of sialic acid per mole of protein. 
     
     
         15 . The method of  claim 14 , wherein the total sialic acid content of the initial population of recombinant polypeptides is about 13-14 moles of sialic acid per mole of protein. 
     
     
         16 . The method of any one of  claims 1 - 15 , wherein the recombinant polypeptide comprises an Fc domain. 
     
     
         17 . The method of  claim 16 , wherein the recombinant polypeptide comprises an antibody. 
     
     
         18 . The method of  claim 16 , wherein the recombinant polypeptide comprises an Fc fusion polypeptide comprising a ligand binding domain of a receptor. 
     
     
         19 . The method of  claim 18 , wherein the receptor is a TNF receptor. 
     
     
         20 . The method of  claim 19 , wherein the recombinant polypeptide is etanercept. 
     
     
         21 . The method of any one of  claims 1 - 18 , wherein the recombinant polypeptide comprises a clotting factor. 
     
     
         22 . The method of  claim 21 , wherein the recombinant polypeptide is a monomer-dimer hybrid. 
     
     
         23 . The method of  claim 21  or  22 , wherein the clotting factor is selected from Factor VII (FVII), FVIIa, Factor VIII (FVIII), Factor IX (FIX), or FIXa (FIX). 
     
     
         24 . The method of  claim 23 , wherein the FVIII is full-length FVIII or B-domain deleted FVIII. 
     
     
         25 . The method of  claim 23  or  24 , wherein the FVIII is single chain FVIII or dual chain FVIII. 
     
     
         26 . The method of any one of  claims 1 - 25 , wherein the contacting occurs at a load ratio between about 30 and about 100 mg total protein/ml CEX medium. 
     
     
         27 . The method of  claim 26 , wherein the contacting occurs at a load ratio between about 33 and about 54 mg total protein/ml CEX medium. 
     
     
         28 . The method of  claim 27 , wherein the contacting occurs at a load ratio of at least about 41 mg total protein/ml CEX medium. 
     
     
         29 . The method of any one of  claims 1 - 28 , wherein the contacting occurs at a pH between about 4 and about 7. 
     
     
         30 . The method of  claim 29 , wherein the contacting occurs at a pH between about 5 and about 6. 
     
     
         31 . The method of  claim 30 , wherein the contacting occurs at a pH between about 5.5 and about 5.8 
     
     
         32 . The method of  claim 30 , wherein contacting occurs at a pH of at least about 5.6. 
     
     
         33 . The method of any one of  claims 1 - 32 , wherein the contacting occurs at a conductivity between about 8 and about 12 mS/cm. 
     
     
         34 . The method of  claim 33 , wherein the contacting occurs at a conductivity of between about 9.5 and about 11 mS/cm. 
     
     
         35 . The method of  claim 34 , wherein the contacting occurs at a conductivity of at least about 10 mS/cm. 
     
     
         36 . The method of any one of  claims 1 - 35 , wherein the recombinant polypeptides that do not bind the CEX medium comprise about 25% to about 80% of the initial population of recombinant polypeptides. 
     
     
         37 . The method of  claim 36 , wherein the recombinant polypeptides that do not bind the CEX medium comprise about 55% to about 80% of the initial population of recombinant polypeptides. 
     
     
         38 . The method of any one of  claims 1 - 37 , wherein the CEX medium comprises a ligand selected from the group consisting of sulfoethyl; sulphopropyl; sulfopropyl; CH 2 —SO 3   − ; CH 2 CH 2 CH 2 SO 3   − ; SO 3   − ; and CH 2 —COO −1 . 
     
     
         39 . The method of  claim 38 , wherein the CEX medium comprises a sulfoethyl ligand. 
     
     
         40 . The method of  claim 39 , wherein the CEX medium comprises a binding capacity of between about 120 and about 160 mg lysozyme/ml resin. 
     
     
         41 . The method of any one of  claims 1 - 40 , wherein the recombinant polypeptide is produced by a eukaryotic host cell. 
     
     
         42 . The method of  claim 41 , wherein the eukaryotic host cell is a mammalian host cell. 
     
     
         43 . The method of any one of  claims 1 - 42 , wherein the contacting is performed at a manufacturing scale. 
     
     
         44 . The method of any one of  claims 1 - 43 , wherein the composition further comprises at least one impurity. 
     
     
         45 . The method of  claim 44 , wherein the impurity comprises a DNA, RNA, lipid or protein. 
     
     
         46 . The method of  claim 45 , wherein the impurity comprises a protein. 
     
     
         47 . The method of  claim 46 , wherein the protein impurity comprises a truncated form of the recombinant polypeptide, an aggregated form of the recombinant polypeptide, or a misfolded form of the recombinant polypeptide. 
     
     
         48 . The method of any one of  claims 44 - 47 , providing a final composition comprising the recombinant polypeptides that do not bind the CEX medium, wherein the final composition comprises less impurities than the composition that comprised the initial population of polypeptides.