Enhanced capacity and purification of protein by mixed mode chromatography in the presence of aqueous-soluble nonionic organic polymers

Abstract

This invention relates to the use of mixed mode chromatography for purification of a protein from a mixture containing other materials, including fragmented or aggregated antibodies, host cell proteins, DNA, endotoxin, and/or virus. This invention further relates to the integration of such a method into a multi-step procedure with other fractionation methods for purification of antibodies or other proteins suitable for in vivo applications.

1 . A mixed mode chromatography support in contact with a protein preparation, a target protein and an aqueous-soluble nonionic organic polymer, wherein the preparation does not comprise antibodies. 
     
     
         2 . The mixed mode chromatography support of  claim 1 , wherein the nonionic organic polymer is from the group consisting of dextran, starch, cellulose, polyvinylpyrrolidone, polypropylene glycol and polyethylene glycol (PEG). 
     
     
         3 . The mixed mode chromatography support of  claim 1 , wherein the nonionic organic polymer comprises two or more nonionic organic polymers. 
     
     
         4 . The mixed mode chromatography support of  claim 1 , wherein the nonionic organic polymer has an average molecular weight of 100 to 10,000 daltons. 
     
     
         5 . The mixed mode chromatography support of  claim 1 , wherein the support exploits a combination of two or more of the following functionalities to adsorb components of the preparation: cation exchange, anion exchange, hydrophobic interaction, hydrophilic interaction, hydrogen bonding, pi-pi bonding, and metal affinity. 
     
     
         6 . The mixed mode chromatography support of  claim 1 , wherein the support comprises hydroxyapatite. 
     
     
         7 . The mixed mode chromatography support of  claim 6 , wherein the support is selected from the group consisting of hydroxypatite CHT Type I, 20 micron; hydroxypatite CHT Type I, 40 micron; hydroxypatite CHT Type I, 80 micron; hydroxypatite CHT Type II, 20 micron; hydroxypatite CHT Type II, 40 micron; and hydroxypatite CHT Type II, 80 micron. 
     
     
         8 . The mixed mode chromatography support of  claim 1 , wherein the support comprises fluoroapatite. 
     
     
         9 . The mixed mode chromatography support of  claim 8 , wherein the support comprises fluoroapatite CFT Type I, 40 micron or fluoroapatite CFT Type II, 40 micron. 
     
     
         10 . The mixed mode chromatography support of  claim 1 , wherein the mixed mode support comprises a ligand selected from the group consisting of Capto-MMC, Capto-Adhere, Capto-S, Capto-Q, MEP Hypercel, and ABx. 
     
     
         11 . The mixed mode chromatography support of  claim 1 , wherein separation of virus from a target protein on the support operated in bind elute mode is enhanced compared to the separation that would occur in the absence of the aqueous-soluble nonionic organic polymer. 
     
     
         12 . The mixed mode chromatography support of  claim 1 , wherein retention of aggregates, virus, and other contaminants larger than the target protein is enhanced to a greater degree than retention of the target protein, and wherein separation of the aggregates, virus, and other contaminants from the target protein is thereby increased. 
     
     
         13 . The mixed mode chromatography support of  claim 1 , wherein retention of aggregates, virus, and other contaminants larger than the target protein is enhanced to a greater degree than retention of the target protein and wherein the method allows for increased capacity for contaminant removal from the target protein. 
     
     
         14 . The mixed mode chromatography support of  claim 1 , wherein retention of the target protein is enhanced to a greater degree than retention of molecules smaller than the target protein, and wherein separation of the molecules from the target protein is thereby increased.