Process for the purification of fc-containing proteins

Abstract

The invention relates to a process for the purification of an Fc-containing protein based on cation exchange chromatography.

1 . A method for separating and purifying an Fc-containing protein from a fluid comprising at least a cation exchange chromatography purification step comprising:
 (a) binding an Fc protein containing fluid comprising an Fc-containing protein to a cation exchange chromatography resing;   (b) washing the cation chromatography resin with a buffer at a pH about 1 unit below the isoelectric point of the Fc-containing protein, the buffer having a conductivity of about 2 to 6 mS/cm;   (c) eluting the Fc-containing protein with a buffer at a pH about 1 unit below the isoelectric point of the Fc-containing protein with an increasing salt gradient; and   (d) applying the eluent of step (c) to an anionic exchange chromatography matrix or a hydrophobic interaction chromatography matrix.   
     
     
         2 . The method according to  claim 1 , wherein the method comprises applying the eluted Fc-containing protein to an anion exchange chromatography matrix, eluting the Fc-containing protein from the anion exchange chromatography matrix, applying the eluted Fc containing protein to a hydrophobic interaction chromatography matrix and eluting the Fc-containing protein from the hydrophobic interaction chromatography matrix. 
     
     
         3 . The method according to  claim 1 , wherein the method comprises applying the eluted Fc-containing protein to a hydrophobic interaction chromatography matrix, eluting the Fc-containing protein from the hydrophobic interaction chromatography matrix, applying the eluted Fc-containing protein to an anion exchange chromatography matrix and eluting the Fc-containing protein from the anion exchange chromatography resin. 
     
     
         4 . The method according to  claim 1 , wherein the binding of the Fc-containing protein in step (a) is carried out at pH below 5. 
     
     
         5 . The method according to  claim 1 , wherein the Fc-containing protein is diluted in water to a conductivity of less than 4 mS/cm at about pH 7.0 prior to its binding to the cation exchange resin in step (a). 
     
     
         6 . The method according to  claim 1 , wherein the washing in step (b) is carried out at a pH from about 7 to about 8.5 at a conductivity of about 2 to 6 mS/cm. 
     
     
         7 . The method according to  claim 1 , wherein the washing in step (b) is carried out with a phosphate buffer at about pH 8, having a conductivity of about 3.5 mS/cm. 
     
     
         8 . The method according to  claim 1 , wherein the Fc-containing protein is eluted from the cation exchange resin with an increasing salt gradient at a conductivity ranging from about 2 to about 15 mS/cm at a pH of about 7 to about 8.5. 
     
     
         9 . The method according to  claim 1 , wherein the Fc-containing protein is eluted from the cation exchange resin with an increasing NaCl gradient ranging from about 0 to about 150 mM at a pH ranging from about 7 to about pH 8.5. 
     
     
         10 . The method according to  claim 1 , wherein cutting out the tail of the elution peak in step (c) is performed. 
     
     
         11 . The method according to  claim 1 , wherein the cation exchange resin in step (a) is a strong cation exchange resin. 
     
     
         12 . The method according to  claim 11 , wherein the strong cation exchange resin is Fractogel EMD SE Hicap (M) resin. 
     
     
         13 . The method according to  claim 11 , wherein the resin is loaded at about pH 4, at a conductivity of about 15 mS/cm and at a dynamic capacity of about 40 to 47 g of Fc-containing protein per liter of packed cation exchange resin. 
     
     
         14 . The method according to  claim 1 , wherein the eluate of the cation exchange resin resulting from step (c) has an HCP level of less than 10,000 ppm or of less than 5,000 ppm. 
     
     
         15 . The method according to  claim 1 , wherein the eluate of the cation exchange resin resulting from step (c) has an aggregate level of less than 1%. 
     
     
         16 . The method according to  claim 1 , wherein the eluate of the cation exchange resin resulting from step (c) has levels of incomplete Fc-containing protein that are undetectable by SDS-PAGE under non-reducing conditions and silver staining when loading 1 mcg of Fc-containing protein. 
     
     
         17 . The method according to  claim 1 , wherein the incomplete Fc-containing protein fragment comprises free antibody heavy and/or light chains. 
     
     
         18 . The method according to  claim 1 , wherein the Fc-containing fluid is clarified harvest. 
     
     
         19 . The method according to  claim 1 , wherein the Fc-containing protein has an isoelectric point between about 7.5 and about 9.5. 
     
     
         20 . The method according to  claim 1 , further comprising formulating the purified Fc-containing protein into a pharmaceutical composition.