Ion exchange chromatography with improved selectivity for the separation of polypeptide monomers, aggregates and fragments by modulation of the mobile phase

Abstract

Herein is reported a method for producing a polypeptide in monomeric form comprising the following step: recovering the polypeptide in monomeric form from an ion exchange chromatography material by applying a solution comprising a non-ionic polymer and an additive.

1 . A method for producing an antibody of the IgG class in monomeric form comprising the following steps:
 applying a solution comprising poly (ethylene glycol) and one or more additives selected from the group comprising sorbitol, xylitol, glycerol, glycine, Betaine, proline, fructose, glucose, and sucrose to a cation exchange chromatography material to which the antibody of the IgG class had been adsorbed, whereby the antibody of the IgG class in monomeric form remains adsorbed to the cation exchange chromatography material, and   recovering the antibody of the IgG class in monomeric form from the cation exchange chromatography material by applying a solution comprising poly (ethylene glycol) and the one or more additives,   
       whereby the poly (ethylene glycol) polymer has a concentration of from about 5% to 15% by weight and/or the additive has a concentration of from 3% to 25% by weight. 
     
     
         2 . A method for producing an antibody of the IgG class preparation with reduced host cell protein content comprising the following steps:
 applying a solution comprising poly (ethylene glycol) and one or more additives selected from the group comprising sorbitol, xylitol, glycerol, glycine, Betaine, proline, fructose, glucose, and sucrose to a cation exchange chromatography material to which the antibody of the IgG class had been adsorbed, whereby the antibody of the IgG class in monomeric form remains adsorbed to the cation exchange chromatography material, and   recovering the antibody of the IgG class in monomeric form from the cation exchange chromatography material by applying a solution comprising poly (ethylene glycol), one or more additives selected from the group comprising sorbitol, xylitol, glycerol, glycine, Betaine, proline, fructose, glucose, and sucrose, and an elution compound, and thereby producing the antibody of the IgG class preparation with reduced host cell protein content,   
       whereby the poly (ethylene glycol) has a concentration of from about 5% to 15% by weight and/or the additive has a concentration of from 3% to 25% by weight. 
     
     
         3 . The method according to  claim 1 , characterized in that the poly (ethylene glycol) has a concentration of from about 7% to 13% by weight and/or the additive has a concentration of from 3% to 25% by weight. 
     
     
         4 . The method according to  claim 1 , characterized in that the poly (ethylene glycol) has a concentration of from about 7% to 13% by weight and/or the additive has a concentration of from 12% to 25% by weight. 
     
     
         5 . The method according to  claim 1 , characterized in that the poly (ethylene glycol) has a concentration of from about 7% to 13% by weight and/or the additive has a concentration of from 12% to 20% by weight. 
     
     
         6 . The method according to  claim 1 , characterized in that the poly (ethylene glycol) has a molecular weight of about 3,500 Da+/−20%. 
     
     
         7 . The method according to  claim 1 , characterized in that the additive is sorbitol. 
     
     
         8 . The method according to  claim 1 , characterized in that the monomeric antibody has a molecular weight of from 100 kDa to 200 kDa. 
     
     
         9 . The method according to  claim 1 , characterized in that the method further comprises the following steps:
 applying a first solution that optionally comprises poly (ethylene glycol) and one or more additives selected from the group comprising sorbitol, xylitol, glycerol, glycine, Betaine, proline, fructose, glucose, and sucrose, to a cation exchange chromatographic material and thereby equilibrating the material,   applying the solution comprising the antibody of the IgG class to the equilibrated chromatography material and thereby loading the chromatography material,   producing the antibody of the IgG class in monomeric form by applying a solution to the chromatographic material comprising poly (ethylene glycol) and one or more additives selected from the group comprising sorbitol, xylitol, glycerol, glycine, Betaine, proline, fructose, glucose, and sucrose, and thereby desorbing/eluting the antibody of the IgG class in monomeric form from the chromatographic material.   
     
     
         10 . The method according to  claim 1 , wherein the recovered antibody of the IgG class in monomeric form is efficiently separated from the antibody in aggregated form, antibody fragments, host cell protein or host cell nucleic acid. 
     
     
         11 . The method according to  claim 1 , wherein the recovered antibody of the IgG class in monomeric form is biologically active.