Protein purification using hcic and ion exchange chromatography

Abstract

The present invention provides methods for purifying proteins. In particular, the methods employ a two-step non-affinity chromatography process without the use of an in-process tangential flow filtration step.

1 . A method for purifying a target protein from a mixture, which comprises the target protein and one or more contaminants, comprising:
 (a) subjecting the mixture to a cation exchange chromatography step and a hydrophobic charge induction chromatography step, in either order, wherein there is no in-process tangential flow filtration step; and   (b) isolating the target protein.   
     
     
         2 - 3 . (canceled) 
     
     
         4 . The method  claim 1 , wherein the target protein is isolated to a purity of about 100 parts per million (ppm) or less of host cell protein and about 10 pg/mg or less of nucleic acids. 
     
     
         5 . The method of  claim 1 , which further includes a viral inactivation step. 
     
     
         6 . The method of  claim 5 , wherein the viral inactivation step is an in-process step. 
     
     
         7 . The method of  claim 1 , wherein the mixture is prepared from a cell culture supernatant. 
     
     
         8 . The method of  claim 1 , wherein the cation exchange chromatography is performed at a pH range from 3 to 10. 
     
     
         9 . The method of  claim 1 , wherein the cation exchange chromatography is performed at a pH range from 4.0 to 8.0. 
     
     
         10 . The method of  claim 1 , wherein the hydrophobic charge induction chromatography step is performed at a pH range from 3 to 10. 
     
     
         11 . The method of  claim 1 , wherein the hydrophobic charge induction chromatography is performed using at a pH range from 4.0 to 9.0. 
     
     
         12 . The method of  claim 1 , wherein the target protein binds to a cation exchange chromatography column at pH 3 to 9 and at a conductivity range from 0.1 to 40 mS/cm. 
     
     
         13 . The method of  claim 1 , wherein the target protein binds to a cation exchange chromatography column at pH 4.0 to 8.0 and at a conductivity from 0.5 to 10 mS/cm. 
     
     
         14 . The method of  claim 1 , wherein the target protein binds to a hydrophobic charge induction chromatography column at pH 5 to 10 and at a conductivity range from 0 to 90 mS/cm. 
     
     
         15 . The method of  claim 1 , wherein the target protein binds to a hydrophobic charge induction chromatography column at pH 6 to 9 and conductivity range from 2 to 9 mS/cm. 
     
     
         16 . The method of  claim 1 , wherein the target protein is bound to the cation exchange chromatography column and the column is washed using a wash buffer having a pH range from 3 to 9 and a conductivity range from 0.1 to 40 mS/cm. 
     
     
         17 . The method of  claim 1 , wherein the target protein is bound to a cation exchange chromatography column and the column is washed using a wash buffer having a pH range from 4.0 to 8.0 and a conductivity range from 0.5 to 10 mS/cm. 
     
     
         18 . The method of  claim 1 , wherein the target protein is bound to a hydrophobic charge induction chromatography column and the column is washed using a wash buffer having a pH range from 5 to 10 and a conductivity range from 0 to 90 mS/cm. 
     
     
         19 . The method of  claim 1 , wherein the target protein is bound to a hydrophobic charge induction chromatography column and the column is washed using a wash buffer having a pH range from 6 to 9 and a conductivity range from 0.1 to 9 mS/cm. 
     
     
         20 . The method of  claim 1 , wherein the target protein elutes from the cation exchange chromatography column at a pH range from 3 to 10 and a conductivity range from 0.1 to 40 mS/cm. 
     
     
         21 . The method of  claim 1 , wherein the target protein elutes from the cation exchange chromatography column at a pH range from 4.0 to 8.0 and a conductivity range from 5 to 15 mS/cm. 
     
     
         22 . The method of  claim 1 , wherein the target protein elutes from the hydrophobic charge induction exchange chromatography column at a pH range from 3.0 to 7.0 and a conductivity range from 0 to 20 mS/cm. 
     
     
         23 . The method of  claim 1 , wherein the target protein elutes from the hydrophobic charge induction exchange chromatography column at a pH range from 4.0 to 6.0 and a conductivity range from 0.1 to 2.0 mS/cm. 
     
     
         24 - 29 . (canceled) 
     
     
         30 . The method of  claim 1 , wherein the target protein is a monoclonal antibody or antibody fragment. 
     
     
         31 . (canceled) 
     
     
         32 . The method of  claim 30  wherein the antibody is a fully human antibody. 
     
     
         33 . The method of  claim 30  wherein the antibody is selected from anti-CTLA4 antibody and anti-CD30 antibody. 
     
     
         34 - 36 . (canceled) 
     
     
         37 . The method of  claim 1 , further comprising the step of formulating the isolated protein into a pharmaceutical composition.