Protein purification

Abstract

A method for purifying a polypeptide by ion exchange chromatography is described which involves changing the conductivity and/or pH of buffers in order to resolve a polypeptide of interest from one or more contaminants.

1 . A method for purifying a polypeptide from a composition comprising the polypeptide and a contaminant, which method comprises the following steps performed sequentially:
 (a) binding the polypeptide to an ion exchange material using a loading buffer, wherein the loading buffer is at a first conductivity and pH;   (b) washing the ion exchange material with an intermediate buffer at a second conductivity and/or pH so as to elute the contaminant from the ion exchange material;   (c) washing the ion exchange material with a wash buffer which is at a third conductivity and/or pH, wherein the change in conductivity and/or pH from the intermediate buffer to the wash buffer is in an opposite direction to the change in conductivity and/or pH from the loading buffer to the intermediate buffer; and   (d) washing the ion exchange material with an elution buffer at a fourth conductivity and/or pH so as to elute the polypeptide from the ion exchange material.   
     
     
         2 . The method of  claim 1  wherein the ion exchange material comprises a cation exchange resin. 
     
     
         3 . The method of  claim 1  wherein the ion exchange material comprises an anion exchange resin. 
     
     
         4 . The method of  claim 2  wherein the conductivity and/or pH of the intermediate buffer is/are greater than the conductivity and/or pH of the loading buffer and the conductivity and/or pH of the wash buffer is/are less than the conductivity and/or pH of the intermediate buffer. 
     
     
         5 . The method of  claim 1  wherein the conductivity and/or pH of the wash buffer is/are about the same as the conductivity and/or pH of the loading buffer. 
     
     
         6 . The method of  claim 2  wherein the conductivity and/or pH of the elution buffer is/are greater than the conductivity and/or pH of the intermediate buffer. 
     
     
         7 . The method of  claim 1  wherein elution of the contaminant and of the polypeptide is achieved by modifying the conductivity of the intermediate buffer and of the elution buffer, respectively. 
     
     
         8 . The method of  claim 7  wherein the pH remains approximately constant for each of steps (a)-(d). 
     
     
         9 . The method of  claim 1  wherein the polypeptide and the contaminant have slightly different pI's. 
     
     
         10 . The method of  claim 1  wherein the contaminant is a deamidated variant of the polypeptide. 
     
     
         11 . The method of  claim 7  wherein the conductivity of the intermediate buffer and of the elution buffer is modified by changing the salt concentration therein. 
     
     
         12 . The method of  claim 11  wherein the conductivity of the intermediate buffer and of the elution buffer is modified by changing the NaCl concentration therein. 
     
     
         13 . The method of  claim 1  further comprising washing the ion exchange material with a regeneration buffer after step (d). 
     
     
         14 . The method of  claim 2  wherein the cation exchange resin comprises sulphopropyl immobilized on agarose. 
     
     
         15 . The method of  claim 1  wherein the polypeptide is an antibody. 
     
     
         16 . The method of  claim 15  wherein the antibody binds HER2. 
     
     
         17 . The method of  claim 1  wherein the contaminant is a deamidated variant of the polypeptide. 
     
     
         18 . The method of  claim 1  further comprising subjecting the composition comprising the polypeptide to one or more further purification steps either before, during, or after the ion exchange chromatography method so as to obtain a homogeneous preparation of the polypeptide. 
     
     
         19 . The method of  claim 18  further comprising conjugating the purified polypeptide with a heterologous molecule. 
     
     
         20 . The method of  claim 19  wherein the heterologous molecule is polyethylene glycol, a label or a cytotoxic agent. 
     
     
         21 . The method of  claim 18  further comprising preparing a pharmaceutical composition by combining the homogeneous preparation of the polypeptide with a pharmaceutically acceptable carrier. 
     
     
         22 . A polypeptide which has been purified according to the method of  claim 1 . 
     
     
         23 . A method for purifying a polypeptide from a composition comprising the polypeptide and a contaminant, which method comprises the following steps performed sequentially:
 (a) binding the polypeptide to a cation exchange material using a loading buffer, wherein the loading buffer is at a first conductivity and pH;   (b) washing the cation exchange material with an intermediate buffer at a second conductivity and/or pH which is greater than that of the loading buffer so as to elute the contaminant from the ion exchange material;   (c) washing the cation exchange material with a wash buffer which is at a third conductivity and/or pH which is less than that of the intermediate buffer; and   (d) washing the cation exchange material with an elution buffer at a fourth conductivity and/or pH which is greater than that of the intermediate buffer so as to elute the polypeptide from the ion exchange material.   
     
     
         24 . A method for purifying an antibody from a composition comprising the antibody and a contaminant, which method comprises loading the composition onto a cation exchange resin, wherein the amount of antibody loaded onto the cation exchange resin is from about 20 mg to about 35 mg of the antibody per mL of cation exchange resin. 
     
     
         25 . The method of  claim 24  wherein the cation exchange resin comprises sulphopropyl immobilized on agarose. 
     
     
         26 . The method of  claim 24  further comprising eluting the antibody from the cation exchange resin. 
     
     
         27 . The method of  claim 26  further comprising eluting the contaminant from the cation exchange resin in an intermediate wash step prior to eluting the antibody from the cation exchange resin. 
     
     
         28 . A composition comprising a mixture of anti-HER2 antibody and one or more acidic variants thereof, wherein the amount of the acidic variants(s) is less than about 25%. 
     
     
         29 . The composition of  claim 28  further comprising a pharmaceutically acceptable carrier. 
     
     
         30 . The composition of  claim 28  wherein the anti-HER2 antibody is humMAb4D5-8. 
     
     
         31 . The composition of  claim 30  which is a lyophilized formulation. 
     
     
         32 . The composition of  claim 30 , which is a liquid formulation. 
     
     
         33 . The composition of  claim 32 , which is an aqueous formulation. 
     
     
         34 . A composition comprising a mixture of anti-HER2 antibody and one or more acidic variants thereof, wherein the amount of the acidic variants(s) is less than about 25%, and wherein the anti-HER2 antibody is conjugated to a cytotoxic molecule. 
     
     
         35 . The composition of  claim 33 , wherein the anti-HER2 antibody is humMAb4D5-8.