Protein purification

Abstract

A method for purifying a polypeptide by ion exchange chromatography is described in which a gradient wash is used to resolve a polypeptide of interest from one or more contaminants.

What is claimed is: 
     
         1 . A method for purifying a polypeptide from a composition comprising the polypeptide and contaminants, which method comprises the sequential steps of:
 (a) loading the composition onto an ion exchange resin with an equilibration buffer having a first salt concentration;   (b) washing the ion exchange resin with a wash buffer until a predetermined protein concentration is measured in the flowthrough, wherein the salt concentration of the wash buffer increases from an initial, second salt concentration that is greater than the salt concentration of the equilibration buffer, to a final, third salt concentration;   (c) passing a fixed volume of wash buffer at the final, third salt concentration over the cation exchange resin; and   (d) eluting the polypeptide from the ion exchange resin with elution buffer that has a salt concentration that is greater than the final salt concentration of the wash buffer.   
     
     
         2 . The method of  claim 1  wherein the ion exchange resin is amnion exchange 3.  resin. 
     
     
         3 . The method of  claim 1  wherein the ion exchange resin in a cation exchange resin. 
     
     
         4 . The method of  claim 3  wherein the cation exchange resin comprises sulphopropyl immobilized on agarose. 
     
     
         5 . The method of  claim 1  wherein the elution buffer has a higher conductivity than the equilibration buffer. 
     
     
         6 . The method of  claim 1  wherein the elution buffer comprises about 145 mM Na/HOAc and the equilibration buffer comprises about 70 mM Na/HOAc. 
     
     
         7 . The method of  claim 1  wherein the elution buffer comprises about 100 mM NaCl and the equilibration buffer comprises about 45 mM NaCl. 
     
     
         8 . The method of  claim 1  wherein the wash buffer comprises a mixture of equilibration buffer and elution buffer. 
     
     
         9 . The method of  claim 8  wherein the increase in the salt concentration of the wash buffer during step (b) is achieved by increasing the proportion of elution buffer in the wash buffer. 
     
     
         10 . The method of  claim 9  wherein the proportion of elution buffer in the wash buffer increases at a constant rate. 
     
     
         11 . The method of  claim 10  wherein the increase in the proportion of elution buffer causes the salt concentration of the wash buffer to increase at a constant rate of from about 1 mM to about 3 mM per column volume of wash buffer. 
     
     
         12 . The method of  claim 9  wherein the percentage of elution buffer in the wash buffer increases at two or more different rates during the course of washing in step (b). 
     
     
         13 . The method of  claim 12  wherein the percentage of elution buffer in the wash buffer increases at a first rate for a first segment of the washing, at a second rate for a second segment of the washing and at a third rate for a third segment of the washing. 
     
     
         14 . The method of  claim 1  wherein the polypeptide is an antibody. 
     
     
         15 . The method of  claim 14  wherein the antibody binds HER2. 
     
     
         16 . The method of  claim 14  wherein the contaminant is a deamidated variant of the antibody. 
     
     
         17 . The method of  claim 14  wherein the amount of antibody in the composition loaded onto the ion exchange resin is from about 15 mg to about 45 mg per mL of cation exchange resin. 
     
     
         18 . The method of  claim 1  wherein the predetermined protein concentration in step (b) corresponds to an OD of 0.6 measured at 280 nm. 
     
     
         19 . The method of  claim 1  wherein from about 0.4 to about 1 column volumes of wash buffer are passed over the ion exchange resin in step (c). 
     
     
         20 . The method of  claim 1  wherein the pH of the equilibration buffer, wash buffer and elution buffer is approximately the same. 
     
     
         21 . The method of  claim 23  wherein the pH of the equilibration buffer, wash buffer and elution buffer is approximately 5.5. 
     
     
         22 . 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 steps (a) through (d) so as to obtain a homogeneous preparation of the polypeptide. 
     
     
         23 . The method of  claim 22  further comprising preparing a pharmaceutical composition by combining the homogeneous preparation of the polypeptide with a pharmaceutically acceptable carrier. 
     
     
         24 . The method of  claim 22  further comprising conjugating the purified polypeptide with a heterologous molecule. 
     
     
         25 . The method of  claim 24  wherein the heterologous molecule is polyethylene glycol, a label or a cytotoxic agent. 
     
     
         26 . A polypeptide which has been purified according to the method of  claim 1 . 
     
     
         27 . A method for purifying an antibody from a composition comprising the polypeptide and a contaminant, which method comprises the following steps performed sequentially:
 (a) binding the antibody to a cation exchange material with an equilibration buffer at a first conductivity;   (b) washing the cation exchange material with a wash buffer, wherein the conductivity of the wash buffer increases from a second conductivity that is higher than the first conductivity to a third conductivity during the washing;   passing a fixed volume of wash buffer at the third conductivity over the cation exchange material; and   (d) eluting the antibody from the cation exchange material with an elution buffer at a fourth conductivity that is higher than the third conductivity.   
     
     
         28 . The method of  claim 27  wherein the cation exchange resin comprises sulphopropyl immobilized on agarose. 
     
     
         29 . The method of  claim 27  wherein the conductivity of the wash buffer increases at a constant rate from the second conductivity to the third conductivity. 
     
     
         30 . The method of  claim 27  wherein the conductivity of the wash buffer increases at two or more different rates from the second conductivity to the third conductivity. 
     
     
         31 . The method of  claim 30  wherein the conductivity of the wash buffer increases at a first rate for a first segment of the washing, at a second rate for a second segment of the washing and at a third rate for a third segment of the washing. 
     
     
         32 . The method of  claim 31  wherein the wash buffer comprises a mixture of equilibration buffer and elution buffer. 
     
     
         33 . The method of  claim 32  wherein the conductivity of the wash buffer is increased by increasing the proportion of elution buffer in the wash buffer. 
     
     
         34 . The method of  claim 33  wherein the proportion of elution buffer in the wash buffer increases at a constant rate of about 6% during the first segment, at a constant rate of about 3.5% during the second segment and at a constant rate of about 2% during the third segment. 
     
     
         35 . The method of  claim 33  wherein the proportion of elution buffer in the wash buffer increases from about 26% to about 54% during the first segment, from about 54% to about 61% during the second segment and from about 61% to about 74% during the second segment. 
     
     
         36 . The method of  claim 31  wherein the cation exchange material is washed with about 5 column volumes of wash buffer in the first segment, about 2 column volumes of wash buffer in the second segment and about 6 column volumes of wash buffer in the third segment. 
     
     
         37 . The method of  claim 27  wherein the conductivity of the wash buffer is increased by increasing the percentage of elution buffer in the wash buffer. 
     
     
         38 . The method of  claim 27  wherein the conductivity of the wash buffer is increased by increasing the salt concentration therein. 
     
     
         39 . The method of  claim 27  wherein the fixed volume of wash buffer passed over the cation exchange material in step (c) is between about 0.4 column volumes and about 1.0 column volumes. 
     
     
         40 . The method of  claim 27  further comprising washing the ion exchange material with a regeneration buffer after step (d). 
     
     
         41 . A method for purifying an antibody from a composition comprising the antibody and a contaminant, which method comprises the following steps performed sequentially:
 (a) loading the composition onto a cation exchange material;   (b) washing the cation exchange material with a wash buffer with a conductivity that increases at a first rate from a first conductivity to a second conductivity, at a second rate from the second conductivity to a third conductivity and at a third rate from the third conductivity to a fourth conductivity; and   (c) eluting the antibody from the ion exchange material, wherein the amount of antibody in the composition loaded onto the cation exchange material is from about 15 mg to about 45 mg of the antibody per ml of cation exchange material.   
     
     
         42 . A method for purifying a polypeptide from a composition comprising the polypeptide and a contaminant, which method comprises the following steps performed sequentially:
 (a) loading the composition onto an ion exchange material;   (b) washing the cation exchange material with wash buffer using a multi-slope gradient until a predetermined protein concentration is measured in the flowthrough; and   eluting the polypeptide from the ion exchange material.   
     
     
         43 . The method of  claim 42  wherein the multi-slope gradient comprises two or more segments. 
     
     
         44 . The method of  claim 43  wherein each segment of the multi-slope gradient has a shallower slope. 
     
     
         45 . The method of  claim 42 , additionally comprising the step between steps (b) and (c) of washing the column with from 0.4 to 1 column volumes of wash buffer. 
     
     
         46 . The method of  claim 45  wherein the wash buffer has the composition of the wash buffer at the end of step (b).