Antibody purification by cation exchange chromatography

Abstract

A method for purifying an antibody by cation exchange chromatography is described in which a high pH wash step is used to remove of contaminants prior to eluting the desired antibody using an elution buffer with increased conductivity.

1 . A method for purifying an antibody from a composition comprising the antibody and at least one contaminant, which method comprises the sequential steps of:
 (a) loading the composition onto a cation exchange material wherein the composition is at a first pH;   (b) washing the cation exchange material with a first wash buffer at a pH which is greater than that of the composition in (a), wherein the pH of the first wash buffer is from about 6.8 to about 9.0;   (c) washing the cation exchange material with a second wash buffer at a pH which is less than that of the first wash buffer; and   (d) eluting the antibody from the cation exchange material with an elution buffer at a conductivity which is substantially greater than that of the second wash buffer.   
     
     
         2 . The method of  claim 1  wherein the pH of the second wash buffer and the pH of the elution buffer are approximately the same. 
     
     
         3 . The method of  claim 1  wherein the antibody binds to human CD20. 
     
     
         4 . The method of  claim 1  wherein the antibody binds to human vascular endothelial growth factor (VEGF). 
     
     
         5 . The method of  claim 1  wherein the pH of the composition in (a) is from about 4.0 to about 6.0, the pH of the first wash buffer is from about 6.8 to about 8.0, the pH of the second wash buffer is from about 5.0 to about 6.0, and the pH of the elution buffer is from about 5.0 to about 6.0. 
     
     
         6 . The method of  claim 1  wherein the conductivity of the elution buffer is from about 10 mS/cm to about 100 mS/cm. 
     
     
         7 . The method of  claim 1  wherein the elution buffer comprises about 100 to about 300 mM NaCl. 
     
     
         8 . The method of  claim 1  wherein the cation exchange material comprises cross-linked poly(styrene-divinylbenzene) flow-through particles coated with a polyhydroxylated polymer functionalized with sulfopropyl groups. 
     
     
         9 . The method of  claim 1  wherein the contaminant is selected from the group consisting of Chinese Hamster Ovary Proteins (CHOP), leached protein A, DNA, aggregated antibody, cell culture media component, garamycin, and viral contaminant. 
     
     
         10 . The method of  claim 1  further comprising subjecting the composition comprising the antibody 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 antibody. 
     
     
         11 . The method of  claim 10  further comprising conjugating the purified antibody with a heterologous molecule. 
     
     
         12 . The method of  claim 10  or  11  further comprising preparing a pharmaceutical composition by combining the homogeneous preparation of the antibody or conjugated antibody with a pharmaceutically acceptable carrier. 
     
     
         13 . A method for purifying an antibody that binds human CD20 from a composition comprising the antibody and one or more contaminants selected from the group consisting of Chinese Hamster Ovary Proteins (CHOP), leached protein A, DNA, and aggregated CD20 antibody, which method comprises the sequential steps of:
 (a) loading the composition onto a cation exchange material wherein the composition is at a pH from about 4.0 to about 6.0;   (b) washing the cation exchange material with a first wash buffer at a pH from about 6.8 to about 9.0;   (c) washing the cation exchange material with a second wash buffer at a pH from about 5.0 to about 6.0; and   (d) eluting the antibody from the cation exchange material using an elution buffer with a pH from about 5.0 to about 6.0 and a conductivity from about 10 mS/cm to about 100 mS/cm.   
     
     
         14 . The method of  claim 13  wherein the antibody is rituximab. 
     
     
         15 . The method of  claim 13  wherein the elution buffer comprises about 100 to about 300 mM NaCl. 
     
     
         16 . A method for purifying an antibody that binds human vascular endothelial growth factor (VEGF) from a composition comprising the antibody and one or more contaminants selected from the group consisting of a cell culture media component, garamycin, Chinese Hamster Ovary Proteins (CHOP), DNA, viral contaminant, and aggregated VEGF antibody, which method comprises the sequential steps of:
 (a) loading the composition onto a cation exchange material wherein the composition is at a pH from about 4.0 to about 6.0;   (b) washing the cation exchange material with a first wash buffer at a pH from about 6.8 to about 8.0;   (c) washing the cation exchange material with a second wash buffer at a pH from about 5.0 to about 6.0; and   (d) eluting the antibody from the cation exchange material using an elution buffer with a pH from about 5.0 to about 6.0 and a conductivity from about 10 mS/cm to about 100 mS/cm.   
     
     
         17 . The method of  claim 16  wherein the antibody is bevacizumab. 
     
     
         18 . The method of  claim 16  wherein the elution buffer comprises about 100 to about 300 mM NaCl. 
     
     
         19 . A composition comprising rituximab in a buffer comprising about 25 mM HEPES, at a pH of about 7.8. 
     
     
         20 . A composition comprising bevacizumab in a buffer comprising about 25 mM MOPS at a pH of about 7.0.