Method for purifying immunoglobulin solutions

Abstract

Herein is reported a method for purifying cell cultivation supernatants either directly after fermentation or after one or more preliminary purification steps, such as protein A affinity chromatography. By adjusting the pH value in the acid range and subsequent incubation of the acidified solution host cell nucleic acid and host cell protein can be precipitated but the target polypeptide remains in solution. Thereafter the precipitate and therewith the contaminating host cell components can be removed by a simple physical separation step.

1 - 18 . (canceled) 
     
     
         19 . A method for producing an immunoglobulin comprising the following steps:
 i) adding a solution consisting of an acid and water to a cell cultivation broth for adjusting the pH to a value of below 5.5,   ii) incubating the pH adjusted cell cultivation broth, and   iii) removing the precipitate from the incubated cell cultivation broth and thereby producing the immunoglobulin,   wherein the cell cultivation broth comprises the immunoglobulin at a concentration of not more than 10 mg/ml,   wherein the concentration of the added acid is 5.5 mol/l or lower.   
     
     
         20 . A method for purifying a cell cultivation broth comprising the following steps:
 i) adding a solution consisting of an acid and water to the cell cultivation broth for adjusting the pH value to a value of below 5.5,   ii) incubating the pH adjusted cell cultivation broth, and   iii) removing the precipitate from the incubated cell cultivation broth and thereby purifying the cell cultivation broth,   wherein the cell cultivation broth comprises the immunoglobulin at a concentration of not more than 10 mg/ml,   wherein the concentration of the added acid is 5.5 mol/l or lower.   
     
     
         21 . The method according to  claim 19 , wherein at least 90% of the immunoglobulin remains in solution during the incubating step. 
     
     
         22 . The method according to  claim 19 , wherein the incubating is at a temperature of from 2° C. to 10° C. 
     
     
         23 . The method according to  claim 22 , wherein the incubating of the pH adjusted cell cultivation broth is at a temperature of about 4° C. 
     
     
         24 . The method according to  claim 19 , wherein the incubating the pH adjusted broth is for about at least 2 hours. 
     
     
         25 . The method according to  claim 24 , wherein the incubating is for about 2-72 hours, about 2-48 hours, or about 24 hours. 
     
     
         26 . The method according to  claim 19 , wherein the removing is by a method selected from sedimentation, decantation, filtration, settlement, and centrifugation. 
     
     
         27 . The method according to  claim 19 , wherein the acid is selected from acetic acid, citric acid, hydrochloric acid, and phosphoric acid. 
     
     
         28 . The method according to  claim 19 , wherein the concentration of the acid is of from 1.5 mol/l to 5.5 mol/l. 
     
     
         29 . The method according to  claim 27 , wherein the acid is acetic acid with a concentration of from 1.5 mol/l to 4 mol/l. 
     
     
         30 . The method according to  claim 28 , wherein the acid is acetic acid with a concentration of from 1.5 mol/l to 4 mol/l. 
     
     
         31 . The method according to  claim 19 , wherein the concentration of the immunoglobulin is of from 1 mg/ml to 5 mg/ml. 
     
     
         32 . The method according to  claim 19 , wherein the cell is a CHO cell. 
     
     
         33 . A method for producing a polypeptide comprising the following steps:
 i) providing a mammalian cell comprising a nucleic acid encoding the polypeptide,   ii) cultivating the cell under serum free conditions,   iii) recovering the cell cultivation broth, without removing cells and cell debris from the cell cultivation broth, and   iv) purifying the cell cultivation broth by the following steps and thereby producing a polypeptide:
 a) adding a solution consisting of an acid and water to the cell cultivation broth for adjusting the pH to a value below 5.5, 
 b) incubating the pH adjusted cell cultivation broth, and 
 c) removing the precipitate from the incubated cell cultivation broth, 
   wherein the cell cultivation broth comprises the polypeptide at a concentration of not more than 10 mg/ml,   wherein the concentration of the added acid is 5.5 mol/l or lower.