Method for the Purification of Antibodies

Abstract

A method for the purification of immunoglobulins by ion exchange chromatography is described. The chromatographic method uses a weak ion exchange resin and a single step elution process for the purification of an immunoglobulin. Additionally a method for the determination of the salt concentration for the single step elution of an immunoglobulin from an ion exchange resin is described.

1 . Method for purifying an immunoglobulin, wherein the method comprises
 a) providing a solution comprising an immunoglobulin, a buffer substance, and optionally a salt;   b) bringing the solution and a weak ion exchange material in contact under conditions whereby the immunoglobulin binds to the weak ion exchange material;   c) recovering the immunoglobulin from the weak ion exchange material in a single step by using a solution comprising a buffer substance and a salt.   
   
   
       2 . Method according to  claim 1 , characterized in that the weak ion exchange material is a weak cation exchange material. 
   
   
       3 . Method according to any one of the  claims 1  to  2 , characterized in that the buffer substance is citric acid or a salt thereof or phosphoric acid or a salt thereof. 
   
   
       4 . Method according to any one of the  claims 1  to  3 , characterized in that the method is a chromatographic or a batch method. 
   
   
       5 . Method according to any one of the  claims 1  to  4 , characterized in that the salt in step c) is selected from the group consisting of sodium chloride, sodium sulphate, potassium chloride, potassium sulfate, salts of citric acid, salts of phosphoric acid, and mixtures of these components. 
   
   
       6 . Method according to any one of the  claims 1  to  5 , characterized in that the immunoglobulin is a member of the immunoglobulin class G or E. 
   
   
       7 . Method according to any one of the  claims 1  to  6 , characterized in that the solution in the recovering step c) has a pH value of from pH 3.0 to pH 7.0. 
   
   
       8 . Method for determining the concentration of a salt for eluting a polypeptide from an ion exchange chromatography material in a single step purification process, comprising the following two steps
 a) step one comprising the following sub-steps
 a1) providing a solution comprising a polypeptide, a buffer substance, and optionally a salt; 
 a2) bringing a first aliquot of the solution containing the polypeptide and an ion exchange material in contact under conditions whereby the polypeptide binds to the ion exchange material; 
 a3) recovering the polypeptide from the ion exchange material by using a solution comprising a buffer substance and a salt whereby the concentration of the salt is increased linearly; 
 a4) determining the starting concentration of the salt where the first fraction of the polypeptide starts to elute from the ion exchange column; 
   b) step two comprising the following sub-steps
 b1) bringing a second aliquot of the solution containing the polypeptide and an ion exchange material in contact under conditions whereby the polypeptide binds to the ion exchange material; 
 b2) recovering the polypeptide from the ion exchange material by using a three step elution method, whereby
 i) the salt concentration of the first elution step is calculated as the sum of
 the product of the starting concentration of the salt as determined in step a4) and the total number of monovalent cations different from hydrogen denoted in the molecular formula of the salt and 
 the product of the concentration of the buffer salt and the total number of monovalent cations different from hydrogen denoted in the molecular formula of the buffer salt; 
 
 ii) the salt concentration of the second elution step is the product of the salt concentration of the first elution step and a factor between 1.25 and 1.35; 
 iii) the salt concentration of the third elution step is the product of the salt concentration of the first elution step and a factor between 1.50 and 1.70; 
 
 whereby the factors of step ii) and iii) are determined as follows: at a starting concentration of between 10 mM and 40 mM the factors are 1.35 and 1.70 respectively, at a starting concentration of between 40 mM and 70 mM the factors are 1.30 and 1.60 respectively, and at a starting concentration of more than 70 mM the factors are 1.25 and 1.50 respectively. 
 b3) determining at which sub-step of the three step elution method of step b2) the polypeptide is eluted from the ion exchange column thereby determining the concentration of a salt for eluting a polypeptide from an ion exchange chromatography material in a single step purification process. 
   
   
   
       9 . Method according to  claim 8 , characterized in that the ion exchange chromatography material is a weak ion exchange chromatography material. 
   
   
       10 . Method according to any one of the  claims 8  to  9 , characterized in that the polypeptide is an immunoglobulin.