Method for the production of a glycosylated immunoglobulin

Abstract

Herein is reported a method for the production of an immunoglobulin comprising the following steps: a) providing a eukaryotic cell comprising a nucleic acid encoding the immunoglobulin, b) cultivating the eukaryotic cell in a cultivation medium wherein the amount of glucose available in the cultivation medium per time unit is kept constant and limited to less than 80% of the amount that could maximally be utilized by the cells in the cultivation medium per time unit, and c) recovering the immunoglobulin from the culture.

1 . Method for the production of an immunoglobulin comprising
 a) cultivating a eukaryotic cell comprising a nucleic acid encoding the immunoglobulin in a cultivation medium wherein the ratio of the current specific glucose consumption rate of a single cell in a cultivation medium to the maximum known specific glucose consumption rate of the kind of cell is kept constant, and   b) recovering the immunoglobulin from the culture.   
     
     
         2 . Method according to  claim 1 , wherein the ratio is from 0.8 to 0.2. 
     
     
         3 . Method according to  claim 2 , wherein the ratio is from 0.6 to 0.4. 
     
     
         4 . Method for the production of an immunoglobulin comprising
 a) cultivating a eukaryotic cell comprising a nucleic acid encoding the immunoglobulin in a cultivation medium wherein the amount of glucose available in the cultivation medium per time unit is kept constant and limited to a constant value of less than 80% of the amount that could maximally be utilized by the cells in the cultivation medium per time unit, and   b) recovering the immunoglobulin from the culture.   
     
     
         5 . Method according to  claim 4 , wherein the constant value is less than 80% and more than 20%. 
     
     
         6 . Method according to  claim 4 , wherein the cultivating is a fed-batch cultivating. 
     
     
         7 . Method according to  claim 6 , wherein the cultivating is a fed-batch cultivating wherein the feeding is started at day 2 or day 3 of the cultivating. 
     
     
         8 . Method according to  claim 1 , wherein the cultivating is at a pH value from pH 6.5 to 7.5. 
     
     
         9 . Method according to  claim 8 , wherein the cultivating is at a pH value from pH 6.9 to 7.3. 
     
     
         10 . Method according to  claim 9 , wherein the cultivating is at a pH value from pH 6.95 to pH 7.05 or at a pH value from pH 7.15 to pH 7.25. 
     
     
         11 . Method according to  claim 1 , wherein the immunoglobulin is an immunoglobulin of class G or class E. 
     
     
         12 . Method according to  claim 1 , wherein the eukaryotic cell is selected from the group consisting of CHO cells, NS0 cells, BHK cells, hybridoma cells, PER.C6® cells, insect cells, and Sp2/0 cells. 
     
     
         13 . Method according to  claim 12 , wherein the eukaryotic cell is a Chinese Hamster Ovary (CHO) cell. 
     
     
         14 . Method according to  claim 1 , wherein cultivating the eukaryotic cell is performed for six to twenty days. 
     
     
         15 . Method according to  claim 1 , wherein the immunoglobulin is an anti-IL-6R antibody. 
     
     
         16 . Use of a method according  claim 1 , for decreasing the amount of an immunoglobulin with a mannose-5 glycostructure (M5) content in an immunoglobulin preparation. 
     
     
         17 . Composition comprising an immunoglobulin, characterized in that the composition has been prepared by a method according to  claim 1 . 
     
     
         18 . The composition according to  claim 17 , wherein the immunoglobulin is an anti-IL-6R antibody. 
     
     
         19 . The composition according to  claim 18 , wherein the anti-IL-6R antibody comprises Tocilizumab. 
     
     
         20 . The composition according to  claim 18 , wherein the mannose-5 glycostructure (M5) content in the composition is 8% or less. 
     
     
         21 . The composition according to  claim 20 , wherein the M5 content is 6% or less. 
     
     
         22 . The composition according to  claim 21 , wherein the M5 content is 4% or less. 
     
     
         23 . A composition comprising an antibody that binds human interleukin 6 receptor (anti-IL-6R antibody) with oligosaccharide attached thereto, wherein mannose-5 glycostructure (M5) content in the composition is 8% or less. 
     
     
         24 . The composition according to  claim 23 , wherein the anti-IL-6R antibody is Tocilizumab. 
     
     
         25 . The composition according to  claim 23 , wherein the anti-IL-6R antibody has been produced by a recombinant Chinese Hamster Ovary (CHO) cell. 
     
     
         26 . The composition according to  claim 23 , wherein the M5 content is less than 5%. 
     
     
         27 . The composition according to  claim 26 , wherein the M5 content is 4% or less.