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 .- 35 . (canceled) 
     
     
         36 . A composition comprising Tocilizumab protein with mannose-5 glycostructure (M5) attached to Asn 297  of the Tocilizumab protein, wherein the fraction of M5 is in a range from 2.8% to 10% of the sum comprising M5, G(0), G(1), and G(2) oligosaccharide attached to Asn 297  of the Tocilizumab protein, wherein the fraction equals area % fraction determined in a liquid chromatography method. 
     
     
         37 . The composition according to  claim 36 , wherein the Tocilizumab protein with M5 attached thereto has been produced by a recombinant Chinese Hamster Ovary (CHO) cell. 
     
     
         38 . The composition according to  claim 37 , wherein the CHO cell is cultured in cell culture at an initial cell density of at least 10 5  cells/ml. 
     
     
         39 . The composition according to  claim 38 , wherein the initial cell density comprises 8−12×10 5  cells/ml. 
     
     
         40 . The composition according to  claim 37 , wherein the relative antibody concentration produced by the CHO cell is greater than 100% of the antibody concentration resulting from constant feeding of CHO cells. 
     
     
         41 . The composition according to  claim 37 , wherein the Tocilizumab protein is produced by a cell culture of CHO cells at a cultivation volume from 10,000 L-50,000 L. 
     
     
         42 . The composition according to  claim 36 , wherein the M5 fraction is in a range from 2.8 to 8%. 
     
     
         43 . The composition according to  claim 36 , wherein the M5 fraction is in a range from 2.8 to 6%.