Methods for controlling the galactosylation profile of recombinantly-expressed proteins

Abstract

The present invention relates to methods for modulating the glycosylation profile of recombinantly-expressed proteins. In particular, the present invention relates to methods of controlling the galactosylation profile of recombinantly-expressed proteins by supplementing production medium, e.g., a hydrolysate-based or a chemically defined medium, with manganese and/or D-galactose

What is claimed is: 
     
         1 . A method for controlling the galactosylation profile of a recombinantly-expressed protein comprising supplementing a production media used in the recombinant expression of said protein with a manganese supplement and/or a galactose supplement. 
     
     
         2 . The method of  claim 1  wherein the recombinantly-expressed protein is an antibody. 
     
     
         3 . The method of  claim 2  wherein the antibody is an anti-TNFα antibody. 
     
     
         4 . The method of  claim 3  wherein the anti-TNFα antibody is adalimumab. 
     
     
         5 . The method of  claim 1  wherein the manganese supplement is a biologically-acceptable manganese salt. 
     
     
         6 . The method of  claim 5  wherein the biologically-acceptable manganese salt is manganese (II) chloride. 
     
     
         7 . The method of  claim 1  wherein galactose supplement can take the form of any biologically-acceptable galactose-containing compound. 
     
     
         8 . The method of  claim 7  wherein the biologically-acceptable galactose-containing compound is D-(+)-galactose. 
     
     
         9 . The method of  claim 1  wherein the production media is supplemented with a sufficient amount of a manganese and/or a manganese-containing supplement to achieve a manganese concentration selected from 0-100 μM. 
     
     
         10 . The method of  claim 9  wherein the production media is supplemented with a sufficient amount of a manganese and/or a manganese-containing supplement to achieve a manganese concentration selected from the following: 0.1, 0.2, 0.5, 1.0, 10, 20, 25, 40, 50, 60, 75, 80, and 100 μM. 
     
     
         11 . The method of  claim 1  wherein the production media is supplemented with a sufficient amount of a galactose and/or galactose-containing supplement to achieve a galactose concentration selected from the following: 0-100 mM. 
     
     
         12 . The method of  claim 11  wherein the production media is supplemented with a sufficient amount of a galactose and/or galactose-containing supplement to achieve a galactose concentration selected from the following: 1, 4, 5, 10, 15, 20, 30, 40, 60, and 100 mM. 
     
     
         13 . The method of  claim 1  wherein the production media is supplemented with a sufficient amount of a manganese and/or a manganese-containing supplement and galactose and/or galactose-containing supplement to achieve manganese and galactose concentrations selected from 0-100 μM manganese and 0-100 mM galactose. 
     
     
         14 . The method of  claim 13  wherein the production media is supplemented with a sufficient amount of a manganese and/or a manganese-containing supplement and galactose and/or galactose-containing supplement to achieve a manganese (Mn) and a galactose (Gal) concentration selected from the following presented as Mn (μM)/Gal (mM): 0/1, 0/4, 0/5, 0/10, 0/15, 0/20, 0/30, 0/40, 0/60, 0/100, 0.1/0, 0.2/0, 0.5/0, 1.0/0, 10/0, 20/0, 25/0, 40/0, 50/0, 75/0, 80/0, 100/0, 0.2/1, 0.2/4, 0.2/30, 0.5/1, 0.5/4, 0.5/30, 10/10, 10/20, 10/40, 20/10, 20/20, 20/40, 25/15, 40/10, 40/20, 40/40, 40/100, 50/30, 60/20, 60/40, 60/100, 80/20, 80/40, 80/100, 100/20, 100/40, 100/100. 
     
     
         15 . The method of  claim 14  wherein the production media is supplemented with a sufficient amount of a manganese and/or a manganese-containing supplement and galactose and/or galactose-containing supplement to achieve a manganese (Mn) and a galactose (Gal) concentration selected from the following presented as Mn (μM)/Gal (mM): 0.2/1, 0.2/4, 0.2/30, 0.5/1, 0.5/4, 0.5/30, 10/10, 10/20, 10/40, 20/10, 20/20, 20/40, 25/15, 40/10, 40/20, 40/40, 40/100, 50/30, 60/20, 60/40, 60/100, 80/20, 80/40, 80/100, 100/20, 100/40, 100/100.