Use of metal ions for modulation of protein glycosylation profiles of recombinant proteins

Abstract

Protein glycosylation greatly influences the structure, function, and pharmacokinetics of recombinant proteins. Here, growth media supplemented with metal ions is shown to modulate the protein glycosylation profile of recombinant proteins expressed in a variety of eukaryotic cell lines. In particular, millimolar amounts of ferric salts (e.g. ferric nitrate, ferric citrate, etc.) significantly increased the percentage of galactosylated N-glycan species. The effect on protein glycosylation was concentration-dependent manner up to 1.0 mM across multiple cell lines without adverse effects on cell viability. The use of iron supplements in cell culture media provides an efficient and effective approach towards the specific re-targeting of N-glycan glycoform profiles of recombinantly expressed proteins.

1 . A method for modulating the glycosylation profile of a recombinant protein expressed in a eukaryotic cell, comprising:
 culturing a eukaryotic cell in a protein-free, chemically defined culture medium supplemented with an effective amount of a metal salt; and   expressing a recombinant protein in the eukaryotic cell,   wherein the amount of the metal salt is effective at modulating the glycosylation profile of the recombinant protein.   
     
     
         2 . The method for modulating the glycosylation profile of a recombinant protein according to  claim 1 , wherein the metal salt is an iron salt. 
     
     
         3 . The method for modulating the glycosylation profile of a recombinant protein according to  claim 2 , wherein the iron salt is selected from the group consisting of ferric nitrate, ferric citrate and ferrous sulfate. 
     
     
         4 . The method for modulating the glycosylation profile of a recombinant protein according to  claim 1 , wherein the effective amount of the metal salt is about 0.1 mM to about 1 mM. 
     
     
         5 . The method for modulating the glycosylation profile of a recombinant protein according to  claim 1 , wherein the chemically defined culture medium contains less than about 1 μM of metal ions. 
     
     
         6 . The method for modulating the glycosylation profile of a recombinant protein according to  claim 1 , wherein the eukaryotic cell is a mammalian cell. 
     
     
         7 . The method for modulating the glycosylation profile of a recombinant protein according to  claim 6 , wherein the mammalian cell is selected from the group consisting of Chinese hamster ovary (CHO) cells, CHO DUX-B 11 cells, mouse myeloma SP2/0, human HT-1080, NSO myeloma cell line, mouse L cells, mouse A9 cells, baby hamster kidney cells, C127 cells, COS cells and PC8 cells. 
     
     
         8 . The method for modulating the glycosylation profile of a recombinant protein according to  claim 1 , wherein the recombinant protein is an antibody, a fragment or variant thereof, or a fusion protein comprising an antibody, a fragment or variant thereof. 
     
     
         9 . The method for modulating the glycosylation profile of a recombinant protein according to  claim 8 , wherein the recombinant antibody is humanized. 
     
     
         10 . The method for modulating the glycosylation profile of a recombinant protein according to  claim 1 , wherein the addition of the metal ion to the chemically defined cell culture medium reduces NGA2F and NGA2F-GlcNAc glycosylation of the expressed recombinant protein. 
     
     
         11 . The method for modulating the glycosylation profile of a recombinant protein according to  claim 1 , wherein the addition of the metal ion to the chemically defined cell culture medium reduces NGA2F and NGA2F-GlcNAc glycosylation of the expressed recombinant protein by at least 15%. 
     
     
         12 . The method for modulating the glycosylation profile of a recombinant protein according to  claim 1 , wherein the addition of the metal ion to the chemically defined cell culture medium increases galactosylaton of the expressed recombinant glycoprotein. 
     
     
         13 . The method for modulating the glycosylation profile of a recombinant protein according to  claim 1 , wherein the addition of the metal ion to the chemically defined cell culture medium increases NA1F and NA2F glycosylation of the expressed recombinant protein. 
     
     
         14 . The method for modulating the glycosylation profile of a recombinant protein according to  claim 1 , wherein the addition of the metal ion to the chemically defined cell culture medium increases NA1F glycosylation of the expressed recombinant protein by at least about 4 fold. 
     
     
         15 . The method for modulating the glycosylation profile of a recombinant protein according to  claim 1 , wherein the addition of the metal ion to the chemically defined cell culture medium increases NA2F glycosylation of the expressed recombinant protein by at least about 7 fold. 
     
     
         16 . A cell culture medium for modulating the glycosylation profile of a recombinantly-expressed protein, comprising:
 a protein-free chemically defined culture medium supplemented with an effective amount of a metal salt effective at modulating the glycosylation profile of a recombinantly-expressed protein,   wherein the chemically defined culture medium does not contain ferric citrate.   
     
     
         17 . The cell culture medium according to  claim 16 , wherein the metal salt is an iron salt. 
     
     
         18 . The cell culture medium according to  claim 17 , wherein the iron salt is ferric nitrate. 
     
     
         19 . The cell culture medium according to  claim 16 , wherein the effective amount of the metal salt is about 0.1 mM to about 1 mM. 
     
     
         20 . The cell culture medium according to  claim 16 , wherein the recombinantly-expressed protein is an antibody, a fragment or variant thereof, or a fusion protein comprising an antibody, a fragment or variant thereof. 
     
     
         21 . An antibody or dual-variable-domain-immunoglobulin (DVD-Ig) produced by the process for modulating a glycosylation profile of  claim 1 . 
     
     
         22 . The antibody or dual-variable-domain-immunoglobulin (DVD-Ig) of  claim 21 , wherein the addition of the metal ion to the chemically defined cell culture medium reduces NGA2F and NGA2F-GlcNAc glycosylation of the expressed antibody or dual-variable-domain-immunoglobulin (DVD-Ig). 
     
     
         23 . The antibody or dual-variable-domain-immunoglobulin (DVD-Ig) of  claim 21 , wherein the addition of the metal ion to the chemically defined cell culture medium increases NA1F and NA2F glycosylation of the expressed antibody or dual-variable-domain-immunoglobulin (DVD-Ig). 
     
     
         24 . The antibody or dual-variable-domain-immunoglobulin (DVD-Ig) of  claim 21 , wherein the addition of the metal ion to the chemically defined cell culture medium increases galactosylaton of the expressed antibody or dual-variable-domain-immunoglobulin (DVD-Ig). 
     
     
         25 . An antibody or DVD-Ig molecule having a glycoprofile as disclosed herein.