Method for increasing the galactose content of recombinant proteins

Abstract

The present invention relates to a method for increasing the galactose content of a recombinant protein produced in mammalian cells, wherein during the cultivation of said cells the pH of the cell culture is changed and a composition comprising nucleosides, transition metal salts and/or sugars is fed.

1 . A method for increasing the galactose content of a recombinant protein produced in mammalian cells, said method comprising:
 a) culturing mammalian cells transformed with at least one recombinant nucleic acid molecule encoding the recombinant protein in a cell culture medium at a first pH for a first period of time;   b) culturing said mammalian cells in said cell culture medium at a second pH which is different from the first pH for a second period of time; and   c) feeding a composition comprising at least two of the following components:
 (i) one or more nucleoside(s); 
 (ii) one or more transition metal salt(s); and 
 (iii) one or more sugar(s); 
    to the culture of (b).   
     
     
         2 . A method for producing a recombinant protein in mammalian cells, said method comprising:
 a) culturing mammalian cells transformed with at least one recombinant nucleic acid molecule encoding the recombinant protein in a cell culture medium at a first pH for a first period of time;   b) culturing said mammalian cells in said cell culture medium at a second pH which is different from the first pH for a second period of time; and   c) feeding a composition comprising at least two of the following components:
 (i) one or more nucleoside(s); 
 (ii) one or more transition metal salt(s); and 
 (iii) one or more sugar(s) 
    to the culture of (b);   d) harvesting the cell culture fluid comprising the recombinant protein; and   e) obtaining the recombinant protein.   
     
     
         3 . The method according to  claim 2 , wherein the recombinant protein is produced at a large scale. 
     
     
         4 . The method according to  claim 1 , wherein the mammalian cells are Chinese hamster ovary cells. 
     
     
         5 . The method according to  claim 1 , wherein the recombinant protein is an Fc-containing protein. 
     
     
         6 . The method according to  claim 1 , wherein the second pH is lower than the first pH. 
     
     
         7 . The method according to  claim 6 , wherein the second pH is 0.05 to 0.3 pH units lower than the first pH. 
     
     
         8 . The method according to  claim 1 , wherein the nucleoside is uridine. 
     
     
         9 . The method according to  claim 8 , wherein the concentration of uridine within the composition is 1 to 20 mM. 
     
     
         10 . The method according to  claim 1 , wherein the transition metal salt is manganese (II) chloride. 
     
     
         11 . The method according to  claim 10 , wherein the concentration of manganese (II) chloride within the composition is 0.002 mM to 0.1 mM. 
     
     
         12 . The method according to  claim 1 , wherein the sugar is galactose. 
     
     
         13 . The method according to  claim 12 , wherein the concentration of galactose within the composition is 5 mM to 100 mM. 
     
     
         14 . A method for producing a rituximab biosimilar antibody in Chinese hamster ovary cells, said method comprising:
 a) culturing Chinese hamster ovary cells transformed with one or more recombinant nucleic acid molecules encoding the light and the heavy chain of the antibody in a cell culture medium at a pH of 7.15 for a first period of time;   b) culturing said Chinese hamster ovary cells in a cell culture medium at a pH of 7.00 for a second period of time;   c) feeding a composition comprising the following components:
 (i) 1 to 20 mM uridine; 
 (ii) 0.002 mM to 0.1 mM manganese (II) chloride; and 
 (iii) 5 mM to 100 mM galactose 
    to the culture of (b);   d) harvesting the cell culture fluid comprising the rituximab; and   e) obtaining the rituximab.   
     
     
         15 . A method for improving the biosimilarity of a therapeutic antibody produced by Chinese hamster ovary cells to its reference antibody, said method comprising the steps of:
 a) culturing Chinese hamster ovary cells transformed with one or more recombinant nucleic acid molecules encoding the light and the heavy chain of the therapeutic antibody in a cell culture medium at a pH of 7.15 for a first period of time;   b) culturing said Chinese hamster ovary cells in said cell culture medium at a pH of 7.00 for a second period of time; and   c) feeding a composition comprising the following components:
 (i) uridine; 
 (ii) manganese (II) chloride; and 
 (iii) galactose 
    to the culture of (b).   
     
     
         16 . The method according to  claim 1 , wherein the cells are cultured at the first pH until the viable cell density is 4.5 to 6.0×10 6  cells/ml. 
     
     
         17 . The method according to  claim 1 , wherein the cells are cultured at the second pH for 6 to 7 days. 
     
     
         18 . The method according to  claim 1 , wherein the temperature is kept constant during steps (a), (b) and (c). 
     
     
         19 . The method according to  claim 1 , wherein the composition further contains at least one amino acid selected from the group consisting of L-valine, L-cysteine, L-phenylalanine and L-serine. 
     
     
         20 . The method according to  claim 1 , wherein the feeding of step (c) is performed at least twice. 
     
     
         21 . The method according to  claim 1 , wherein the feeding of step (c) is preceded by a feeding step with a composition to which the components (i) and (iii) have not been added. 
     
     
         22 . The method according to  claim 1 , wherein the culture medium in steps (a) and (b) does not contain uridine and galactose. 
     
     
         23 . The method according to  claim 1 , wherein the composition of step (c) does not contain one or more of thymidine, fructose, mannose, sucrose and N-acetylmannosamine. 
     
     
         24 . The method according to  claim 1 , wherein the osmolality of the culture in steps (a), (b) and (c) is lower than 400 mOsm/kg.