Fucosylation-deficient cells

Abstract

An isolated nucleic acid encoding an FX protein having a serine at position 79, a lysine at position 90, a leucine at position 136, an arginine at position 211, a serine at position 289, and a combination thereof is provided. Cells having a gene encoding a modified FX protein are provided, wherein the cells exhibit a reduced ability to fucosylate a glycoprotein at a first temperature, but exhibit the ability to fucosylate the glycoprotein at a second temperature. Methods and compositions for making glycoproteins with reduced fucosylation are provided.

1 .- 15 . (canceled) 
     
     
         16 . A method of culturing cells comprising:
 a) providing mammalian cells which
 (i) comprise a salvage fucosylation pathway, 
 (ii) comprise a de novo fucosylation synthesis pathway characterized by a modified GDP-4-keto-6-deoxy-mannose-3,5-epimerase-4-reductase (FX) protein, and 
 (iii) comprise and ectopically express a polynucleotide encoding an Fc-containing glycoprotein, 
 wherein the cells produce the Fc-containing glycoprotein in a fucosylated form when the cells are cultured in media comprising external fucose, and 
 wherein the cells produce the Fc-containing glycoprotein in a reduced fucosylated form when compared to a mammalian cell comprising an unmodified Fx protein; and 
   b) culturing the mammalian cells in a cell culture medium that:
 (i) comprises external fucose; or 
 (ii) is devoid of external fucose. 
   
     
     
         17 . The method of claim  1 , further comprising step c) isolating the Fc-containing glycoprotein from the culture of step b)(i) or step b)(ii). 
     
     
         18 . The method of  claim 16 , wherein the external fucose is L-fucose. 
     
     
         19 . The method of  claim 16 , wherein said culturing the cells of step b)(i) comprises supplementing cell culture medium with 5 mM fucose or 10 mM fucose. 
     
     
         20 . The method of  claim 16 , wherein the mammalian cells are selected from the group consisting of a COS, Chinese Hamster Ovary (CHO), 293, BHK, HeLa and Vero cells. 
     
     
         21 . The method of  claim 20 , wherein the mammalian cells are CHO cells. 
     
     
         22 . The method of  claim 16 , wherein the Fc-containing glycoprotein is an antibody. 
     
     
         23 . The method of  claim 16 , wherein the modified FX protein comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 1. 
     
     
         24 . The method of  claim 23 , wherein the modified FX protein comprises amino acid substitution L289S. 
     
     
         25 . The method of  claim 24 , wherein the modified FX protein further comprises at least one additional amino acid substitution selected from the group consisting of N90K, N79S, P136L and G211R. 
     
     
         26 . The method of  claim 24 , wherein the modified FX protein comprises amino acid substitutions L289S and N90K. 
     
     
         27 . The method of  claim 16 , further comprising detecting the amount of fucosylated Fc-containing glycoprotein from the culture of step b)(i) or step b)(ii). 
     
     
         28 . The method of  claim 27 , wherein said detecting the amount of fucosylated Fc-containing glycoprotein comprises:
 isolating the Fc-containing glycoproteins from a sample of cells from the cell culture of step b)(i) or step b)(ii);   contacting the isolated glycoproteins with a label under conditions that permit the glycoproteins to bind the label; and   detecting the amount of labeled glycoprotein in the sample by Mass Spectrometry.   
     
     
         29 . The method of  claim 27 , wherein said detecting the amount of fucosylated Fc-containing glycoprotein comprises:
 isolating the Fc-containing glycoproteins from a sample of cells from the cell culture of step b)(i) or step b)(ii);   deglycosylating the glycoproteins to remove glycans from the glycoproteins; and   detecting the amount of fucosylated glycans in the sample by high performance liquid chromatography (HPLC).   
     
     
         30 . The method of  claim 27 , wherein said detecting the amount of fucosylated Fc-containing glycoprotein comprises:
 staining a sample of cells from the cell culture of step b)(i) or step b)(ii) with lectin; and   detecting the amount of stained cells in the sample to determine the amount of fucosylated glycoprotein in the sample.   
     
     
         31 . The method of  claim 27 , wherein at least 90% of said Fc-containing glycoproteins are fucosylated in step b)(i). 
     
     
         32 . The method of  claim 31 , wherein between 93% and 96% of said Fc-containing glycoproteins are fucosylated in step b)(i). 
     
     
         33 . The method of  claim 27 , wherein no more than 10% of the Fc-containing glycoproteins are fucosylated in step b)(ii). 
     
     
         34 . The method of  claim 17 , further comprising step d) determining the biological activity of the isolated Fc-containing glycoprotein. 
     
     
         35 . The method of  claim 16 , wherein said culturing further comprises culturing the cells in the cell medium at 37° C. for at least 3 days.