FUCOSYLATION-DEFICIENT CELLS
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.