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 for detecting cells with a reversible ability to fucosylate glycoproteins comprising:
a) culturing mammalian cells in the absence of external fucose, wherein the cells comprise a nucleic acid encoding a modified fucosylation pathway enzyme, and wherein the cells express a glycoprotein;
b) detecting the amount of fucosylated glycoprotein in the cell culture of step a);
c) culturing the cells of step a) in the presence of external fucose;
d) detecting the amount of fucosylated glycoprotein the cell culture of step c); and
e) comparing the amount of fucosylated glycoprotein detected in the cell culture of step b) and the cell culture of step d), wherein cells from step a) that exhibit a reduced amount of fucosylated glycoprotein in the cell culture when compared to the cells from step d) are cells with a reversible ability to fucosylate glycoprotein.
17 . The method of claim 16 , wherein the external fucose is L-fucose.
18 . 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, Vero.
19 . The method of claim 18 , wherein the mammalian cells are CHO cells.
20 . The method of claim 16 , wherein the glycoprotein is an antibody.
21 . The method of claim 16 , wherein the modified fucosylation pathway enzyme is a modified GDP-4-keto-6-deoxy-mannose-3,5-epimerase-4-reductase (FX) protein.
22 . The method of claim 21 , wherein the modified FX protein comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 1
23 . The method of claim 22 , wherein the modified FX protein comprises amino acid substitution L289S.
24 . The method of claim 23 , 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.
25 . The method of claim 24 , wherein the modified FX protein comprises amino acid substitutions L289S and N90K.
26 . The method of claim 16 , wherein said detecting the amount of fucosylated glycoprotein comprises:
isolating the glycoproteins from a sample of cells from the cell culture of step a) or the cell culture of step c);
contacting the 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.
27 . The method of claim 16 , wherein said detecting the amount of fucosylated glycoprotein comprises:
staining a sample of cells from the cell culture of step a) or the cell culture of step c); with lectin; and
detecting the amount of stained cells in the sample to determine the amount of fucosylated glycoprotein in the sample.
28 . The method of claim 27 , wherein the lectin is biotinylated, and said staining further comprises contacting the cells with fluorescently-labeled streptavidin.
29 . The method of claim 28 , wherein the lectin is Lens culinaris agglutinin (LCA).
30 . The method of claim 16 , wherein said detecting the amount of fucosylated glycoprotein comprises:
isolating the glycoprotein from a sample of the cell culture of step a) or the cell culture of step c);
deglycosylating the glycoprotein to remove glycans from the protein; and
detecting the amount of fucosyl-containing glycans in the sample by high performance liquid chromatography (HPLC).
31 . The method of claim 16 , further comprising:
e) modulating the amount of fucosylated glycoprotein by controlling the amount of the external fucose present in the cell culture of step c).
32 . The method of claim 16 , wherein no more than 10% of said glycoprotein is fucosylated in step b).
33 . The method of claim 32 , wherein between 1% and 6% of said glycoprotein is fucosylated.
34 . The method of claim 32 , wherein at least 90% of said glycoprotein is fucosylated in step d).
35 . The method of claim 34 , wherein between 93% and 96% of said glycoprotein is fucosylated in step d).
36 . The method of claim 16 , wherein said culturing the cells in the presence of external fucose of step c) comprises supplementing cell culture medium with 5 mM fucose or 10 mM fucose.
37 . The method of claim 36 , wherein said culturing further comprises culturing the cells at 37° C. for at least 3 days.
38 . A method of determining the level of fucosylation of a glycoprotein by cells comprising:
a) culturing the cells in the absence of external fucose, wherein the cells comprise a modified fucosylation pathway enzyme, and wherein the cells express a glycoprotein;
b) detecting the amount of fucosylated glycoprotein in the cell culture of step a);
c) culturing the cells of step a) in the presence of external fucose;
d) detecting the amount of fucosylated glycoprotein in the cell culture of step c); and
e) determining whether the level of fucosylation of the glycoprotein is reduced in the absence of external fucose.
39 . The method of claim 38 , wherein the external fucose is L-fucose.
40 . The method of claim 38 , wherein the cells are mammalian cells.
41 . The method of claim 40 , wherein the mammalian cells are CHO cells.
42 . The method of claim 38 , wherein the glycoprotein is an antibody.
43 . The method of claim 38 , wherein the modified fucosylation pathway enzyme is a modified GDP-4-keto-6-deoxy-mannose-3,5-epimerase-4-reductase (FX) protein
44 . The method of claim 43 , wherein the modified FX protein comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 1
45 . The method of claim 44 , wherein the modified FX protein comprises amino acid substitution L289S.
46 . The method of claim 45 , 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.
47 . The method of claim 46 , wherein the modified FX protein comprises amino acid substitutions L289S and N90K.
48 . The method of claim 38 , wherein said detecting the amount of fucosylated glycoprotein comprises:
isolating the glycoproteins from a sample of cells from the cell culture of step a) or the cell culture of step c);
contacting the 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.
49 . The method of claim 38 , wherein said detecting the amount of fucosylated glycoprotein comprises:
staining a sample of cells from the cell culture of step a) or the cell culture of step c); with lectin; and
detecting the amount of stained cells in the sample to determine the amount of fucosylated glycoprotein in the sample.
50 . The method of claim 49 , wherein the lectin is biotinylated, and said staining further comprises contacting the cells with fluorescently-labeled streptavidin.
51 . The method of claim 50 , wherein the lectin is Lens culinaris agglutinin (LCA).
52 . The method of claim 38 , wherein said detecting the amount of fucosylated glycoprotein comprises:
isolating the glycoprotein from a sample of the cell culture of step a) or the cell culture of step c);
deglycosylating the glycoprotein to remove glycans from the protein; and
detecting the amount of fucosyl-containing glycans in the sample by high performance liquid chromatography (HPLC).
53 . The method of claim 38 , wherein said determining whether the level of fucosylation is reduced comprises comparing the amount of fucosylated glycoprotein detected in step b) to the amount of fucosylated glycoprotein detected in step d).
54 . The method of claim 38 , wherein no more than 10% of said glycoprotein is fucosylated in step b).
55 . The method of claim 54 , wherein between 1% and 6% of said glycoprotein is fucosylated.
56 . The method of claim 55 , wherein at least 90% of said glycoprotein is fucosylated in step d).
57 . The method of claim 56 , wherein between 93% and 96% of said glycoprotein is fucosylated in step d).
58 . The method of claim 38 , wherein said culturing the cells in the presence of external fucose of step c) comprises supplementing cell culture medium with 5 mM fucose or 10 mM fucose.
59 . The method of claim 58 , wherein said culturing further comprises culturing the cells at 37° C. for at least 3 days.