Genomically modified cell neutralized to serum-free system
Development of a host cell capable of producing a glycoprotein composition such as an antibody composition which is useful in development of medicaments is desired. The present invention provides a cell in which a genomic gene encoding an enzyme relating to a sugar chain modification in which 1-position of fucose is bound to 6-position of N-acetylglucosamine in the reducing end through α-bond in a complex type N-glycoside-linked sugar chain is knocked out, wherein the cell is naturalized in a serum-free medium and a process for producing a glycoprotein composition using the cell.
1 . A cell in which a genomic gene encoding an enzyme relating to a sugar chain modification in which 1-position of fucose is bound to 6-position of N-acetylglucosamine in the reducing end through α-bond in a complex type N-glycoside-linked sugar chain is knocked out, wherein the cell is naturalized in a serum-free medium.
2 . The cell according to claim 1 , wherein all of alleles on a genome encoding an enzyme relating to modification of a sugar chain in which 1-position of fucose is bound to 6-position of N-acetylglucosamine in the reducing end through α-bond in a complex type N-glycoside-linked sugar chain are knocked out, and wherein the cell is naturalized in a serum-free medium.
3 . The cell according to claim 1 , wherein an exon region containing an initiation codon of the genomic gene encoding an enzyme relating to modification of a sugar chain in which 1-position of fucose is bound to 6-position of N-acetylglucosamine in the reducing end through α-bond in a complex type N-glycoside-linked sugar chain is deleted, and wherein the cell is naturalized in a serum-free medium.
4 . The cell according to claim 1 , wherein the enzyme relating to modification of a sugar chain in which 1-position of fucose is bound to 6-position of N-acetylglucosamine in the reducing end through α-bond in a complex type N-glycoside-linked sugar chain is α-1,6-fucosyltransferase.
5 . The cell according to claim 4 , wherein the α-1,6-fucosyltransferase is a protein encoded by a DNA selected from the following (a) or (b):
(a) a DNA comprising the nucleotide sequence represented by SEQ ID NO:1;
(b) a DNA which hybridizes with a DNA consisting of the nucleotide sequence represented by SEQ ID NO:1 under stringent conditions and encodes a protein having α-1,6-fucosyltransferase activity.
6 . The cell according to claim 4 , wherein the α-1,6-fucosyltransferase is a protein selected from the group consisting of the following (a), (b) and (c):
(a) a protein comprising the amino acid sequence represented by SEQ ID NO:5;
(b) a protein consisting of an amino acid sequence in which one or more amino acid(s) is/are deleted, substituted, inserted and/or added in the amino acid sequence represented by SEQ ID NO:5 and having α-1,6-fucosyltransferase activity;
(c) a protein consisting of an amino acid sequence which has at least 80% amino acid sequence homology to the amino acid sequence represented by SEQ ID NO:5 and having α-1,6-fucosyltransferase activity.
7 . The cell according to claim 1 , which is resistant to a lectin which recognizes a sugar chain structure in which 1-position of fucose is bound to 6-position of N-acetylglucosamine in the reducing end through α-bond in a complex type N-glycoside-linked sugar chain.
8 . The cell according to claim 7 , wherein said resistance is resistance in which the cell survives at a higher ratio than a cell in which the genomic gene has not been knocked out when the cells are cultured in a medium containing the lectin which recognizes a sugar chain structure in which 1-position of fucose is bound to 6-position of N-acetylglucosamine in the reducing end through α-bond in a complex type N-glycoside-linked sugar chain.
9 . The cell according to claim 1 , wherein the serum-free medium is a protein-free medium.
10 . The cell according to claim 1 , which comprises a gene encoding a glycoprotein.
11 . The cell according to claim 10 , wherein the glycoprotein is a glycoprotein having no sugar chain structure in which 1-position of fucose is bound to 6-position of N-acetylglucosamine in the reducing end through α-bond in a complex type N-glycoside-linked sugar chain.
12 . The cell according to claim 10 , wherein the glycoprotein is an antibody.
13 . The cell according to claim 12 , wherein the antibody belongs to an IgG class.
14 . A process for producing a glycoprotein composition, which comprises using the cell according to claim 1 .
15 . A process for producing a glycoprotein composition, which comprises culturing the cell according to claim 1 in a medium to form and accumulate the glycoprotein composition in the culture, and recovering and purifying the glycoprotein composition from the culture.
16 . The process for producing a glycoprotein composition according to claim 14 or 15 , wherein the process is carried out by batch culture, fed-batch culture or perfusion culture.
17 . The process according to claim 14 or 15 , wherein at least one selected from a nutrient factor and a physiologically active substance is added to the medium during culturing.
18 . The process according to claim 17 , wherein the nutrient factor is at least one selected from a glucose, an amino acid and a vitamin.
19 . The process according to claim 17 , wherein the physiologically active substance is at least one selected from an insulin, an insulin-like growth factor, transferrin and albumin.
20 . The process according to claim 14 or 15 , wherein the glycoprotein composition is an antibody composition.
21 . A method for naturalizing a cell in which a genomic gene encoding an enzyme relating to a sugar chain modification in which 1-position of fucose is bound to 6-position of N-acetylglucosamine in the reducing end through α-bond in a complex type N-glycoside-linked sugar chain is knocked out in a serum-free medium, which comprises inoculating the cell into a medium for naturalization to give a cell density of 1×10 5 to 1×10 6 cells/ml.
22 . A method for obtaining a clone in which a genomic gene encoding an enzyme relating to a sugar chain modification in which 1-position of fucose is bound to 6-position of N-acetylglucosamine in the reducing end through α-bond in a complex type N-glycoside-linked sugar chain is knocked out, which comprises naturalizing the cell in a serum-free medium by the method according to claim 21 , and then cloning the cell.
23 . A cell in which a genomic gene encoding an enzyme relating to a sugar chain modification in which 1-position of fucose is bound to 6-position of N-acetylglucosamine in the reducing end through α-bond in a complex type N-glycoside-linked sugar chain is knocked out, wherein the cell is naturalized in a serum-free medium, which is obtainable by the method according to claim 21 .
24 . A clone in which a genomic gene encoding an enzyme relating to a sugar chain modification in which 1-position of fucose is bound to 6-position of N-acetylglucosamine in the reducing end through α-bond in a complex type N-glycoside-linked sugar chain is knocked out, wherein the clone is naturalized in a serum-free medium, which is obtainable by the method according to claim 22 .
25 . The method according to claim 21 or 22 , wherein the serum-free medium is a protein-free medium,
26 . The cell according to claim 23 , wherein the serum-free medium is a protein-free medium.
27 . The clone according to claim 24 , wherein the serum-free medium is a protein-free medium.