Afucosylation modification
AssigneeGenentech, Inc.
InventorsNatarajan Vijayasankaran, Gavin Christian Barnard, Jason Goodrick, Shahram Misaghi, Eleanor Oates
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Abstract
The present application relates to methods of producing a protein in fucosylated and afucosylated forms at a predetermined ratio.
Claims50
A method of producing a protein with a desired ratio of fucosylated to afucosylated glycan forms, the method comprising:
- (a) culturing a host cell capable of expressing a protein of interest in a culture medium under conditions suitable to express the protein of interest, wherein the host cell has been engineered to (i) produce the protein of interest, and (ii) to possess reduced or inactivated GDP-keto-6-deoxymannose-3,5-epimerase, 4-reductase (FX) activity compared to wild type,
- wherein the amount of FX activity in the host cell influences the ratio of fucosylated to afucosylated glycan forms of the expressed protein.
A method of producing a protein, wherein the protein is produced in fucosylated and afucosylated forms in a desired ratio, comprising:
- culturing a host cell capable of expressing a protein of interest in fucosylated and afucosylated forms in a desired or near-desired ratio in a culture medium, wherein the host cell has a partial knock-out of the FX gene, optionally wherein the near-desired ratio of fucosylated to afucosylated forms is adjusted to the desired ratio by increasing or decreasing the amount of fucose in the culture medium and adjusting the timing of the fucose addition.
The method according to any one of the preceding claims, wherein the cell engineered to possess reduced GDP-keto-6-deoxymannose-3,5-epimerase,4-reductase (FX) activity compared to wild type is capable of expressing the protein of interest.
The method of any one of the preceding claims, wherein the host cell capable of expressing a protein of interest has been produced by the method of claim 22.
The method according to any one of the preceding claims, wherein the culture medium comprises a fucose source.
The method according to any one of the preceding claims, wherein the cell is cultured in a fed-batch process, an intensified fed-batch process, a continuous or a semicontinuous process.
The method according to any one of the preceding claims, wherein the protein is an Fc-containing protein.
The method according to any one of the preceding claims, wherein the host cell is a eukaryotic cell, and/or wherein the host cell is a mammalian cell, in particular wherein the mammalian cell is a Chinese hamster ovary (CHO) cell.
The method according to any one of the preceding claims, further comprising purifying the protein from the culture medium.
The method according to any one of the preceding claims, wherein the host cell has been engineered to possess reduced FX activity compared to wild type by introducing a sequence deletion, a sequence addition or a sequence substitution into the FX gene in the host cell causing a partial knock out of the FX gene.
The method according to claim, wherein the sequence deletion, sequence addition or sequence substitution to the FX gene has been created using:
- (a) a clustered, regularly interspaced, short palindromic repeats (CRISPR) system;
- (b) a transcription activator-like effector nuclease (TALEN) system;
- (c) a zinc-finger nuclease (ZFN) system;
- (d) a transposase system;
- or (e) a meganuclease system.
A method of producing a cell capable of expressing a protein of interest, wherein the protein of interest can be produced in fucosylated and afucosylated forms in a desired ratio, comprising:
- (i) subjecting isolated host cells capable of expressing a protein of interest to gene editing to produce individual gene-edited cell clones with complete to partial knock-out of the FX gene;
- (ii) selecting the gene-edited cell which produces the protein of interest in fucosylated and afucosylated forms at the desired ratio;
- or
- optionally,
- (i) transfecting a clonal population of an isolated host cell that has been gene edited to have reduced FX activity compared to wild type with a polynucleotide sequence encoding a protein of interest, wherein the isolated host cell has been selected for a particular level of FX activity which results in an expressed protein being produced in a particular ratio of fucosylated and afucosylated forms;
- (ii) selecting a transfected cell clone which expresses the protein of interest in fucosylated and afucosylated forms at a desired ratio.
The method according to any one of the preceding claims, wherein the level of fucosylation or the ratio of fucosylated and afucosylated protein produced by a cell clone is determined using LCA-FITC staining and FACS profile analysis.
A method of producing a protein, wherein the protein is produced in fucosylated and afucosylated forms at a desired ratio, comprising
- (i) determining the period of time required for culturing a host cell having substantially no FX activity in a culture medium lacking a fucose source prior to culturing said host cell in a culture medium comprising a fucose source sufficient to produce fucosylated and afucosylated forms of the protein in a desired ratio;
- (ii) culturing the host cell having substantially no FX activity in a culture medium lacking a fucose source for the period of time determined in step (i);
- and
- (iii) culturing the host cell in a culture medium comprising a fucose source for a period of time sufficient to produce the protein in fucosylated and afucosylated forms at the desired ratio.
Dependencies are inferred from the claim text.