Patent ID: 9574003
Date: 2017-02-21
CPC Classifications: C07K,C12N,C12Y

Claim:
1. A method of selecting a Chinese hamster ovary (CHO) cell having a zero fucose level useful as a host cell for expressing a recombinant protein, the method comprising: (a) introducing a genetic mutation into a coding region for a GDP-mannose 4,6-dehydratase (GMD) in a CHO cell of a population of CHO cells by contacting the cells with methotrexate (MTX), thereby generating a CHO cell with a mutated GMD protein, (b) contacting the population of CHO cells comprising at least one CHO cell with a mutated GMD protein with a non-toxic fucose binding agent for an amount of time that allows binding of the fucose binding agent to a fucose moiety on a cell membrane of one or more cells of the population of cells, wherein the amount of time does not allow killing of the cells; and (c) depleting from the population of cells comprising mutated cells, a subpopulation of one or more cells which bind the fucose binding agent, thereby selecting cells useful as host cells for expressing recombinant proteins, the selected cells having zero fucose content in the absence of external fucose, wherein the expressed mutated GMD protein lacks amino acids encoded by exons 3 and 4 or lacks amino acids encoded by exons 8 and 9, and further wherein the cells exhibit a stable fucose phenotype for more than 100 population doublings.