Preparation of functional human factor VIII in mammalian cells using methotrexate based selection
AssigneeGenentech, Inc.
InventorsDaniel J. Capon, Richard M. Lawn, Arthur D. Levinson, Gordon A. Vehar, William I. Wood
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Abstract
A method for producing factor VIII in recombinant mammalian host cells utilizing an expression vector containing a selectable marker DNA and an amplifiable marker DNA. The initial selection is based upon the selectable marker and subsequent amplification of factor VIII DNA and amplifiable marker DNA is conducted in cells not deficient in the amplifiable marker.
Claims13
A process for producing factor VIII which comprises:
- (a) cotransfecting a mammalian host cell with a DNA sequence encoding factor VIII, a second DNA sequence encoding an amplifiable marker, and a third DNA sequence encoding a selectable marker;
- (b) growing the transfected cell in a non-selective medium;
- (c) transferring the transfected cells to a selection medium and selecting for such selectable marker resistant cells;
- and
- (d) amplifying said amplifiable marker DNA sequence by culturing the selected cells of step (c) in media containing increasing amounts of selection agent, wherein the host cell is not deficient in the amplifiable marker.
A mammalian host cell cotransfected with a DNA sequence encoding factor VIII, a second DNA sequence encoding an amplifiable marker and a third DNA sequence encoding a selectable marker, wherein the host cell is not deficient in the amplifiable marker.
Dependencies are inferred from the claim text.