Expression vector organization, novel production cell generation methods and their use for the recombinant production of polypeptides
AssigneeHoffmann-La Roche Inc.
InventorsPeter Michael Huelsmann, Hendrik Knoetgen
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Abstract
Herein is reported an expression vector comprising—an antibody light chain expression cassette,—an antibody heavy chain expression cassette, and—a selection marker expression cassette, wherein the expression cassettes are arranged unidirectional, and wherein the expression cassettes are arranged in the 5′ to 3′ sequence of antibody heavy chain expression cassette, antibody light chain expression cassette and selection marker expression cassette. Further are reported herein methods for the generation of antibody producing cells and the use of these cells for the recombinant production of antibodies.
Claims65
1-52. (canceled)
An expression vector comprising:
- a) an antibody light chain expression cassette;
- b) an antibody heavy chain expression cassette;
- and
- c) a selection marker expression cassette,
- wherein the expression cassettes are arranged unidirectional, and
- wherein the expression cassettes are arranged in the 5′ to 3′ sequence of antibody heavy chain expression cassette, antibody heavy chain expression cassette, antibody light chain expression cassette and selection marker expression cassette.
A method for the transfection of a eukaryotic cell with an expression vector, said method comprising using an expression vector that is linearized prior to transfection by cleavage in the prokaryotic origin of replication.
A method for the selection of a eukaryotic cell said method adding a selection agent comprising:
- a nucleic acid encoding an antibody, to the cultivation medium for the first time about 24 hours after transfection, wherein a nucleic acid encoding an antibody is located.
A method for the production of an antibody said method comprising the following step:
- a) cultivating a eukaryotic cell, which has been obtained by the transfection with an expression vector encoding an antibody that has been linearized prior to the transfection by cleavage in the prokaryotic origin of replication;
- and
- b) recovering the antibody from the cell or the cultivation medium.
A method for the transfection of a eukaryotic cell with an expression vector encoding an antibody, said method comprising:
- use prokaryotic and eukaryotic nucleic acid sequences, wherein the prokaryotic nucleic acid sequences are removed from the expression vector prior to the transfection of the eukaryotic cell with the expression vector.
A method for transfecting an eukaryotic cell, said method comprising using a linearized expression vector encoding an antibody with no prokaryotic nucleic acid sequences.
A method for the recombinant production of an antibody, said method comprising using an expression vector encoding an antibody with only eukaryotic nucleic acid sequences for the generation of a eukaryotic cell.
The expression vector wherein the expression vector comprises:
- a) an antibody light chain expression cassette;
- b) a first antibody heavy chain expression cassette;
- c) a second antibody heavy chain expression cassette;
- and
- d) a selection marker expression cassette,
- wherein at least one of the antibody heavy chain expression cassettes and the antibody light chain expression cassette and the selection marker expression cassette are arranged unidirectional;
- and
- wherein the unidirectional expression cassettes are arranged in the 5′ and 3′ sequence of antibody heavy chain expression cassette, antibody light chain expression cassette and selection marker expression cassette.
The expression vector according to claim 1, wherein the expression vector comprises:
- a) a first antibody light chain expression cassette;
- b) a second antibody light chain expression cassette;
- c) a first antibody heavy chain expression cassette;
- d) a second antibody heavy chain expression cassette;
- and
- e) a selection marker expression cassette,
- wherein at least one of the antibody heavy chain expression cassettes and the antibody light chain expression cassette and the selection marker expression cassette are arranged unidirectional;
- and
- wherein the unidirectional expression cassettes are arranged in the 5′ and 3′ sequence of antibody heavy chain expression cassette, antibody light chain expression cassette and selection marker expression cassette.
Dependencies are inferred from the claim text.