Methods Of Gene Amplification And Expression
Disclosed are methods relating to amplification and expression of a nucleic acid sequence encoding a polypeptide of interest in recombinant cells, and cell lines and polypeptides produced from such methods. The methods disclosed herein permit the amplification of cell lines that express a polypeptide of interest in a relatively short period of time through the use of a bioreactor.
1 . A method for producing a recombinant cell line that expresses a polypeptide of interest, comprising (a) culturing a host cell in a bioreactor in media, wherein the host cell is transfected with (i) a nucleic acid sequence encoding a polypeptide of interest and (ii) a nucleic acid sequence encoding an amplifiable marker; and (b) adding an increasing amount of an amplification agent to the media to produce the recombinant cell line.
2 . The method of claim 1 wherein the nucleic acid sequence encoding a polypeptide of interest and the nucleic acid sequence encoding an amplifiable marker are contained on a single vector.
3 . The method of claim 1 wherein the amplifiable marker encodes a DHFR protein.
4 . The method of claim 1 wherein the host cells are mammalian cells.
5 . The method of claim 1 wherein the host cells are Cl-JO cells.
6 . The method of claim 1 wherein the polypeptide of interest is an antibody.
7 . The method of claim 1 wherein the bioreactor is a perfusion bioreactor.
8 . The method of claim 1 wherein the increasing amount of the amplification agent is added to the media when % cell viability is increasing or stable.
9 . A method for producing a polypeptide of interest, which comprises culturing host cells transfected with (i) a nucleic acid sequence encoding a polypeptide of interest and (ii) a nucleic acid sequence encoding an amplifiable marker in a bioreactor in media containing increasing amounts of an amplification agent, thereby expressing the polypeptide of interest.
10 . The method of claim 9 wherein the nucleic acid sequence encoding a polypeptide of interest and the nucleic acid sequence encoding an amplifiable marker are contained on a single vector.
11 . The method of claim 9 wherein the amplifiable marker encodes a DHFR protein.
12 . The method of claim 9 wherein the host cells are mammalian cells.
13 . The method of claim 9 wherein the host cells are CHO cells.
14 . The method of claim 9 wherein the polypeptide of interest is an antibody.
15 . The method of claim 9 wherein the bioreactor is a perfusion bioreactor.
16 . The method of claim 9 wherein the amplification agent is added in increasing amounts to the media when % cell viability is increasing or stable.
17 . The method of claim 9 , wherein the host cells are derived from the same single clone.
18 . The method of claim 9 , wherein the host cells are pooled multiple clones.
19 . The method of claim 9 , wherein the host cells are bulk transfectants.