Methods of gene amplification and expression
View Patent ↗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 highly expresses a polypeptide of interest, comprising:
(a) inoculating host cells in a perfusion bioreactor, wherein the host cells are transfected with (i) a nucleic acid sequence encoding a polypeptide of interest and (ii) a nucleic acid sequence encoding an amplifiable marker;
(b) culturing the host cells in the presence of an increasing amount of an amplification agent in the perfusion bioreactor to amplify the nucleic acid sequence encoding a polypeptide of interest in the host cells; and
(c) subcloning the host cells from (b) to produce a cell line that highly expresses a polypeptide of interest.
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 CHO cells.
6. The method of claim 1 wherein the polypeptide of interest is an antibody.
7. 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.
8. The method of claim 1 , wherein the host cells are derived from the same single clone.
9. The method of claim 1 , wherein the host cells are pooled multiple clones.
10. The method of claim 1 , wherein the host cells are bulk transfectants.
11. The method of claim 1 , wherein the amplification agent is methotrexate.