Methods Of Gene Amplification And Expression

Abstract

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.

What is claimed is: 
     
         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.