System and method for high-yield transient expression in mammalian cells

Abstract

High-yield mammalian transient expression systems can include a cell culture media (particularly serum free, non-animal derived, and/or chemically defined media) for introducing macromolecules and compounds (e.g., nucleic acid molecules) into cells (e.g., eukaryotic cells). Cells containing such introduced materials can then be cultured in the cell culture media. In particular, the invention allows introduction of nucleic acid molecules (e.g., vectors) into cells (particularly mammalian cells) and expression of proteins encoded by the nucleic acid molecules in the cells. The invention obviates the need to change the cell culture medium each time a different procedure is performed with the cells (e.g., culturing cells vs. transfecting cells). The invention also relates to compositions and kits useful for culturing and transforming/transfecting cells.

1 . A method for producing a recombinant protein is cultured eukaryotic cells, said method comprising:
 obtaining a suspension culture comprising cells in a high density culture medium, said suspension culture having a cell density of between about 2×10 6  to about 2×10 7  cells/ml;   transfecting said cells with an expressible nucleic acid comprising an expression vector containing a genetic sequence capable of producing an expressed protein;   incubating said transfected cells for a first period of time;   contacting said transfected cells with at least one expression enhancer composition;   incubating said transfected cells in the presence of said transfection enhancer for a second period of time such that said vector expresses said protein; and   harvesting said transfected cells after said second period of time.   
     
     
         2 . The method according to  claim 1 , wherein said cultured eukaryotic cells are a suspension culture adapted for growth under high density conditions. 
     
     
         3 . The method according to  claim 1 , wherein said suspension culture comprises 293 cells, a derivative of 293 cells, 293F cells, a derivative of 293F cells, PER-C6 cells, a derivative of PER-C6 cells, CHO cells, a derivative of CHO cells, CapT cells, a derivative of CapT cells, COS cells, a derivative of COS cells, COS-7 cells, a derivative of COS-7, Sp2/0 cells, or a derivative of Sp2/0 cells, wherein said cells have been adapted for growth under high density conditions. 
     
     
         4 . The method according to  claim 3 , wherein said suspension culture comprises a derivative of 293 F cells. 
     
     
         5 . The method according to  claim 1 , wherein the volume of the suspension culture is in the range of about 200 μL to about 5 L. 
     
     
         6 . The method according to  claim 1 , wherein said expression composition comprises at least one of valproic acid (VPA, acid and sodium salt), sodium propionate, lithium acetate, dimethyl sulfoxide (DMSO), sugars including galactose, amino acid mixtures, or butyric acid, or any combinations of the aforementioned. 
     
     
         7 . The method according to  claim 6 , wherein the expression composition comprises valproic acid, 
     
     
         8 . The method according to  claim 7 , wherein the concentration of valproic acid (VPA) is in the range of about 0.20 mM to about 25 mM. 
     
     
         9 . (canceled) 
     
     
         10 . The method according to  claim 6 , wherein the expression composition comprises sodium propionate. 
     
     
         11 . The method according to  claim 10 , wherein the final concentration of sodium propionate in the culture is in the range of about 0.2 mM to about 100 mM. 
     
     
         12 . (canceled) 
     
     
         13 . The method according to  claim 6 , wherein the expression composition comprises lithium acetate. 
     
     
         14 . The method according to  claim 13 , wherein the final concentration of LiAc in the culture is in the range of about 0.25 to about 25 mM. 
     
     
         15 . (canceled) 
     
     
         16 . The method according to  claim 6 , wherein the expression composition comprises butyric acid. 
     
     
         17 - 25 . (canceled) 
     
     
         26 . The method according to  claim 1 , wherein said method does not require replacement, replenishment, or supplementation of the high density culture medium following the transfection step. 
     
     
         27 . The method according to  claim 1 , wherein said high density culture medium is not replaced, replenished or supplemented following the transfection step. 
     
     
         28 . The method according to  claim 1 , wherein said high density culture medium is a serum-free/protein-free chemically defined culture medium capable of promoting the growth of transfected cells at cell densities in excess of 2.5×10 6  cells/ml with cell viability remaining in excess of 80%. 
     
     
         29 . (canceled) 
     
     
         30 . The method according to  claim 1 , further comprising harvesting said transfected cells after said second period of time. 
     
     
         31 . The method according to  claim 30 , further comprising purifying said expressed protein. 
     
     
         32 . The method according to  claim 1 , wherein said cells express one or more expression enhancing proteins. 
     
     
         33 . The method according to  claim 32 , wherein said expression enhancing proteins are selected from the list consisting of AKT, P18, P21, Bcl-X L , and PKBa. 
     
     
         34 - 37 . (canceled)