Early post-transfection isolation of cells (epic) for biologics production

Abstract

Provided herein are methods for selecting a population of cells expressing a target polypeptide. In some aspects, the disclosure provides methods for sorting and selecting populations of transfected host cells based on their early expression of a selectable polypeptide. In certain embodiments, the sorting is performed using fluorescence-activated cell sorting or magnetic-activated cell sorting based on the selectable polypeptide. Such selection methods can be further utilized to generate clonal populations of producer cells, e.g. for large-scale manufacturing of a target polypeptide of interest.

1 . A method of producing a population of producer cells expressing a target polypeptide, the method comprising:
 (a) transfecting host cells with one or more vectors that encode one or more mRNAs, wherein the one or more mRNAs encode a selectable polypeptide and the target polypeptide;   (b) isolating from the transfected host cells, within 2 to 15 days of transfection, a sub-population of early-expressing transfected host cells which express the selectable polypeptide; and   (c) expanding the sub-population of transfected host cells, thereby producing a population of producer cells expressing the target polypeptide.   
     
     
         2 . The method of  claim 1 , wherein steps (b) and (c) are each performed in drug-selection-free medium. 
     
     
         3 . The method of  claim 1 , further comprising isolating the target polypeptide from the population of producer cells. 
     
     
         4 . The method of  claim 1 , further comprising isolating one or more single transfected host cells from the expanded sub-population and culturing the one or more single transfected host cells to produce clonal populations of the one or more single transfected host cells. 
     
     
         5 . The method of  claim 4 , wherein at least one of the clonal populations of the one or more single transfected host cells yields a 2- to 30-fold improvement in production of the target polypeptide compared to that of a stable pool of transfected but uncloned host cells obtained at step (c). 
     
     
         6 . (canceled) 
     
     
         7 . (canceled) 
     
     
         8 . The method of  claim 1 , wherein the isolating in step (b) is performed between two and four days, two days, or three days after transfection. 
     
     
         9 - 11 . (canceled) 
     
     
         12 . The method of  claim 1 , wherein the expanding in step (c) is for between 4-31 days. 
     
     
         13 . (canceled) 
     
     
         14 . (canceled) 
     
     
         15 . The method of  claim 1 , wherein the isolating in step (b) employs magnetic activated cell sorting (MACS), fluorescence activated cell sorting (FACS), or ClonePix. 
     
     
         16 . The method of  claim 1 , wherein the selectable polypeptide is a FACS=selectable polypeptide and the isolating in step (b) employs FACS. 
     
     
         17 . The method of  claim 1 , wherein the target polypeptide and the selectable polypeptide form a fusion polypeptide. 
     
     
         18 . The method of  claim 1 , wherein the target polypeptide and the selectable polypeptide are encoded by a single multicistronic mRNA. 
     
     
         19 . The method of  claim 18 , wherein the multicistronic mRNA comprises a first open reading frame (ORF) that encodes the selectable polypeptide and a second ORF that encodes the target polypeptide, wherein the first ORF is 5′ to the second ORF. 
     
     
         20 . The method of  claim 19 , wherein the first ORF has a non-AUG start codon. 
     
     
         21 . The method of  claim 20 , wherein the non-AUG start codon is a UUG, GUG, or CUG in a Kozak consensus sequence. 
     
     
         22 . The method of  claim 19 , wherein the second ORF has an AUG start codon. 
     
     
         23 . The method of  claim 19 , wherein the ORF that encodes the selectable polypeptide is devoid of any AUG sequences. 
     
     
         24 . The method of  claim 1 , wherein the selectable polypeptide is CD52 or CD59. 
     
     
         25 . The method of  claim 1 , wherein the target polypeptide is a therapeutic agent, a secreted protein or an antibody or an Fc fusion protein. 
     
     
         26 . (canceled) 
     
     
         27 . (canceled) 
     
     
         28 . The method of  claim 1 , wherein the host cells are selected from the group consisting of CHO cells, HEK293 cells, and HeLa cells. 
     
     
         29 . A clonal population of transfected host cells that express a selectable polypeptide and a target polypeptide obtainable by the method of  claim 1 . 
     
     
         30 . (canceled)