Early post-transfection isolation of cells (EPIC) for biologics production
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, wherein the mRNA encoding the target polypeptide and the mRNA encoding the selectable polypeptide are both encoded on one vector;
(b) isolating from the transfected host cells, within 2 to 5 days of transfection, a sub-population of early-expressing transfected host cells which express the selectable polypeptide, wherein the isolating employs magnetic activated cell sorting (MACS) or fluorescence activated cell sorting (FACS) on the selectable polypeptide; and
(c) expanding the isolated sub-population of early-expressing transfected host cells, thereby producing a population of producer cells expressing the target polypeptide,
wherein the population of producer cells expresses an enhanced level of the target polypeptide compared to an unselected population of the transfected host cells, and
wherein steps (b) and (c) are each performed in drug-selection-free medium.
2. The method of claim 1 , further comprising isolating the target polypeptide from the population of producer cells.
3. The method of claim 1 , further comprising isolating one or more single transfected host cells from the population of producer cells expressing the target polypeptide and culturing the one or more single transfected host cells to produce clonal populations of the one or more single transfected host cells.
4. The method of claim 3 , 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 derived from the population of producer cells expressing the target polypeptide obtained at step (c).
5. The method of claim 1 , wherein the transfected host cells subject to selection in (b) contains at least 80-120×10 6 cells.
6. The method of claim 1 , wherein the isolating in step (b) is performed between two and four days after transfection.
7. The method of claim 1 , wherein the sub-population of transfected host cells contains 0.5-6.0×10 6 cells prior to expansion in step (c).
8. The method of claim 1 , wherein the expanding in step (c) is for between 4-31 days.
9. The method of claim 1 , wherein the selectable polypeptide is a FACS selectable polypeptide and the isolating in step (b) employs FACS.
10. The method of claim 1 , wherein the target polypeptide and the selectable polypeptide form a fusion polypeptide.
11. The method of claim 1 , wherein the target polypeptide and the selectable polypeptide are encoded by a single multicistronic mRNA.
12. The method of claim 11 , 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.
13. The method of claim 12 , wherein the first ORF has a non-AUG start codon.
14. The method of claim 12 , wherein the second ORF has an AUG start codon.
15. The method of claim 12 , wherein the ORF that encodes the selectable polypeptide is devoid of any AUG sequences.