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 a vector that encodes 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, between two and four days after transfection, a highly positive transient expression sub-population of early-expressing transfected host cells which express the selectable polypeptide, wherein the highly positive transient sub-population of early-expressing transfected host cells comprises about the top 10 percent of transfected host cells in terms of their expression of the selectable polypeptide; and
(c) expanding the isolated highly positive transient expression sub-population of early-expressing transfected host cells, thereby producing a population of producer cells expressing the target polypeptide, 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 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.
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 obtained at step (c).
5. The method of claim 1 , wherein the isolating in step (b) is performed four days after transfection.
6. The method of claim 1 , wherein the isolating in step (b) is performed two days after transfection.
7. The method of claim 1 , wherein the isolating in step (b) is performed three days after transfection.
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 isolating in step (b) employs magnetic activated cell sorting (MACS), or fluorescence activated cell sorting (FACS).
10. The method of claim 1 , wherein the selectable polypeptide is a FACS-selectable polypeptide and the isolating in step (b) employs FACS.
11. The method of claim 1 , wherein the target polypeptide and the selectable polypeptide form a fusion polypeptide.
12. The method of any of claim 1 , wherein the target polypeptide and the selectable polypeptide are encoded by a single multicistronic mRNA.
13. The method of claim 12 , 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.
14. The method of claim 13 , wherein the first ORF has a non-AUG start codon.
15. The method of claim 14 , wherein the non-AUG start codon is a UUG, GUG, or CUG in a Kozak consensus sequence.
16. The method of claim 13 , wherein the second ORF has an AUG start codon.
17. The method of claim 13 , wherein the ORF that encodes the selectable polypeptide is devoid of any AUG sequences.
18. The method of claim 1 , wherein the selectable polypeptide is CD52 or CD59.
19. The method of claim 1 , wherein the target polypeptide is a therapeutic agent.
20. The method of claim 1 , wherein the target polypeptide is an antibody or an Fc fusion protein.
21. The method of claim 1 , wherein the host cells are selected from the group consisting of CHO cells, HEK293 cells, and HeLa cells.