US 11,685,943 B2
Early post-transfection isolation of cells (EPIC) for biologics production
Victor R. Cairns, Bridgewater, NJ (US); Christine DeMaria, Bridgewater, NJ (US); and Jason Vitko, Bridgewater, NJ (US)
Assigned to GENZYME CORPORATION, Cambridge, MA (US)
Filed by Genzyme Corporation, Cambridge, MA (US)
Filed on Nov. 14, 2019, as Appl. No. 16/683,837.
Application 16/683,837 is a continuation of application No. 15/727,272, filed on Oct. 6, 2017, abandoned.
Claims priority of provisional application 62/405,392, filed on Oct. 7, 2016.
Prior Publication US 2020/0299743 A1, Sep. 24, 2020
Int. Cl. C12N 15/00 (2006.01); C12P 21/00 (2006.01); C12N 15/85 (2006.01); C07K 14/705 (2006.01); C07K 14/435 (2006.01); C12N 5/00 (2006.01); C12N 5/10 (2006.01); G01N 15/14 (2006.01)
CPC C12P 21/00 (2013.01) [C07K 14/435 (2013.01); C07K 14/43504 (2013.01); C07K 14/70592 (2013.01); C07K 14/70596 (2013.01); C12N 5/0087 (2013.01); C12N 5/10 (2013.01); C12N 15/85 (2013.01); G01N 15/14 (2013.01); G01N 2015/149 (2013.01)] 21 Claims
 
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.