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US 11,635,363 B2Granted
FLARE (flow cytometry attenuated reporter expression) technology for rapid bulk sorting
AssigneeGenzyme Corporation
InventorsVictor R. Cairns, Jose Ignacio Sancho Chavida, Christine DeMaria
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Abstract
Provided herein are methods and compositions for batch production of producer cells using fluorescence activated cell sorting (FACS). In some aspects, the disclosure provides a drug-selection-free method for batch production of producer cells using FACS. Such batch production methods and compositions can be further utilized to generate clonal populations of producer cells, e.g., for large-scale manufacturing of a polypeptide of interest.
Claims11
A method of fluorescence activated cell sorting (FACS) to batch select producer cells expressing a target polypeptide, the method comprising:
- (a) transfecting Chinese Hamster Ovary (CHO) cells with a vector comprising a multicistronic mRNA that encodes a FACS selectable polypeptide and a target polypeptide, wherein the multicistronic mRNA comprises a first open reading frame (ORF) that encodes the FACS selectable polypeptide and a second ORF that encodes the target polypeptide, wherein the first ORF is 5′ to the second ORF, wherein the first ORF has a non-AUG start codon and the second ORF has an AUG start codon,
- (b) growing the transfected CHO cells to select for a heterogeneous population of producer cells that express varying levels of the FACS selectable polypeptide and the target polypeptide,
- (c) selecting a first heterogeneous sub-population of producer cells from the heterogenous population of producer cells using FACS, wherein the producer cells in the first heterogeneous sub-population express the FACS selectable polypeptide at a level that is higher than the level of at least 80% of the producer cells in the heterogeneous population in (b), and
- (d)expanding the first heterogeneous sub-population of producer cells, without cloning, thereby producing an expanded first heterogeneous sub-population of uncloned producer cells,
- (e) selecting a second heterogeneous sub-population of uncloned producer cells from the expanded first heterogeneous sub-population of uncloned producer cells in (d) using FACS, wherein the uncloned producer cells in the second heterogeneous sub-population express the FACS selectable polypeptide at a level that is higher than the level of at least 80% of the producer cells in the expanded first heterogeneous sub-population of uncloned producer cells in (d), and
- (f) expanding the second heterogeneous sub-population of uncloned producer cells thereby producing an expanded second heterogeneous sub-population of uncloned producer cells,
- wherein the CHO cells are not subject to selection with methotrexate (MTX) or any other selection agent.
4 more elements
Dependencies are inferred from the claim text.