title: Slashing the timelines: Opting to generate high-titer clonal lines faster via viability-based single cell sorting.
abstract: Chinese hamster ovary (CHO) cell line development (CLD) is a long and laborious process, which requires up to 5 - 6 months in order to generate and bank CHO lines capable of stably expressing therapeutic molecules. Additionally, single cell cloning of these production lines is also necessary to confirm clonality of the production lines. Here we introduce the utilization of viability staining dye in combination with flow cytometer to isolate high titer clones from a pool of selected cells and single cell deposit them into the wells of culture plates. Our data suggests that a stringent selection procedure along with viability dye staining and flow cytometry-based sorting can be used to isolate high expressing clones with titers comparable to that of traditional CLD methods. This approach not only requires less labor and consumables, but it also shortens CLD timelines by at least 3 weeks. Furthermore, single cell deposition of selected cells by a flow sorter can be regarded as an additional clonality assurance factor that in combination with Day 0 imaging can ensure clonality of the production lines.
authors.affiliation: Dept. of Early Stage Cell Culture, Genentech, Inc, 1 DNA Way, South San Francisco, CA, 94080.
authors.forename: Shahram
authors.initials: S
authors.lastname: Misaghi
authors.affiliation: Dept. of Early Stage Cell Culture, Genentech, Inc, 1 DNA Way, South San Francisco, CA, 94080.
authors.forename: David
authors.initials: D
authors.lastname: Shaw
authors.affiliation: Dept. of Early Stage Cell Culture, Genentech, Inc, 1 DNA Way, South San Francisco, CA, 94080.
authors.forename: Salina
authors.initials: S
authors.lastname: Louie
authors.affiliation: Dept. of Early Stage Cell Culture, Genentech, Inc, 1 DNA Way, South San Francisco, CA, 94080.
authors.forename: Adrian
authors.initials: A
authors.lastname: Nava
authors.affiliation: Dept. of Early Stage Cell Culture, Genentech, Inc, 1 DNA Way, South San Francisco, CA, 94080.
authors.forename: Laura
authors.initials: L
authors.lastname: Simmons
authors.affiliation: Dept. of Early Stage Cell Culture, Genentech, Inc, 1 DNA Way, South San Francisco, CA, 94080.
authors.forename: Brad
authors.initials: B
authors.lastname: Snedecor
authors.affiliation: Dept. of Immunology, Genentech, Inc, 1 DNA Way, South San Francisco, CA, 94080.
authors.forename: Chungkee
authors.initials: C
authors.lastname: Poon
authors.affiliation: Dept. of Immunology, Genentech, Inc, 1 DNA Way, South San Francisco, CA, 94080.
authors.forename: Jonathan S
authors.initials: JS
authors.lastname: Paw
authors.affiliation: Dept. of Immunology, Genentech, Inc, 1 DNA Way, South San Francisco, CA, 94080.
authors.forename: Laurie
authors.initials: L
authors.lastname: Gilmour-Appling
authors.affiliation: Dept. of Immunology, Genentech, Inc, 1 DNA Way, South San Francisco, CA, 94080.
authors.forename: James E
authors.initials: JE
authors.lastname: Cupp
mesh_terms: D000818:Animals; D016466:CHO Cells / Q000166:cytology*; D018929:Cell Culture Techniques / Q000379:methods*; D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival / Q000235:genetics; D006224:Cricetinae; D003412:Cricetulus; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D059010:Single-Cell Analysis
keywords: flow cytometry; single cell deposition; viability staining dye