title: A single-step FACS sorting strategy in conjunction with fluorescent vital dye imaging efficiently assures clonality of biopharmaceutical production cell lines.
abstract: The development of biopharmaceutical production cell lines typically starts with generation of heterogeneous populations of cells, from which then single cell clones are established. Several regulatory guidelines require that production cell lines are clonal, and the actual demonstration of clonality has been increasingly demanded by regulatory authorities over the last years. Here, the authors describe the relative contribution of flow cytometry mediated deposition of single cells in multiwell plates and subsequent imaging to assurance of clonality in a state of the art approach to single cell generation. Within the flow cytometry step, two unit operations are evaluated separately, doublet discrimination during event selection for deposition and droplet deposition accuracy. The imaging procedure is evaluated for the accuracy of detection of non-clonal populations. By employing mixing experiments of cell populations, the authors demonstrate that doublet discrimination is highly efficient, and that an appropriately set up flow cytometry system already can generate >99.5% true single cell clones. The efficiency of the described imaging process depends on several factors, reaching an optimal detection rate of non-clonal wells of about 99.8%. Our results demonstrate that one well characterized cloning step generate biopharmaceutical production cell lines with a probability of clonality of >99.99%.
authors.affiliation: Early Stage Bioprocess Development, Boehringer Ingelheim GmbH & Co. KG, Biberach, Germany.
authors.forename: Jürgen
authors.initials: J
authors.lastname: Fieder
authors.affiliation: Cell Culture Development CMB, Boehringer Ingelheim GmbH & Co. KG, Biberach, Germany.
authors.forename: Patrick
authors.initials: P
authors.lastname: Schulz
authors.affiliation: Early Stage Bioprocess Development, Boehringer Ingelheim GmbH & Co. KG, Biberach, Germany.
authors.forename: Ingo
authors.initials: I
authors.lastname: Gorr
authors.affiliation: Cell Culture Development CMB, Boehringer Ingelheim GmbH & Co. KG, Biberach, Germany.
authors.forename: Harald
authors.initials: H
authors.lastname: Bradl
authors.affiliation: Global NBE/Biosimilar CMC experts, Boehringer Ingelheim GmbH & Co. KG, Biberach, Germany.
authors.forename: Till
authors.initials: T
authors.lastname: Wenger
mesh_terms: D000818:Animals; D001688:Biological Products / Q000378:metabolism*; D016466:CHO Cells; D018929:Cell Culture Techniques; D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival; D003412:Cricetulus; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes; D059010:Single-Cell Analysis
chemical_list: D001688:Biological Products; D005456:Fluorescent Dyes
keywords: Biopharmaceutical production cell line; Clonality; FACS; imaging