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Mitochondrial membrane potential identifies cells with high recombinant protein productivity.
- Source :
-
Journal of immunological methods [J Immunol Methods] 2019 Jan; Vol. 464, pp. 31-39. Date of Electronic Publication: 2018 Oct 17. - Publication Year :
- 2019
-
Abstract
- Development of cell lines for biotherapeutic protein production requires screening large numbers of clones to identify and isolate high producing ones. As such, stable cell line generation is a time- and resource-intensive process. There is an increasing need to enhance the selection efficiency of high-yielding clonal cell lines for cell line development projects by using high throughput screening of live cells for markers predictive of productivity. Single cell deposition by fluorescence activated cell sorting (FACS) is a commonly performed method for cloning to generate cell lines derived from a single recombinant cell. We have developed a novel strategy to identify higher productivity cells at the FACS step by leveraging a simple viable cell staining method that detects mitochondrial membrane potential (Ψm), a key indicator of cellular metabolic activity. We chose a dual-emission dye (Mito-ID, Enzo Life Sciences) that fluoresces green and orange in living cells with the intensity of the orange fluorescence being dependent on the cells Ψm status. Using available clonal cell lines with known productivity, or stable transfectant pools, we evaluated Ψm of cell populations with Mito-ID dye. We determined that the intensity of the Ψm fluorescent signal correlates with the known fed-batch titers of the producer clones, and that cell sorting based on an optimal Ψm staining intensity selectively enriches for higher producing clones from nonclonal transfectant pools. These clones are phenotypically stable for recombinant protein production. Furthermore, the strategy has been successfully applied to identifying higher producing cell lines for a range of antibody molecular formats. Using this method, we can combine an enriching step with the cloning step for high producers, thereby saving time and resources in cell line development.<br /> (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)
- Subjects :
- Animals
Antibodies genetics
Antibody Formation
CHO Cells
Clone Cells
Cloning, Molecular
Cricetulus
Fluorescent Dyes
Phenotype
Recombinant Proteins biosynthesis
Antibodies metabolism
Cell Separation methods
Energy Metabolism
Flow Cytometry
Membrane Potential, Mitochondrial
Mitochondria metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1872-7905
- Volume :
- 464
- Database :
- MEDLINE
- Journal :
- Journal of immunological methods
- Publication Type :
- Academic Journal
- Accession number :
- 30342010
- Full Text :
- https://doi.org/10.1016/j.jim.2018.10.007