title: Efficient enrichment of high-producing recombinant Chinese hamster ovary cells for monoclonal antibody by flow cytometry.
abstract: To screen a high-producing recombinant Chinese hamster ovary (CHO) cell from transfected cells is generally laborious and time-consuming. We developed an efficient enrichment strategy for high-producing cell screening using flow cytometry (FCM). A stable pool that had possibly shown a huge variety of monoclonal antibody (mAb) expression levels was prepared by transfection of an expression vector for mAb production to a CHO cell. To enrich high-producing cells derived from a stable pool stained with a fluorescent-labeled antibody that binds to mAb presented on the cell surface, we set the cell size and intracellular density gates based on forward scatter (FSC) and side scatter (SSC), and collected the brightest 5% of fluorescein isothiocyanate (FITC)-positive cells from each group by FCM. The final product concentration in a fed-batch culture of cells sorted without FSC and SSC gates was 1.2-1.3-times higher than that of unsorted cells, whereas that of cells gated by FSC and SSC was 3.4-4.7-fold higher than unsorted cells. Surprisingly, the fraction with the highest final product concentration indicated the smallest value of FSC and SSC, and the middle value of fluorescence intensity among all fractionated cells. Our results showed that our new screening strategy by FCM based on FSC and SSC gates could achieve an efficient enrichment of high-producing cells with the smallest value of FSC and SSC.
authors: R&D Division, Daiichi Sankyo Co., Ltd., Gunma 370-0503, Japan; Institute of Technology and Science, The University of Tokushima, 2-1 Minamijosanjima-cho, Tokushima 770-8506, Japan. Electronic address: okumura.takeshi.m3@daiichisankyo.co.jp.
authors: Takeshi
authors: T
authors: Okumura
authors: R&D Division, Daiichi Sankyo Co., Ltd., Gunma 370-0503, Japan.
authors: Kenji
authors: K
authors: Masuda
authors: R&D Division, Daiichi Sankyo Co., Ltd., Gunma 370-0503, Japan.
authors: Kazuhiko
authors: K
authors: Watanabe
authors: R&D Division, Daiichi Sankyo Co., Ltd., Gunma 370-0503, Japan.
authors: Kenji
authors: K
authors: Miyadai
authors: R&D Division, Daiichi Sankyo Co., Ltd., Gunma 370-0503, Japan.
authors: Koichi
authors: K
authors: Nonaka
authors: R&D Division, Daiichi Sankyo Co., Ltd., Gunma 370-0503, Japan.
authors: Masayuki
authors: M
authors: Yabuta
authors: Institute of Technology and Science, The University of Tokushima, 2-1 Minamijosanjima-cho, Tokushima 770-8506, Japan.
authors: Takeshi
authors: T
authors: Omasa
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D061249:Batch Cell Culture Techniques; D016466:CHO Cells; D002469:Cell Separation / Q000379:methods*; D048429:Cell Size; D006224:Cricetinae; D003412:Cricetulus; D005434:Flow Cytometry / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D014162:Transfection
chemical_list: D000911:Antibodies, Monoclonal; D016650:Fluorescein-5-isothiocyanate
keywords: Cell line development; Chinese hamster ovary cell; Flow cytometry; Forward scatter; Monoclonal antibody; Side scatter