title: Development of a highly-efficient CHO cell line generation system with engineered SV40E promoter.
abstract: Chinese hamster ovary (CHO) cells have been one of the most widely used host cells for the manufacture of therapeutic recombinant proteins. An effective and efficient clinical cell line development process, which could quickly identify those rare, high-producing cell lines among a large population of low and non-productive cells, is of considerable interest to speed up biological drug development. In the glutamine synthetase (GS)-CHO expression system, selection of top-producing cell lines is based on controlling the balance between the expression level of GS and the concentration of its specific inhibitor, l-methionine sulfoximine (MSX). The combined amount of GS expressed from plasmids that have been introduced through transfection and the endogenous CHO GS gene determine the stringency and efficiency of selection. Previous studies have shown significant improvement in selection stringency by using GS-knockout CHO cells, which eliminate background GS expression from the endogenous GS gene in CHOK1SV cells. To further improve selection stringency, a series of weakened SV40E promoters have been generated and used to modulate plasmid-based GS expression with the intent of manipulating GS-CHO selection, finely adjusting the balance between GS expression and GS inhibitor (MSX) levels. The reduction of SV40E promoter activities have been confirmed by TaqMan RT-PCR and GFP expression profiling. Significant productivity improvements in both bulk culture and individual clonal cell line have been achieved with the combined use of GS-knockout CHOK1SV cells and weakened SV40E promoters driving GS expression in the current cell line generation process. The selection stringency was significantly increased, as indicated by the shift towards higher distribution of producing-cell populations, even with no MSX added into cell culture medium. The potential applications of weakened SV40E promoter and GS-knockout cells in development of targeted integration and transient CHO expression systems are also discussed.
authors.affiliation: Bioprocess Research and Development, Eli Lilly and Company, Indianapolis, IN 46221, United States. Electronic address: Fanlianchun@lilly.com.
authors.forename: Lianchun
authors.initials: L
authors.lastname: Fan
authors.forename: Ibrahim
authors.initials: I
authors.lastname: Kadura
authors.forename: Lara E
authors.initials: LE
authors.lastname: Krebs
authors.forename: Jeffery L
authors.initials: JL
authors.lastname: Larson
authors.forename: Daniel M
authors.initials: DM
authors.lastname: Bowden
authors.forename: Christopher C
authors.initials: CC
authors.lastname: Frye
mesh_terms: D000818:Animals; D016466:CHO Cells / Q000166:cytology / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D015870:Gene Expression / Q000187:drug effects; D055786:Gene Knockout Techniques; D005822:Genetic Vectors; D005974:Glutamate-Ammonia Ligase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008717:Methionine Sulfoximine / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D013539:Simian virus 40 / Q000235:genetics; D014162:Transfection
chemical_list: D011994:Recombinant Proteins; D008717:Methionine Sulfoximine; D005974:Glutamate-Ammonia Ligase
keywords: Bispecific antibody; Chinese hamster ovary (CHO) cell; Glutamine synthetase (GS); Mammalian cell line development; SV40 E promoter