title: Recombinase-mediated cassette exchange (RMCE) for monoclonal antibody expression in the commercially relevant CHOK1SV cell line.
abstract: To meet product quality and cost parameters for therapeutic monoclonal antibody (mAb) production, cell lines are required to have excellent growth, stability, and productivity characteristics. In particular, cell line generation stability is critical to the success of a program, especially where high cell line generation numbers are required for large in-market supply. However, a typical process for developing such cell lines is laborious, lengthy, and costly. In this study, we applied a FLP/FRT recombinase-mediated cassette exchange (RMCE) system to build a site-specific integration (SSI) system for mAb expression in the commercially relevant CHOK1SV cell line. Using a vector with a FRT-flanked mAb expression cassette, we generated a clonal cell line with good productivity, long-term production stability, and low mAb gene-copy number indicating the vector was located in a 'hot-spot.' A SSI host cell line was made by removing the mAb genes from the 'hot-spot' by RMCE, creating a 'landing pad' containing two recombination cassettes that allow targeting of one or two copies of recombinant genes. Cell lines made from this host exhibited excellent growth and productivity profiles, and stability for at least 100 generations in the absence of selection agents. Importantly, while clones containing two copies had higher productivity than single copy clones, both were stable over many generations. Taken together, this study suggests the use of FLP-based RMCE to develop SSI host cells for mAb production in CHOK1SV offers significant savings in both resources and overall cell line development time, leading to a shortened 'time-to-clinic' for therapeutic mAbs.
authors.affiliation: Cell Line Development, World Wide Pharmaceutical Sciences, BioTherapeutics Research and Development, Pfizer Inc., Andover, MA 01810.
authors.forename: Lin
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Cell Line Development, World Wide Pharmaceutical Sciences, BioTherapeutics Research and Development, Pfizer Inc., Andover, MA 01810.
authors.forename: Mara C
authors.initials: MC
authors.lastname: Inniss
authors.affiliation: Cell Line Development, World Wide Pharmaceutical Sciences, BioTherapeutics Research and Development, Pfizer Inc., Andover, MA 01810.
authors.forename: Shu
authors.initials: S
authors.lastname: Han
authors.affiliation: Cell Line Development, World Wide Pharmaceutical Sciences, BioTherapeutics Research and Development, Pfizer Inc., Chesterfield, MO, 63017.
authors.forename: Mark
authors.initials: M
authors.lastname: Moffat
authors.affiliation: Cell Line Development, World Wide Pharmaceutical Sciences, BioTherapeutics Research and Development, Pfizer Inc., Chesterfield, MO, 63017.
authors.forename: Heather
authors.initials: H
authors.lastname: Jones
authors.affiliation: BioTX Clinical Research, Pfizer Inc., Cambridge, MA, 02140.
authors.forename: Baohong
authors.initials: B
authors.lastname: Zhang
authors.affiliation: Cell Culture Development, Lonza Biologics, Slough, SL1 4DX, U.K.
authors.forename: Wendy L
authors.initials: WL
authors.lastname: Cox
authors.affiliation: Development Services Singapore, Lonza Biologics Tuas Pte Ltd, Singapore.
authors.forename: James R
authors.initials: JR
authors.lastname: Rance
authors.affiliation: New Expression Technologies Group, Research & Technology, Lonza Biologics, Granta Park, Great Abington, Cambridge, CB21 6GS, U.K.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Young
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000378:metabolism*; D016466:CHO Cells; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods*; D006224:Cricetinae; D003412:Cricetulus; D004254:DNA Nucleotidyltransferases / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics*
chemical_list: D000911:Antibodies, Monoclonal; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase
keywords: RMCE; cell line development; monoclonal antibody; site specific integration; stability