title: A mechanistic understanding of production instability in CHO cell lines expressing recombinant monoclonal antibodies.
abstract: One of the most significant problems in industrial bioprocessing of recombinant proteins using engineered mammalian cells is the phenomenon of cell line instability, where a production cell line suffers a loss of specific productivity (qP). This phenomenon occurs with unpredictable kinetics and has been widely observed in Chinese hamster ovary (CHO) cell lines and with all commonly used gene expression systems. The underlying causes (both genetic and physiological) and the precise molecular mechanisms underpinning cell line instability have yet to be fully elucidated, although recombinant gene silencing and loss of recombinant gene copies have been shown to cause qP loss. In this work we have investigated the molecular mechanisms underpinning qP instability over long-term sub-culture in CHO cell lines producing recombinant IgG1 and IgG2 monoclonal antibodies (Mab's). We demonstrate that production instability derives from two primary mechanisms: (i) epigenetic--methylation-induced transcriptional silencing of the CMV promoter driving Mab gene transcription and (ii) genetic--progressive loss of recombinant Mab gene copies in a proliferating CHO cell population. We suggest that qP decline resulting from loss of recombinant genes is a consequence of the inherent genetic instability of recombinant CHO cell lines.
authors.affiliation: Department of Chemical and Biological Engineering, University of Sheffield, Mappin St., Sheffield S1 3JD, UK; telephone: +44-114-222-7505; fax: +44-114-222-7501.
authors.forename: Minsoo
authors.initials: M
authors.lastname: Kim
authors.forename: Peter M
authors.initials: PM
authors.lastname: O'Callaghan
authors.forename: Kurt A
authors.initials: KA
authors.lastname: Droms
authors.forename: David C
authors.initials: DC
authors.lastname: James
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000235:genetics; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D019175:DNA Methylation / Q000235:genetics; D018628:Gene Dosage; D015870:Gene Expression; D020868:Gene Silencing; D006801:Humans; D007074:Immunoglobulin G / Q000096:biosynthesis* / Q000235:genetics; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014158:Transcription, Genetic
chemical_list: D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D011994:Recombinant Proteins