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.

Microarray profiling of preselected CHO host cell subclones identifies gene expression patterns associated with in‐creased production capacity

“…The latter can be caused by both genomic changes and epigenetic regulation [17][18][19]. This genomic and phenotypic heterogeneity has advantages and disadvantages: On the one hand it allows the effective selection of cells with different physiological and process relevant properties during screening [6,[20][21][22][23][24][25], on the other hand it limits the stability of these properties [21,26,27] and requires large numbers of subclones to be tested over prolonged periods of time to identify the few stable clones suited for production. The endogenous heterogeneity present in CHO cells is further enhanced by the process of recombination and gene amplification during cell line development, where the gene of interest and selection markers are integrated at a random site into the host cell genome, which may cause knockout of genes as well as changes in their expression levels either by separation of genes from their genetic control elements or by co-amplification with the recombinant gene.…”

Effects of passage number on growth and productivity of hybridoma secreting MRSA anti-PBP2a monoclonal antibodies

“…Whereas hybridoma B6.1 cells maintained at high passages reduced the protective capacity of their secreted mAb (Xin and Cutler 2006). The production instability in recombinant mAb in CHO cells can occur through gene silencing mechanism, or else the progressive loss in the number of copies of the recombinant gene, reducing mAb yield as of P14 or P95 (Beckmann et al 2012;Kim et al 2011).…”

“…Considering cost-effectiveness, it is important to have a cell line secreting proteins or antibodies of interest for development of diagnostics kits (Barnes et al 2003;Schmid et al 1990;Xin and Cutler 2006;Beckmann et al 2012;Kim et al 2011;Li et al 2010;Lee et al 1991). …”

Building a Cell Culture Process with Stable Foundations: Searching for Certainty in an Uncertain World

“…This phenomenon affects mammalian cell lines created using the commonly exploited DHFR and GS gene expression systems (for a schematic diagram of a GS vector see Fig. 12.2), and can occur even when cells are continuously cultured under chemical selection (MTX and MSX, respectively), a state that should theoretically serve to maintain the presence of the integrated genes (Beckmann et al 2012;Kim et al 2011;HellerHarrison et al 2009;Jun et al 2006;Fann et al 2000;Strutzenberger et al 1999;Kim et al 1998a, b). How can recombinant genes be lost from the genome?…”