Selection of CHO host cell subclones with increased specific antibody production rates by repeated cycles of transient transfection and cell sorting

Abstract

Optimization of host cell lines both for transient and stable protein production is typically hampered by the inherent heterogeneity of cells within a population. This heterogeneity is caused not only by "hard fact" gene mutations, but also by subtle differences in the cellular network of regulation, which may include epigenetic variations. Taking advantage of this heterogeneity, we sorted for naturally occurring variants of CHO-K1 and CHO-S host cells that possess an improved cellular machinery for transient antibody production. The long-term goal of this study was both to identify host cells that yield recombinant cell lines with on average higher productivity, but also to study the molecular differences that characterize such cells, independent of the site of gene integration or gene amplification. To identify such cells we optimized the procedure for transient transfection by electroporation to a degree that gave uniform transfer of plasmid DNA into nearly 100% of the cells and resulted in reproducible average productivities, with a standard deviation of 16% between independent experiments. Using this optimized protocol, the 1% of cells with the highest specific productivity was sorted and subcloned with a cold capture secretion assay. Upon re-transfection, the resulting subclones showed the same specific productivity as their respective parental cell line. To enrich for cells with potentially stable improved properties, the 1% highest producers were sorted three times, 2 days after transient transfection each, and the enriched population was again sorted into microtiter plates for subcloning. For each of the two parental cell lines tested, three subclones were obtained that had a threefold higher specific productivity after transient transfection. This property was stable for approximately 3 months, indicating that the changes in productivity were regulatory and not mutational.

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

“…Generation of the subclones of CHO-K1 and CHO-S host cells by a FACS-based directed evolution approach is described in Pichler et al [21]. Subclones K1/1D9, K1/4F10, S/D2 and S/4F11 as well as the parental CHO-K1 and CHO-S host cell lines were transfected with a mixture of the plasmids carrying the light and heavy chain genes of an IgG1 human monoclonal antibody ("transfected"), or with the same amount of an empty plasmid, lacking any product genes ("mock"), as previously described [21].…”

“…Subclones K1/1D9, K1/4F10, S/D2 and S/4F11 as well as the parental CHO-K1 and CHO-S host cell lines were transfected with a mixture of the plasmids carrying the light and heavy chain genes of an IgG1 human monoclonal antibody ("transfected"), or with the same amount of an empty plasmid, lacking any product genes ("mock"), as previously described [21]. In addition, a sample of non-transfected cells was taken 48 h after seeding ("untransfected").…”

“…In addition, a sample of non-transfected cells was taken 48 h after seeding ("untransfected"). Titer was determined by ELISA as described in Pichler et al [21] 96 h after transfection to include the entire period of high productivity after transfection.…”

“…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.…”

“…In the present work, we evaluate the gene expression patterns of four host cell subclones obtained by repeated cell sorting [21], which exhibit a three-fold increased transient production rate compared to the parental cell lines. This plasmid-based setup avoids the side effects from genomic integration discussed above.…”