Characterization of phenotypic and genotypic diversity in subclones derived from a clonal cell line

Abstract

Regulatory guidelines require the sponsors to provide assurance of clonality of the production cell line, and when such evidence is not available, additional studies are typically required to further ensure consistent long‐term manufacturing of the product. One potential approach to provide such assurance of clonal derivation of a production cell line is to characterize subclones generated from the original cell line and assess their phenotypic and genotypic similarity with the hypothesis that cell lines derived from a clonal bank will share performance, productivity and product quality characteristics. In this study, a production cell line that was cloned by a validated FACS approach coupled with day 0 imaging for verification of single‐cell deposition was subcloned using validated FACS and imaging methods. A total of 46 subclones were analyzed for growth, productivity, product quality, copy number, and integration site analysis. Significant diversity in cell growth, protein productivity, product quality attributes, and copy number was observed between the subclones, despite stability of the parent clone over time. The diversity in protein productivity and quality of the subclones were reproduced across time and production scales, suggesting that the resulting population post sub‐cloning originating from a single cell is stable but with unique properties. Overall, this work demonstrates that the characteristics of isolated subclones are not predictive of a clonally derived parental clone. Consequently, the analysis of subclones may not be an effective approach to demonstrate clonal origin of a cell bank. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:613–623, 2018

Genome‐wide analysis of single nucleotide variants allows for robust and accurate assessment of clonal derivation in cell lines used to produce biologics

“…Over the last years, however, it has become increasingly clear that even clonally derived cell lines can gradually become genetically heterogeneous. Indeed, several studies have shown that subclones from the same cell line can display different phenotypic behaviors (Ko et al, 2018; Patel et al, 2018; Tharmalingam et al, 2018). In addition, clonally derived cell lines have been shown to display genetic evolution affecting their genome at various scales (from the accumulation of point mutations to chromosomal rearrangements), as well as epigenetic changes (Feichtinger et al, 2016; Vcelar et al, 2018).…”

Accelerating HIV‐1 VLP production using stable High Five insect cell pools

“…[10] On the other hand, several studies have brought into debate the relevance of clonality by reporting the appearance of differentiated expression patterns during the culture of cells derived from a clonal cell line. [11,12] Cell-to-cell differences have even been detected in master cell banks of CHO cells, but this heterogeneous cell population performed consistently in terms of product quality. [13] In the same line, the development of a clonal CHO cell line did not provide an improved homogeneity of future cell progeny.…”

Nanoscale integration of single cell biologics discovery processes using optofluidic manipulation and monitoring

“…To select a cell line that will give high productivity, long‐term stability, and consistent product and process consistency, subcloning and extensive screening is required on hundreds of clonal cell lines, measuring growth, productivity and product quality in multiple screening assays (Wurm, ). In addition, regulatory agencies require that clinical trials be initiated with material from clonally derived cell lines and banks, so documentation that the cell line is derived from a single cell is required to provide that assurance of clonality (Tharmalingam et al, ).…”