Discussion
Here, we describe the principle of PTSelect™ technology and demonstrate its ability to establish stable CHO-K1 clones producing either EpoFc or adalimumab (Figure 3b and 5b). A stable cell pool expressing EpoFc was established in 11 days (Figures 2c and 3c, a significant time reduction compared to current antibiotic or metabolic selection methods. Notably, the PTSelect™-siRNA activity correlates with the relative GOI expression (Figure 6a). Thus, PTSelect™ can be employed to monitor production instability due to clonal drift by simple flow cytometric analysis (Figure 6b) as well as to enable clonal enrichment (Figure 6c).
A critical pre-requirement for FDA approval of a clonal line is to demonstrate that the entire cell line population is derived from a single cell progenitor (ICH Q5D) (Plavsic, 2017; Welch & Arden, 2019), given the high propensity of CHO cells to undergo clonal variation or clonal drift(Ko et al., 2018). In addition to techniques that were developed to monitor clonal drift by the analysis of sub-clones (Aebischer-Gumy et al., 2018; Tharmalingam et al., 2018; Vcelar et al., 2018) and cell surface staining(Pilbrough et al., 2009), strategies have been explored to minimize clonal drift by introducing transgenes into certain target genomic sites identified to be stable(J. Y. Kim et al., 2012; Sakuma et al., 2015; Zhang et al., 2015; Zhou et al., 2010). The target sites were identified by utilizing CHO genome-based multiomics and in silico models (Ghorbaniaghdam et al., 2014; Lee et al., 2019). These approaches are time-consuming, expensive, laborious, or require expertise in a specific field. PTSelect™ technology enables monitoring productivity variation in a single cell in 6 hours, requiring simple transfection of cells with fluorescent/siRNA-mRNA, followed by flow cytometry analysis. Thus, PTSelect™ technology provides a breakthrough to monitor clonal drift that has been long sought in the biotechnology industry, single-cell productivity monitoring that is simple, rapid, and inexpensive.
To establish stable clones using PTSelect™– first, the PTSelect™-siRNA cassette is cloned into an existing plasmid containing the GOI. Our data indicate that the presence of antibiotic/metabolic selection genes does not interfere with PTSelect™ technology (Figures 3c & 5b). After transfecting cells with the plasmid-GOI-siRNA, performing three depletions is recommended (days 3, 7 and 10). On day 10, cells with the lowest fluorescent signal (lowest 1% or 0.5% based on stringency) can be plated using flow cytometry-based sorting into ten 96-well plates. High producers from the 96-well plates (at 4 weeks) can be enriched by FACS two to three times in a 3-week period, as demonstrated in Figure 6c. Thus, a clone enriched for high producers can be generated in 7 to 8 weeks.
PTSelect™-siRNA activity also offers the convenience and serves as an indicator for GOI expression for those GOIs that lack antibodies for detection. Thus, PTSelect™ accelerates stable pool establishment, permits enrichment of high producers, and provides an alternative to screen for GOIs without an antibody. Overall, we have described an innovative siRNA-based technology to establish stable cell lines more rapidly compared with existing technologies. This technology further offers a simple and a reliable solution to monitor single-cell productivity and population drift in productivity due to clonal variation.
Acknowledgements
Funding for this work was provided by the National Institutes of Health, Phase II STTR grant (R42GM114935). We would like to thank Nicholas St. John for his assistance with mRNA synthesis and cell culture maintenance.