Introduction
Chinese hamster ovary (CHO) cells are preferred for biologics production for their safety as production hosts (Biological Approvals by Year—2017 Biological License Application Approvals , n.d.;Biologics-revolution-in-the-production-of-drugs.pdf , n.d.;Monoclonal Antibodies Approved by the EMA and FDA for Therapeutic Use – ACTIP , n.d.; Research, n.d.), high specific productivity (Qp), human-like protein glycosylation, and adaptability to grow in suspension in animal component (e.g. serum)-free growth media (Fischer et al., 2015; J. Y. Kim et al., 2012). Current technology for establishing stable CHO cell lines utilizing antibiotic/metabolic selection is time-consuming and expensive. In addition, metabolic selection requires expensive, auxotrophic cell lines that are eitherDHFR (dihydrofolate reductase) or GS (glutamine synthetase) negative (Bebbington et al., 1992; Gallagher & Kelly, 2017; Kaufman & Sharp, 1982). Finally, production instability caused by clonal drift affects the productivity of clones (Bandyopadhyay et al., 2018; Lee et al., 2018; Vcelar et al., 2018; Wurm & Wurm, 2017). While clonal variation is inevitable in rapidly proliferating immortalized cell lines, the dearth of tools to monitor clonal drift impacts productivity during CHO cell line expansion (Frye et al., 2016; Li et al., 2010; Mirasol, n.d.).
To overcome these issues, we developed PTSelect™ technology. It employs the cellular post-transcriptional machinery and utilizes an siRNA to select and establish stable cell populations, replacing the need for selection markers. Here, we describe the principle of PTSelect™ technology and demonstrate its ability to establish stable CHO cell lines producing either EpoFc (erythropoietin-conjugated to immunoglobulin Fc region) (Lattenmayer et al., 2007) or the monoclonal antibody, adalimumab (Choi et al., 2014). In parallel, we established stable CHO cell lines using conventional antibiotic (EpoFc) and/or metabolic selection (EpoFc and adalimumab). Productivity, doubling time and stability were compared between clones produced by both methodologies.
We demonstrate that PTSelect™ can generate a stable cell pool within 11 days, increase the fraction of positive clones, and generate clones with greater stability compared to methotrexate (MTX)-selected clones, with no significant difference in the productivity. Most importantly, PTSelect™ technology provides a rapid, simple solution to monitor clonal drift; correlation between productivity and PTSelect™-siRNA activity enables real-time, single-cell monitoring of productivity and population drift due to clonal variation.