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.