Keywords
Selection system, asparaginase, glutamine synthetase, Chinese hamster
ovary cells, biotherapeutic production, mammalian cell line
The integration of exogenous DNA into cultured cells is often
facilitated by co-introduction of the desired DNA alongside a selectable
element and growth under the corresponding selective pressure (e.g.,
transfection of the desired gene + a gene imparting antibiotic
resistance combined with growth in medium containing the antibiotic). In
the biopharmaceutical industry, metabolic selection systems that restore
nutritional prototrophies are routinely used to generate mammalian cell
lines producing high quantities of life-saving biotherapeutic protein
drugs. Dihydrofolate reductase (Dhfr) or glutamine synthetase (Gs) are
the most commonly leveraged metabolic selectable markers
(Cockett et al., 1990;
Kaufman & Sharp, 1982). However, the cell line generation process
using these is time-consuming and laborious, often requiring one (Gs) or
several (Dhfr) rounds of gene amplification driven by the addition of
inhibitory compounds (e.g., methotrexate and methionine sulfoximine)
during selection as well as the screening of 100s to 1000s of clones to
identify clones with the desired production and quality profiles. It has
been shown that utilizing these two systems simultaneously increases the
probability that a highly productive cell will be generated–as well as
improving the maximum product titer
(Li et al., 2010). Recent
work has demonstrated the feasibility of additional metabolic selection
systems in CHO
(Budge et
al., 2021; Capella Roca et al., 2019; Pourcel et al., 2020; Sun et al.,
2020; Zhang et al., 2020) with one study
(Zhang et al., 2022) showing
that using 8 selectable markers simultaneously can significantly
increase the productivity of the resulting cell lines (although the
cells grew very slowly). The orthogonality of these new selection
systems (e.g., each requiring the dropout of a different medium
component for selective pressure) and/or need for multiple genetic edits
led us to explore whether it was possible to increase the selective
stringency of glutamine deprivation in a simpler manner, hereby
enhancing the selective pressure of one of the most established tools in
the clinical cell line generation workflow, without requiring changes
from established selection conditions.
Using a CRISPR-Cas9 knockout screen targeting metabolic genes, we
identified asparaginase (Aspg) as putatively essential for CHO cell
growth in medium lacking glutamine
(Karottki et al., 2021). As
Gs was also identified as essential in that condition, we hypothesized
that a dual selection system based on Aspg together with Gs could
enhance selection without requiring alteration of the selective
pressure.
To confirm the importance of Aspg for growth in glutamine-free media and
assess its viability for use as an additional selectable marker
simultaneously with Gs, we generated three clonal knockout (KO) cell
lines using CRISPR/Cas9: Gs-KO, Aspg-KO, and Gs/Aspg-KO. Knockouts had
verified frameshift insertions or deletions in all alleles and also
showed decreased mRNA expression (Supplementary Figure 1). Both clones
lacking Aspg showed decreased maximum viable cell density when grown in
a glutamine-containing medium (Supplementary Figure 2A) but with
comparable growth rates to the Gs KO cell line (Supplementary Table 1).
When grown without glutamine, Aspg knockout cells showed negligible
growth, but remained viable. Gs/Aspg-KO cells, on the other hand, showed
a dramatic decrease in cell viability–even more quickly than Gs-KO
cells (Supplementary Figure 2B). This suggested that a double selection
system using Gs and Aspg simultaneously would be more stringent than Gs
alone–while still using only glutamine deprivation as the sole
selective pressure.
We generated Enbrel-producing cells from the different knockout cell
lines via 5 different transfections: 1) GS-KO cells with a Gs+Enbrel
plasmid, 2) GS-KO cells with both Gs+Enbrel and Aspg+Enbrel plasmids, 3)
Aspg-KO cells with Aspg+Enbrel plasmid, 4) Aspg-KO cells with both
Gs+Enbrel and Aspg+Enbrel plasmids, and 5) double Gs/Aspg-KO cells with
both Gs+Enbrel and Aspg+Enbrel plasmids. We tested selection in both
static minipools (192/transfection) and bulk suspension format
(duplicates in 6 well suspension, permitting quantification of recovery
profiles). Following recovery, surviving minipools were split 1:2 and
evaluated for terminal cell count and titer. After 5 days of culture,
minipools derived from clones with Aspg knockouts showed lower cell
density and product titer, however, minipools derived from the Gs/Aspg
double knockout transfected with both plasmids showed
~3-4-fold higher median cell-normalized product titer
than Gs knockout derived minipools (Figure 1, Supplementary Table 2). We
observed no change in recovery timelines in the bulk suspension format
and, after characterization of recovered pools in batch culture, the
Gs/Aspg double knockout cells transfected with both plasmids again
showed decreased growth, but significantly (~16-fold)
higher titer and specific productivity (Supplementary Figure 3B and
Supplementary Table 3).
We then tested if minipools could obtain improved performance after
being transitioned to suspension culture. Top minipools from all
transfections were expanded and characterized in 6 well suspension
culture. The trend of higher titer and specific productivity in Gs/Aspg
double knockout derived minipools was maintained, but minipools derived
from cells with Aspg knocked out showed low VCD (Supplementary Figure
4). We thus continued expanding the top 3 Gs/Aspg knockout derived and
Gs knockout derived minipools (based on titer) to test if prolonged time
in suspension culture would improve the performance of the former.
After expansion in shake flask culture, the growth and viability of
Gs/Aspg double knockout derived minipools were still decreased compared
to minipools derived from Gs knockout cells (Figure 2A), but both were
improved compared to their performance in 6-well plates (Supplementary
Figure 4). We again saw significantly higher production of Enbrel, both
in titer (2-4-fold higher in the best performing Gs/Aspg-KO derived
minipool) and specific productivity (10-15-fold improvement in
Gs/Aspg-KO derived minipools) (Figure 2B and Supplementary Table 4).
The improvement in growth and viability from 6-well to shake flask led
us to explore whether further adaptation of minipools derived from
Gs/Aspg knockout cells would improve performance in selection conditions
designed for Gs knockout derived cells. Following ~1
month of adaptation (Supplementary Figure 5), we evaluated the top
minipool from each transfection: The Gs/Aspg KO derived minipool showed
significant improvements in growth and viability while still
outperforming the Gs knockout derivatives in titer and specific
productivity (Figure 3 and Supplementary Table 5). We further assessed
the long-term stability of the dual selection strategy and found that
after an additional month of passaging, minipool performance remained
stable (Figure 4 and Supplementary Table 6).
Finally, we explored the cause of poor growth in the Gs/Aspg-KO derived
pools. Cell growth in this selection system depends on the rescue of
both knocked out enzymes (Gs and Aspg) through the uptake and
integration of both transfected plasmids. It is possible that the
observed low cell growth results from low expression of either or both
plasmids after selection. However, both Gs and Aspg expression levels in
the Gs/Aspg-KO derived pools were at least as high as that of Gs-KO
derived pools following selection and recovery, prior to adaptation
(Supplementary Figure 6); thus, the expression should be sufficient for
robust growth. As adaptation partially recovered growth (and
considerably improved viability) we anticipate that additional media
and/or platform optimization (e.g., altering the plasmid ratio) could
further improve the performance of this system.
The dual Gs/Aspg selection system thus is an intriguing option to
generate more highly productive cell lines. As such it only requires a
single additional genetic edit to the starting cell line and does not
require changes to the traditional Gs-based selection workflow.
Furthermore, it has the potential to be used as an alternative system
for not only the production of proteins but also the expression of
several genes of interest without the use of antibiotics (as seen with
Dhfr/Mtx co-selection (Lee et
al., 2018)). Continued work with cells generated by this approach,
e.g., single-cell cloning, expansion, and characterization in fed-batch
bioreactors, will further demonstrate the value of this system for cell
line generation for biotherapeutic protein production.