Materials and Methods
Plasmid Construction. The Gs-Enbrel plasmid and Aspg-Enbrel
plasmid were constructed by uracil-specific excision reagent (USER)
cloning using flexible assembly sequence tags as previously described
(Lund et al., 2014;
Pristovšek et al., 2018). Each plasmid was generated using 4 input PCR
products: 3 common inputs–a backbone, the Enbrel gene, and an early
SV40 promoter–in addition to the selectable marker (Gs or Aspg). All
inputs were generated via PCR amplification of DNA fragments
(Supplementary Table 7) with Phusion U Hot Start DNA polymerase (cat.
no. F533S, Thermo Fisher Scientific) and uracil-containing primers
(Integrated DNA Technologies, inc.) shown in Supplementary Table 8. The
PCR settings used for the amplification are described in Supplementary
Table 9. The plasmids were constructed by assembling the DNA bricks with
USER Enzyme (cat. no. M5505S, New England Biolabs, Ipswich, MA) and
CutSmart R Buffer (cat. no. M5505L, New England Biolabs) according to
the manufacturer’s protocol. Constructed plasmids were transformed into
E. coli Mach1 competent cells (Thermo Fisher Scientific). All the
constructs were verified through Sanger sequencing by Eurofins Genomics
(Eurofins Scientific, Luxembourg). Two primers were used for each
construct to verify the Enbrel sequence (5’-CGAAATCGGCAAAATCCC-3’) and
the selection gene (Aspg or Gs) (5’- TTTTATTTATGCAGAGGCCGAG-3’).
Confirmed constructs were purified using NucleoBond Xtra Midi EF kit
(Macherey-Nagel) according to the manufacturer’s instructions.
Cell line generation and culture maintenance. CHO-S (Life
Technologies, Carlsbad, CA) cell lines were established using
CRISPR/Cas9 as previously described
(Grav et al., 2015) to
knockout Aspg and/or Gs. The Gs/Aspg-KO was established by knocking out
Aspg in the Gs-KO cell line. All primers for gRNA plasmid construction
and sequence verification are listed in Supplementary Table 10. All cell
lines were cultured in CD-CHO Medium (cat. No 10743029, Thermo Fisher
Scientific, Waltham, MA) with 0.2 % Anti-Clumping Agent (cat. no.
0010057AE, Gibco, Waltham, MA) and 8 mM glutamine unless otherwise
specified. Cells were passaged every 2-3 days in 30 ml of medium in
125-ml shake flasks (Corning, Corning, NY). Viable cell densities and
viabilities were measured using NucleoCounter® NC-200™ or NucleoCounter®
NC-250™ (ChemoMetec, Allerod, Denmark). All cultures were incubated at
37ºC, 80% humidity, and 5% CO2; suspension cultures
with working volumes >500 µl were shaken at 120 rpm.
Batch culture experiments and cell line characterization. Cells
were seeded at an initial cell density of 3 x 105cells/mL in 125 mL shake flasks (Corning) containing 30 mL medium with
or without 8 mM glutamine.
Quantitative real-time polymerase chain reaction (qRT-PCR). The
expression level of Gs and Aspg genes was evaluated by qRT-PCR as
described previously
(Kallehauge et al., 2017).
Oligos for qRT-PCR are listed in supplementary table 11. Gapdh was used
as a housekeeping gene in all the calculations.
96 well-based minipool generation. Cells were passed into
medium without Anti-Clumping Agent two days before transfection. Cells
were seeded at an initial cell density of 1 x 106cells/mL in 6-well plates. Afterward, transfection was performed with
FreeStyleTM MAX Reagent (cat. no. 16447100, Gibco) according to the
manufacturer’s protocol. After 24h, VCD and viability of transfected
cells were analyzed using NucleoCounter® NC-200™. Two 96-well plates for
each transfection condition were seeded with an initial cell density of
5000 cells/well. Transfected cells were seeded in cloning media (20% of
CD CHO Medium + 80% EX CELL® CHO Cloning (Sigma-Aldrich, St. Louis, MO)
with 0.2% Anti-Clumping Agent) after a previous wash step to remove all
the media with glutamine.
Bulk pool generation. 24hr after transfection, cells were
seeded in 6 well plates into selection medium (without glutamine) at a
cell density of 0.3 x 106 cells/mL. Every 2-3 days
cells were passed or spun and resuspended into fresh selection medium
until viability exceeded 90 %.
Adaptation. Cells were inoculated at a concentration of 0.5 x
106 cells/mL in 125 mL flasks with 30 mL of culture
medium without glutamine. Every 2 – 3 days, cells were passed into
fresh culture medium without glutamine until the cell viability reached
over 90%.
Stability testing. Cells were passed every 2 – 3 days at a
concentration of 0.3 x 106 cells/mL in 125 mL
Erlenmeyer flasks with 30 mL of culture medium without glutamine for a
month. Batch culture was performed simultaneously for cells with and
without one-month passaging time.
Titer measurement. The mAb concentration was measured using an
Octet RED96 (Pall, Menlo Park, CA, USA), as described previously
(Kallehauge et al., 2017).
Statistical analysis. Values are expressed as mean ± standard
deviation unless otherwise noted. The data were analyzed with a
two-tailed Student’s t -test and differences were considered
statistically significant at p < 0.05.