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.