MATERIALS AND METHODS
Cell Cultures and Chemical
Chaperones
Expi293F cells were cultured in Expi293 Expression medium (ThermoFisher)
in Erlenmeyer flasks maintained at 37°C, 125 rpm under 8%
CO2, 85% humidity. CHO-S clonal isolate cells (C1-80;
Fernandez-Martell et al., 2018) were cultured in CD CHO medium
(ThermoFisher) supplemented with 8 mM L-glutamine maintained at 37°C,
140 rpm under 5% CO2, 85% humidity. Cells were seeded
at 2×105 viable cells/mL and were sub-cultured every
3‒4 days. Cell viability and VCD were measured using the Vi-CELL XR
(Beckman Coulter). VPA, NaBu, DMSO, glycerol, betaine, TMAO, proline and
FAC were obtained from Sigma while TUDCA was obtained from Merck.
Transient Production in HEK and CHO
Cells
pCAGGS-spike was provided by the
Krammer Laboratory (Icahn School of Medicine at Mount Sinai), amplified,
and purified using QIAGEN Plasmid Plus kit (Qiagen). For Expi293F
transfection, cells were grown to 1.75×106 cells/mL,
centrifuged and resuspended at a density of 3.5×106cells/mL, followed by sequential addition of 0.85 μg of DNA and 2.55 μl
of PEI MAX (each pre-diluted in 10 μL of 150 mM NaCl) per million cells.
At 24 h post-transfection, the cells were diluted 2× by adding fresh
medium, and where applicable culture was shifted to 32°C. For CHO
transfection, cells were seeded one day before transfection and grown to
1.5×106 cells/mL. For every 1.5×106cells, 1.3 μg of DNA and 4.55 μL of PEI MAX (each pre-diluted in 15 μL
of 150 mM NaCl) were combined and incubated at RT for 2 min before being
added into culture. Where applicable, culture was shifted to 32°C at 4 h
post-transfection. For fed-batch production, 5% v/v CHO CD
EfficientFeed B (ThermoFisher) was added at Days 2, 4, 6 and 8.
Generation of Stable CHO Pools and Fed-batch
Production
A stable vector containing an SV40 promoter-driven GS gene was provided
by AstraZeneca, UK. The spike gene was cloned by PCR, inserted into the
vector downstream of 40RPU or 100RPU synthetic promoter (Brown et al.,
2017) and the plasmid constructs were confirmed by DNA sequencing.
10×106 cells per cuvette were electroporated with 7 μg
linearized DNA using Cell Line Nucleofector Kit V system (Lonza) and
transferred to a TubeSpin containing 10 mL glutamine-free culture medium
with the addition of 25 or 50 μM MSX after 48 h. The cells were left to
recover under suspension conditions and recovered pools were
cryopreserved when the cell viability reached >90%. For
fed-batch production, 5% v/v CHO CD EfficientFeed B was added at Days
2, 4, 6 and 8.
Western Blotting
Proteins in culture supernatant were precipitated by TCA/DOC,
resuspended in LDS loading buffer with BME and heated to 70°C. SDS-PAGE
was performed using 4–12% NuPAGE Bis-tris gels and resolved proteins
were transferred to nitrocellulose membranes by iBlot system
(ThermoFisher). Membranes were blocked in 5% milk/TBS-T before being
incubated with HRP-conjugated anti-HisTag antibody (Bio-Rad) and
visualized by enhanced chemiluminescence (ECL; ThermoFisher).
Recombinant Protein Purification and
Quantification
Spike protein was harvested by centrifugation at 3,000×g for 20 min at
4°C and supernatant was filtered through a 0.22 μm filter. Protein was
purified using the ÄKTA Pure system (Cytiva) and a 5-mL HisTrap HP
column (Cytiva). Eluted protein fractions were pooled and buffer
exchanged into storage buffer (20 mM Tris, 200 mM NaCl, 10% v/v
glycerol, pH 8.0) using a PD-10 desalting column (Cytiva). Protein was
quantified using the Pierce Coomassie Plus (Bradford) Assay kit
(ThermoFisher) and analyzed by reducing SDS-PAGE. A complementary
quantification of spike in culture supernatant was performed using
CR3022 antibody ELISA (Supplementary Figure S4).