TITLE: Artisanal Production of Prefusion-Stabilized SARS-CoV-2 Spikes
AUTHORS: Jean-Francois Rivest, Claudia Goupil, Yannick Doyon

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">The two-part protocol presented here describes the establishment of a stable pool of 293-F cells expressing the HexaPro variant of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) spike (S) protein and its production and purification from the culture medium. CRISPR-Cas9-driven targeted integration of the expression cassette at the </span><span style = "font-weight:bold;font-style:italic;">AAVS1 </span><span style = "font-weight:bold;">safe harborlocus streamlines cell line production. Engineered 293-F cells grown as suspension cultures in animal origin-free, chemically defined, protein-free medium allow purification to be performed directly from the culture medium with minimal cell processing steps. Yields of native and purified Spikes typically average 30-50 mg per liter of culture medium following tandem affinity purification using nickel-coupled and Strep-Tactin resins. Purified proteins bind recombinant ACE2 and mAb CR3022 in ELISA-based assays. Establishing an engineered cell line from cryopreserved 293-F cells takes approximately 4 weeks, and subsequent production and purification take between 9 and 14 days. As shown for the D614G mutation, this system can be readily adapted to study Spike variants.</span></div></div>

[STEPS]
?. [Thawing and general handling of 293-F cells before transfection (Timing: 11 days)]
Before thawing the cells, prewarm 30 ml of FreeStyle 293 Expression Medium in a 250 ml tissue culture shaker flask at 37°C, preferably in a humidified atmosphere with 8% CO2. Also, prewarm a 9 ml aliquot of culture medium at 37°C in a 50 ml sterile polypropylene conical tube.
Note: While we used commercial 293-F cells from Gibco, it is likely that this protocol can be adapted for other 293-derived cell lines adapted for suspension and serum-free growth. We recommend culturing 293-F cells in medium without antibiotic, as we have seen reduced growth fitness when using Pen-Strep. Cells should be tested for the absence of mycoplasma contamination before use.
?. [Thawing and general handling of 293-F cells before transfection (Timing: 11 days)]
Rapidly thaw the cells in a 37°C water bath, disinfect with 70% ethanol and resuspend in the 9 ml medium aliquot. Centrifuge the cells at 340g for 5 minutes and discard the supernatant. Resuspend the cell pellet in the 30 ml aliquot of prewarmed medium.
?. [Thawing and general handling of 293-F cells before transfection (Timing: 11 days)]
Assess post-thaw cell count and viability using trypan blue and a hemocytometer or an automated cell counter. Incubate the cells in a 37°C incubator in a humidified atmosphere with 8% CO2 and orbital shaking at 120 RPM.
?. [Thawing and general handling of 293-F cells before transfection (Timing: 11 days)]
Monitor cell growth and viability everyday, until cells reach a confluency of about 2-3 x 106 cells/ml. At this point, cells should be split at 0,5 x 106 cells/ml.
?. [Thawing and general handling of 293-F cells before transfection (Timing: 11 days)]
We recommend passaging the cells at least twice after thawing to ensure proper fitness before transfection. We also recommend transfecting cells at most at 10 passages post-thaw to create a stable cell line, hence the importance of creating a stock of low-passage frozen cells after the initial thaw.
?. [Lipofectamine transfection of 293-F cells (Timing: 3 days)]
Prepare the two plasmid vectors ordered from Addgene (eSpCas9(1.1)_No_FLAG_AAVS1_T2 and AAVS1_puro_CAG_HexaPro) as high-quality DNA preps. We recommend transfecting a control vector such as a mammalian GFP expression plasmid (750 ng) to monitor transfection efficiency and cell death during the subsequent puromycin selection step.
Note: Lipofection conditions, such as the ratios of Lipofectamine 3000 and P3000 reagents, may benefit from further optimisation. However, we found that the conditions presented here were acceptable for generating stable cell lines using 293-F cells. The transfection conditions used here can be scaled up or down.
?. [Lipofectamine transfection of 293-F cells (Timing: 3 days)]
Count and plate 2 x 105 cells in a volume of 2ml per well in a 12-wells cell culture plate for each transfection condition.
?. [Lipofectamine transfection of 293-F cells (Timing: 3 days)]
Using Opti-MEM medium prewarmed at 37°C, prepare the following mixes in two tubes:  Tube A    Reagent    Amount    Lipofectamine 3000    3 µl    Opti-MEM medium    Complete to 50 µl    Tube B    Reagent    Amount    eSpCas9(1.1)_No_FLAG_AAVS1_T2    250 ng    AAVS1_puro_CAG_HexaPro    500 ng    P3000 reagent    2 µl    Opti-MEM medium    Complete to 50 µl
Tube A    Reagent    Amount    Lipofectamine 3000    3 µl    Opti-MEM medium    Complete to 50 µl
Tube B    Reagent    Amount    eSpCas9(1.1)_No_FLAG_AAVS1_T2    250 ng    AAVS1_puro_CAG_HexaPro    500 ng    P3000 reagent    2 µl    Opti-MEM medium    Complete to 50 µl
?. [Lipofectamine transfection of 293-F cells (Timing: 3 days)]
Transfer the content of tube A to tube B and mix thoroughly by pipetting. Incubate this mix 10 minutes at room temperature.
?. [Lipofectamine transfection of 293-F cells (Timing: 3 days)]
Add the content of tube B to the corresponding well of the 12-wells plate.
?. [Lipofectamine transfection of 293-F cells (Timing: 3 days)]
Incubate the cells in a 37°C incubator in a humidified atmosphere with 8% CO2 without shaking for 72h.
?. [Puromycin selection of the transfected 293-F cells (Timing: 8-11 days)]
Prewarm 8 ml FreeStyle Expression Medium in a sterile 50 ml Erlenmeyer flask. Transfer the transfected cells to the flask. Cells may clump and attach to the bottom of the wells, in which case gentle up and down pipetting is generally sufficient to resuspend them. Incubate the cells overnight in a 37°C incubator in a humidified atmosphere with 8% CO2 and orbital shaking at 125 RPM. We recommend lightly unscrewing the cap from the flasks during incubation, as narrow flask caps such as these are usually not vented.
?. [Puromycin selection of the transfected 293-F cells (Timing: 8-11 days)]
The next day, add 10 µl of the diluted puromycin solution to the medium so that the final concentration is 0,5 µg/ml in a 10 ml total volume.
?. [Puromycin selection of the transfected 293-F cells (Timing: 8-11 days)]
Monitor cell death and growth every 2-3 days until a confluency of 2-2,5 x 106 cells/ml, generally 7-10 days after adding the antibiotic. At this point, the puromycin selection is generally complete. If cells reach confluency too early, an additional 1:2 to 1:3 split may be performed.
?. [Puromycin selection of the transfected 293-F cells (Timing: 8-11 days)]
Once the pool of puromycin-selected cells reaches confluency, split the cells in culture medium without puromycin. At this point, we highly recommend taking an aliquot of medium from a confluent culture to confirm the secretion of the spike protein by western blot with a Strep-Tactin HRP conjugate.
?. [Production and harvest from a stable spike-expressing cell line (Timing: 7-12 days)]
To start a production, split the cells in a 2,8L Fernbach baffled culture flask to obtain 0,5 x 106 cells/ml in a total volume of 500ml fresh 293 FreeStyle Expression medium.
Note: Volumes of medium and reagents are provided for protein production from 500ml of culture medium and may be scaled up or down accordingly. As with other chromatography columns, ensure that the volume of liquid inside the column is always sufficient to keep the resin submerged.
?. [Production and harvest from a stable spike-expressing cell line (Timing: 7-12 days)]
Incubate the cells 7-12 days in a 37°C incubator in a humidified atmosphere with 8% CO2 and orbital shaking at 55 RPM. Figure 2A illustrates spike accumulation in the culture medium at several time points.
?. [Production and harvest from a stable spike-expressing cell line (Timing: 7-12 days)]
Once the desired time point has been reached, harvest the cultures, and divide equally between two 500 ml PPCO Nalgene bottles. Centrifuge at 1850g for 20 minutes. Transfer the supernatant to two new Nalgene bottles and repeat centrifugation.
?. [Production and harvest from a stable spike-expressing cell line (Timing: 7-12 days)]
Harvest the supernatant from both bottles after the second centrifugation step and pass through a 0,45 µm sterile filter. Use filtered supernatant for subsequent purification.
?. [Histidine tag purification of the spike protein from filtered culture supernatant (Timing: 1 day)]
Transfer the filtered supernatant to a clean 1 L container and add 30 ml of Ni Sepharose 6 Fast Flow resin pre-rinsed with PBS 1X. Incubate overnight on a rocking platform at room temperature. Optional: save an aliquot of the initial filtered supernatant, referred to here as “Hisinput”.
?. [Histidine tag purification of the spike protein from filtered culture supernatant (Timing: 1 day)]
Transfer the supernatant mixed with the resin to a clean 2,5 cm x 20 cm chromatography column and wait for the beads to settle down. Next, collect the flowthrough. Optional: save an aliquot of this fraction, referred to here as “His flowthrough”.
?. [Histidine tag purification of the spike protein from filtered culture supernatant (Timing: 1 day)]
Add 150 ml of 1X PBS to wash the column and collect the flowthrough. Optional: save an aliquot of this fraction, referred to here as “His wash 1”.
?. [Histidine tag purification of the spike protein from filtered culture supernatant (Timing: 1 day)]
Add 150 ml of His wash buffer and collect the flowthrough. Optional: save an aliquot of this fraction, referred to here as “His wash 2”.
?. [Histidine tag purification of the spike protein from filtered culture supernatant (Timing: 1 day)]
Add 150 ml of His elution buffer and collect the following fractions:E1: 20ml (E1 His)E2: 40ml (E2 His)E3: 30ml (E3 His)E4: 30ml (E4 His)E5: 30 ml (E5 His)
?. [Histidine tag purification of the spike protein from filtered culture supernatant (Timing: 1 day)]
Wash the column with 240 ml PBS and store at 4°C.
?. [Histidine tag purification of the spike protein from filtered culture supernatant (Timing: 1 day)]
Assess protein recovery and purification efficiency by loading the different fractions onto an 8% polyacrylamide gel and performing polyacrylamide gel electrophoresis (PAGE). Total proteins can be visualized by staining the gel with colloidal Coomassie Blue. A typical gel showing the fractions obtained at the different steps of purification is shown in Figure 2. Total proteins can also be rapidly visualized by employing Stain-Free gels and a stain-free enabled imager. A less expensive alternative to commercial Stain-Free gels can be achieved by adding 0,5% (v/v) 2,2,2-Trichloroethanol (TCE) to a handcast polyacrylamide gel and visualizing total proteins on a stain-free enabled imager or a UV gel imager26. In our experience, the second elution (E2 His) contains most of the purified protein and is selected for subsequent Twin-Strep tag purification.
?. [Twin-Strep tag purification of the spike protein from a histidine purified sample (Timing: 1 day)]
Add 10ml of Strep-Tactin XT 4Flow high-capacity resin to the selected His eluate fraction(s) in a clean 50-100 ml container. Incubate overnight on a rocking platform at room temperature.
?. [Twin-Strep tag purification of the spike protein from a histidine purified sample (Timing: 1 day)]
Transfer the His eluate mixed with the resin on a 1,5 cm x 10 cm chromatography column and wait for the beads to settle down. Collect the flowthrough. Optional: save an aliquot of this fraction, referred to here as “Strep-Tactin flowthrough”.
?. [Twin-Strep tag purification of the spike protein from a histidine purified sample (Timing: 1 day)]
Wash the column with 5 x 10 ml of 1X buffer W and collect the flowthrough. Optional: save an aliquot of each of these five fractions, referred to here as “Strep-Tactin wash 1-5”.
?. [Twin-Strep tag purification of the spike protein from a histidine purified sample (Timing: 1 day)]
Elute with 30 ml of 1X buffer BXT and collect the following fractions:E1: 5 ml (E1 ST)E2: 20 ml (E2 ST)E3: 5 ml (E3 ST)
?. [Twin-Strep tag purification of the spike protein from a histidine purified sample (Timing: 1 day)]
Pass 150 ml of 3M MgCl2 to regenerate the column, and re-equilibrate using 80 ml of 1X buffer W. Store the column at 4°C.
?. [Twin-Strep tag purification of the spike protein from a histidine purified sample (Timing: 1 day)]
Assess protein recovery and purification efficiency by loading the different fractions onto an acrylamide gel as described in step 26. Protein yield may be assessed by methods such as Bradford assay and absorbance at 280nm using a UV spectrophotometer. We found that both resins performed well when reused to purify up to 3-4 batches. The resulting purified HexaPro spike protein can be used in downstream applications. We observed that the protein seemed to remain stable at 4°C on the short term but advise flash-freezing followed by -80°C keeping for long term storage.
?. [Anticipated results]
By following this protocol, we consistently obtain yields of 30-50 mg of purified protein per liter of culture supernatant as determined by Bradford assay and A280 absorbance. Most of the purified protein is eluted in the second (E2 ST) and third (E3 ST) fractions of the final elution step, at concentrations of up to 2 mg/ml. Figure 1C shows the consistency in purity obtained from different production lots using the protocol described here. Figure 3 shows that the purified protein binds both to recombinant ACE2 and mAb CR3022 and that this protocol can be adapted to produce a D614G HexaPro spike variant. The vectors used in this protocol are available from Addgene (#79888 and #164077).
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