Superose 6 increase column

Stocks of Rituximab and Trastuzumab (from therapeutic preparations dialyzed against PBS) were diluted at 1 mg/mL in PBS and subject—or not—to ferrous treatment (500 µM FeSO₄ final concentration) or to NaOCl treatment (200 µM NaOCl final concentration). After 30 min incubation on ice, 100 µL of native and treated Abs (not dialysed) were injected on a Superose 6 Increase column (Cytiva, Marlborough, MA, USA) mounted on an Äkta Purifier 10 (Cytiva) and previously equilibrated with at least 3 column volumes of PBS. Elution was carried out with PBS at 0.5 mL/min and monitored at 280 nm. Raw data were exported from Unicorn software (Cytiva) and elution profiles were overlaid using GraphPad Prism v.9 software.

SARS-CoV-2 S subunits and domains were produced in Expi293F Cells (ThermoFisher Scientific) grown in suspension using Expi293 Expression Medium (ThermoFisher Scientific) at 37°C in a humidified 8% CO2 incubator rotating at 130 rpm. Cells grown to a density of 3 million cells per mL were transfected using the ExpiFectamine 293 Transfection Kit (ThermoFisher Scientific) and cultivated for 3–5 days. Proteins were purified from clarified supernatants using a nickel HisTrap HP affinity column (Cytiva) and washed with ten column volumes of 20 mM imidazole, 25 mM sodium phosphate pH 8.0, and 300 mM NaCl before elution on a gradient to 500 mM imidazole. To produce SARS-CoV-2 S in the postfusion state, SARS-CoV-2 S D614G was incubated with 1:1 w/w S2X58-Fab (16 (link)) and 10 ug/mL trypsin for one hour at 37°C before size exclusion on a Superose 6 Increase column (Cytivia). Proteins were buffer exchanged into 20 mM sodium phosphate pH 8 and 100 mM NaCl and concentrated using centrifugal filters (Amicon Ultra) before being flash frozen.

For the preparations of Ag‐S‐Dps complexes, comprising RBD‐S‐Dps, NP‐S‐Dps and Spike‐S‐Dps the antigens: RBD‐SpyT2, SpyT2‐NP and Spike/Spike‐SpyT2, and the scaffold protein SpyC‐Dps were diluted in PBS buffer + 250 mm NaCl to 0.2–1 mg·mL⁻¹ and mixed. To achieve full occupancy of SpyC‐Dps with the antigens, the molar ratio for SpyC‐Dps to RBD‐SpyT2 was 1 : 1.3, for SpyT2‐NP 1 : 2 and for trimeric Spike/Spike‐SpyT2 1 : 2.5. Reactions were left for ˜ 5 min at RT, and covalent coupling between SpyCather2 and SpyTag2 was checked by SDS/PAGE. Subsequently, samples were concentrated using Vivaspin Turbo concentrators (100 000 MWCO). Antigen‐decorated SpyC‐Dps complexes were separated from the excess antigens by SEC in PBS + 250 mm NaCl on a Superose 6 Increase column (Cytiva). Fractions were checked again for purity by SDS/PAGE, frozen in LN2 and stored at −80 °C.