Minidawn mals detector

For SEC‐MALS measurements, a Superdex 75 Increase 10/300 GL column (Cytiva Life Sciences) coupled to an ÄKTA pure™ protein purification system was used, which was further connected to a miniDAWN MALS detector and an Optilab differential refractive index (dRI) detector (Wyatt Technology). Measurements were performed at room temperature using buffer C with the addition of 0.02% sodium azide, 0.8 mL min⁻¹ flow rate, and protein concentrations of 1, 2, and 3 mg mL⁻¹ with an injection volume of 100 μL. Reproducibility of the SEC‐MALS runs was ensured by obtaining identical results in the measured BSA (bovine serum albumin) samples (Pierce) at 2 mg mL⁻¹ as the first and last sample of each day's measurements. The first BSA injection was used for detector normalization, peak alignment, and band broadening. The dRI signal was used as a concentration source for molar mass determination in all cases. Data were collected and analyzed using ASTRA 8.0.2.5 software (Wyatt Technology).

Recombinant SARS-CoV-2 RBDs hu-RBD and Lt-RBD (20 μl of 1 mg/ml) were injected into a Protein KW-802.5 analytical size-exclusion column (Shodex) and separated with a flow rate of 1 ml min^–1 in PBS using a high-pressure liquid chromatography system (Shimadzu Prominence). For MW characterization, light scattering was measured with a miniDAWN MALS detector (Wyatt), connected to a differential refractive index detector (Shimadzu RID-20A) for quantitation of the total mass, and to a UV detector (Shimadzu SPD-20A), for evaluation of the sole protein content. Chromatograms were collected and analyzed using the glycoconjugate analysis algorithm available in the ASTRA7 software (Wyatt, using an estimated dn/dc value of 0.185 ml/g for proteins and 0.140 ml/g for glycans). Calibration of the instrument was verified by injection of 10 μl of monomeric bovine serum albumin (3 mg/L) (Sigma-Aldrich).

The molar mass, oligomerization state, and degree of glycosylation of S1 were determined by SEC-MALS. The S1 protein from the scaled-up production run was purified by Strep-Tactin purification followed by removal of the strep-tag by TEV protease incubation and SEC. After SEC, the second peak on the chromatogram (Fig. S2, elution 99 to 117 ml) was selected for SEC-MALS analyses to exclude large aggregates. An Infinity 1260 II high-performance liquid chromatography system (Agilent) was coupled to an Optilab dRI detector and miniDawn MALS detector (Wyatt Technologies, USA). The column thermostat and autosampler were both set to 20°C. The protein was diluted to 0.83 mg/ml in PBS (Sigma-Aldrich) and was then resolved in duplicate experiments on a Superdex 200 increase 10/30 GL column (GE Life Sciences) equilibrated in PBS. With ASTRA software (Wyatt Technologies), the absorption at 280 nm, light scattering, and refractive index properties of the eluates were collected and analyzed. The contribution of protein and glycosylation to the eluting species was determined by the conjugate analysis method in ASTRA using dn/dc values of 0.185 ml/g and 0.14 ml/g for protein and glycans, respectively, and an extinction coefficient for the protein at 280 nm of 1.2 ml/(mg·cm). The dn/dc value obtained for the conjugate was used for further molecular weight determination.