Steritop filter

HEK293F cells (Invitrogen) were transiently transfected with SARS-CoV-2 S-Foldon pPPI4. Cells were grown in Freestyle medium (Life Technologies) and transfected at a density of 0.8–1.2 million cells/mL. A mixture of PEImax (1 µg/mL) and expression plasmid (312.5 µg/mL) in OptiMEM (GIBCO) was made on the day of transfection and added to the cells. After 6 days, cells and growth medium were transferred into centrifuge buckets and spun down at 3000 × g for 30 min. Supernatants were filtered through 0.22-µm Steritop filters (Merck Millipore). After filtration, spikes were purified using Ni-NTA agarose beads. Eluted proteins were concentrated and the buffer was changed to PBS using 100 kDa cutoff Vivaspin filters (GE Healthcare). Next, proteins were applied to a Superose 6 increase 10/300 GL column linked to a NGC chromatography system (BIO-RAD) in PBS. The appropriate size-exclusion fractions were pooled and proteins were stored at −80 °C.

All constructs were transiently transfected into HEK 293F cells (Invitrogen) maintained in Freestyle medium (Life Technologies) at 0.8–1.2 million cells/mL. For transfection, a mix of expression plasmid (312.5 μg/L cells) and PEImax (937.5 μg/L cells) was made in OptiMEM (Gibco) and was added to the cells. Six days after transfection, supernatants were collected by centrifuging cell cultures at 3000 × g for 30 min. Supernatants were filtered using 0.22 μm Steritop filters (Merck Millipore) and subjected to Ni-NTA agarose beads for affinity purification. Eluted proteins were concentrated and buffer exchanged to PBS using Vivaspin (GE Healthcare) filters with a 100.000 Da cutoff. Protein concentrations were measured using Nanodrop using the proteins peptidic molecular weight.

HEK293F cells (0.8-1.2 million cells per mL) were transiently transfected with the coronavirus S constructs. Cells were maintained in Freestyle medium (Life Technologies). A mix of PEImax (937.5 μg/L cells) and expression plasmid (312.5 μg/L cells) was prepared in OptiMEM (Gibco) and added to the cells. Six days after the transfection the supernatants were collected by centrifuging the cell cultures at 3,000 x g for 30 min and were filtered through 0.22 μm Steritop filters (Merck Millipore). Supernatants were subjected to Ni-NTA agarose beads for affinity purification. Proteins were eluted and buffer exchanged to PBS and concentrated using Vivaspin filters (GE Healthcare) with a 100,000 Da cut-off. Additionally, proteins were applied to a Superose 6 increase 10/300 GL column (GE healthcare) in PBS for size exclusion chromatography. Appropriate size fractions were collected and subsequently pooled and concentrated using Vivaspin filters if necessary. Concentrations were measured using the peptidic molecular weight with Nanodrop. Proteins were stored at -80°C. The BtKY72 S protein was acquired from ACRO Biosystems, while influenza A [A/Victoria/2570/2019 (H1N1)pdm09-like virus] hemagglutinin was obtained from The Native Antigen Company. HIV-1 BG505 Env was produced as described elsewhere.⁵⁷

The nanobody-IgG1 constructs were transiently expressed in HEK-293F cells (Invitrogen). Cells were cultured in Freestyle medium (Life Technologies) at a density of 1×10⁶ cells/mL. At day 1, the cells were transfected using the antibody plasmid and 1 µg/µl PEImax (Polysciences) in a 3:1 ratio in OptiMEM. For the production of the bispecific antibodies, HEK-293F cells were transfected with the J3 IgG1-knob plasmid and either the 2E7-IgG1-hole or 1F10-IgG1-hole plasmid in a 1:1 ratio, with PEImax (1 µg/µl) in a 3:1 ratio in OptiMEM. Supernatant was harvested at day 6, centrifuged (30 minutes, 4000 rpm) and filtered using 0.22 µm Steritop filters (Merck Millipore). Antibodies were purified using protein A/G (Pierce) affinity chromatography. Antibodies were eluted from the column using 0.1 M glycine (pH 2.5) and neutralized using neutralization buffer 1M Tris (pH 8.7). The eluates were concentrated and buffer exchanged to PBS using 50 kDa Vivaspin filters (GE Healthcare). In all assays we correct for protein size using the following calculation: $C=\frac{m}{V}x\frac{1}{MW}$ . Where C is the molar concentration in mol/L or M, m is mass of the protein in grams (g), V is volume of solution in liters (L) and MW is the molecular weight of the protein in g/mol.

All antibodies were transiently expressed in HEK293F cells similarly to the proteins. Suspension HEK293F cells (Invitrogen, cat no. R79009) at a density of 0.8–1.2 million cells/mL were co-transfected with two plasmids expressing IgG1 heavy chain (HC) and light chain (LC) in a 1:1 ratio using 1 mg/mL PEI MAX (Polysciences) (1:3 plasmid:PEI MAX). Supernatants containing the antibodies were harvested five days post-transfection, centrifuged for 30 min at 4000 × g and filtered using 0.22 μm Steritop filters (Merck Millipore). The filtered supernatant was run over a 1 mL protein A/G column (Pierce) followed by two column volumes of PBS wash. The antibodies were eluted with 18 mL 0.1 M glycine pH 2.5 directly captured in 2 mL neutralization buffer (1 M TRIS pH 8.7). The purified antibodies were buffer exchanged to PBS using 100 kDa VivaSpin20 columns (Sartorius). The IgG concentration was determined using a NanoDrop 2000 and the antibodies were stored at 4 °C until further analyses.