Pellicon xl device

For purification, the supernatants were filtered through 0.45 μm membrane filters (Merck Millipore HAWP04700), concentrated and diafiltrated against 20 mM phosphate buffer containing 500 mM NaCl and 20 mM imidazole (pH 7.4) using a Labscale TFF system equipped with a 5 kDa cut‐off PelliconTM XL device (Merck Millipore, PXC005C50). His‐tagged trimer Spike and monomeric RBD were captured using a 5 ml HisTrap FF crude column (Cytiva, 17528601) connected to an ÄKTA pure chromatography system (Cytiva). Bound proteins were eluted by applying a linear gradient of 20–500 mM imidazole over 20 column volumes. Fractions containing the protein of interest were pooled, concentrated using Vivaspin 20 Ultrafiltration Units (Sartorius, VS2011) and dialyzed against PBS (pH 7.4) at 4°C overnight using a SnakeSkin Dialysis Tubing (Thermo Fisher Scientific, 68100). The RBD was further polished by size exclusion chromatography (SEC) using a HiLoad 16/600 Superdex 200 pg column (Cytiva, 28‐9893‐35) equilibrated with PBS (pH 7.4). Both purified proteins were stored at −80°C until further use.

Pooled cell culture harvests were centrifuged (4800 g, 15 min), filtered (0.45 μM Supor^® membrane filter, Pall Corporation, United States), 20-fold concentrated and diafiltered with 20 mM phosphate buffer containing 500 mM NaCl and 40 mM imidazole (pH 7.4) using a Labscale TFF System equipped with a 30 kDa cut-off Pellicon^TM XL device (Merck, Germany). The ÄKTA pure chromatography system (GE Healthcare, United States) was used for the following purification steps. The diafiltrated solution was loaded onto a 1 mL HisTrap HP column (GE Healthcare, United States) and bound protein eluted applying a linear gradient of 40 to 500 mM imidazole over 20 column volumes. Fractions containing CD19-AD2 were pooled and dialyzed against 20 mM phosphate buffer containing 200 mM NaCl (pH 7.4) using a Spectra/Por Dialysis tubing (Spectrum Chemical Mfg. Corp., United States) at 4°C overnight. Dialyzed protein was concentrated using Amicon Ultrafilters with a cut-off of 10K (Merck, Germany) and subjected to SEC on a HiLoad 16/600 Superdex 200 pg column (GE Healthcare, United States) equilibrated with the same buffer used for dialysis. Fractions containing only monomeric CD19-AD2 were pooled, concentrated and stored at −80°C for all further analysis steps.