Superose 6 increase 10 300 gl column

Attachment of CBU1910-(His)₆ to tNTA-E2 nanoparticles
followed similar procedures established using other protein-(His)_n antigens.^{56 (link)} Briefly, a 10×
molar excess of NiCl₂ was incubated with tNTA-E2 for 2
h at room temperature and subsequently purified from unchelated Ni
using Zeba spin desalting columns into 20 mM HEPES + 360 mM NaCl buffer
pH 7.3. To test optimal conjugation, varying molar ratios of (His)₆-tagged CBU1910 (previously synthesized^{47 (link)}) were added to the Ni-tNTA-E2 and incubated at room temperature
for 2 h. The Ni-tNTA-E2 + CBU1910-(His)₆ reaction required
optimization to yield unaggregated/precipitated constructs, the details
of which are described in the Supporting Information. After conjugation, solutions were purified with size exclusion
chromatography (SEC) using a Superose 6 Increase 10/300 GL column
(Cytiva) on a FPLC (AKTA, Cytiva). Fractions from the SEC were run
on an SDS-PAGE gel and stained with a Pierce Silver Stain Kit (Thermo
Fisher Scientific) to determine the presence of CBU1910-E2, E2, and
CBU1910, and conjugation efficiencies were estimated by evaluating
band intensities with standards. Fractions containing CBU1910-E2 were
combined and concentrated with a centrifuge concentrator (Vivaspin
6, 10,000 MWCO). Protein concentration was measured via a bicinchoninic
acid assay kit (Pierce). Nanoparticle size was assessed via dynamic
light scattering (DLS).

The culture supernatant of selected SARS-CoV-2 S mutants produced in a 1-liter scale was directly loaded through 5 ml of nickel–nitrilotriacetic acid Excel column (Cytiva Life Sciences). The column was washed with 300 mM sodium chloride and 50 mM imidazole in 20 mM Hepes buffer (pH 7.5) and captured S mutants were eluted with 300 mM sodium chloride and 250 mM imidazole in 20 mM Hepes buffer (pH 7.5). The collected samples from appropriate elution fractions were pooled and concentrated before further purification of the S trimer by gel filtration using a Superose 6 increase 10/300 GL column (Cytiva Life Sciences) with 1× phosphate-buffered saline (PBS), pH 7.4, as a running buffer. The fractions corresponding to the targeted S mutants based on SDS–polyacrylamide gel electrophoresis analysis were pooled together and quantitated using the absorbance at 280 nm.

The gene for mGITRL was cloned into the pEZT-BM expression vector (39 (link)) with a Strep tag added in frame at the N terminus. GITRL protein was produced in the same way as for the receptor protein except 10% (v/v) P2 virus was added when the suspension cells reached a density of 3.5 × 10⁶ cells/ml and cells were pelleted down at 96 hours after transduction. The whole purification was performed on ice or in the cold room at 4°C. The membrane pellet and the lysate supernatant were prepared the same way as for the receptor. The lysate supernatant was filtered through a 0.45-μm filter and loaded on a pre-equilibrated StrepTrap HP column (Cytiva) with purification buffer containing 20 mM tris (pH 8.0), 300 mM NaCl, and 0.5 mM DDM. The column was washed with 10 column volumes of purification buffer and eluted with 5 column volumes of purification buffer containing 20 mM desthiobiotin (IBA). The main elution fractions were concentrated to 700 μl using a centrifugal filter unit (10-kDa MWCO, Amicon) and injected on a Superose 6 Increase 10/300 GL column (Cytiva) equilibrated with purification buffer. The peak fractions were verified by SDS-PAGE gel and then pooled and concentrated.

The biophysical characterization of SARS-CoV-2 HexaPro S protein was conducted using SDS-PAGE analysis (4–12% NuPage™ SDS gel, ThermoFisher, Victoria, Australia) and size-exclusion chromatography (SEC) on a Superose 6 Increase 10/300 GL column (Cytiva, Marlborough, MA, USA) with Gel Filtration Calibration Kits containing a mixture of proteins (thyroglobulin, ferritin, aldolase, conalbumin, ovalbumin, carbonic anhydrase, Cytiva, Marlborough, MA, USA) as standards. The accuracy of the antigenic structure of HexaPro S protein was probed by indirect enzyme-linked immunosorbent assay (ELISA) using a panel of S-specific monoclonal antibodies (1047, 2M-10B11, CR3022, S309, hACE2, 2-17, 1-22, mAb 2.8, and mAb 18C2) [47 (link),48 (link),49 (link),50 (link),51 (link),52 (link)].

Purified S protein was subjected to size-exclusion chromatography using a superose 6 increase 10/300 GL column (Cytiva) in a 1 × PBS buffer. Thyroglobulin (669 kDa), Ferritin (440 kDa), and Aldolase (158 kDa) were used as molecular weight markers.

An ultra-high-performance liquid chromatography system (UltiMate 3000 BioRS, Thermo Scientific) coupled to an FLD-3400RS fluorescence detector (Thermo Scientific) was used for performing the analytical-scale SE-UHPLC fractionation. Flucelvax monovalent drug substance (3mg) was applied to a Superose 6 Increase 10/300 GL column, (Cytiva, Cat# 29-0915-96) equilibrated in running buffer (0.37 g/L monobasic potassium phosphate, 1.29 g/L disodium phosphate, 0.1 g/L magnesium chloride, 0.2 g/L potassium chloride and 8.0 g/L sodium chloride, pH 7.2) and run through the column at a rate of 0.12 mL/min (column temperature 30°C). Antigen peaks were collected as 16 x 1mL fractions with an AKTA pure chromatography system (Cytiva) and pooled to represent HA species: high molecular weight (MW) HA oligomers A (fractions 1-5), high MW HA oligomers B (fractions 9 and 10), HA trimer (fraction 12) and HA monomer (fraction 15). In-line fluorescence data (FLD excitation 280nm, emission 345nm) was assessed using Chromeleon 7.2 software package. The molecular weight of SE-UHPLC separated HA species was determined using gel filtration standards (Bio-RAD, cat# 151-1901). The 4 fractions including Monovalent control antigens were normalized by total protein nitrogen before being assayed via SRID and ELISA methods.