Lc 2010 liquid chromatograph

A recombinant antigen of SARS-CoV-2 RBD-SD1-Fc and a previously prepared adjuvant in 0.05 M Tris-HCl buffer solution with addition of 0.15 M NaCl (pH 7.5) were mixed, so that the final concentration of RBD antigen was 40 μg/mL or 10 μg/mL, and the adjuvant is 400 μg/mL. The components were thoroughly mixed and placed in a shaker in the refrigerator for 3 h. The vials with RBD-SD1-Fc and adjuvant were then stored in a refrigerator at 2–8 °C.
The completeness of RBD-SD1-Fc sorption on betulin particles was assessed using HPLC (Shimadzu LC-2010 liquid chromatograph, “Shimadzu”, Kyoto, Japan) and a Superdex 200 Increase 10/300GL analytical column (“Merck KgaA”, Darmstadt, Germany). Preliminarily, the vaccine samples were centrifuged at 4000× g for 30 min.

To prepare the adjuvant, we used a betulin solution in tetrahydrofuran (TGF) provided by LLC “Berezovyy Mir” (Moscow, Russia). At the first step, sterilizing filtration was carried out through a nylon membrane (NRG Pall N66+, “Pall Corporation”, New York, NY, USA) with 0.22 µm pore diameter. To obtain a homogenous dispersion of the adjuvant and reduce the concentration of TGF, 1% betulin solution was mixed with a 25-fold volume of sterile 0.05 M Tris-buffer solution (pH 9.0). The suspension was sonicated (35–40 kHz) for 10 min.
The zeta potential of betulin nanoparticles was measured using electrophoretic light scattering (ELS) with Zetasizer Nano (“Malvern Panalytical”, Malvern, Worcestershire, UK). The size of adjuvant nanoparticles was determined using Laser Dynamic Light Scattering (Zetasizer Nano ZS, “Malvern Panalytical”, Malvern, Worcestershire, UK) with a scattering angle of 170° and λ = 633 nm. The calculation of nanoparticle size was performed on the basis of the Stokes–Einstein equation.
The presence of betulin was analyzed using reverse phase HPLC (Shimadzu LC-2010 liquid chromatograph, “Shimadzu”, Kyoto, Japan) and Kromasil 100-C18 (150 × 4.8 mm) analytical column (“Nouryon”, Amsterdam, The Netherlands) with isocratic elution and detection at λ = 210 nm.