Binary ethylenimine bei

Baby hamster kidney-21 (BHK-21) suspension cells were developed from the original adherent cell line, BHK-21 (C-13) (ATCC, Manassas, VA, USA) by the Animal and Plant Quarantine Agency and the Korea Research Institute of Bioscience & Biotechnology for use in suspension culture with serum-free media [10 ]. The cells reached at least 1.5 × 10⁶ cells/mL from a seeding density of 3 × 10⁵ cells/mL within 72 h. The type O FMDV strains (O/Boeun/SKR/2017 and O/Andong/SKR/2010), isolated in Korea, were used to inoculate BHK-21 suspension cells (3 × 10⁶ cells/mL) at a multiplicity of infection (MOI) of 0.001 on a shaking platform in an incubator held at 37 °C. Viruses were harvested at 16 h post-infection and clarified by centrifugation at approximately 3000× g for 20 min at 4 °C to remove cell debris. Binary ethylenimine (BEI, Sigma-Aldrich, St. Louis, MO, USA) was added at 3 mM to the virus culture supernatant to inactivate FMDV, followed by incubation at 100 rpm for 28 h at 26 °C. Subsequently, the BEI was neutralized by adding 2% sodium thiosulfate (Daejung Chemicals & Metals, Siheung, Korea).

FMDV O/SKR/Boeun/2017 (O BE) [15 (link)], which is a Korean isolate, was used in this study. FMDV O BE was inoculated in BHK21 suspension cells at a multiplicity of infection of 0.005 and incubated at 37 °C in a 5% CO₂ shaking incubator at 110 rpm. Subsequently, CVIS was harvested via centrifugation (4000× g, 20 min) at 16 h post-infection and inactivated by the addition of 3 mM binary-ethylenimine (BEI) (Sigma-Aldrich, St. Louis, MO, USA). The CVIS was then incubated in a shaking incubator at 26 °C for 24 h. Residual BEI was quenched using 2% sodium thiosulfate (Daejung Chemicals, Siheung-si, Korea). The inactivated CVIS was used by itself or concentrated ten times (10×) using a tangential flow filtration system (Merck KGaA, Darmstadt, Germany) with a 100 kDa molecular weight cut-off filter to analyze the proteins and dsDNA contamination of the target peak fractions. Otherwise, inactivated CVIS was concentrated up to 50 times (50×) by mixing it with a final concentration of 7.5% (w/v) PEG 6000 (Sigma-Aldrich) and 0.5 M NaCl (Sigma-Aldrich). PEG-P was obtained by centrifugation (10,000× g for 30 min). It was diluted to the original concentration (1×) or 10 times (10×) of the original concentration by Tris-KCl buffer variant with a low salt concentration (20 mM Tris, 150 mM KCl) to allow benzonase to work effectively.

Four strains of FMDV were used in this study. FMDV O SKR/JC/2014 (O JC), O SKR/BE/2017 (O BE), and A SKR/YC/2017 (A YC) strains were Korean isolates, while FMDV Asia 1 Shamir/ISR/1989 (As 1 Shamir) was of foreign origin. Each strain of FMDV was inoculated in BHK21 suspension cells at a multiplicity of infection (MOI) of 0.002 and was incubated at 37 °C in a 5% CO₂ shaking incubator at 110 rpm. Subsequently, the clarified virus culture supernatant was harvested via centrifugation (4000× g, 20 min) at 16 h post-infection.
Then, viruses were inactivated by the addition of 3 mM binary-ethylenimine (BEI) (Sigma-Aldrich, St. Louis, MO, USA) and incubated in a shaking incubator at 26 °C for 24 h [15 (link)]. Residual BEI was quenched using 2% sodium thiosulfate (Daejung Chemicals, Siheung-si, Korea). The inactivated FMDV culture supernatant was concentrated by mixing it with a final concentration of 7.5% (w/v) polyethylene glycol (PEG) 6000 (Sigma-Aldrich) and 0.5 M NaCl (Sigma-Aldrich). The precipitate was obtained by centrifugation (10,000× g for 30 min) and further purified by sucrose gradient ultracentrifugation as described in a previous study [16 (link)]. The purified 146S antigen was then resuspended in each buffer and excipient composition.