Capto core 700

For the testing of different solutions, MMC was performed with 1 mL HiTrap columns loaded with Capto Core 700 (Cytiva) and an ÄKTA pure 25 FPLC system (Cytiva) to automate the process. Each column was equilibrated with 10 column volumes (CV) of one of eight buffered solutions (see Table 1) followed by the application of 20 CV of bacterial lysate (previously dialyzed in the same solvent, see above) containing recombinant γPFD. The flow-through was collected in 1 mL volume fractions. After each run, the columns were cleaned with 10 CV of distilled water, 10 CV of a cleaning-in-place solvent (1 M sodium hydroxide in 30% (v/v) isopropanol), 10 CV of distilled water, and re-equilibrated with one of the eight buffers being tested (Table 1). All steps were performed at 0.5 mL/min flow rate. In all other experiments, MMC was performed by gravity flow using Poly-Prep Chromatography Columns (Bio-Rad) loaded with up to 2 mL of Capto Core 700 resin. The procedure was essentially the same, except all steps were performed manually.

The processes of YFV production and purification are similar to those described by Pato and coworkers [10 (link)]. The working virus seed of YF 17D-213 infects the Vero cells (WHO-approved cell line) and undergoes bioreactor culturing. The supernatant of the culture medium containing the virus was treated with β-propriolactone for 24 h at room temperature to inactivate virus, and then concentrated 100 times by using a molecular weight cutoff of 300 kDa membrane filter (Sartorius, Germany), followed by downstream chromatography purification. Chromatography was performed using Akta pilot, operated with the UNICORN software (https://www.cytivalifesciences.com/en/us/shop/chromatography/software/unicorn-7-p-05649) (Cytiva, Marlborough, MA, USA). Ion exchange (Q Sepharose Fast Flow) and Capto Core700 (Cytiva, USA) resin prepacked in an XK50 Column were used for capturing and polishing, respectively, and the entire purification process. The purified sample was subsequently filtered using 0.45 μm filter units (Sartorius, Goettingen, Germany).

rVSVs expressing the SARS-CoV S (GenBank: AY278554.2) (termed as rVSV-SARS), the SARS-CoV-2 prototype strain S (GenBank: MN908947) (termed as rVSV-SARS2), SARS-CoV-2 VOCs S, or 55 SARS-CoV-2 S with different single point mutations were generated as previously described (33 (link)). Briefly, SARS-CoV or SARS-CoV-2 genes encoding for the S protein were cloned separately into the pCAG eukaryotic expression plasmid (Addgene). These genes had an 18–amino acid C-terminal truncation. rSARS-CoV and rSARS-CoV2 were rescued by VSVdG-EGFP-G (Addgene, 31842) from the Vero E6 cells transfected with plasmids pCAG-SARS1-Sdel18 and pCAG-SARS2-Sdel18 (48 (link)), respectively. Supernatants were harvested and purified by Capto Core 700 (Cytiva) multimodal chromatography. The viral particles were collected in the column flowthrough.

CHIKV-181/25 was propagated on serum-free Vero cells, with harvests clarified and treated with Benzonase to minimize host-cell DNA/RNA contamination prior to concentration and buffer-exchange using tangential flow filtration (TFF) followed by CaptoCore 700 chromatography (Cytiva). All vaccine approaches utilized the same high-purity live CHIKV-181/25 material as their starting point. HydroVax-based inactivation conditions were optimized for CHIKV-181/25 and included 0.0003% H₂O₂, 2 μM CuCl₂, 20 μM MZ and 0.06% formaldehyde, in a buffer matrix containing a protective level of polyatomic oxyanions [22 ] (150 mM Na₂HPO₄) at pH = 7.5, for 20 hours at room temperature. Other inactivation approaches included heat inactivation (HI; 1 hour at 80°C), ultraviolet irradiation (UV, 10 Joules at room temperature using a Spectrolinker XL-1000 UV crosslinker), β-propiolactone (BPL, 0.10% for 20 hours at room temperature, supplemented with addition of 50 mM HEPES), and formaldehyde (0.01% for 20 days at 37°C as previously described [24 (link)]). Following inactivation, chemical components were removed using ion-exchange chromatography, TFF or dialysis with complete inactivation confirmed through cell culture-based residual live virus testing. Vaccine antigens were formulated with 0.2% aluminum hydroxide (Alhydrogel, InvivoGen) prior to use.