Lyophilization of YS1646-derived Vaccine Candidates

YS1646-derived vaccine candidates and control strains were first cultured in LB medium and then formulated in a lyophilization buffer consisting of 28% (w/v) sucrose (BioShop, Burlington, Canada) and 1% gelatin (Sigma-Aldrich, St. Louis, MO) in 25 mM potassium phosphate (KPO₄) at pH 7^{75 (link)}. Samples were aliquoted and frozen at −80 °C overnight. The following day, the strains were freeze-dried using a ModulyoD Freeze Dryer (Thermo Fisher Scientific) for 24 h. Samples were later stored at room temperature for various times ranging from 2 weeks to 4 months and cultured on LB plates to determine viability through the colony-forming unit (cfu) counts. A sample that did not undergo the freeze-drying process was cultured to determine the initial viability of strains (determined by cfu counts) prior to lyophilization.