Stability Assays for Vaccatide vH2

The thermal, acidic, endopeptidase, and exopeptidase stability assays were performed as previously described (Wong et al., 2016 (link)). Trypsin and carboxypeptidase A were acquired from Sigma–Aldrich (MO, United States). Briefly, the purified vaccatide vH2 was incubated with or without enzyme at the optimal temperature and with a buffer solution as recommended by the manufacturer. At each specific time point, the sample was aliquoted and analyzed by an Aeris peptide XB-C₁₈ column (particle size 3.6 μm, 100 mm × 2.7 mm; Phenomenex, CA, United States) attached to an Acquity ultra-performance liquid chromatography H-class system (Waters, Milford, Japan). The areas under the peaks before and after treatment were determined to evaluate the stability.