Fragmentation and Purification of Vi Capsular Polysaccharide

Vi, freeze dried as the sodium salt, was solubilized in water and H₂O₂ was added to give a final concentration of 2.5 mg/mL Vi and 5% (wt/v) H₂O₂ in water. The mixture was heated at 80±0.5°C for 2h. The mixture was then injected into a Hiscreen Capto Q [GE Healthcare] column (4.7 mL of resin loading up to 100 mg of fragmented Vi mixture) equilibrated with buffer A and populations of different average size were separated using a gradient step method. NaH₂PO₄ 20 mM pH 7.2 and NaH₂PO₄ 20 mM NaCl 1M pH 7.2 were used as buffer A and B respectively. Pools at average size Vi of 8.6 and 43 kDa were eluted at 25 and 37% of buffer B respectively. Each pool was desalted on a Sephadex G-25 column [GE Healthcare] equilibrated with water. The average size of the fragmented Vi pools was determined by HPLC-SEC equipped with a TSK gel 3000 PW_XL column and a TSK gel PW_XL guard column (Tosoh Bioscience). Dextrans (5, 25, 50, 80, 150 kDa) were used as standards (Sigma Aldrich). The mobile phase was 0.1 M NaCl, 0.1 M NaH₂PO₄, 5% CH₃CN, pH 7.2, at the flow rate of 0.5 mL/min (isocratic method for 30 min). HPAEC-PAD was used to measure Vi content [10 (link), 21 (link)]. ¹H NMR was used to verify Vi identity and confirm O-acetylation levels were >60% [10 (link), 21 (link)].