Capto s impact

Candidate antibodies were expressed with Expi-CHO Expression system(gibco) for 12days and the supernatant was harvested and purified by protein A resin (GE healthcare). The antibodies were further purified by Q FF (GE healthcare) and Capto S ImpAct (GE healthcare) sequentially and then changed to buffer containing 10 mM CH₃COONa·3H₂O, 30 mM NaCl, 5% sucrose, 0.03% tween-20, pH6.0.

The eluate of benzamidine affinity chromatography was adjusted to pH 6.5 immediately, and then loaded onto a Capto S ImpAct (5.0 × 1.2 cm, GE Healthcare, Sweden) column equilibrated in Buffer E (25 mmol/L phosphate buffer, pH 6.5) in advance. The column was subsequently eluted with a step gradient of 25% and 27% Buffer F (0.5 mol/L NaCl, 25 mmol/L phosphate buffer, pH 6.5). The eluate with a gradient of 27% Buffer F was then collected for subsequent Cibacron Blue affinity chromatography.

The chromatographic column GE XK 16/40 and GE XK 26/40 were separately filled with Capto S ImpAct (GE Healthcare) gel and Capto Q (GE Healthcare) gel. 5 column volumes of chromatography buffer A and chromatography buffer B (20 mM sodium phosphate, 1 mM EDTA, 1 M NaCl, pH 6.5) were used respectively for regeneration. The hydrophobic chromatographic eluent was separately sampled on Capto S ImpAct and Capto Q gel for purification. Capto S ImpAct was sampled at 6.0 mm/min and 15.0 mm/min, the column was equilibrated with chromatography buffer A after loading, and the flow-through liquid was collected to detect the purity. The 4D5Fv-PE25 on Capto Q was eluted with chromatography buffer C (20 mM sodium phosphate, 1.0 mM EDTA, 10% NaCl, pH 6.5), and elution peaks were collected to detect protein purity by SDS-PAGE.