Empower 3 pro software

Automated affinity capture via Protein A chromatography was performed using a Hamilton Vantage liquid handler. Briefly, approximately 28 ml of each clarified cell culture supernatant was loaded onto separate ProPlus PhyTips (1 ml pipet tip containing 80 μl resin; Biotage) at a flow rate of 0.16 ml/min. The resin was sequentially washed with 1 ml of Dulbecco’s phosphate-buffered saline (DPBS), pH 7.2 (Invitrogen), DPBS plus 1 M NaCl, and DPBS at the flow rate of 0.5 ml/min. Captured antibodies were eluted thrice, each with 0.35 ml of 30 mM sodium acetate, pH 3.6, at the flow rate of 0.5 ml/min. The three elution pools were combined, and pH was adjusted to 5 with addition of 0.03 ml of 1 M sodium acetate, pH ∼9. Sample concentrations were measured using UV spectroscopy on a Lunatic Plate Reader (Unchained Labs) with calculated extinction coefficients. Analytical size-exclusion chromatography was performed on an Agilent 1290 Infinity II LC UPLC system with a Waters BEH200 SEC column (200 Å, 1.7 μm, 4.6 mm × 150 mm); 10-μg injections with 50 mM sodium phosphate, pH 6.8, 200 mM arginine, and 0.05% sodium azide as the mobile phase and a 0.5-ml/min constant flow rate. Chromatograms were analyzed within Empower 3 Pro software (Waters).