2.5. Characterization of ADCs: SEC and HIC Method

To determine aggregation, conjugates were analyzed by size-exclusion chromatography (SEC) (column: TSKgel G3000SWxl 7.8 mm × 30 cm, 5 μm (Tosoh Bioscience; P/N: 08541)) using 0.2 M sodium phosphate 0.2 M potassium chloride, pH 6.5 with 15% (v/v) isopropyl alcohol as mobile phase. An injection volume of 10 μL was loaded to the column at a constant flow rate of 0.35 mL/min. Chromatographs were integrated based on elution time to calculate the purity of monomeric conjugate species.

The drug-to-antibody ratio (DAR) was evaluated by HIC on a high-performance liquid chromatography (HPLC) system (Agilent 1260 HPLC system, TSKgel Butyl-NPR column 4.6 mm × 3.5 cm, 2.5 μm (Tosoh Bioscience; P/N: 14947)). The HIC method used 1.5 M ammonium sulfate in 25 mM potassium phosphate pH 7.0 (mobile phase A) and 25 mM potassium phosphate pH 7.0 containing 25% isopropanol v/v (mobile phase B) run at a flow rate of 0.8 mL/min over a 12-min linear gradient with UV monitoring at 254 and 280 nm.