4.5. Characterization of the ADC

The aggregation of the final ADC was evaluated by size exclusion chromatography (SEC), and the drug-to-antibody ratio (DAR) was measured by hydrophobic interaction chromatography (HIC). SEC analyses were carried out on a 1260 HPLC (Agilent Technologies, Santa Clara, CA, USA) and TSK-gel G3000SWXL analytical column (7.8 mm × 300 cm; TOSOH Bioscience, Tokyo, Japan). The column was equilibrated with 0.22 μm-filtered 40 mM sodium phosphate and 150 mM sodium chloride at pH 7 at a flow rate of 0.5 mL/min, and the UV absorbance at 280 nm was detected for 35 min. HIC analyses were carried out on a TSK-gel Butyl-NPR column (2.5 μm, 4.6 mm × 3.5 cm, TOSOH Bioscience) in HIC buffer A (50 mM potassium phosphate, pH 7.0, and 1.5 M ammonium sulfate) and HIC buffer B (50 mM potassium phosphate, pH 7.0, 20% isopropanol). The gradient was 0% to 100% buffer B over 15 min, flow rate was 0.8 mL/min, and the UV detection wavelength was 280 nm. In addition, the endotoxin levels were determined using tachypleus amebocyte lysate (0.125 EU/mL, Zhanjiang bokang marine biological CO., LTD, Zhanjiang, China) according to the manufacturer’s instructions.