2.8. SEC-HPLC Analysis

Size exclusion chromatography (SEC)-HPLC analysis was performed based on previously reported [22]. Each ADCs was analyzed AdvanceBio SEC 300 Å, 4.6 × 150 mm, 2.7 µm column (Agilent), (Tosoh Bioscientific), connected to an Agilent 1260 HPLC system containing a binary gradient pump, a temperature-controlled column compartment, an autosampler, and a diode array detector. The system conditions were as follows: flow rate = 0.25 mL/min at 30 °C; mobile phase A (MPA) = 100 mM NaHPO₄/NaH₂PO₄, 250 mM NaCl, 10% v/v IPA, pH 6.8 (mobile phase, MP). The absorbance was monitored at 280 nm (reference wavelength at 450 nm). Each ADC (1 mg/mL, 40 μL) was injected into the system and eluted over an 11 min run consisting of MP.