Antibody–drug conjugate preparation

A solution of naked mAb (10 mg/mL in PBS (7.0) + 2 mmol/L EDTA) was treated with 7 molar equivalents of tris(2-carboxyethyl)phosphine (TCEP) for 2 hours at 37°C. Then 14 molar equivalents of linker-drug (from a 10 mmol/L DMA stock solution) were added to the fully reduced antibody, while keeping residual DMA concentration below 10% (v/v). The mixture was incubated at room temperature for an hour followed by purification over a Zeba spin desalting column (Thermo Fisher Scientific) to remove excess reagents. In this step, the resulting ADC was also buffer exchanged into formulation buffer (20 mmol/L histidine, 150 mmol/L NaCl, pH5.5). Moreover, the excess linker-drug could be removed thoroughly by treating the product with activated charcoal (30 mg of charcoal to 1 mL ADC solution) followed by vortexing for 2 hours at room temperature. The charcoal was then removed via sterile filtration (0.2 μm PES filters) and the final ADC was stored at −80°C.