Site-specific antibody conjugation

HIPS-azide^44,45 was resuspended in DMSO to 5 mg/ml and was added to aldehyde-tagged antibody in citrate buffer. The reaction was agitated overnight at 37 °C protected from light. Upon reaction completion, the reaction mixture was filtered using a 40 kDa Zeba size exclusion column. Tri-GalNAc DBCO (50 equiv.) was then added to antibody labeled with HIPS-azide (>1 mg/ml) and the reaction mixture was incubated at rt for 3 days in dark. The reaction mixture was filtered using a 40 kDa Zeba size exclusion column and the protein concentration was determined from A₂₈₀ using Nanodrop 200 Spectrophotometer.