ADC concentration, DAR, and aggregation measurement

The concentration of mAb and conjugated drug in the antibody–drug conjugate was calculated by measuring UV absorbance of an aqueous solution of the antibody–drug conjugate at two wavelengths of 280 nm and 370 nm, as the total absorbance at any given wavelength is equal to the sum of the absorbance of all light-absorbing chemical species that are present in a system (additivity of absorbance). Equations were as follows.

A280 = AD,280 + AA,280 = εD,280CD +εA,280CA (1) and

A370 = AD,370 + AA,370 = εD,370CD +εA,370CA (2).

The values of εA,280, εA,370, εD,280, and εD,370 were estimated based on calculating the amino acid sequence of the antibody or obtained by UV measurement of the compound according to Lambert-Beer's law. Solving the simultaneous equations (1) and (2) by substitution of the above values and then CA and CD were determined. Dividing CD by CA, then the average number of conjugated drug molecules per antibody was determined.

Aggregation was assessed by size exclusion chromatography which was performed on an Agilent 1260 Infinity II HPLC system with UV detection at 280 nm. Column was a TSKgel G3000SWXL column (Tosoh Bioscience). Samples were injected at 50 μg load. Mobile phase was 200 mmol/L sodium phosphate and 150 mmol/L sodium chloride (pH 7.0). Also, 15% (v/v) isopropanol was added to the mobile phase to minimize secondary hydrophobic interactions with the stationary phase and furthermore prevent bacterial growth. The flow rate was 0.75 mL/minutes and column temperature was set at room temperature.

Antibody–drug conjugates with a different number of drugs per antibody were separated using a butyl HIC column (TSK-gel Butyl-NPR 4.6 × 35 mm 2.5 μm, Tosoh Bioscience). HIC was also performed on an Agilent 1260 Infinity II HPLC system with UV detection at 280 nm. Mobile phase A was 1.5 M (NH₄)₂SO₄, 50 mmol/L K₂HPO₄, pH7.0 and mobile phase B was 21.3 mmol/L KH₂PO₄, 28.6 mmol/L K₂HPO₄, 25% (v/v) isopropanol, pH7.0. Gradient program was as follows: B %: 0%–25% (0–1 minute, 0.8 mL/minute), 25% (1–3 minutes, 0.6 mL/minute), 25%–80% (3–13 minutes, 0.6 mL/minute), 80% (13–17 minutes, 0.6 mL/minute), 80%–0% (17–17.10 minutes, 0.5 mL/minute), 0% (17.10–25 minutes, 0.7 mL/minute).