Butyl npr column

HIC HPLC was used to determine levels of the molar ratio of drug substitution: 1260 HPLC (Agilent; Wilmington, DE, USA); Butyl-NPR column (2.5 μm, 4.6 × 35 mm, #14947, TOSOH Bioscience; Tokyo, Japan). Here, HIC buffer A was 50 mM potassium phosphate, pH 7.0, and 1.5 M ammonium sulfate; and HIC buffer B was 50 mM potassium phosphate, pH 7.0, 20% isopropanol. The gradient was 100% to 100% B over 15 min; flow rate was 1 mL/min; and UV detection wavelength was 280 nm. The DAR was determined by peak area integration according to the reported method [32 ].
SEC HPLC was used to determine levels of aggregation within each ADC: 1260 HPLC (Agilent; Wilmington, DE, USA); G3000SWXL analytical column (7.8 mm × 30 cm, #08541, TOSOH Bioscience; Tokyo, Japan). The SEC buffer contained 40 mM sodium phosphate and 150 mM sodium chloride (pH 7.0, 1 mL/min flow rate); and the UV detection wavelength was 280 nm. We performed a needle wash after each injection and include blank runs between each analyte. The aggregation was determined by peak area integration according to the reported method [33 ].

A BsAb was mixed in a 1:1:1 molar ratio with the two parental mAbs prior to differential protein A purification. Differential protein A purification was carried out using a 1 mL mAbSelect Sure column (GE Healthcare). The mixture was eluted in 3 steps using buffers containing either 50 mM citrate pH 4.7, pH 4.2, or pH 3.4. Elution fractions were collected and concentrated to >1 mg/mL prior to analysis. The pooled elution peaks from the differential protein A purification were analyzed by hydrophobic interaction chromatography (HIC) using a butyl NPR column (Tosoh Biosciences). Approximately 30 ug of each sample were injected onto the column and eluted using a 0 to 100% gradient of buffers containing 100 mM sodium phosphate pH 6.0, 1.5 M (NH₄)₂SO₄, or 100 mM sodium phosphate.

HIC was performed using a TSKgel Butyl‐NPR column (3.5 cm × 4.6 mm, 2.5‐µm particle size, TOSOH, Joint analytical systems) on a Waters ACQUITY ARC (U)HPLCystem equipped with a PDA detector. Following a 5‐µL injection of 1‐mg/mL sample diluted in mobile phase A (MPA) (25‐mM phosphate, 1.5‐M ammonium sulfate at pH 6.95), components were eluted using a gradient of 0% – 100% mobile phase B (MPB) (25‐mM phosphate, pH 6.95 in 20% isopropanol) in MPA over 20 min at 27°C at a flowrate 0.4 mL/min. UV absorbance was detected at both 214 and 330 nm. Chromatograms were acquired and analyzed using the Empower chromatography data system (Waters).