LC-multiple reaction monitoring (MRM) quantitation of PLBD2
Both purified antibody mAb-3 with spiked-in PLBD2 standard and mAb drug substance (mAb-3, mAb-4, mAb-5, mAb-6, mAb-7, mAb-8) were digested by trypsin and then were subjected to LC-MRM analysis. LC-MRM analysis was performed on an Agilent 6495A QQQ Mass Spectrometry (Wilmington, DE) equipped with an Agilent 1290 infinity HPLC (Wilmington, DE). 20 µL of the digested samples were injected onto an Acquity BEH C18 column (2.1×50 mm, 1.7μm) at 40°C pre-equilibrated with 12% mobile phase B (0.1% formic acid in acetonitrile, mobile phase A is 0.1% formic acid in water) at a flow rate of 0.4 mL/min. Post sample injection the gradient was maintained isocratically at 12%B for 0.5 min, followed by a linear increase to 15%B over 6 min, and then increased to 90%B in 0.1 min after which the gradient was kept at 90%B for 2.5 min. In the end the gradient was decreased to 12% B to allow the column to be re-equilibrated for 3 min. Eluent between 2-13 min was analyzed using an ESI source operating under positive mode, with gas temperature 250°C, gas flow 12 L/min, nebulizer gas 20 psi, sheath gas temperature 300°C, sheath gas flow 11 L/min, capillary voltage 3500V and nozzle voltage 500 V. PLBD2 were monitored at 615.35/ 817.41 (SVLLDAASGQLR) for quantitation and 427.7/ 450.3 (YQLQFR) for confirmation. Peak integration was performed by Skyline(MacLean et al., 2010), and PLBD2 concentrations were calculated based on the calibration curved created by spiked-in PLBD2 standards.