Using Hydrophilic Interaction Chromatography - Mass Spectrometry for Heightened Product Characterization to Overcome Challenges with Hydrophobic Antibody Drug Conjugates and Monoclonal Antibodies

摘要

Hydrophilic Interaction Chromatography is a compelling and complementary alternative to Reversed-Phase Ultrahigh Performance Liquid Chromatography Reversed-phase chromatography often does not yield full recovery of the hydrophobic subunits from derived monoclonal antibodies or antibody drug conjugates. HILIC is a promising alternative because the most hydrophobic subunit elutes first without extreme conditions. HILIC yields full recovery for the two molecules tested here, and does so without signs of on-column hydrolysis. Based on a loading study, the optimal on-column injection producing the highest chromatographic peak resolution and recovery was 5 μg. Both mAb and ADC subunits/domains can be effectively separated and identified by ultrahigh-resolution MS using accurate mass measurements. HILIC is complementary to RP-UHPLC for assessing N-glycosylation profiles; orthogonal information can be obtained by simply reversing common RP gradients, and using a wide-pore 300A amide-bonded stationary phase (ACQUITY UPLC Glycoprotein BEH Amide Column, 300A, 1.7 μm, 2.1 mm × 150 mm).