Native Reversed-Phase Liquid Chromatography: A Technique for LCMS of Intact Antibody–Drug Conjugates

The synthesis of antibody–drug conjugates (ADCs) using the interchain cysteines of the antibody inherently gives a mixture of proteins with varying drug-to-antibody ratio. The drug distribution profiles of ADCs are routinely characterized by hydrophobic interaction chromatography (HIC). Because HIC is not in-line compatible with mass spectrometry (MS) due to the high salt levels, it is laborious to identify the constituents of HIC peaks. An MS-compatible alternative to HIC is reported here: native reversed phase liquid chromatography (nRPLC). This novel technique employs a mobile phase 50 mM ammonium acetate for high sensitivity in MS and elution with a gradient of water/isopropanol. The key to the enhancement is a bonded phase giving weaker drug–surface interactions compared to the noncovalent interactions holding the antibody–drug conjugates together. The hydrophobicity of the bonded phase is varied, and the least hydrophobic bonded phase in the series, poly(methyl methacrylate), is found to resolve the...