End-to-End One-Pot Preclinical Immunoaffinity-Liquid Chromatography-Tandem Mass Spectrometry Workflow for Simultaneous Quantitation of Total and Conjugated Antibody Levels of Antibody-Drug Conjugates with Protease-Cleavable Linkers.

The share of the oncology drug pipeline represented by antibody-drug conjugates (ADCs) continues to grow at a remarkable pace. To support the preclinical screening and characterization of early ADC candidates at scale, innovative bioanalytical approaches that improve turnaround times and data quality are sorely needed. This article presents a novel, streamlined one-pot immunoaffinity-liquid chromatography-tandem mass spectrometry (LC-MS/MS) workflow employing proteinase K for the simultaneous quantitation of total and conjugated antibody levels in human IgG1-based ADCs with protease-cleavable linkers by generating both surrogate analytes in a single digestion. Peptide mapping was used to select a sensitive and selective surrogate peptide, followed by optimization of reaction pH, temperature, and digestion time. The method was qualified according to nonregulated acceptance criteria and demonstrated robust linearity, accuracy, and precision, performing comparably to the conventional trypsin assay in both toxicokinetic and pharmacokinetic studies. Real-world applications highlighted the utility of this method as a sound alternative when troubleshooting the trypsin assay. By consolidating a dual-protease workflow into a single-protease workflow, this one-pot assay minimizes sample handling and variability, reduces the usage of samples and consumables, and increases LCMS uptime by halving the number of samples to be analyzed.