Increased Throughput of Combined Stability Testing and Metabolite Identification Using a Sample Multiplexing Strategy for the Optimization of Engineered Cyclic Peptide Drugs

Engineered cyclic peptides (ECPs) have been in the spotlight as novel drug modalities for challenging therapeutic targets. Oral delivery of engineered cyclic peptides benefits from ease of administration. However, one of the main hurdles in developing orally effective peptide drugs is their potential metabolic instability due to enzymatic degradation. To that end, <i>in vitro</i> experiments with simulated intestinal fluid (SIF) have been used to assess drug metabolic stability in the gastrointestinal tract. Currently, metabolic stability evaluations and biotransformation assessments are performed separately, which can be time-consuming and result in complex data analysis. Presented here is a sample multiplexing strategy to address these challenges by leveraging a Thermo Scientific Orbitrap Astral mass spectrometer with two complementary analyzers, enabling the simultaneous analysis of metabolic stability from the Orbitrap full scan and biotransformation from the Astral analyzer from one sample injection. Furthermore, we demonstrate that 10 engineered cyclic peptides can be pooled into one sample injection without compromising the data quality to decrease the instrument run time and improve the throughput of the assay.