Development of Flow-NMR Spectroscopy for Real-Time Monitoring and Kinetics Studies of Biomolecules: Case Study of Liraglutide Oligomerization

We report the development of a comprehensive flow-NMR methodology for mechanistic studies of biomolecules. This approach allows for systematic kinetic investigation via precise sample condition modulations. Traditionally utilized for reaction monitoring and kinetic studies of small molecules, the application of flow-NMR to larger biomolecules, such as peptide oligomers and proteins, has remained unexplored. Here, we present a pioneering study using flow-NMR to examine the pH-dependent oligomeric interconversion of liraglutide, a glucagon-like peptide 1 (GLP-1) receptor agonist known for its efficacy in managing type 2 diabetes and obesity. Liraglutide molecules are prone to forming distinct oligomers and even fibrils under certain conditions, influencing their stability, absorption, and bioavailability─factors critically important in pharmaceutical applications. The developed methodologies and suite of flow-NMR experiments collectively yield comprehensive insights into the interconversion process of liraglutide without resorting to combining multiple other techniques. It incorporates various 1D and pseudo-2D proton NMR experiments, including GUPPY-DOSY, a newly developed version of a flow-compatible DOSY experiment, to monitor critical parameters such as diffusion coefficients (<i>D</i>), transverse relaxation (<i>R</i>₂), and structural similarity. The relative ease of setting up and executing this set of flow-NMR experiments offers a straightforward path to extending their application to the characterization of other complex systems, including therapeutic proteins and biologic drugs.