An Orthogonal Framework for Higher-Order Structural and In Vitro Functional Comparability of Chemically Synthesized...

Synthetic peptide test products referencing recombinant-origin products present unique regulatory challenges in demonstrating higher-order structural (HOS) comparability beyond primary sequence sameness. Liraglutide, a lipidated glucagon-like peptide-1 (GLP-1) receptor agonist with defined self-association behavior, represents an important model for such assessments.

A head-to-head comparability study was performed between a chemically synthesized liraglutide product and its recombinant reference listed drug (RLD). An orthogonal analytical framework integrating spectroscopic, biophysical, nuclear magnetic resonance (NMR)-based, and in vitro functional methods was applied to assess secondary structure, supramolecular assembly, aggregation propensity, molecular conformational fingerprints, and biological activity.

The test products and RLD demonstrated highly comparable structural and functional profiles across all evaluated dimensions. Secondary structural characteristics, NMR fingerprints, and spatial conformational features were highly consistent. Solution-state analyses showed comparable diffusion behavior and a dominant low-oligomeric assembly, with apparent aggregation numbers close to the hexamer-to-heptamer range and without evidence of abnormal aggregation or fibrillation. Functional studies further confirmed comparable GLP-1 receptor activity and stability profiles.

Orthogonal structural and functional characterization can substantially reduce residual uncertainty for synthetic peptide generics referencing recombinant products and provides a practical framework for comparability assessment of complex peptide drugs.