Multi-Technique Profiling to Reveal the Batch-to-Batch Variability in the Assembly of Supramolecular Polymers

Supramolecular polymers are formed through reversible interactions, making their assembly sensitive to subtle variations in molecular composition and formulation. Here, we investigate how batch-to-batch variability affects the assembly behavior of a supramolecular system. Using ureido-pyrimidinone-glycinamide (UPy-Gly) as a model, ten samples collected over a four-year period were analyzed by high-performance liquid chromatography–UV/mass spectrometry and nuclear magnetic resonance spectroscopy to identify and quantify trace components. Distinct profiles were observed for each batch, arising from differences in purification method, storage conditions, and the chemist who performed the synthesis. Correlating the relative abundance of trace components with multiple analytical readouts, including UV–visible absorbance, cryo-TEM, small-angle X-ray scattering, and tactoid formation via liquid–liquid phase separation, identified component-specific associations with assembly properties and tactoid geometry. To assess whether these batch-dependent differences translate to biological responses, we additionally evaluated UPy-Gly–induced phenotypic shifts using the Cell Painting assay. Subtle, batch-dependent variations in trace components were associated with measurable differences in supramolecular assembly behavior without abolishing tactoid formation, while Cell Painting profiles and viability remained unchanged under the conditions tested, highlighting both the sensitivity and robustness of this supramolecular polymer system.