Rapid Insights into the Stabilization of Interferon Alpha by Two Surfactants Revealed by STD-NMR Spectroscopy

Surfactants are commonly used in biopharmaceutical formulations to stabilize proteins against aggregation. However, the choice of a suitable surfactant for a particular protein is decided mostly empirically, and their mechanism of action on molecular level is largely unknown. Here we show that a straightforward label-free method, saturation transfer difference (STD) nuclear magnetic resonance (NMR) spectroscopy, can be used to detect protein-surfactant interactions in formulations of a model protein, interferon alpha. We find that polysorbate 20 binds with its fatty acid to interferon, and that the binding is stronger at pH closer to the iso-electric point of the protein. In contrast, we did not detect interactions between poloxamer 407 and inter-feron alpha. Neither of the two surfactants affected the tertiary structure and the thermal stability of the protein as evident from circular dichroism and nanoDSF measurements. Interestingly, both surfactants inhib-ited the formation of subvisible particles during long-term storage, but only polysorbate 20 reduced the amount of small soluble aggregates detected by size-exclusion chromatography. This proof-of-principle study demonstrates how STD-NMR can be employed to quickly assess surfactant-protein interactions and support the choice of surfactant in protein formulation.(c) 2022 The Authors. Published by Elsevier Inc. on behalf of American Pharmacists Association. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)

Automated summary

This study demonstrates the application of saturation transfer difference (STD) nuclear magnetic resonance (NMR) spectroscopy in detecting protein-surfactant interactions. It was found that polysorbate 20 binds with interferon alpha, with stronger binding at pH closer to the protein's iso-electric point. In contrast, no interactions were detected between poloxamer 407 and interferon alpha. This research highlights the potential of STD-NMR for quickly assessing surfactant-protein interactions and aiding in surfactant selection for protein formulation.