Insights into Folding and Molecular Environment of Lyophilized Proteins Using Pulsed Electron Paramagnetic Resonance Spectroscopy.

Abstract

There is still an insufficient understanding of how the characteristics of protein drugs are maintained in the solid state of lyophilizates, including aspects such as protein distances, local environment, and structural preservation. To this end, we evaluated protein folding and the molecules' nearest environment by electron paramagnetic resonance (EPR) spectroscopy. Double electron-electron resonance (DEER) probe distances of up to approximately 200 Å and is suitable to investigate protein folding, local concentration, and aggregation, whereas electron spin echo envelope modulation (ESEEM) allows the study of the near environment within approximately 10 Å of the spin label. We spin-labeled human serum albumin (HSA) and freeze-dried different concentrations with 100 g/L deuterated sucrose. DEER showed distinct local concentration behaviors for two folding states, directly correlating folding percentage with the interprotein distance, reaching 2 nm at an HSA concentration of 84 g/L. Interestingly, 50% of the HSA molecules showed partial structural perturbation already at 2.6 g/L, which corresponds to a molar ratio Suc/HSA of 7469. This percentage increased to 97% with an increase in the HSA concentration to 84 g/L. The degree of protein perturbation cannot be told, and no signs of unfolding are found after reconstitution. ESEEM demonstrated a higher sucrose concentration around the protein label compared to the HSA environment in highly concentrated sucrose solutions. The partial unfolding detected in DEER could lead to label exposure and explain the enhanced sucrose detection in the intimate shell. Our work provides new insights regarding sucrose enrichment in the nearest shell of proteins upon lyophilization. In addition, the results indicate substantial partial structural perturbation, even in the presence of enormous supplies of stabilizing sugars. Thus, pulse EPR spectroscopy allows additional understanding of the solid state of protein lyophilizates, which is complementary to SANS, FTIR, or ssNMR.