Insights\ninto Folding and Molecular Environment of\nLyophilized Proteins Using Pulsed Electron Paramagnetic Resonance\nSpectroscopy

There\nis still an insufficient understanding of how the characteristics\nof protein drugs are maintained in the solid state of lyophilizates,\nincluding aspects such as protein distances, local environment, and\nstructural preservation. To this end, we evaluated protein folding\nand the molecules’ nearest environment by electron paramagnetic\nresonance (EPR) spectroscopy. Double electron–electron resonance\n(DEER) probe distances of up to approximately 200 Å and is suitable\nto investigate protein folding, local concentration, and aggregation,\nwhereas electron spin echo envelope modulation (ESEEM) allows the\nstudy of the near environment within approximately 10 Å of the\nspin label. We spin-labeled human serum albumin (HSA) and freeze-dried\ndifferent concentrations with 100 g/L deuterated sucrose. DEER showed\ndistinct local concentration behaviors for two folding states, directly\ncorrelating folding percentage with the interprotein distance, reaching\n2 nm at an HSA concentration of 84 g/L. Interestingly, 50% of the\nHSA molecules showed partial structural perturbation already at 2.6\ng/L, which corresponds to a molar ratio Suc/HSA of 7469. This percentage\nincreased to 97% with an increase in the HSA concentration to 84 g/L.\nThe degree of protein perturbation cannot be told, and no signs of\nunfolding are found after reconstitution. ESEEM demonstrated a higher\nsucrose concentration around the protein label compared to the HSA\nenvironment in highly concentrated sucrose solutions. The partial\nunfolding detected in DEER could lead to label exposure and explain\nthe enhanced sucrose detection in the intimate shell. Our work provides\nnew insights regarding sucrose enrichment in the nearest shell of\nproteins upon lyophilization. In addition, the results indicate substantial\npartial structural perturbation, even in the presence of enormous\nsupplies of stabilizing sugars. Thus, pulse EPR spectroscopy allows\nadditional understanding of the solid state of protein lyophilizates,\nwhich is complementary to SANS, FTIR, or ssNMR.