Abstract
Lyophilization can extend protein drugs stability and shelf life, but it also can lead to protein degradation in some cases. The development of safe freeze-drying approaches for sensitive proteins requires a better understanding of lyophilization on the molecular level. The evaluation of the freezing process and its impact on the protein environment in the nm scale is challenging because feasible experimental methods are scarce. In the present work we apply pulse EPR as a tool to study the local concentrations of the solute in the 20 nm range and of the solvent in the 1 nm range for a spin labeled 27 kDa monomeric green fluorescent protein, mEGFP, and the 172 Da TEMPOL spin probe, frozen in different water/glycerol-d5 mixtures. For average glycerol volume fractions, φgly-d5avg, ≥ 0.4 we observed transparent glassy media; the local concentration and the 1 nm solvent shell of TEMPOL and the protein correspond to those of a uniform vitrified glass. At φgly-d5avg ≤ 0.3 we observed partial ice crystallization, which led to ice exclusion of glycerol and TEMPOL with freeze-concentration up to the glycerol maximal-freeze local volume fraction, φgly-d5loc, of 0.64. The protein concentration and its shell behavior was similar except for the lowest φgly-d5avg (0.1), which showed a 4.7-fold freeze-concentration factor compared to sevenfold for TEMPOL, and also a smaller φgly-d5loc. We explain this behavior with an increased probability for proteins to get stuck in the ice phase during fast freezing at higher freeze-concentration and the related large-scale mass transfer.