The purpose of the present studies was to systematically investigate protein-mannitol interactions using vacuum drying, to obtain a better understanding of the effect of protein/mannitol wt/wt ratios on the physical state of mannitol and protein secondary structure in the dried state. Solutions containing beta-lactoglobulin (betaLg):mannitol (1:1-1:15 wt/wt) were vacuum dried at 5 degrees C under 3000 mTorr of pressure. The physical state of mannitol was studied using x-ray powder diffractometry (XRPD), polarized light microscopy (PLM), Fourier-transform infrared (FTIR) spectroscopy, and modulated differential scanning calorimetry (MDSC). XRPD studies indicated that mannitol remained amorphous up to 1:5 wt/wt betaLg:mannitol ratio, whereas PLM showed the presence of crystals of mannitol in all dried samples except for the 1:1 wt/wt betaLg:mannitol dried sample. FTIR studies indicated that a small proportion of crystalline mannitol was present along with the amorphous mannitol in dried samples at lower (less than 1:5 wt/wt) betaLg:mannitol ratios. The T(g) of the dried 1:1 wt/wt betaLg:mannitol sample was observed at 33.4 degrees C in MDSC studies, which indicated that at least a part of mannitol co-existed with protein in a single amorphous phase. Evaluation of the crystallization exotherms indicated that irrespective of the betaLg:protein wt/wt ratio in the initial sample, the protein to amorphous mannitol ratio was below 1:1 wt/wt in all dried samples. Second-derivative FTIR studies on dried betaLg and recombinant human interferon alpha-2a samples showed that mannitol affected protein secondary structure to a varying degree depending on the overall mannitol content in the dried sample and the type of protein.