Inhalation of spray-dried particles is a promising delivery route for proteins as an alternative to injection. Changes in the protein structure and aggregation have to be avoided. The effect of mannitol, a stabilizing agent typically used in both liquid and lyophilized protein formulations, on an antibody (IgG1) in a spray-dried powder was studied using different biophysical and chromatographic techniques. At first, different solutions composed of antibody (IgG1) and mannitol at a ratio between 20/80 and 100/0 IgG1/mannitol (100 mg/ml total solid) were investigated for their stability. Protein solutions containing the IgG1 showed mannitol-dependent aggregation. High amounts of mannitol (50-80%) exerted a destabilizing effect on the antibody and the aggregate 9 level increased to 2.6-4.2%. In contrast, solutions with only 20-40% mannitol showed the same amount of aggregates as the pure antibody solution. The antibody mannitol solutions were investigated by circular dichroism (CD) and Fourier transform infrared spectroscopy (FTIR) to evaluate whether changes in the protein secondary structure can be correlated with aggregation. Considering the sensitivity of the used methods and data evaluation, FTIR spectra did not reveal structural changes in the IgG1/mannitol solutions compared to the pure antibody, despite varying aggregate levels. Thermal stress was reflected in perturbations of the secondary structure, but mannitol-dependent aggregation could not be correlated to detectable alterations in the FTIR spectra. Analyzing the CD spectra revealed no distinctive change in the shape of the CD curve, indicating that the protein secondary structure is mainly retained. This is in agreement with the infrared data. Subsequently, the IgG1/mannitol solutions were spray-dried at Tin/Tout of 90/50 degrees C. Using ATR-FTIR for the investigation of the protein amide I band in the spray-dried powder revealed changes in the sub-components of the amide I band. This indicates that the peptide groups (CO and NH) of the protein are found in a different environment in the solid state, compared to the liquid protein formulation. After redissolution of the powders, the native structure of the pure antibody solution was found identical to the protein secondary structure before spray-drying, indicating that the protein secondary structure is not strongly altered in the dry state, and not affected by the spray-drying process. Thus, from the presented study it can be concluded that the formation of antibody aggregates in mannitol formulations cannot be correlated with significant perturbations of the protein secondary structure elements.