Freeze-dry microscopy of protein/sugar mixtures: drying behavior, interpretation of collapse temperatures and a comparison to corresponding glass transition data.

ABSTRACT The purpose of this study is to investigate the change in collapse appearance and temperature of protein/sugar mixtures as a function of nucleation temperature ( T n ), sublimation velocity ( V sub ) and the sugar/protein mole ratio when performing freeze-dry microscopy experiments. BSA and HSA were used as sample proteins and mixed with either sucrose or trehalose. Differential scanning calorimetry was used to determine the corresponding glass transition temperatures ( T ′ g ). To allow a more representative comparison between these analytical methods, a collapse midpoint temperature ( T c-50 ) was introduced. While there was no distinct correlation between T n and the onset of collapse ( T oc ) for either mixture, V sub was found to correlate with the measured collapse temperature which is important for comparability of experiments. Furthermore, V sub could be used to qualitatively investigate the product resistance to water vapor flow. A dramatic change in the appearance of collapse was found for high sugar/protein mole ratios (≥362:1) which needs to be considered to avoid a misinterpretation of T oc data. At low protein concentrations midpoint T ′ g data showed good agreement with T oc values but were found significantly lower at higher protein concentrations. Application of the Gordon–Taylor equation failed to predict the critical temperature for any of the protein/sugar mixtures studied