Optimization of a Raman Microscopy Technique to Efficiently Detect Amorphous–Amorphous Phase Separation in Freeze‐Dried Protein Formulations

ABSTRACTA confocal Raman microscopic technique was optimized to more efficiently detect amorphous–amorphous phase separation in freeze‐dried protein formulations. A Renishaw Raman inVia confocal microscope was used to collect 100–200 μm line maps (2 μm step size) of freeze‐dried protein–excipient formulations. At each point across the line map, the composition was evaluated from the intensity of the nonoverlapping peaks representative of each component. Collection aperture, scan time, and line map length significantly contributed to the phase‐separation analysis, whereas different sample preparation methods did not affect the analysis. Using the optimized parameters (i.e., large aperture 5 s scan time, 200 μm line map), phase separation was successfully detected in binary polymer formulations and was comparable to the previously developed Raman method. However, the previous method required 2.5 h/sample, whereas the optimized method only requires 0.5 h/sample. Phase separation was detected in the following protein–excipient formulations: lysozyme–trehalose (1:1), lysozyme–isomaltose (1:1), β‐lactoglobulin–dextran (1:1), β‐lactoglobulin–dextran (1:3), and β‐lactoglobulin–trehalose (1:1). Phase separation was not detected in lysozyme–sucrose (1:1) and β‐lactoglobulin–sucrose (1:1) formulations. The optimized method successfully detected phase separation in several protein formulations, where phase separation was previously suspected, and promised to be a useful tool for detection of phase separation in amorphous therapeutic formulations. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:2749–2758, 2014