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Using the Fluorescence Red Edge Effect to Assess the Long-Term Stability of Lyophilized Protein Formulations
journal contribution
posted on 2015-04-06, 00:00 authored by Ken K. Qian, Pawel
J. Grobelny, Madhusudan Tyagi, Marcus T. CiceroneNanosecond
relaxation processes in sugar matrices are causally
linked through diffusional processes to protein stability in lyophilized
formulations. Long-term protein degradation rates track mean-squared
displacement (⟨u2⟩) of hydrogen
atoms in sugar glasses, a parameter describing dynamics on a time
scale of picoseconds to nanoseconds. However, measurements of ⟨u2⟩ are usually performed by neutron scattering,
which is not conducive to rapid formulation screening in early development.
Here, we present a benchtop technique to derive a ⟨u2⟩ surrogate based on the fluorescence
red edge effect. Glycerol, lyophilized trehalose, and lyophilized
sucrose were used as model systems. Samples containing 10–6 mole fraction of rhodamine 6G, a fluorophore, were excited at either
532 nm (main peak) or 566 nm (red edge), and the ⟨u2⟩ surrogate was determined based the corresponding
Stokes shifts. Results showed reasonable agreement between ⟨u2⟩ from neutron scattering and the surrogate
from fluorescence, although deviations were observed at very low temperatures.
We discuss the sources of the deviations and suggest technique improvements
to ameliorate these. We expect that this method will be a valuable
tool to evaluate lyophilized sugar matrices with respect to their
ability to protect proteins from diffusion-limited degradation processes
during long-term storage. Additionally, the method may have broader
applications in amorphous pharmaceutical solids.