Solid state fluorescence of lyophilized proteins

• Published in: Analytical biochemistry 2008-05, Vol.376 (2), p.173-182
• Main Authors: Ramachander, Ranjini, Jiang, Yijia, Li, Cynthia, Eris, Tamer, Young, Meagan, Dimitrova, Mariana, Narhi, Linda
• Format: Article
• Language: English
• Subjects: Chromatography, Gel; Circular Dichroism; Fluorescence; Fluorescence spectroscopy; Freeze Drying; Lyophilization; Protein Conformation; Protein stability; Protein Structure, Tertiary; Proteins - chemistry; Solid state fluorescence; Spectrometry, Fluorescence - methods; Tertiary structure
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2008.02.008

Fluorescence spectroscopy has been used to measure changes in the tertiary structure of proteins in the solution state. The sensitivity of fluorescence to the protein tryptophan environment has made it a useful tool for studying protein conformation and stability. Using fluorescence spectroscopy to probe structural alterations in lyophilized proteins has been limited due to technical challenges and overwhelming background light scattering. We have investigated the possibility of analyzing lyophilized proteins using the Cary-Eclipse spectrofluorometer by monitoring the fluorescence of the protein therapeutic after subjecting the lyophilized cake to heat-induced accelerated degradation. We have been able to obtain reproducible fluorescence spectra, detecting possible structural changes under these conditions. Fluorescence and circular dichroism spectroscopic analyses of the reconstituted proteins indicated that changes in fluorescence intensities observed in the solid state could be correlated to that in solution and to possible tertiary structural changes. Size exclusion chromatography analysis of protein Y subject to accelerated degradation showed a correlation between decreasing fluorescence intensity and increasing protein Y tetramer in solution, consistent with long-term stability. This suggests that solid state, intrinsic protein fluorescence measurements using the Cary-Eclipse holder may be feasible for long-term stability studies and formulation development.