Date of Completion


Embargo Period



Raman, Lyophilization, Freeze-dried, Solid-state, protein, stability, PCA, PLS, FTIR, CD, Conformation

Major Advisor

Dr. Robin H. Bogner

Associate Advisor

Dr. Devendra Kalonia

Associate Advisor

Dr. Carl Anderson

Field of Study

Pharmaceutical Science


Doctor of Philosophy

Open Access

Open Access


Therapeutic proteins are freeze-dried to improve storage stability. However, any changes in conformation of the protein molecule induced by stresses encountered during freeze-drying can influence the storage stability. Fourier Transform Infrared spectroscopy (FTIR) is very useful for monitoring secondary structural changes in protein solid state, but is much less sensitive to smaller tertiary structural changes associated with tryptophan, tyrosine and phenylalanine residues, that may be more predictive of stability. The aim of the present study was development and evaluation of a solid-state Raman spectroscopic method capable of detecting both tertiary and secondary structural solid-state changes in the spectral region from 600 to 1850 cm-1.

Conformational changes in α-lactalbumin, induced at various pH levels, were detectable in the Raman spectra in both the solution and freeze-dried solid states. Principle component analysis (PCA) was able to distinguish the pH induced spectral differences that were visually obscured by large peaks from sucrose, an often-used stabilizer. Principal components (PCs) separated spectral variation in the samples largely due to sample state, protein structural differences, and the presence of sucrose. Using this model protein, the new method proved capable of detecting the protein structural changes in the solid state.

Raman spectral analysis of lyophilized human serum albumin (HSA) detected protein structural changes that occurred during storage and were related to protein instability. Use of chemometrics tools (PCA) identified many small spectral differences, even with overlapping peaks

from stabilizers. A PLS regression model was used to evaluate correlation of spectral changes in the entire region to aggregation rate (R2 > 0.98). Protein stability followed the same rank order as the extent of Raman spectral changes in HSA formulations immediately after lyophilization. Similar data suggested that a material-sparing approach reusing protein samples is representative of protein stored as lyophilized cake.

Changes in Raman scattering associated with tertiary structural changes in the solid-state analyzed by PLS and PCA provides an additional tool for identifying the formulation of therapeutic proteins providing optimal stability. The ultimate goal is a high-throughput screening method to predict rank order in protein storage stability from the Raman scattering spectra of product in situ.

Development of a Raman Spectroscopic Method to Detect Protein Conformation in Amorphous Solids: Application to Protein Formulation and Stability

August 09, 2019

Lauren Kathleen Fontana

B.S., Framingham State University, 1999

Ph.D., University of Connecticut, 2019

Directed by: Professor Robin H. Bogner