Date of Award


Degree Type


Degree Name

Master of Science in Pharmaceutical Sciences




Biomedical Sciences

First Advisor

Christopher T. Rhodes


Maintenance of the structural integrity of a therapeutic protein is essential for its efficacy in relation to physiological and pharmacological activity. Therefore, a major challenge confronting the pharmaceutical scientist working with protein formulation is the instability of the protein during processing, handling, storage, and use. Hence well-defined preformulation studies for proteins need to be performed to understand the stability issues relevant to their production, formulation, and use. Vast numbers of drugs available in the market are rnicromolecular dugs, which have molecular weight less than 1000 D. There is a good reason to believe that polypeptides and proteins offer different challenges from conventional molecules. Protein drugs are probably the wave of the future.

Fluorescence spectroscopy has found wide use in studying denaturation of proteins by monitoring its folding-unfolding pattern. Protein conformational changes are usually accompanied by changes in the fluorescence ermss1on spectrum. Fluorescence spectroscopy is not a normal component of preformulation scan of new substances. However, it may have the potential for that purpose, particularly for polypeptides and proteins.

The conformational changes of a humanized monoclonal antibody have been characterized by intrinsic and extrinsic fluorescence measurements m aqueous solutions of pH 3 to 8 in the temperature range of 5 °C to 70 °C. 1- anilinonapthalene 8-sulfonate (ANS) was employed as a polarity-sensitive extrinsic probe. ANS is a compound that fluoresces strongly in hydrophobic non- aqueous environments, but is almost non-fluorescent in water. Because of these characteristics, it has proved to be a very useful probe of accessible protein interior.

Changes in the fluorescence spectrum of the protein are observed with increasing temperature. The intensity of the trytophan peak decreased linearly with temperature. Additionally, the tryptophan emission peak displayed a red shift from 335 nm to about 345 nm over the temperature range studied. The onsets of temperature of transition for the protein were estimated at each pH. The protein at pH 6, 7 and 8 was characterized by an onset of transition of about 60 °C, which was higher than at pH 3 to 5. Since higher onset of transition temperature reflects greater thermal stability, the preformulation studies suggest the protein is likely to be most thermally stable in an aqueous solution formulated at pH 6, 7, and 8.



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