Date of Completion

12-10-2013

Embargo Period

12-10-2013

Major Advisor

Robin H. Bogner

Co-Major Advisor

Michael J. Pikal

Associate Advisor

Devendra S. Kalonia

Associate Advisor

Evgenyi Y. Shalaev

Associate Advisor

Marcus T. Cicerone

Field of Study

Pharmaceutical Science

Degree

Doctor of Philosophy

Open Access

Open Access

Abstract

Lyophilized protein pharmaceuticals are not infinitely stable and have a limited shelf-life often necessitating refrigeration. Excipients are added to protect the product during both processing and storage; however, the mechanisms of stabilization are still not fully understood. The goal of this research was to better understand critical factors in stabilization of dried proteins, such that long-term, ambient-temperature storage can be achieved. There were two main objectives:

1) Investigate amino acids as excipients to potentially extend the shelf-life of lyophilized proteins.

2) Refine a method for detection of amorphous-amorphous phase separation to better assess potential long-term stabilization issues.

First, the stability of sucrose-based protein formulations was evaluated in the presence of amino acids. Stability of two proteins were improved by the addition of all amino acids studied, over a wide concentration range. Molar volume and polarity of the amino acids influenced the stabilization of the proteins. There was no correlation between measures of global mobility and stability; however, in some cases decreased local mobility was accompanied by improved stability. Stabilization was not well correlated with a decrease

in the system free volume. While no clear predictive measures were identified, amino acids in general showed a notable ability to improve storage stability of dried proteins.

Second, a confocal Raman microscopic technique was optimized to more efficiently detect amorphous-amorphous phase separation in lyophilized protein formulations. By assessing various instrument settings, a previous method was refined to significantly reduce experimental time. Phase separation was detected in protein systems where phase separation had previously been suspected. Raman microscopy has the potential to be used as an early indicator of suboptimal stability by identifying phase separation without the need for long-term stability studies.

The establishment of amino acids as stabilizers for lyophilized proteins and the identification of an indicator for potentially poor stability in these formulations contribute to the long-term goal of achieving more stable proteins. Greater stability of therapeutic proteins will facilitate their distribution and use globally by military troops and by patients in less-developed countries, where product quality is often compromised by the lack of a cold-chain and proper storage conditions.

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