Date of Award

Spring 1-1-2014

Document Type


Degree Name

Doctor of Philosophy (PhD)


Chemistry & Biochemistry

First Advisor

Theodore Randolph

Second Advisor

John Carpenter

Third Advisor

Xuedong Liu

Fourth Advisor

Amy Palmer

Fifth Advisor

Joel Kaar


Lyophilization is often the choice for therapeutic proteins when an aqueous formulation is not sufficiently stable to achieve the desired shelf life. Lyophilization incorporates molecules in rigid and inert glassy matrices by added excipients, where proteins generally exhibit slower degradation kinetics. Unfortunately, both physical and chemical degradations may still occur at unacceptable rates, and the factors that control the rates of these degradations are not well understood. There are a few properties often considered critical to the stability of proteins: a) the extent of native structural retention during lyophilization, b) the dynamic properties of the glassy matrix, c) phase separation, and d) protein adsorption onto glass-air interface.

In this work, we investigated the mechanisms of protein stability in lyophilized sample using two model proteins: recombinant human growth hormone (rhGH) and keratinocyte growth factor-2 (KGF-2). First, we examined formulations of rhGH (helix dominant protein) combined with disaccharides (sucrose or trehalose) and various amounts of hydroxyethyl starch (HES), which upon lyophilization yielded glasses with a wide range of retention of native protein structure and glass transition temperatures, inverse mean square displacements -1 for hydrogen atoms (fast β relaxation), and the relaxation time τ β, which correlates with relaxation due to fast Johari-Goldstein motions. We found that protein structure and local mobility in the glass were strongly correlated with rate constants for aggregation, deamidation and oxidation. Second, we tested lyophilized formulations of KGF-2 (beta trefoil structure). Although degradation rates were generally decreased in formulations with greater native state structural retention and with reduced fast &beta relaxations, these two factors could not account quantitatively for the aggregation and chemical degradation rates of KGF-2 observed. Rather, it appears that the fraction of protein found at the glass solid-air interface is a dominant factor governing protein degradation in freeze-dried formulations. Finally, we lyophilized rhGH using five different methods, which yielded glassy solids with different surface protein contents, glassy state mobilities and degrees of retention of native secondary structure. We found that the extent of rhGH degradation during storage can largely be ascribed to the resulting levels of rhGH found at the solid-air interface after lyophilization.