Date of Award


Document Type


Degree Name

Doctor of Philosophy (Ph.D.)


Pharmaceutical and Chemical Sciences

First Advisor

Xiaoling Li

First Committee Member

Bhaskara R. Jasti

Second Committee Member

Xin Guo

Third Committee Member

Liang Xue

Fourth Committee Member

Narang Ajit


Biologics are large, complex therapeutic agents generally produced from living organisms. One group of biologics is peptide/protein based. Biological agents offer unique advantages over traditional therapeutics including longer half-lives, higher specificity, greater efficacy, and reduced off-target effects. However, protein/peptide based drugs suffer from both delivery and stability issues. The higher order of protein structures (secondary, tertiary, etc.) derive ~80% of their conformational stability from paltry hydrophobic effects, with net stabilization of 5-15 kcal/mole observed for many proteins. Loss of conformational stability can lead to increased aggregation, precipitation, and degradation; and reduced activity and side effects. To increase stability and improve other properties of the therapeutic agent, additives, referred to as excipients, are included in their formulation. Generally, stabilizing effects from excipients work by imposing enthalpic or entropic penalties on protein/peptide unfolding, increasing the free energy of the denatured state. How excipient stabilizes by what thermodynamic mechanism for a given protein/peptide is not always clear, requiring careful study and optimization for prospective agents. Much effort has gone into understanding excipient protection mechanisms and identifying potential liable regions like amino acid sequence and hydrophobic patches. One area that has received relatively little attention has been the effect of excipients on secondary structure (SS) thermodynamic stabilization/destabilization. SS features are major components of biologic conformation in which deviations, even temporary, can lead to aggregation and precipitation. In this study, an experimental system is proposed to quantify and classify helix stabilization in a model peptide and protein. Thermodynamic stability was evaluated via helix unfolding in the peptide, or protein through use of circular dichromism (CD) and nuclear magnetic resonance (NMR) for model peptide polyL-lysine (PLL) and CD and differential scanning calorimetry (DSC) for model protein bovine serum albumin (BSA). The chosen molecular weight of the PLL polymer, adopts a helical structure, is neutral and a monomeric under tested conditions, making it an ideal model to evaluate excipient effects on helix stability. BSA is largely helical in nature, with most changes and aggregation behavior resulting from loss of helicity, making it a logical extension from the model peptide. Results showed stabilization from mannitol and trehalose being mainly enthalpically driven in both peptide and protein. Enthalpic destabilization was observed for PLL and BSA at low to mid concentrations but stabilizing for PLL and destabilizing for BSA at high concentrations, respectively. Moreover, use of entropy-enthalpy compensation (EEC) plots revealed primary stabilization mechanisms at varying excipient concentrations and types allowing for a classification system to be established under different conditions. Peptide/protein based therapeutics typically exist in a complex milieu of additives designed to enhance stability and performance, or allow novel delivery methods (oral, pulmonary, etc.) not typically available to such agents. Ultimately, this work provides a model for understanding excipient effects on helix stability in a complex system. Further work into other SS, higher order structures, as well as complex formulation systems in the model framework described in this work will help to improve the formulation optimization process.





Rights Statement

Rights Statement

In Copyright. URI:
This Item is protected by copyright and/or related rights. You are free to use this Item in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you need to obtain permission from the rights-holder(s).