| dc.description.abstract | The conformational stability of a protein is a fundamental concern in formulating protein drug products, and development of analytical methods for stability characterization is critical for this purpose. Because of the complexity, relatively lower stability and variety of degradation pathways of protein molecules, the selection of appropriate analytical methods is important and needs to meet specific requirements for stability testing, screening formulations, as well as for product release. To characterize the stability of proteins comprehensively, the conformational stability of proteins is typically investigated using a variety of biophysical measurements as a function of environmental stresses such as pH and temperature. These multiple techniques typically require a variety of single function instruments. Each technique, however, provides information on only a particular aspect of a protein's higher order structural integrity (secondary, tertiary, and quaternary). The combination of different techniques provides a better understanding of the overall conformational stability of a protein, although these measurements typically require large sample quantities and long experimental times. Therefore, the development of high throughput methods to comprehensively characterize protein stability with a single multifunctional instrument is needed to save both materials and time. In the first part of my work (Chapters 2-4), a new methodology is developed to obtain protein conformational stability data simultaneously, including UV absorption, light scattering, and near- and far- UV CD by employing a multimodal spectrometer (Chapter 2). Fluorescence spectral data are also collected on the same instrument although not simultaneously. The method is developed by examining the thermal and pH stability of four model proteins. Results show reproducible and accurate data using this single instrument, and data collection is rapid with minimal protein sample requirements. This study also illustrates the application of this method for generating empirical phase diagrams (EPDs) to better characterize the overall conformational stability of proteins. This new approach facilitates the rapid characterization of protein structure and stability data in a single instrument. It is useful for analysis of unknown proteins, as well as screening of solution conditions to optimize stability for protein therapeutic drug candidates. I also describe the application of this new method (Chapter 2) to characterize the stability of the recombinant anthrax protective antigen (rPA) and its 2-fluorohistidine (2-FHis) labeled analogue (Chapter 3). Protective antigen (PA) is a key, non-toxic component of the anthrax toxin. It is also a primary antigenic component of the current formaldehyde inactived anthrax vaccine. An analogue of PA has been recently shown to block key steps in pore formation in the process of inducing cytotoxicity in cells. Thus, it can potentially be used as an antitoxin or a vaccine. This analogue is synthesized by incorporating the unnatural amino acid 2-fluorohistidine (2-FHis). In this study, the effects of 2-FHis labeling on rPA antigen stability and its dynamic properties are investigated by various experimental techniques. Physical stability profiles of the two proteins (rPA and 2-FHis rPA) are created by using the empirical phase diagram (EPD) approach, and stability differences between them are identified. Results show that rPA and 2-FHis rPA have similar stability at pH 7-8. With decreasing pH, however, 2-FHis rPA is found to be more stable. Intramolecular dynamics sensitive measurements of the proteins at pH 5 find that 2-FHis rPA has increased internal mobility under acidic pH conditions. The stability and dynamics characterization data provide information useful for the formulation development of 2-FHis rPA as a more stable antigen for vaccine development. In addition, the new multimodal spectrometer method (Chapter 2) is coupled to other techniques such as SDS-PAGE, static light scattering, dynamic light scattering, isoelectric focusing, size-exclusion liquid chromatography as well as micro flow imaging to characterize the stability of dominant-negative inhibitor (DNI) protein (Chapter 4). DNI is a translocation-deficient mutant of PA with double mutations of K397D and D425K. It is a promising candidate for treatment or prevention of anthrax disease, which can potentially provide high immunogenicity in vaccines and therapeutic activity in antitoxic therapy. The lyophilized protein formulated with common excipients was stored for about 8 years at refrigerated temperature. In my study, samples were placed at two selected temperatures (40 and 70 oC) for one to four weeks, and analyzed by the above mentioned methods. The chemical stability (deamidation) was also evaluated using capillary isoelectric focusing (cIEF). Results show that the thermally stressed DNI protein retained its native conformation at temperatures as high as 70 oC for one month. The long-term stability of DNI was also demonstrated for samples stored at 4 oC for about 8 years. These data provide valuable and encouraging information for development of an alternative recombinant anthrax vaccine using a mutated anthrax protective antigen. In the second part of my work (Chapters 5, 6), the research focuses on stability characterization and formulation of a pneumolysin mutant (L460D), a candidate for a pneumococcal vaccine (Chapter 5), and two virus-like particles (VLPs) as candidates for use against human filovirus infection (Ebola and Marburg, Chapter 6). In these studies, a three step experimental design is employed in the characterization and formulation processes. First, the conformational stability of the pneumolysin mutant and VLPs are characterized under stressed environmental conditions (pH 3-8 and temperature 10-87.5 oC) by different analytical methods. Second, different types of biophysical data are summarized by using the empirical phase diagram (EPD) method, which is also used to define conditions for an excipients screening study. Finally, the pharmaceutical excipients are screened and potential stabilizers are identified by appropriate assays. In these studies, critical parameters (such as solution pH, temperature, ionic strength and stabilizers) affecting the stability of proteins are defined. These studies provide a basis for selection of optimized solution conditions for further long-term stability studies of pneumococcal antigens and Ebola and Marburg virus-like particles, and illustrate the utility of the multimodal spectrometer and EPD analysis for vaccine formulation development. | |
