| dc.description.abstract | Protein based vaccine antigens and adjuvants offer several advantages over inactivated or live attenuated viruses and bacteria including ease to manufacture and improved safety. Due to the structural complexity and inherent marginal stability of protein based antigens and adjuvants, however, extensive analytical characterization and robust formulation development approaches are required to develop a stable, potent and safe vaccine dosage form for use in patients. In addition, the elucidation of physical and chemical instability pathways of protein antigens plays a key role in their formulation design, and are generally studied using numerous biophysical and analytical techniques, during forced degradation as well as accelerated and real-time stability studies. Furthermore, analytical comparability assessments form a cornerstone for assessing vaccine quality (including stability) between pre-change versus post-change drug products during development and scale-up of manufacturing processes. This Ph.D. thesis research work is aimed to better understand vaccine stability from three different pharmaceutical development aspects including analytical characterization, formulation development and comparability assessments. In addition, this work contributes towards the ongoing efforts to discern interrelationships of a protein antigen’s physicochemical properties to critical quality attributes of various vaccine candidates. As a part of this work, we developed and utilized improved analytical tools for formulation development and comparability assessments. For example, a scaled down micro-polyethylene glycol (PEG) induced precipitation assay was developed to determine apparent solubility of proteins using less than a milligram of material (as described in Chapter 2). This assay will be helpful in apparent solubility measurements during pharmaceutical development of proteins, especially, in early stages where limited material is available. In addition, analytical characterization of three different protein virus-like particle antigens (equine encephalitis virus-like particles, EEV) and a protein adjuvant (double mutant heat labile toxin, dmLT) by various biophysical and biochemical techniques was performed. The analytical characterization of the physical stability profile of the VLPs and physicochemical stability profile of dmLT, along with the role of pharmaceutical excipients in stabilizing their respective formulations, was evaluated. Lastly, an analytical comparability assessment involving five CRM197 carrier protein molecules was performed to better understand the effect of different manufacturing processes on the physicochemical and in vitro antigenic properties of the carrier protein. Currently, the protein adjuvant dmLT is an oral vaccine candidate for enterotoxigenic E.coli (ETEC) and is under clinical development. For this project, the primary and higher-order structures, physicochemical and conformational stability profiles of dmLT were assessed as described in Chapter 3. The physicochemical degradation pathways of dmLT included protein aggregation, glycation and oxidation. By identifying the physicochemical degradation pathways of dmLT using newly developed stability-indicating analytical methods, a more stable candidate bulk formulation of dmLT was developed that protected dmLT against conformational destabilization, freeze-thaw stress, aggregation/particle formation and chemical degradation. As described in Chapter 4, this work contributed to ongoing efforts to develop a vaccine against three strains of equine encephalitis (Eastern, Western and Venezuelan). Here, analytical characterization of three different monovalent VLPs was performed to identify structural alterations induced by thermal and pH stress. A candidate formulation mitigating thermal and aggregation instabilities of the VLPs was also developed. The candidate formulation showed a good maintenance of stability at all storage temperatures (40 to -80oC), with the exception of a distinct instability at -20°C; the mechanism of which is discussed in detail in Chapter 4. The interaction of both monovalent and trivalent VLP formulation with aluminum salt adjuvants was also studied to better understand the binding interactions of the VLPs with the adjuvant and its implications on future drug product development are discussed. Lastly, a comprehensive and detailed analytical characterization of CRM197 molecules from different manufacturers and expression hosts was executed as described in Chapter 5. This work provides an initial basis to eventually develop global manufacturing specifications to ensure the quality of the bulk CRM197 proteins from a variety of manufacturers. Furthermore, the high similarities between five different recombinant CRM197 expressed in the native host (C.diptheriae) vs. alternative hosts (E.coli or P.fluorescens), as demonstrated in this work, is a first step that will help facilitate lower cost CRM197 bulk production, with the long term goal to develop lower-cost polysaccharide conjugate vaccines for the developing world. | |
