| dc.description.abstract | One of the predominant concerns with protein therapeutics is their tendency to aggregate at various stages of protein production, purification, filling, transportation, and administration. This occurrence has biological significance; while there is no definite, general cause and effect relationship for all protein drugs, many studies suggest that protein aggregates in certain biotherapeutics can decrease efficacy or cause untoward immune responses in human patients. Current research suggests that certain types of protein aggregates may be more immunogenic than others. In this Ph.D. thesis research work, three different IgG monoclonal antibodies (2 IgG1 mAbs, one in solution and one in lyophilized form and one IgG2 mAb in solution) were stressed by a variety of different conditions and the resulting aggregates and particles were characterized using a broad array of methods. Some of the characteristics examined included aggregate/ particle size, count, and morphology, as well as the covalent cross-linking and structural integrity of the protein within the aggregates. In all cases, accelerated stability studies, similar to those performed in the biopharmaceutical industry, were utilized to generate aggregates. In the first study, an IgG1 mAb in solution was subjected to freeze-thaw, shaking, stirring, and heat stress in the presence and absence of NaCl. Depending on the solution and stress conditions, very different types of aggregates and particles formed. In the second study, an IgG1 mAb in lyophilized form was shaken to mimic worst-case shipping condition, which led to extensive cake breakage and upon reconstitution, displayed increased turbidity and subvisible particles compared to the unstressed sample. This study highlights potential stability concerns regarding lyophilized protein undergoing various shipping processes. In the third study, the impact of protein particle size on inducing an early and late phase immune response in an in-vitro assay using human peripheral blood mononuclear cells (PBMC) was investigated. Stir-induced IgG2 mAb aggregates were size-enriched using fluorescence activated cell sorting (FACS) and tested for their ability to induce PBMC cytokine responses, at two phases of the immune response. The size-enriched particles were simultaneously characterized to determine traits, other than size, that may be responsible for the in-vitro assay responses. Amorphous subvisible particles 5-10 μm in size, containing protein with partially altered secondary structure and elevated surface hydrophobicity (compared to controls), and containing elemental fluorine, displayed relatively elevated cytokine release profiles compared to other size ranges. Studies carried out as part of this Ph.D. thesis highlight the importance of 1) comprehensively characterizing protein aggregates and particles to better understand their formation, 2) the need for closer evaluation of the effects of shaking stress on lyophilized protein formulations during shipping, and 3) studying the potential biological implications of a subset of protein particles in an in vitro system, along with developing a better understanding these aggregate's physicochemical properties, should provide improved insights into why some protein aggregates elicit higher immune responses than others in vivo. | |
