Sonje, Jayesh2024-01-192024-01-192022-03https://hdl.handle.net/11299/260146University of Minnesota Ph.D. dissertation. March 2022. Major: Pharmaceutics. Advisors: Raj Surayanarayanan, Ronald Siegel. 1 computer file (PDF); x, 195 pages.Freeze-thaw and freeze-drying are common unit operations in manufacturing of biotherapeutics. Excipients such as sugars, buffers, and surfactants, each with an intended functionality, aid in preventing protein destabilization against stresses encountered during processing or storage. Sugars, specifically added to stabilize proteins, can only be effective if retained in the amorphous state. Selective crystallization of a buffer component during freezing, drying or storage can result in in a pH shift. The first objective of this thesis was to investigate the impact of stress associated with pH shift on protein (lactate dehydrogenase, LDH) conformation in presence of a crystallizing (sodium phosphate) and non-crystallizing (histidine) buffer, during the freezing and thawing stages. This was accomplished using small angle neutron and dynamic light scattering. The significant findings were: (i) LDH, at high concentration, had self-stabilizing effect and exhibited reversible aggregation after 5 freeze-thaw cycles, irrespective of buffer used. (ii) At low LDH concentrations, only with the selection of an appropriate buffer, irreversible aggregation could be avoided. Crystallization of certain excipients such as mannitol, is desirable, from a processing as well as product quality perspective. Mannitol is widely used in freeze-dried formulations and is known to crystallize as an unstable mannitol hemihydrate (MHH). Although, the conditions of formation of MHH are well established, its dehydration kinetics and the impact of the release of lattice water on the formulation stability is not known. The next set of objectives were (i) to investigate MHH dehydration kinetics at different conditions (relative humidity and temperature) in lyophilized mannitol formulations (with and without sucrose), and (ii) to determine the impact of MHH dehydration followed by sucrose crystallization on protein stability. Finally, in protein drug substances which are stored frozen, the use of mannitol as a crystallizing excipient and its role in generating a homogeneous freeze-concentrate was explored. Overall, the thesis highlights the importance of a multidisciplinary approach, using a variety of complementary characterization tools, to gain insights into protein stability.enExcipient crystallizationFreeze-drying/Freeze-thawMannitol hemihydrateProtein stabilitySmall angle neutron scatteringSolid state characterization (DSC XRD)Thesis or Dissertation