| Protein unfolding and aggregation during industrial purification steps can complicate process development efforts and compromise product yields. Often, the underlying biophysical mechanisms that compromise protein stability are poorly understood, and optimization of processing conditions proceeds in a trial-and-error fashion. Amide hydrogen-deuterium exchange (HX) is a versatile isotopic labeling technique that allows direct analysis of protein conformational changes in solution and in the adsorbed phase. In this work, HX was successfully employed to characterize protein conformational changes in two industry-relevant environments---hydrophobic interaction chromatography (HIC) and assembly of a recombinant virus-like particle (VLP).; In the first part of the project, the stability of a model unstable protein, bovine alpha-lactalbumin, was investigated during adsorption to three commercial HIC resins. Specifically, the effects of salt type, salt concentration, and protein loading on unfolding behavior were evaluated. It was observed that alpha-lactalbumin unfolding decreased markedly with increases in loading. Also, the protein appeared significantly more stable during adsorption to a phenyl resin than a butyl resin. The results obtained here underscore important differences between aggregation and surface-induced unfolding mechanisms in chromatography.; The last part of the project focused on a recombinant vaccine candidate for human papillomavirus (HPV). During production of the vaccine, pentameric subunits of the major capsid protein, L1, are assembled to form full-size virus-like particles in vitro. Assembly is controlled by changing buffer conditions such as pH and salt concentration. Here, we used peptide-level HX analysis to determine how the assembly process affects the tertiary structure of L1. It was found that the structure of L1 was largely unchanged upon assembly, but that there were significant increases in solvent exposure in one region of the molecule. These results build on an existing atomic structural model and help address remaining questions about the nature of inter-pentameric contacts in the VLP. |
