The value of fluorescence and B22 techniques as complementary approaches in protein stability analyses

Recombinant proteins gain more and more importance in the pharmaceutical market. One major challenge is to assure the protein integrity during manufacturing, shipping and storage. Complementary analytical approaches are deemed necessary to fully characterize and understand structural changes of the protein and the quality of formed aggregates. Hence, the focus of this thesis was the investigation of protein unfolding and protein-protein interactions as first steps of protein aggregation. Using extrinsic fluorescence analysis and B22 techniques, protein unfolding as well as attractive or repulsive protein-protein interactions could be determined for both the protein aggregates as well as the monomer, due to prior size-separation, simultaneously and individually. As both fluorescence and B22 analyses can be conducted in combination with SEC, the most widely used and best established technique, the developed methods represent easily accessible and complementary approaches to analyze protein unfolding, interactions and aggregation. In sum, this allows to shed more light on the induction factors and the sequence of events in protein aggregation during formulation development, manufacturing and storage.