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.