A Model Study to Assess Fibrillation and Product Stability to Support Peptide Drug Design

The fibrillation of therapeutic peptides can present
significant
quality concerns and poses challenges for manufacturing and storage.
A fundamental understanding of the mechanisms of fibrillation is critical
for the rational design of fibrillation-resistant peptide drugs and
can accelerate product development by guiding the selection of solution-stable
candidates and formulations. The studies reported here investigated
the effects of structural modifications on the fibrillation of a 29-residue
peptide (PepA) and two sequence modified variants (PepB, PepC). The
C-terminus of PepA was amidated, whereas both PepB and PepC retained
the carboxylate, and Ser16 in PepA and PepB was substituted with a
helix-stabilizing residue, α-aminoisobutyric acid (Aib), in
PepC. In thermal denaturation studies by far-UV CD spectroscopy and
fibrillation kinetic studies by fluorescence and turbidity measurements,
PepA and PepB showed heat-induced conformational changes and were
found to form fibrils, whereas PepC did not fibrillate and showed
only minor changes in the CD signal. Pulsed hydrogen–deuterium
exchange mass spectrometry (HDX-MS) showed a high degree of protection
from HD exchange in mature PepA fibrils and its proteolytic fragments,
indicating that most of the sequence had been incorporated into the
fibril structure and occurred nearly simultaneously throughout the
sequence. The effects of the net peptide charge and formulation pH
on fibrillation kinetics were investigated. In real-time stability
studies of two formulations of PepA at pH’s 7.4 and 8.0, analytical
methods detected significant changes in the stability of the formulations
at different time points during the study, which were not observed
during accelerated studies. Additionally, PepA samples were withdrawn
from real-time stability and subjected to additional stress (40 °C,
continuous shaking) to induce fibrillation; an approach that successfully
amplified oligomers or prefibrillar species previously undetected
in a thioflavin T assay. Taken together, these studies present an
approach to differentiate and characterize fibrillation risk in structurally
related peptides under accelerated and real-time conditions, providing
a model for rapid, iterative structural design to optimize the stability
of therapeutic peptides.