Fibril Nucleation Kinetics of a Pharmaceutical Peptide: The Role of Conformation Stability, Formulation Factors, and Temperature Effect.

Peptide aggregation, such as the formation of fibrils, could pose a significant challenge for the stability of parenteral peptide drugs. To ensure a robust peptide formulation, a thorough understanding of aggregation kinetics and the development of appropriate accelerated testing conditions are necessary. The present research investigated factors that impact the fibrillation kinetics of a helical 29mer pharmaceutical peptide (peptide A) and attempts to correlate results of accelerated kinetic studies with real time kinetics. Conformational flexibility of the peptide and its potential impact on aggregation kinetics were thoroughly evaluated. Three orthogonal approaches to evaluate aggregation kinetics were assessed, thioflavin T fluorescence, turbidity, and soluble peptide concentration. The results from the methods demonstrated that peptide A showed nucleated polymerization kinetics. The lag time of the fibrillation process depends heavily on pH, ionic strength, temperature, agitation, and substrate interface. The temperature-dependent fibril nucleation kinetics follow Arrhenius behavior, despite a helical fold in the peptide structure. This finding suggests a potential opportunity to leverage accelerated testing conditions to project the long-term performance at storage temperatures. The present study provides both fundamental understanding and practical approaches to mitigate the aggregation risk for pharmaceutical peptides with a strong tendency to form fibrils.