Elucidation of the Mechanism of Endo-Xaa^C‑terminal Peptide Impurity Formation in SPPS through DoE Investigation, Their Control, and Suppression

Standard peptide acid syntheses on the CTC resin frequently experience the formation of impurities with redundant incorporation of the C-terminal residues, annotated as endo-Xaa^C‑terminal in this paper. Mechanism of the formation of endo-Xaa^C‑terminal impurity was elucidated through dedicated design of experiment (DoE) investigations in two separate Ferring Pharmaceuticals’ peptide projects. Significant models have been established in the DoE studies, implicating an identical pattern of the correlation between endo-Xaa^C‑terminal impurity formation and the investigated process parameters. Pre-activation time of the Fmoc-Xaa^penultimate-OH residues stands out as the most critical process parameter on endo-Xaa^C‑terminal impurity formation. Besides, pre-activation temperature and coupling temperature of Fmoc-Xaa^penultimate-OH are also significant to this impurity. The established models have been verified by multi-batches of corresponding productions and the predicted endo-Xaa^C‑terminal impurity contents were well in line with the actual values. Formations of endo-Xaa^C‑terminal impurities in the process of peptide acid syntheses on the CTC resin are hence well understood through these DoE investigations, and corresponding control strategies are established accordingly as to minimize its occurrence in production.