The Detrimental Effects of Crystalline Excipients: How They Jeopardize the Long-Term Stability of Freeze-Dried...

Background: Despite the growing importance of polypeptide-based drugs in clinical therapy, studies investigating the stability of their freeze-dried formulations remain scarce. Crystalline excipients, such as mannitol, are commonly used in freeze-dried formulations of chemically synthesized drugs, but they often negatively impact the long-term stability of biological macromolecules like monoclonal antibodies (mAbs). This study bridges this knowledge gap by evaluating the effects of crystalline and amorphous excipients, surfactants, and amino acid-based stabilizers on the long-term stability of freeze-dried formulations using model polypeptides, glucagon and insulin. Methods: The freeze-dried formulations were prepared with crystalline and amorphous excipients, surfactants, and amino acid-based stabilizers. The crystallization behavior of the excipients and the thermal stability of the formulations were thoroughly characterized using X-ray powder diffraction (XRPD) and differential scanning calorimetry (DSC). Results: The crystallization of mannitol was directly correlated with a significant reduction in the long-term stability of both model polypeptides. This detrimental effect mirrors the instability observed in mAbs formulations, indicating a common mechanism of protein destabilization induced by crystalline excipients, independent of molecular size. Conclusions: This study provides the first direct evidence that crystalline excipients pose a significant risk to the stability of freeze-dried polypeptides. These findings offer critical insights for the rational design of stable freeze-dried formulations, guiding industrial development strategies for polypeptide-based therapeutics.