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Excipient crystallinity and its protein-structure-stabilizing effect during freeze-drying.

Ken-ichi Izutsu1, Shigeo Kojima

  • 1National Institute of Health Sciences, Kamiyoga 1-18-1, Setagaya, Tokyo 158-8501, Japan. izutsu@nihs.go.jp

The Journal of Pharmacy and Pharmacology
|August 28, 2002
PubMed
Summary

Mannitol crystallization during freeze-drying impacts protein stability. Amorphous excipients like sucrose, not crystalline mannitol, best stabilize protein structures through molecular interactions, preserving enzyme activity.

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Area of Science:

  • Biochemistry
  • Pharmaceutical Sciences
  • Materials Science

Background:

  • Freeze-drying (lyophilization) is crucial for stabilizing proteins but can cause structural damage.
  • Excipients are used to mitigate these effects, with their physical state (crystalline vs. amorphous) potentially influencing efficacy.

Purpose of the Study:

  • To investigate the role of mannitol crystallization during freeze-drying on the structural stability of model proteins.
  • To compare the protective effects of crystalline versus amorphous mannitol and other excipients.

Main Methods:

  • Utilized five model proteins: lysozyme, bovine serum albumin, ovalbumin, beta-lactoglobulin, and lactate dehydrogenase.
  • Employed Fourier Transform Infrared (FT-IR) spectroscopy to analyze protein secondary structure.

Related Experiment Videos

  • Investigated the impact of mannitol crystallinity, buffer composition (potassium phosphate), and pre-treatment temperatures (-10°C).

    • Main Results:

    • Freeze-drying without excipients perturbed protein alpha-helix and beta-sheet structures.
    • Sucrose provided significant structural protection, while mannitol's effect varied with its crystallinity.
    • Higher mannitol crystallinity, induced by heat treatment, correlated with reduced protein stabilization and enzyme activity.
    • Combining mannitol with potassium phosphate buffer decreased mannitol crystallinity and lessened structural changes.

    Conclusions:

    • The stabilizing effect of mannitol during freeze-drying is dependent on its crystalline state; amorphous forms are more protective.
    • Molecular interactions, such as hydrogen bonding between amorphous excipients and proteins, are key to structure stabilization.
    • Mannitol's effectiveness is limited when crystalline, highlighting the importance of excipient form in lyophilization.