Thermodynamic Origin of Differential Excipient-Lysozyme Interactions

Area of Science:

• Protein therapeutics
• Biophysical chemistry
• Computational biophysics

Background:

• Effective purification and stable formulation of protein therapeutics depend on understanding protein interactions.
• Protein-excipient, protein-ion, and protein-water interactions are critical.
• Polyanionic excipients like citrate and tripolyphosphate influence protein behavior.

Purpose of the Study:

• To investigate the molecular mechanisms behind tripolyphosphate-induced lysozyme precipitation.
• To compare the interactions of lysozyme with tripolyphosphate versus citrate.
• To elucidate the role of ions and water in protein-excipient interactions.

Main Methods:

• Multiple-walker metadynamics simulations were employed.
• Free energy calculations were performed for lysozyme interacting with excipients, ions, and water.
• Multiscale decomposition of energy and entropy components was utilized.

Main Results:

• Tripolyphosphate stabilizes lysozyme through interactions with basic residues, leading to sodium ion release.
• Citrate interactions with lysozyme result in water molecule release.
• Differences in ion and water release mechanisms explain precipitation behavior.

Conclusions:

• The study reveals distinct interaction mechanisms between lysozyme and tripolyphosphate versus citrate.
• Tripolyphosphate's interaction with basic residues and sodium ion release are linked to lysozyme precipitation.
• Understanding these mechanisms is crucial for designing stable protein formulations and effective purification strategies.