Manipulation of lyophilization-induced phase separation: implications for pharmaceutical proteins

Area of Science:

• Pharmaceutical Sciences
• Biophysical Chemistry
• Protein Formulation

Background:

• Lyophilization (freeze-drying) is crucial for pharmaceutical protein stability.
• Protein damage during lyophilization can occur due to formulation incompatibilities.
• Component interactions during freezing and drying are often overlooked.

Purpose of the Study:

• To investigate liquid-liquid phase separation in poly(ethylene glycol) (PEG) and dextran mixtures during lyophilization.
• To determine the impact of this phase separation on protein structural integrity.
• To identify formulation strategies to prevent phase separation and associated protein damage.

Main Methods:

• Formulation of PEG and dextran solutions with buffering salts.
• Induction and monitoring of liquid-liquid phase separation during a lyophilization cycle.
• Assessment of protein unfolding using a model protein (recombinant hemoglobin).
• Comparative analysis of NaCl vs. KCl in preventing phase separation and damage.

Main Results:

• Liquid-liquid phase separation occurs in PEG/dextran solutions during freeze concentration in lyophilization.
• This phase separation leads to unfolding of recombinant hemoglobin.
• Replacing NaCl with KCl in the buffer effectively inhibits phase separation and prevents protein structural damage.
• pH changes were ruled out as the cause of protein damage.

Conclusions:

• Formulation component incompatibilities, specifically liquid-liquid phase separation, are a significant cause of protein damage during lyophilization.
• KCl inclusion kinetically prevents phase separation by forming a glass upon rapid cooling, thereby preserving protein structure.
• Strategic salt selection is critical for robust protein formulation during freeze-drying.