Protein Aggregation in Frozen Trehalose Formulations: Effects of Composition, Cooling Rate, and Storage Temperature

Area of Science:

• Biopharmaceutical formulation
• Protein stabilization
• Cryoprotectant characterization

Background:

• Trehalose is a common cryoprotectant in biopharmaceutical formulations.
• Understanding trehalose phase behavior is critical for protein stability during freezing.
• Factors influencing trehalose crystallization and protein aggregation require detailed investigation.

Purpose of the Study:

• To investigate the impact of cooling rate, storage temperature, and formulation composition on trehalose phase distribution.
• To assess the effects of these parameters on protein stability in frozen solutions.
• To identify optimal conditions for stabilizing monoclonal antibody (mAb) formulations during frozen storage.

Main Methods:

• Fourier Transform Near-Infrared (FT-NIR) spectroscopy to analyze trehalose phase distribution.
• Size-exclusion chromatography to determine protein aggregation.
• Systematic variation of cooling rates, storage temperatures, and trehalose-mAb ratios.

Main Results:

• Faster cooling rates (>100°C/min) induced trehalose crystallization and protein aggregation.
• Slower cooling rates (≤1°C/min) maintained amorphous trehalose and protein stability.
• Storage temperature influenced aggregation rates, with -14°C showing faster initial aggregation but -20°C showing higher cumulative aggregation.
• An optimal trehalose-to-mAb ratio (0.2-2.4 w/w) was identified for stabilizing mAb formulations.

Conclusions:

• Cooling rate and formulation composition significantly impact trehalose phase behavior and protein stability.
• Optimal trehalose-mAb ratios can mitigate protein aggregation, even under rapid cooling conditions.
• These findings provide critical insights for developing robust frozen biopharmaceutical formulations.