Supercritical fluid processing of proteins: lysozyme precipitation from aqueous solution

Area of Science:

• Biotechnology
• Materials Science
• Chemical Engineering

Background:

• Protein precipitation and size reduction are crucial for pharmaceutical and biotechnological applications.
• Supercritical fluid technology offers a tunable and environmentally friendly approach for particle formation.
• Hen egg lysozyme is a model protein frequently studied for processing techniques.

Purpose of the Study:

• To investigate the efficacy of the Solution Enhanced Dispersion by Supercritical fluid (SEDS) process for precipitating hen egg lysozyme.
• To determine the impact of SEDS process parameters (temperature, pressure, flow rates) on particle characteristics and protein integrity.
• To evaluate the biological activity and structural stability of lysozyme after SEDS processing.

Main Methods:

• Aqueous hen egg lysozyme solutions (3% w/v) were processed using SEDS with a supercritical carbon dioxide-ethanol mixture.
• Particle morphology, size distribution, and biological activity were analyzed.
• High-sensitivity differential scanning calorimetry (HSDSC) and FT-Raman spectroscopy were employed to assess structural integrity.

Main Results:

• The SEDS process produced lysozyme precipitates with primary particle sizes ranging from 1-5 micrometers.
• HSDSC data indicated that the stabilizing physical forces of lysozyme remained largely unchanged post-processing.
• FT-Raman spectroscopy revealed minimal secondary structural disorder, particularly under optimized conditions (200 bar, 40°C).

Conclusions:

• The SEDS process is a viable method for achieving size reduction of hen egg lysozyme from aqueous solutions.
• The process demonstrates potential for preserving the protein's native structure and biological activity.
• Optimized SEDS parameters are key to minimizing structural alterations and ensuring high-quality protein precipitates.