High-throughput process development of purification alternatives for the protein avidin

Area of Science:

• Biochemistry
• Protein Purification
• Process Development

Background:

• Avidin-biotin technology drives demand for purified avidin.
• Current purification methods for chicken egg white avidin require optimization.

Purpose of the Study:

• To explore alternative avidin purification strategies using high-throughput process development (HTPD).
• To identify efficient methods for separating avidin from major egg white impurities.

Main Methods:

• High-throughput process development (HTPD) was employed.
• Evaluated aqueous two-phase extraction, selective precipitation (salts, polyethylene glycol), and chromatography (hydrophobic, mixed-mode).
• Utilized high-throughput tandem high-performance liquid chromatography for protein quantification.

Main Results:

• Identified conditions for separating avidin from ovalbumin, ovomucoid, ovotransferrin, and lysozyme.
• Achieved 77% avidin purity with >90% yield using polyethylene glycol precipitation and two-phase extraction.
• Mixed-mode chromatography yielded a final avidin purity of at least 97% with >90% yield.

Conclusions:

• HTPD successfully identified a novel, efficient purification process for avidin.
• The new method offers a beneficial alternative to traditional ion-exchange chromatography for industrial applications.
• Combined precipitation and mixed-mode chromatography significantly improve avidin purity and reduce process volume.