Downstream processing of virus-like particles: single-stage and multi-stage aqueous two-phase extraction

Area of Science:

• Biotechnology
• Vaccine Development
• Separation Science

Background:

• Virus-like particles (VLPs) are crucial vaccine candidates, but their purification faces challenges with traditional methods like ultracentrifugation and chromatography.
• Current downstream processing for VLPs is often time-intensive and inefficient, necessitating novel separation technologies.

Purpose of the Study:

• To evaluate aqueous two-phase extraction (ATPE) as an alternative purification method for virus-like particles (VLPs).
• To optimize ATPE parameters for high capacity and separation efficiency in the purification of human B19 parvovirus-like particles.

Main Methods:

• A solubility-guided approach was used to design polyethylene (PEG) salt aqueous two-phase systems.
• ATPE performance was assessed by varying PEG molecular weight, pH, and salt additives.
• Multi-stage ATPE was implemented using a centrifugal partition chromatography (CPC) device for enhanced impurity removal.

Main Results:

• Optimized ATPE achieved a high VLP capacity of up to 4.1 mg/mL.
• Single-stage ATPE demonstrated 99.6% DNA clearance, while multi-stage ATPE improved host cell protein (HCP) separation.
• A two-step downstream process yielded 90.6% HPLC purity and 63.9% VLP recovery.

Conclusions:

• ATPE is a viable and efficient technology for VLP purification, offering high capacity and improved impurity removal compared to conventional methods.
• Optimized multi-stage ATPE and a subsequent two-step process provide a scalable and effective downstream purification strategy for VLP-based vaccine candidates.