Application of an aqueous two-phase systems high-throughput screening method to evaluate mAb HCP separation

Area of Science:

• Biotechnology
• Separation Science
• Chemical Engineering

Background:

• Aqueous two-phase systems (ATPSs) are gaining traction in the biopharmaceutical industry as a cost-effective alternative to chromatography.
• Increasing product titers and economic pressures necessitate efficient and scalable separation techniques.
• Current ATPS implementation is resource-intensive, highlighting the need for automated screening methods.

Purpose of the Study:

• To develop and evaluate a miniaturized and automated method for screening ATPS.
• To optimize ATPS for biopharmaceutical separation tasks, focusing on speed and efficiency.
• To assess the influence of various parameters on protein partitioning within ATPS.

Main Methods:

• Automated cloud-point method for binodal determination of PEG4000-PO(4) systems.
• Application of the developed ATPS screening procedure to model and industrial biopharmaceutical separation tasks.
• Investigation of pH, NaCl addition, and tie-line length effects on protein partitioning.

Main Results:

• Automated and manual binodal determinations for PEG4000-PO(4) systems yielded comparable results.
• Monoclonal antibodies were successfully shifted to the upper phase with NaCl addition, influenced by pH relative to protein pI.
• Precipitation of monoclonal antibodies was observed with NaCl addition, PEG4000 increase, and pH changes, indicating capacity limitations.
• Host cell protein reduction of up to 50% was achieved with >95% recovery of target proteins in model systems.

Conclusions:

• The developed automated screening procedure enables rapid evaluation and optimization of ATPS for biopharmaceutical separations.
• The study demonstrates the feasibility of using ATPS for initial purification steps, achieving significant host cell protein reduction.
• Further optimization is required to match the industrial relevance of chromatographic procedures, but the screening method accelerates this process.