Downstream processing of antibodies: single-stage versus multi-stage aqueous two-phase extraction

Area of Science:

• Biochemical Engineering
• Separation Science
• Protein Purification

Background:

• Human antibody purification is critical for biopharmaceutical development.
• Aqueous two-phase systems (ATPSs) offer a promising platform for antibody separation.
• Optimizing ATPS conditions is essential for efficient and cost-effective purification.

Purpose of the Study:

• To evaluate and compare single-stage and multi-stage ATPS strategies for human antibody purification.
• To investigate the impact of system parameters (pH, ligand concentration, volume ratio) on antibody partitioning.
• To develop an optimized multi-stage extraction process for enhanced immunoglobulin G (IgG) recovery and purity.

Main Methods:

• Liquid-liquid extraction using ATPS composed of polyethylene glycol 3350 (PEG 3350), dextran, and triethylene glycol diglutaric acid (TEG-COOH).
• Investigation of single-stage extraction performance by varying pH, TEG-COOH concentration, and volume ratio.
• Simulation of a four-stage cross-current operation and determination of liquid-liquid equilibrium (LLE) data for multi-stage optimization.

Main Results:

• Single-stage extraction showed optimal conditions at 1.3% (w/w) TEG-COOH and a volume ratio of 2.2, yielding 96% IgG recovery with 87% protein purity.
• Multi-stage counter-current extraction simulation predicted a 5-stage process with a volume ratio of 0.4, achieving 95% IgG recovery and 93% protein purity.
• The optimized multi-stage ATPS effectively removed most contaminants, resulting in a total purity of approximately 85%.

Conclusions:

• TEG-COOH acts as an effective ligand for selective IgG extraction in PEG/dextran ATPS.
• Multi-stage counter-current extraction significantly enhances both IgG recovery yield and purity compared to single-stage systems.
• Optimized ATPS provides a viable and efficient method for purifying human antibodies from cell culture supernatant.