AF4 and PEG Precipitation as Predictive Assays for Antibody Self-Association

Area of Science:

• Biopharmaceutical Development
• Protein Chemistry
• Analytical Chemistry

Background:

• High-protein-concentration monoclonal antibody (mAb) formulations can face physical stability challenges like high viscosity and opalescence.
• Early identification of mAbs prone to these issues is crucial for successful drug development.
• Traditional methods for assessing self-association (kD) require large amounts of purified material, often unavailable during early discovery.

Purpose of the Study:

• To demonstrate asymmetric field-flow fractionation (AF4) as a method for measuring mAb self-association and identifying issues at high concentrations.
• To establish a two-step screening strategy combining AF4 and a polyethylene glycol (PEG) precipitation assay for early developability assessment.
• To enable the selection of mAbs compatible with high-concentration formulations early in the discovery pipeline.

Main Methods:

• Utilized asymmetric field-flow fractionation (AF4) to concentrate samples and measure self-association under formulation-relevant conditions (>100 mg/mL).
• Analyzed a library of commercial antibodies using AF4, correlating retention time with high-concentration behavior.
• Employed a polyethylene glycol (PEG) precipitation assay to complement AF4 findings and differentiate opalescence from high viscosity.

Main Results:

• AF4 successfully identified monoclonal antibodies exhibiting high-concentration issues using minimal sample (30 μg) without extensive purification.
• Retention times from AF4 correlated with problematic macroscopic properties.
• PEG precipitation assay results were consistent with AF4 and further distinguished between opalescence and viscosity issues.

Conclusions:

• AF4 serves as an effective early-stage screening tool for identifying problematic monoclonal antibodies for high-concentration formulations.
• A combined AF4 and PEG assay strategy allows for efficient validation and discrimination of stability issues.
• This approach facilitates the selection of developable antibody candidates early in the discovery process, optimizing manufacturing, stability, and delivery.