A sub-two minutes method for monoclonal antibody-aggregate quantification using parallel interlaced size exclusion...

Area of Science:

• Biopharmaceutical Process Development
• Analytical Chemistry
• Protein Aggregation Analysis

Background:

• Monitoring monoclonal antibody (mAb) aggregate levels is mandatory during process development and commercial production.
• Standard size exclusion chromatography (SEC) using HPLC is precise and simple but too time-consuming for high-throughput process development (HTPD) and real-time process monitoring.
• Current SEC methods typically yield only two samples per hour, hindering efficient workflow.

Purpose of the Study:

• To compare different SEC columns for mAb aggregate quantification, focusing on throughput, resolution, and reproducibility.
• To develop and validate a significantly faster SEC method for mAb aggregate analysis.
• To establish a method suitable for HTPD and purification process monitoring.

Main Methods:

• Evaluation of three distinct SEC columns for mAb aggregate quantification.
• Optimization of SEC method by utilizing a short column (150 mm) with sub-two micron particles.
• Implementation of parallel interlaced sample injections and dual-column parallelization to minimize assay time.

Main Results:

• A short SEC column with sub-two micron particles achieved high resolution (~1.5) and precision (CV<1) in under 6 minutes.
• The optimized parallel interlaced SEC (PI-SEC) method, combining interlaced injections and parallel columns, reduced assay time to below 2 minutes.
• Aggregate levels and precision determined by the PI-SEC assay were comparable to the standard single-injection SEC assay.

Conclusions:

• The developed PI-SEC methodology significantly enhances throughput for mAb aggregate analysis.
• This optimized method is a valuable tool for HTPD and real-time purification process monitoring.
• PI-SEC offers a rapid, precise, and reproducible solution for essential mAb aggregate quantification.