Analytical Scale 96-well Protein A Affinity Resin-Based Purification using Andrew+™ Automation Robot Supporting Upstream Bioprocessing

Significance of the Topic

The purification of monoclonal antibodies (mAbs) is a cornerstone in biotherapeutic development and quality control workflows.
Automated, high-throughput affinity purification accelerates process optimization and ensures consistent sample preparation across large experimental sets.

Study Objectives and Overview

This application note describes the development and validation of a 96-well plate Protein A affinity purification protocol using an Andrew+™ Liquid Handling Robot.
Key goals were to enable dynamic sample throughput (1–96), minimize sample volume, and achieve high recovery and reproducibility for downstream critical quality attribute (CQA) analyses.

Methodology and Instrumentation

• Sample generation: HCCF produced by NISTCHO cells, filtered and frozen until use.
• Automated purification: Cytiva MabSelect™ Protein A resin in a filter plate format processed by Andrew+ Robot with on-board Extraction+ vacuum unit and offline mixing on an Eppendorf shaker.
• Elution and neutralization: Three sequential glycine (pH 3.0) elutions followed by Tris (pH 7.2) neutralization to yield a final 100 µL volume.
• Analysis: LC-UV quantification on a Waters BioResolve™ Protein A Affinity Column calibrated with NISTmAb standard.

Main Findings and Discussion

• Total process time for 96 samples: 75 minutes (55 minutes unattended, 20 minutes offline mixing).
• Sample consumption: 120 µL HCCF per well; final eluate ~100 µL (20% mAb concentration enrichment).
• Recovery: 96.8 ± 8.2% (mean ± SD across 96 wells).
• Reproducibility: 8.5% RSD for inter-well recovery; <1% RSD for LC-UV triplicate injections.
• Dynamic protocol interface allows users to specify any sample number up to 96, with optimized use of an 8-channel pipette for multiples of eight.

Benefits and Practical Applications

• Rapid throughput with unattended handling reduces hands-on time and operator variability.
• Minimal sample requirement conserves valuable culture material.
• High recovery and low variability support reliable CQA workflows, including intact and reduced protein analysis, glycan profiling, SEC, and charge variant assays.
• Publicly available protocol in the OneLab™ library under “ProA-resin purification” for seamless adoption.

Future Trends and Potential Applications

• Integration with real-time bioreactor platforms (e.g., Ambr® systems) for closed-loop process monitoring.
• Miniaturization to higher-density formats or single-cell screening applications.
• Coupling automated purification with advanced data analytics and machine learning for predictive process control.
• Expansion to other affinity ligands or multi-modal resins for broader protein purification workflows.

Conclusion

The automated Protein A purification protocol on Andrew+ Robot delivers a robust, flexible, and high-throughput solution for mAb sample preparation.
With excellent recovery, reproducibility, and minimal hands-on time, this method supports accelerated bioprocess development and reliable downstream analytics.
The protocol is freely accessible in the OneLab library for rapid implementation.

References

1. Koza SM, Hanna CM, Jiang AHW, Yu YQ. Automated High-Throughput Analytical-Scale Monoclonal Antibody Purification Using Production-Scale Protein A Affinity Chromatography Resin. Waters Application Note; 2023.
2. Cytiva. MabSelect Antibody Purification Chromatography Resin Product Brochure; 2025.
3. Koza SM, Shiner S, Lauber MA. Lowering Quantitation Limits for mAb Titer Measurements Using Small Volume 3.5 µm Particle-Size Protein-A Affinity Columns. Waters Application Note; 2025.