Automated High-Throughput Analytical- Scale Monoclonal Antibody Purification Using Production-Scale Protein A Affinity Chromatography Resin

Significance of the Topic

High-throughput analytical‐scale purification of monoclonal antibodies (mAbs) is critical for bioprocess development and quality control. By efficiently removing host‐cell proteins, DNA, and other media components, purified mAb samples enable reliable downstream analyses such as size‐exclusion chromatography (SEC) and released N‐glycan profiling.

Objectives and Overview

The study aimed to develop and validate an automated Protein A affinity purification protocol for 96‐well format, capable of processing 120 µg to 240 µg of mAb per well. Key goals included maximizing recovery and final concentration while minimizing manual intervention and compatibility with process‐scale Protein A resins.

Methodology and Used Instrumentation

The workflow employs a 96‐well 0.2 µm filtration plate loaded with Protein A resin, followed by sequential equilibration, binding, washing, elution (100 mM glycine pH 3.0), and neutralization (1 M Tris pH 7.5). Twelve pipetting steps and four incubations per sample are automated on an Andrew+ pipetting robot with Extraction+ module, using the OneLab visual programming interface. The binding step is performed on an orbital shaker at 8 °C for 20 min to maintain resin suspension.

• Robotic platform: Andrew+ Pipetting Robot with Extraction+ module
• Filter plate: Pall AcroPrep Advance 96‐well, 0.2 µm
• Collection plate: Waters QuanRecovery 700 µL 96‐well
• Protein A resin: Cytiva MabSelect slurry (~25%)
• Orbital shaker: Eppendorf ThermoMixer C, 8 °C, 1250 RPM
• Software: OneLab

For downstream analysis, SEC‐UV was performed on an ACQUITY Premier UPLC with TUV detector and Protein SEC 250 Å column, using 200 mM ammonium acetate mobile phase. Released N‐glycan profiling utilized an ACQUITY UPLC I-Class PLUS system with Glycan BEH Amide column and RDa detector, coupled to a BioAccord LC-MS (ESI-ToF).

Key Results and Discussion

Recovery exceeded 85% for 120 µg loads (1.02 µg/µL concentration) and reached ≥94% for 240 µg loads (2.27 µg/µL). A reproducibility study (n=8) achieved 90%±5% recovery for samples in PBS and conditioned media. The method effectively removed most host‐cell contaminants, enabling clear SEC profiles and consistent glycan maps. Minor artifacts of high‐molecular‐weight species (HMWS) were observed at low pH, highlighting potential bias in aggregation quantitation.

Benefits and Practical Applications

• High throughput: ~1 hr for 48 samples with minimal hands‐on time
• Flexible resin choice: Protein A, G, or L process‐scale media
• High recovery (>90%) and concentration suitable for QC assays
• Effective removal of host‐cell proteins for SEC and glycan analysis

Future Trends and Applications

Further enhancements may include adaptation to higher mAb loads (up to 480 µg), integration with other affinity ligands, and optimization to reduce aggregation artifacts. Expanding this approach to peptide mapping or mass‐based titer assays can accelerate bioprocess screening and support process analytical technology (PAT) initiatives.

Conclusion

The automated filter‐plate Protein A purification on the Andrew+ platform delivers robust, reproducible, and high‐recovery mAb samples for downstream analytical methods. This workflow reduces manual workload, supports high‐throughput screening, and ensures sample quality for critical QC and development assays.

Reference

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