High Efficiency Protein A Affinity Columns for the LC-MS and 2D LC-MS Analysis of mAb and msAb

Significance of the Topic

Monoclonal antibodies (mAbs) and multispecific antibodies (msAbs) are critical biotherapeutics requiring precise structural and purity characterization during development and manufacturing. High‐efficiency Protein A (ProA) affinity columns coupled with liquid chromatography–mass spectrometry (LC-MS) workflows enable direct analysis from clarified cell-culture fluids, reducing sample preparation time and improving throughput for quality control and process monitoring.

Objectives and Study Overview

This study introduces a newly developed high-efficiency ProA affinity column (3.5 µm particles, 2.1 × 20 mm) compatible with pressures up to 6500 PSI. The aims are to demonstrate its performance for:

• Subunit analysis after on-line reduction (ProA-RP-MS).
• Intact mass determination via size exclusion (ProA-SEC-MS) and native MS (ProA-MS).
• Two-dimensional LC-MS separations combining ProA affinity capture with reversed-phase or size‐exclusion dimensions.

Methodology and Instrumentation

The workflows encompass one- and two-dimensional LC-MS configurations in heart-cutting mode. Key steps include:

• Affinity capture on the ProA column followed by elution with acidic phosphate buffer gradients.
• Trap desalting or on-line reduction using DTT for subunit chain separation.
• Second-dimension separation on RP or SEC columns under optimized organic gradients.
• ESI-QTOF detection in positive mode with broad mass ranges (m/z 400–7000).

Instrumentation

• ProA affinity column, 3.5 µm, 2.1 × 20 mm (MaxPeak Premier technology).
• BioResolve mAb RP polyphenyl guard and analytical columns (2.1 × 30/50 mm, 2.7 µm, 450 Å).
• ACQUITY Premier Protein SEC column (4.6 × 150 mm, 1.7 µm, 250 Å).
• Waters ACQUITY UPLC system with autosampler, dual column managers, PDA detector.
• Xevo G2-XS and G3 QTOF mass spectrometers, waters_connect software.

Main Results and Discussion

The ProA affinity column demonstrated high binding efficiency and sharp elution profiles for mAbs and msAbs directly from buffer or cell culture medium. Intact mass analysis (ProA-SEC-MS and ProA-MS) provided accurate mass determinations matching predicted glycoforms with sub-ppm errors. Heart-cutting 2D-LC-MS separated high-molecular-weight aggregates, monomer, and fragments with consistent UV280 resolution. On-line reduction enabled clear subunit chain identification and glycoform profiling. High-throughput native MS showed linear response curves for titer quantification in both neat and cell culture samples (R²>0.999).

Benefits and Practical Applications

• Direct analysis from cell-culture supernatant minimizes sample handling.
• Combined size and subunit resolution for simultaneous purity, identity, and glycoform profiling.
• High throughput with rapid cycle times and robust column performance.
• Quantitative titer determination with MS signal linearity across relevant concentration ranges.

Future Trends and Applications

Advancements may include integration with automated sample preparation platforms, development of ultra-high-pressure ProA chemistries for faster separations, and coupling with orthogonal detectors such as ion mobility for deeper structural insights. Real-time process analytical technology (PAT) solutions leveraging online ProA-MS could further accelerate bioprocess monitoring.

Conclusion

The high-efficiency ProA affinity column, when hyphenated with advanced LC-MS configurations, delivers a versatile platform for comprehensive mAb and msAb characterization at intact and subunit levels. This approach streamlines biotherapeutic analysis, providing critical data for quality assurance, process development, and regulatory compliance.

References

None specified in the original document.