Reliable High Resolution Protein SEC Separations for Online Native LC-MS mAb Analysis

Importance of Topic

Native LC-MS combining size exclusion chromatography (SEC) with mass spectrometry is a critical tool for the characterization of protein therapeutics. By preserving protein conformations and using volatile mobile phases, native SEC-MS enables sensitive detection of aggregates, fragments, and noncovalent complexes with simplified charge state distributions and reduced matrix interference.

Goals and Study Overview

This application note evaluates how new SEC column technology—Waters ACQUITY Premier and XBridge Premier Protein SEC 250 Å columns featuring hydroxy-terminated polyethylene oxide (PEO) ligands and MaxPeak High Performance Surfaces—improves native SEC-MS of monoclonal antibodies (mAbs). Key objectives include reducing secondary interactions, optimizing limits of detection, enhancing peak symmetry, and maximizing analyte recovery under ammonium acetate mobile phases suited for online MS detection.

Methodology

Samples:

• NISTmAb Reference Material and Intact mAb Mass Check Standard.
• BEH200 Protein Standard Mix spanning 10–300 kDa.

Chromatography:

• System: ACQUITY UPLC I-Class with TUV detector.
• Columns: XBridge Premier Protein SEC 250 Å 2.5 µm and ACQUITY Premier Protein SEC 250 Å 1.7 µm (4.6×150 mm).
• Mobile Phases: Ammonium acetate from IonHance CX-MS pH concentrates, varied from 20–200 mM with 2 % ACN.
• Flow Rate: 0.2 mL/min; Column Temp: 30 °C; Sample Temp: 4 °C; Injection: 7.5 µL.

Applied Instrumentation

Mass Spectrometry:

• BioAccord System with ACQUITY RDa Detector (Full scan, positive mode, 400–7000 m/z default, capillary 1.5 kV, cone 150 V).
• Xevo G2-XS QTof (full scan, positive mode, high mass range 400–5000 m/z, capillary 2.5 kV, cone 60 V).

Main Results and Discussion

Limit of Detection:

• Using XBridge Premier SEC, mAb detected by UV down to 37.5 ng injected load with symmetrical peaks; a commercially available PEO-bonded column showed poor recovery and tailing below 150 ng.

Salt Tolerance:

• ACQUITY Premier SEC delivered optimal resolution and peak shape at ≥50 mM ammonium acetate; 20 mM induced peak distortion and co-elution issues.

Native SEC-MS Performance:

• PEO ligand columns reduced ionic and hydrophobic interactions, enabling detection of high molecular weight species (~2 % aggregates) and clear monomer signals.
• Background PEO ions at 600–900 m/z were excluded by setting MS acquisition lower limit ≥1000 m/z, improving signal-to-noise.
• Xevo QTof and BioAccord systems both yielded robust total ion and extracted ion chromatograms for mAb with reproducible mass spectra.

Reproducibility:

• Over 250 injections of murine IgG, MS extracted ion peak areas and UV responses showed %RSD ≤3 %, confirming column stability and consistency.

Benefits and Practical Applications

• Enhanced sensitivity and recovery for low-abundance protein species.
• Improved peak symmetry and resolution with volatile mobile phases.
• Compatibility with lower ionic strength ammonium acetate facilitates higher MS response.
• High throughput clone screening and routine QC analysis where sample is limited.

Future Trends and Potential Applications

• Development of even lower background column materials to extend native MS mass range.
• Miniaturized SEC formats for ultra-low flow nano-ESI-MS applications.
• Integration with ion exchange and hydrophobic interaction chromatography for multi-dimensional native separation.
• Automated high-throughput platforms for biopharmaceutical process monitoring.

Conclusion

The combination of high coverage PEO bonding and hydrophilic MaxPeak surfaces in ACQUITY Premier and XBridge Premier Protein SEC columns significantly reduces undesired analyte–surface interactions under native SEC-MS conditions. This yields superior detection limits, peak shapes, and recoveries for mAb analysis with ammonium acetate mobile phases. The technology enables robust, high throughput native LC-MS characterization of protein therapeutics.

Reference

• Farsang E. et al. Coupling Non-denaturing Chromatography to MS for mAb Characterization. J Pharm Biomed Anal. 2020;185:113207.
• Murisier A. et al. On-line Ion Exchange and Native MS for mAb Charge Variants. J Chromatogr A. 2021;1655:462499.
• Chen B. et al. Online Hydrophobic Interaction Chromatography-MS for Top-Down Proteomics. Anal Chem. 2016;88(3):1885–91.
• Lauber MA. Wanted: Native Protein LC-MS. The Analytical Scientist. 2019.
• Goyon A. et al. SEC Coupled to MS for Therapeutic Antibodies. J Chromatogr B. 2017;1065–1066:35–43.
• Shion H. et al. Molecular Mass Analysis of mAb Chains using BioAccord. Waters App Note 720006529EN.