Practical Considerations for Optimizing MS Quality during IEX-MS

Significance of the topic

A high level of mass spectral quality is critical for reliable characterization of protein charge variants by ion-exchange chromatography coupled to mass spectrometry IEX-MS
Contaminants such as sodium or potassium adducts reduce resolution, sensitivity and mass accuracy, especially under native MS conditions
Ensuring MS cleanliness at every step improves data quality and expedites mAb variant profiling

Study objectives and overview

This work presents best practices to optimize MS spectral quality in strong cation-exchange MS analyses of monoclonal antibodies
Key aims include evaluating the impact of reagent grade, container material and sample preparation on adduct formation and signal clarity
Comparative experiments demonstrate improvements achieved by substituting glassware and non-volatile buffers with certified plastics and volatile salts

Methodology and instrumentation

Sample preparation

• IdeS digestion of NIST mAb into Fab and Fc fragments at 37 °C for 30 minutes
• Digestions performed in either phosphate buffered saline or MS-grade ammonium acetate

Chromatography and detection

• ACQUITY UPLC I-Class PLUS system with BioResolve SCX mAb 3 μm, 2.1 × 50 mm column at 30 °C
• ACQUITY TUV and RDa detectors in ESI positive mode, mass range m/z 400–7000
• Mobile phases prepared from IonHance CX-MS pH concentrates in certified LDPE bottles
• Gradient from 80% to 2% ammonium acetate at pH 5.0–8.5 over 21 minutes at 0.1 mL/min

Instrumentation

• ACQUITY UPLC I-Class PLUS
• ACQUITY RDa detector and TUV detector
• BioResolve SCX mAb column
• MS-certified LDPE mobile phase bottles and sample vials
• UNIFI Scientific Information System for data analysis

Main results and discussion

Replacing glass mobile phase bottles with MS-certified LDPE reduced sodium adduct intensity but did not fully eliminate adducts when digestion buffer and vials remained sources of contamination
Switching IdeS digestion from PBS to volatile ammonium acetate significantly lowered salt adducts in the F(ab2) and Fc/2 spectra and improved signal-to-noise
Using plastic autosampler vials further sharpened peaks and increased protonated ion abundance due to minimized surface leaching
Implementing an effluent divert valve during non-retained regions prevents non-volatile buffer components from entering the MS source and reduces maintenance needs

Benefits and practical applications

These optimized practices enhance detection sensitivity, mass accuracy and spectral resolution for charge variant analysis
Improved data quality accelerates development and quality control of therapeutic antibodies by reducing adduct interference
The workflow is directly applicable to biopharmaceutical labs performing routine native or denaturing IEX-MS assays

Future trends and potential applications

Integration of automated sample preparation and microplate-based consumables certified for low metal background
Development of novel low-leaching polymer materials for vials and fluidics
Enhanced source tuning and dynamic divert valve control to further minimize matrix effects
Expansion of IEX-MS to complex multi-attribute method workflows and continuous monitoring in quality control settings

Conclusion

A coherent MS-hygiene strategy encompassing MS-grade reagents, certified thermoplastics and buffer selection yields substantial improvements in IEX-MS performance
Minimizing sources of non-volatile salts and metal ions at every stage is essential for reliable mAb charge variant characterization
The protocols described serve as a blueprint for high-quality MS data in both R&D and QC environments

Reference

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