Optimizing LC-MS Separations of mAbs using a BioResolve SCX mAb Column and IonHance CX-MS pH Concentrates

Significance of Topic

Monoclonal antibodies (mAbs) possess inherent charge heterogeneities due to chemical and enzymatic modifications that affect product efficacy and safety. Charge variant analysis by ion exchange chromatography is a critical quality control measure. Recent advances in volatile salt–mediated pH gradients enable direct coupling of IEX to mass spectrometry (IEX-MS), streamlining variant identification and reducing analysis time.

Goals and Study Overview

This work aimed to develop and optimize a salt-mediated pH-gradient IEX-MS method for robust charge variant profiling of intact and subunit-digested mAbs. By employing IonHance™ CX-MS pH Concentrates and a BioResolve™ SCX mAb column, the study sought to simplify mobile phase preparation, ensure MS compatibility, and achieve high resolution and reproducibility.

Instrumentation Used

• Waters ACQUITY™ UPLC I-Class PLUS System
• Xevo™ G2-S QTof and ACQUITY™ RDa MS Detectors
• BioResolve™ SCX mAb Column, 2.1×50 mm, 3 µm
• IonHance™ CX-MS pH Concentrates (Buffers A and B)
• UNIFI™ Scientific Information System

Methodology

Sample Preparation:
• IdeS digestion of NIST mAb, trastuzumab, or adalimumab at 37 °C for 30 min in ammonium acetate buffer.
Chromatography and MS Conditions:
• Mobile phase A: 50 mM ammonium acetate pH 5.0 (2 % ACN); B: 160 mM ammonium acetate pH 8.5 (2 % ACN) at 10× dilution.
• Flow rate: 0.1 mL/min; column temperature: 30 °C; gradient: 2→98 % B over 21 min with 6 min return.
• Detection via UV at 280 nm and ESI+ mass spectrometry (m/z 400–7,000).

Main Results and Discussion

• IonHance CX-MS pH Concentrates produced a linear pH gradient despite pKa limitations, enabling efficient charge variant separation.
• Method robustness was demonstrated across different reagent and column batches, with retention time RSDs < 2 % for major subunits and variants.
• Alternative dilutions (e.g., 21× or 75 mM pH 8.4) enhanced the resolution of high-pI variants and prevented incomplete elution.
• High-quality native mass spectra were obtained for intact mAb and subunit fragments, facilitating direct identification of deamidation, lysine truncation, and other variants.

Benefits and Practical Applications

• Simplified mobile phase preparation using premade pH concentrates reduces labor and error.
• Direct IEX-MS coupling eliminates offline fractionation or 2D workflows, accelerating charge variant analysis.
• High reproducibility and sensitivity support release testing and comparability studies for biotherapeutic products.

Future Trends and Opportunities

• Expansion of salt-mediated pH-gradient IEX-MS to other classes of biologics and complex glycoproteins.
• Integration with automated data processing and machine learning for variant annotation.
• Development of next-generation IEX column chemistries for enhanced resolution and speed.
• Implementation of multiplexed or online 2D LC setups to further improve throughput and depth of characterization.

Conclusion

The combination of BioResolve™ SCX mAb columns and IonHance™ CX-MS pH Concentrates offers a versatile, reproducible, and MS-compatible platform for mAb charge variant analysis. Flexibility in dilution and gradient design permits fine-tuning for diverse analytes, supporting robust QC and advanced biopharmaceutical research.

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