Development of pH Gradient Mobile Phase Concentrates for Robust, High Resolution mAb Charge Variant Analysis

Significance of the Topic

The analysis of monoclonal antibody (mAb) charge variants is critical for ensuring therapeutic safety, stability, and efficacy. Post‐translational modifications generate heterogeneous charge profiles that can impact drug performance. pH gradient ion‐exchange chromatography offers a reproducible, broadly applicable alternative to traditional salt gradients, simplifying method development and improving robustness.

Objectives and Study Overview

• Establish a universal pH gradient mobile phase system covering mAb isoelectric points from 6 to 10.
• Optimize buffer composition, pH range, and gradient conditions for high resolution on SCX mAb columns.
• Compare performance versus leading competitor technologies.
• Demonstrate method robustness, reproducibility, and lifetime under routine conditions.

Methodology and Instrumentation

Five mAbs (panitumumab, infliximab, trastuzumab, adalimumab, and NIST mAb RM 8671) and a charge variant standard were analyzed using BioResolve CX pH concentrates A and B diluted 10-fold with water. Separations employed BioResolve SCX mAb columns (3 µm, 4.6 × 50 mm), an ACQUITY UPLC H-Class Bio system with Empower 3 Software, and pH monitoring via a GE Healthcare pH/C-900 detector. Gradients ranged from 0 to 100 % B (pH 5.0–10.2) at flow rates up to 1.0 mL/min.

Key Results and Discussion

The optimized mobile phases use four biological buffer salts (succinic acid, BIS-TRIS propane, triethanolamine, CAPSO) to deliver a linear pH gradient with constant capacity. Kinetic studies showed pH linearity depends on gradient steepness (ΔpH/Δcolumn volume ≤ 0.5 recommended). Compared to a competitor system, the BioResolve method achieved higher peak capacity and resolution for high‐pI mAbs, lower carryover, and consistent performance across two concentrate and column batches. A 20 min BioResolve gradient outperformed a 50 min competitor protocol, resolving minor basic variants. Column lifetime testing (500 injections) confirmed stable retention times, pressures, and resolution.

Benefits and Practical Applications

• Platform method requires minimal sample‐specific optimization.
• High resolution and reproducibility for mAbs across wide pI range.
• Flexible throughput: shortened gradients for speed or focused gradients for more detailed profiling.
• Improved laboratory efficiency and reduced development time.

Future Trends and Potential Applications

Integration with automated method development workflows and extension to other biotherapeutics (e.g., antibody–drug conjugates) are anticipated. Coupling pH gradient separations with high‐resolution mass spectrometry will further enhance variant characterization. Ongoing work may refine buffer systems for even steeper gradients and higher throughput in QC and R&D settings.

Conclusion

The combination of BioResolve CX pH concentrates and BioResolve SCX mAb columns establishes a robust, universal pH gradient approach for mAb charge variant analysis. It streamlines implementation, delivers superior resolution and reproducibility, and supports high‐throughput workflows in analytical and QC laboratories.

Used Instrumentation

• ACQUITY UPLC H-Class Bio System
• Empower 3 Software
• GE Healthcare Monitor pH/C-900

Reference

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