Accelerating Charge Variant Analysis of Biotherapeutics with the BioAccord System

Significance of the Topic

The characterization of charge variants in monoclonal antibodies and other biotherapeutics is fundamental to process development, quality control, and regulatory compliance. Ion exchange chromatography (IEX) is widely used for this purpose, but conventional salt gradients limit compatibility with mass spectrometry, reducing the level of structural information obtainable.

Objectives and Study Overview

This work demonstrates a mass spectrometry–compatible IEX method using Waters IonHance CX-MS pH concentrates on the BioAccord LC-MS System. Comparative evaluations with pH and salt gradients on BioResolve SCX mAb columns assess performance in charge variant resolution and downstream applicability for both MS and UV detection.

Methodology and Instrumentation

A dual-detector setup combined UV detection with electrospray ionization mass spectrometry. IEX separations were performed using linear pH gradients delivered by IonHance CX-MS concentrates and BioResolve CX concentrates. Salt-based methods were also tested using MES buffers and sodium chloride gradients. Columns were maintained at 40 °C and samples at 10 °C. Data acquisition and processing utilized waters_connect and UNIFI software platforms.

Instrumentation Used

• BioAccord LC-MS System with BioResolve SCX mAb column
• ACQUITY Premier UPLC with tunable UV detector
• RDa high-resolution mass detector
• UNIFI Application integrated with waters_connect informatics
• Empower 3 software for UV method validation

Key Results and Discussion

Mass spectral data enabled direct identification of C-terminal lysine truncation variants via observed 128 Da mass shifts. Comparative MS analysis differentiated glycosylation profiles between innovator and biosimilar products. UV-based separations using BioResolve CX pH concentrates matched MS-grade pH gradients in resolution. Traditional salt gradient methods on the same column also achieved comparable profiles with high reproducibility (RSD < 1 %).

Benefits and Practical Applications

The combined use of pH concentrates and MS offers rapid, information-rich charge variant profiling without additional sample treatments. Flexibility to switch between pH and salt gradients allows seamless method transfer between research and manufacturing labs. High method robustness supports routine quality control and biopharmaceutical process monitoring.

Future Trends and Opportunities

Advancements may include automated gradient optimization, integration with high-throughput MS workflows, and development of novel buffer formulations to extend pH and ionic strength ranges. Enhanced data analytics and machine learning could further accelerate variant identification and method development.

Conclusion

This study validates a versatile IEX platform combining IonHance CX-MS pH concentrates, BioResolve columns, and the BioAccord system for detailed MS-compatible charge variant analysis. The approach delivers consistent performance across MS and UV detection modes and supports both pH and salt gradient methods, facilitating accelerated development and manufacturing of biotherapeutics.

References

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