SEC-MS with Volatile Buffers for Characterization of Biopharmaceuticals

Importance of the Topic

The characterization of biopharmaceutical proteins, particularly monoclonal antibodies, requires precise size and mass information. Size exclusion chromatography (SEC) coupled directly to mass spectrometry (MS) with volatile buffers overcomes the limitations of high-salt mobile phases and ion-pair reversed-phase chromatography, offering high-resolution separations at ambient temperature and direct molecular weight measurement.

Objectives and Study Overview

This study evaluates the performance of a biocompatible UHPLC system (Thermo Scientific Vanquish Flex) for denaturing SEC separations using volatile, MS-compatible buffers. Key aims include demonstrating effective protein separation, minimizing secondary interactions, and achieving accurate mass determination for standard proteins and therapeutic monoclonal antibodies.

Methodology

Denaturing SEC separations were performed isocratically at 25 °C on a 300 Å, 5 μm SEC column using mobile phases containing low concentrations of trifluoroacetic acid (0.05%) and formic acid in acetonitrile/water (30/70, v/v). Protein standards (cytochrome c, myoglobin, ribonuclease A, transferrin) were injected at 1 μL, and intact and reduced monoclonal antibodies (rituximab, denosumab) were analyzed after disulfide reduction. The volatile buffer composition was optimized via the quaternary pump to balance separation efficiency and MS signal intensity.

Used Instrumentation

• Vanquish Flex UHPLC system with quaternary pump, Split Sampler FT, column compartment, and diode array detector with 10 mm LightPipe flow cell.
• Thermo Scientific Q Exactive HF Hybrid Quadrupole-Orbitrap mass spectrometer with HESI-II source.
• Thermo Scientific Dionex Chromeleon CDS software for data acquisition and ProteinDeconvolution software for deconvolution and mass accuracy evaluation.

Results and Discussion

The SEC-MS method separated the four protein standards with baseline resolution and delivered mass measurements within 0.75–1.23 ppm of theoretical values. Increasing TFA concentration minimized protein complex formation but slightly reduced signal intensity. Separation of intact and reduced rituximab showed clear resolution between intact antibody, light chain, heavy chain, and formulation buffer components, demonstrating effective desalting and impurity profiling.

Benefits and Practical Applications

The volatile buffer SEC-MS approach enables direct, high-accuracy mass determination of biotherapeutics without extensive sample cleanup. It provides rapid, isocratic runs with low carryover and room-temperature operation suitable for temperature-sensitive proteins. This method serves as an alternative to ion-pair reversed-phase chromatography for purity assessment, proteoform profiling, and formulation buffer removal prior to MS.

Future Trends and Potential Applications

Further developments may include integration of online sample preparation, expansion to native MS conditions for higher-order structure analysis, and method scouting workflows leveraging flexible gradient and buffer compositions. Adapting SEC-MS to newer orbitrap and time-of-flight platforms could enhance throughput and detection of minor product variants in quality control environments.

Conclusion

Implementing low-concentration volatile acids in SEC mobile phases on a biocompatible UHPLC platform enables seamless coupling to MS, delivering reliable size separation and precise mass measurement of biopharmaceutical proteins. The approach simplifies sample handling, improves data quality, and broadens analytical capabilities in biotherapeutic characterization.

References

1. Thermo Scientific Application Note 591: LC/MS Analysis of the Monoclonal Antibody Rituximab Using the Q Exactive Benchtop Orbitrap Mass Spectrometer. 2013.
2. Arakawa, T. et al. The critical role of mobile phase composition in size exclusion chromatography of protein pharmaceuticals. J Pharm Sci. 2010;99(4):1674–1692.