Seamless LC-MS method transfer in a biopharmaceutical development laboratory

Importance of the Topic

Native SEC-MS provides critical structural insights into monoclonal antibodies and other biotherapeutics under near-native conditions. In fast-paced biopharmaceutical development, ensuring consistent, high-quality data when upgrading to new high-resolution accurate mass (HRAM) instruments is essential for reliable lead selection and quality assessment.

Objectives and Study Overview

This study aimed to transfer an existing native SEC-MS method from a Thermo Scientific Q Exactive Plus hybrid quadrupole-Orbitrap system to the next-generation Orbitrap Exploris 480 platform without compromising data quality. Key goals included:

• Validating method consistency across instruments
• Optimizing MS resolution and sample load
• Demonstrating throughput and reproducibility for large sample sets

Methodology and Instrumentation

The workflow employed a Vanquish Horizon Duo UHPLC configured for tandem SEC columns, enabling continuous MS acquisition with a 4.7 min run time per sample and automated next-injection preparation.

• LC conditions: 4.6 × 150 mm SEC column, 25 mM ammonium acetate (pH 5.4) mobile phase, 0.3 mL/min at 20 °C.
• MS platforms: Q Exactive Plus (35 000 resolution at m/z 200, 10 µg injection) and Orbitrap Exploris 480 (45 000 resolution, 4 µg injection), both with BioPharma option.
• Data acquisition control in Chromeleon CDS; intact protein deconvolution in Protein Metrics Byos.

Main Results and Discussion

Resolution testing between 7 500 and 60 000 showed that 45 000 provided the best balance of spectral clarity and signal-to-noise for resolving closely spaced antibody glycoforms. Sample loads from 1 to 10 µg yielded consistent relative glycoform abundances, demonstrating flexibility in sample concentration.
Comparative profiling of a system suitability antibody on both platforms produced nearly identical glycoform distributions (<1–2 % absolute difference). Longitudinal robustness tests across multiple studies, operators, and months confirmed low variability (%CV <5 %) on both the legacy and the Exploris 480 systems.

Benefits and Practical Applications

• High-throughput mAb screening with sub-5 min cycles and no downtime between injections.
• Seamless method transfer using the same LC-MS control and data-analysis software.
• Reduced sample consumption on the Exploris 480 while maintaining or improving sensitivity.
• Reliable intact mass and glycoform profiling for lead selection and QC workflows.

Future Trends and Opportunities

Ongoing developments may include further automation of data processing pipelines, expansion to other biotherapeutic formats, and leveraging evolving HRAM technologies to decrease analysis time and sample requirements even further.

Conclusion

The method transfer to the Orbitrap Exploris 480 platform was accomplished seamlessly, preserving data quality, enhancing sensitivity, and maintaining robust, reproducible glycosylation profiling for high-throughput biopharmaceutical screening.

Reference

• Thermo Scientific Application Note 73423
• Thermo Scientific Application Note 73885
• Thermo Scientific Application Note 72348