Increasing throughput of native SEC-MS in biopharmaceutical development using a tandem UHPLC setup

Importance of the topic

Native size exclusion chromatography–mass spectrometry (SEC-MS) under native conditions offers a streamlined approach to assess both aggregation and intact mass of biotherapeutics in a single analysis. This capability is crucial in early drug development where sample volume is limited and rapid, informative screening of monoclonal antibody candidates is required.

Objectives and Study Overview

The study describes the integration of a Vanquish Duo tandem UHPLC system with a Q Exactive Plus Orbitrap mass spectrometer to boost SEC-MS throughput for biopharmaceutical lead selection. The main goals were to reduce analysis time and sample consumption, maintain high analytical robustness, and enable seamless QC-compatible data processing.

Methodology and Instrumentation

Samples were directly injected (≤20 μL, up to 100 μL when diluted) onto a 4.6×150 mm, 1.7 μm SEC column using 25 mM ammonium acetate buffer (pH 5.4) at 0.3 mL/min. A Vanquish Duo UHPLC system performed tandem LC by alternating analytical and reconditioning flow between two columns. Native MS data were acquired on a Q Exactive Plus with BioPharma Option (m/z 2500–8000, resolution 35 000) using HESI source conditions. Chromeleon 7.2.9 software with the Intact Protein Deconvolution tool processed mass spectra.

Results and Discussion

Implementation of the tandem setup reduced per-sample run time from 8 to 4.7 minutes. In a lead selection study of 384 monoclonal antibodies (total 423 runs including system suitability tests), overall analysis time dropped from 158 to 32 hours. Sample consumption was cut by 33 % compared with separate SEC and intact RP-LC-MS assays. Chromatographic performance remained robust (monomer retention time RSD < 0.05 %, peak area RSD < 0.2 %). Mass accuracy across 17 system suitability injections was within ±10 ppm. The Orbitrap MS delivered high-resolution native spectra, enabling precise glycoform and variant identification with minimal deconvolution artifacts.

Benefits and Practical Applications of the Method

• Dramatic reduction in analysis time and sample requirements for early lead selection.
• Simultaneous characterization of aggregation and intact mass in one assay.
• High data quality and robust performance suitable for QC laboratory adoption.

Future Trends and Potential Applications

Extending native SEC-MS to other chromatographic modalities (ion exchange, affinity) can expand proteoform profiling. Advances in UHPLC automation, Orbitrap technology, and data processing algorithms will further increase throughput, sensitivity for low-abundance variants, and compliance readiness for regulated environments.

Conclusion

The native SEC-MS tandem UHPLC approach on an Orbitrap platform achieved nearly fivefold throughput gains while preserving analytical quality and reducing sample use. This robust, high-throughput workflow is well suited for biopharmaceutical lead selection and aligns with future QC demands.

References

1. Füssl et al. Comprehensive characterisation of adalimumab heterogeneity via native charge variant analysis hyphenated to Orbitrap MS. MAbs (2018).
2. Scheffler & Damoc. Full characterization of heterogeneous antibody samples under native and denaturing conditions on Q Exactive BioPharma. App. Note 72348 (2017).
3. Bailey. The Quest for Highly Effective Resolution: Therapeutic Protein Characterization via Native MS. c&en Whitepapers (2018).