Streamlining biotherapeutic characterisation by mass spectrometry

Science
Antibody-based biotherapeutics are large complex proteins which require rigorous structural characterisation to assess their quality and ensure that they will function as intended in the human body. Essential quality attributes can be obtained by analysing the therapeutic protein at multiple structural levels: intact protein, and its subunits and peptides. Liquid Chromatography-Mass Spectrometry (LC-MS), a powerful analytical technique, provides the high resolution and accurate mass necessary for such characterisation, but the conventional use of different column chemistry–and thus separate LC-MS setups–to analyse proteins at different structural levels have led to increased analytical time and cost. To improve efficiency and analytics automatability, we have streamlined the multilevel structural characterisation of therapeutic proteins onto a single-column LC-MS setup with robust and optimised easy-to-operate methods. In particular, the newly developed subunit sequencing method is broadly applicable to antibody-based biotherapeutics, eliminating the need for complex method development tailored to individual proteins by skilled mass spectrometrists and thereby extending the utility of LC-MS for routine quality assessment of therapeutic proteins.
Societal Impact
Streamlining of MS-based workflows for quality assessment of therapeutic proteins onto a single-column LC-MS setup minimises downtime and cost by eliminating the need for different LC-MS configurations. This approach not only enhances the robustness and efficiency of MS analytical workflows but also simplifies the integration of an LC-MS analyser as a process analytical technology tool in biomanufacturing. This also promotes the use of LC-MS in real-time product quality monitoring, enabling Quality by Designing manufacturing and accelerated product release testing, thus ensuring the expected product quality with reduced COGS.
Technical Summary
The robust performance of a single-column LC-MS setup for multilevel structural characterization of biotherapeutic proteins was demonstrated using three structurally distinct antibody-based molecules – NISTmAb (a monoclonal antibody), RG7221 (a 4-chain asymmetric bispecific antibody) and a Fc fusion protein with complex O- and N-glycosylation patterns (etanercept). The proteins were assessed by intact protein and subunit mass analyses, as well as middle-down and bottom-up MS analyses using a C4 column on an LC-coupled ZenoTOF 7600 MS. Notably, high sequence coverage and comprehensive profiling of PTMs were obtained in bottom-up analysis using the C4 column, which is conventionally only used with high molecular mass intact or subunit protein analytes. We have also established a robust and versatile middle-down MS method on the ZenoTOF 7600 MS. The standardised parameters required no further optimisation for individual antibody-based biotherapeutics, and a single LC-MS run was generally sufficient for the middle-down sequencing of all the subunits from each analyte.
References
Low, K. N., Kok, Y. J., Tate, S., & Bi, X. (2025). Multilevel ─ Intact, Subunits, and Peptides ─ Characterization of Antibody-Based Therapeutics by a Single-Column LC-MS Setup. Anal. Chem. 2025, 97, 9, 5118–5125 https://doi.org/10.1021/acs.analchem.4c06350
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