MULTIDIMENSIONAL LC-MS PLATFORMS FOR STRUCTURE-FUNCTION CHARACTERIZATION OF THERAPEUTIC ANTIBODIES

Importance of the topic

Therapeutic monoclonal antibodies are pivotal in treating numerous diseases but pose analytical challenges due to their large size, biosynthetic heterogeneity, and diverse post-translational modifications. Comprehensive characterization of these complex molecules underpins safety, efficacy, and regulatory compliance across biopharmaceutical development and manufacturing.

Study objectives and overview

This work presents the design and application of multidimensional liquid chromatography coupled to mass spectrometry (MD-LC-MS) platforms for structure–function profiling of therapeutic antibodies. Key goals include improved resolution of proteoforms and glycoforms, site-specific characterization of chemical modifications, clone screening based on charge and size variants, and direct assessment of FcRn binding and antigen affinity.

Methodology and instrumentation

MD-LC-MS workflows integrate one-dimensional separations (RPLC, SEC, CEX, HIC) with two- and three-dimensional heart-cutting or comprehensive 2D/L 3D setups. On-line reduction and digestion enable Middle-up and bottom-up peptide mapping. Native MS and hydrogen–deuterium exchange modules probe higher-order structure and receptor interactions. Instruments include Agilent Bio-mAb and peptide mapping columns, Q-TOF mass spectrometers, UV detectors, quaternary and isocratic pumps, automated column selectors, and dedicated FcRn affinity columns.

Main results and discussion

Comprehensive RPLC×RPLC peptide maps resolved over sixty peptides in trastuzumab and detected low-abundance species. Heart-cutting 2D-LC-MS distinguished high-molecular-weight aggregates and low-molecular-weight fragments, facilitating purity assessment. Three-dimensional LC-MS with 2D multi-method capability enabled parallel clone evaluation for tocilizumab, linking glycoform profiles to functional ELISA potency. MD-LC-MS with on-line digestion achieved over 90 % sequence coverage of trastuzumab and pinpointed deamidation and isomerization events through retention time shifts. Advanced MD-LC-MS of Fc fragments tracked site-specific oxidation, correlated deuterium uptake differences via HDX-MS, and measured FcRn binding by native affinity MS. A 2D CEX–FcRn-MS approach mapped charge variants to altered receptor affinity, ranking oxidation variants by functional impact.

Benefits and practical applications

These MD-LC-MS platforms deliver high-resolution, automated attribute monitoring, crucial for clone selection, comparability studies, and quality control in CMC workflows. The fusion of top-down, middle-up, and bottom-up analyses affords comprehensive structural annotation, while native MS and HDX modules connect molecular modifications to binding function, enhancing risk assessment and formulation optimization.

Future trends and opportunities

Emerging directions include higher-order dimensions of separation, integration of microfluidic digestion, real-time multi-attribute monitoring with AI-driven data interpretation, miniaturized high-throughput formats, and broader adoption in regulatory frameworks. Expansion of these strategies to other biotherapeutics, such as fusion proteins and bispecifics, promises deeper insights into critical quality attributes.

Conclusion

Multidimensional LC-MS approaches represent a powerful toolkit for in-depth structure–function analysis of therapeutic antibodies. By resolving complex proteoforms, linking modifications to function, and enabling automated affinity assessment, these platforms drive more robust and efficient biopharmaceutical development.

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