2D-LC-MS Methods for Enhanced Separation and In-depth Characterization of Protein Variants in Monoclonal IgGs

Daniel Saidemberg, Early CMC Biologics, Teva, Netanya, Israel (daniel.saidemberg@teva.co.il)
Gabriela Ridner-Bahar, Early Cmc Biologics, Teva, Netanya, Israel

Background: Monoclonal antibody (mAb) variants can differ in their physicochemical properties such as charge, hydrophobicity, and size, therefore they require tailored separation strategies for comprehensive analysis. Improved separation facilitates high-resolution mass spectrometry for detailed variant characterization. In this study we developed and optimized a robust two-dimensional liquid chromatography-mass spectrometry (2D LC-MS) method to achieve enhanced separation and in-depth characterization of variants present in monoclonal antibody (mAb) samples.

Methods: A 2D Vanquish™ UHPLC system was coupled to a high-resolution Orbitrap Exploris 240™ (Thermo™) mass spectrometer for intact electrospray mass spectrometry (ES-MS) analysis. In the first dimension (1D), mAb variants were separated based on their different physicochemical properties. The on-line second dimension employed reverse-phase (RP) HPLC to further resolve IgG variants according to their hydrophobicity.

Results and Discussion: The optimized methods demonstrated high reproducibility and significantly improved resolution of closely eluting variants, this enabled successful identification and relative quantitation of distinct mAb variants expression containing different post translational modifications. This approach contributed to a deeper understanding of IgG heterogeneity and helped trace potential critical quality attributes (CQAs) of the studied mAbs. The case studies presented in this work support the development of various 2D-LC-MS methods.

Conclusion: The developed multidimensional LC-MS workflow provides a robust and efficient platform for high-resolution characterization of monoclonal IgGs. It offers valuable insights into product quality and heterogeneity; and is broadly applicable in research and development settings to support process development and consistency of mAb-based therapeutics.

 

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Short Biography of Presenting Author

Daniel Saidemberg, Ph.D.

Daniel Saidemberg is a scientist at Teva Pharmaceutical Industries, specializing in protein characterization, LC-MS method development, and biopharmaceutical analysis. His work integrates advanced analytical chemistry with a systems-level understanding of biomolecules. His research interests include peptide mapping, mass spectrometry for characterization of biomolecules, and the optimization of workflows for complex biologics. Daniel is passionate about bridging scientific rigor with innovation in analytical development to advance therapeutic quality and reliability.