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Understanding the pathway and kinetics of aspartic acid isomerization in peptide mapping methods for monoclonal antibodies

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Abstract

Isomerization of aspartic acid (Asp) in therapeutic proteins could lead to safety and efficacy concerns. Thus, accurate quantitation of various Asp isomerization along with kinetic understanding of the variant formations is needed to ensure optimal process development and sufficient product quality control. In this study, we first observed Asp-succinimide conversion in complementarity-determining regions (CDRs) Asp-Gly motif of a recombinant mAb through ion exchange chromatography, intact protein analysis by mass spectrometry, and LC-MS/MS. Then, we developed a specific peptide mapping method, with optimized sample digestion conditions, to accurately quantitate Asp-succinimide-isoAsp variants at peptide level without method-induced isomerization. Various kinetics of Asp-succinimide-isoAsp isomerization pathways were elucidated using 18O labeling followed by LC-MS analysis. Molecular modeling and molecular dynamic simulation provide additional insight on the kinetics of Asp-succinimide formation and stability of succinimide intermediate. Findings of this work shed light on the molecular construct and the kinetics of the formation of isoAsp and succinimide in peptides and proteins, which facilitates analytical method development, protein engineering, and late phase development for commercialization of therapeutic proteins.

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Acknowledgements

The authors would like to acknowledge Dr. Jeff Beckman for his critical review and Drs. Jing Tong, Kai Meng Zhou, and Li Tao for their scientific input. The authors also would like to thank Suzanne Shirar for her assistance in manuscript preparation. Yuanli Song would like to thank Dr. Jie Chen for her administrative support.

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Author notes

  1. Yuanqi Tao

    Present address: Analytical Science Biologics, Takeda Pharmaceutical Company, Lexington, MA, 02421, USA

  2. Yuanli Song

    Present address: Process Development & Manufacture Operations, GSK, MA, 02451, Waltham, USA

  3. Nesredin Mussa

    Present address: Ultragenyx, CA, 94005, Brisbane, USA

  4. June Kuang, Yuanqi Tao and Yuanli Song contributed equally to this work.

Authors and Affiliations

  1. Biologics Development Organization, Bristol-Myers Squibb Company, Devens, MA, 01434, USA

    June Kuang, Yuanqi Tao, Yuanli Song, Letha Chemmalil, Nesredin Mussa, Julia Ding & Zheng Jian Li

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  1. June Kuang
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Kuang, J., Tao, Y., Song, Y. et al. Understanding the pathway and kinetics of aspartic acid isomerization in peptide mapping methods for monoclonal antibodies. Anal Bioanal Chem 413, 2113–2123 (2021). https://doi.org/10.1007/s00216-021-03176-z

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