Abstract
Over recent decades, bispecific antibodies (bsAbs) have garnered significant attention for their superior therapeutic efficacy compared to progenitor monoclonal antibodies, enabling innovative treatment strategies. Despite their potential, the development of bsAbs presents significant challenges, with structural stability playing a pivotal role in manufacturability, therapeutic performance, and safety. Among the factors influencing stability, the design and incorporation of molecular linkers are particularly critical. In this study, we investigated the structural stability and fragmentation profiles of a symmetric bispecific antibody (Sym-bsAb), targeting HER2 and CD3, under forced degradation conditions. The Sym-bsAb exhibited pronounced fragmentation under prolonged thermal stress, particularly when combined with high pH and salt conditions. Intact mass analysis identified key degradation events, including sequential clipping along G4S and G4 linkers, fragmentations at interchain cystinyl residues and cleavage at the C-terminal of asparagine residues. The identification of G4S and G4 linkers as vulnerable regions prone to clipping in Sym-bsAb provided valuable insights into the stability and manufacturability of bsAbs incorporating linker sequences, underscoring critical considerations for their development.
Data availability
The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.
Abbreviations
- BiTE:
-
Bispecific T cell engager
- bsAbs:
-
Bispecific antibodies
- Fab:
-
Fragment antigen-binding
- HCCF:
-
Harvest cell culture fluid
- H:
-
Heavy chain
- HMW:
-
High molecular weight species
- HPLC-SEC:
-
High-performance liquid chromatography-size exclusion chromatography
- L:
-
Light chain
- LMW:
-
Low molecular weight species
- mAb:
-
Monoclonal antibody
- Sym-bsAb:
-
Symmetric bispecific antibody
- (VH)scFv :
-
Variable heavy chain of a single-chain fragment
- (VL)scFv :
-
Variable light chain of a single-chain fragment
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Acknowledgements
This research was supported by the Bioprocessing Technology Institute (BTI), Agency for Science, Technology and Research (A*STAR).
Funding
This work was supported by the Bioprocessing Technology Institute (BTI), Agency for Science, Technology and Research (A*STAR).
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Nattha Ingavat: Methodology, Validation, Formal analysis, Investigation, Data Curation, Writing - Original Draft. Yee Jiun Kok: Methodology, Validation, Formal analysis, Investigation, Data Curation, Writing - Original Draft. Nuruljannah Dzulkiflie: Formal analysis, Data Curation. Liew Jia Min: Formal analysis, Data Curation. Wang Xinhui: Formal analysis, Data Curation. Kia Ngee Low: Data Curation. Ka Pui But: Data Curation. Amihan Anaja: Data Curation. Loh Han Ping: Data Curation. Han Kee Ong: Data Curation. Farouq Bin Mahfut: Data Curation. Yuansheng Yang: Supervision. Xuezhi Bi: Conceptualization, Methodology, Investigation, Writing - Review & Editing, Supervision. Wei Zhang: Conceptualization, Methodology, Investigation, Resources, Writing - Review & Editing, Supervision.
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Ingavat, N., Kok, Y.J., Dzulkiflie, N. et al. Structural stability of symmetric bispecific antibodies: a case study showing potential compromise near linker regions. Sci Rep (2026). https://doi.org/10.1038/s41598-026-40607-2
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DOI: https://doi.org/10.1038/s41598-026-40607-2
