CDR clipping-induced heterodimerization: identification of a novel dimerization mechanism in a co-formulated antibody cocktail via a multifaceted mass spectrometry approach
Co-formulated antibody cocktails are becoming an increasingly popular therapeutic class; however, they present analytical challenges over traditional single monoclonal antibody (mAb) formulations. One paramount concern is the formation of heteromeric species that have unknown impacts on safety and e...
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| Format: | Article |
| Language: | English |
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Taylor & Francis Group
2025-12-01
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| Series: | mAbs |
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| Online Access: | https://www.tandfonline.com/doi/10.1080/19420862.2025.2546074 |
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| author | Andrew J Heindel Yang Shen Timothy N Tiambeng Yuetian Yan Shunhai Wang Ning Li |
| author_facet | Andrew J Heindel Yang Shen Timothy N Tiambeng Yuetian Yan Shunhai Wang Ning Li |
| author_sort | Andrew J Heindel |
| collection | DOAJ |
| description | Co-formulated antibody cocktails are becoming an increasingly popular therapeutic class; however, they present analytical challenges over traditional single monoclonal antibody (mAb) formulations. One paramount concern is the formation of heteromeric species that have unknown impacts on safety and efficacy. Consequently, effective approaches for identifying and characterizing high-molecular weight (HMW) impurities are critical to the successful development of this therapeutic class. In this study, we used a multifaceted mass spectrometry approach to characterize a unique dimer species formed between two co-formulated mAbs under thermal stress, revealing an intriguing dimerization mechanism that is driven by complementarity-determining region clipping-induced domain swap. Size exclusion chromatography-mass spectrometry, complemented by post-column denaturation, was utilized at both intact and subunit levels to pinpoint the dimerization interface. Additionally, by probing the disulfide bond susceptibility changes via limited reduction and middle-down analysis, the structural changes of the involved domains were studied. These results highlight the critical role of sophisticated analytical methods in comprehending and addressing the complexities linked to co-formulated mAb cocktails. |
| format | Article |
| id | doaj-art-d414abc220214dd5b3024ec51f83cd28 |
| institution | Kabale University |
| issn | 1942-0862 1942-0870 |
| language | English |
| publishDate | 2025-12-01 |
| publisher | Taylor & Francis Group |
| record_format | Article |
| series | mAbs |
| spelling | doaj-art-d414abc220214dd5b3024ec51f83cd282025-08-20T03:36:58ZengTaylor & Francis GroupmAbs1942-08621942-08702025-12-0117110.1080/19420862.2025.2546074CDR clipping-induced heterodimerization: identification of a novel dimerization mechanism in a co-formulated antibody cocktail via a multifaceted mass spectrometry approachAndrew J Heindel0Yang Shen1Timothy N Tiambeng2Yuetian Yan3Shunhai Wang4Ning Li5Analytical Chemistry Group, Regeneron Pharmaceuticals, Inc., Tarrytown, NY, USAAntibody Engineering, Regeneron PharmaceuticalsInc, Inc., Tarrytown, NY, USAAnalytical Chemistry Group, Regeneron Pharmaceuticals, Inc., Tarrytown, NY, USAAnalytical Chemistry Group, Regeneron Pharmaceuticals, Inc., Tarrytown, NY, USAAnalytical Chemistry Group, Regeneron Pharmaceuticals, Inc., Tarrytown, NY, USAAnalytical Chemistry Group, Regeneron Pharmaceuticals, Inc., Tarrytown, NY, USACo-formulated antibody cocktails are becoming an increasingly popular therapeutic class; however, they present analytical challenges over traditional single monoclonal antibody (mAb) formulations. One paramount concern is the formation of heteromeric species that have unknown impacts on safety and efficacy. Consequently, effective approaches for identifying and characterizing high-molecular weight (HMW) impurities are critical to the successful development of this therapeutic class. In this study, we used a multifaceted mass spectrometry approach to characterize a unique dimer species formed between two co-formulated mAbs under thermal stress, revealing an intriguing dimerization mechanism that is driven by complementarity-determining region clipping-induced domain swap. Size exclusion chromatography-mass spectrometry, complemented by post-column denaturation, was utilized at both intact and subunit levels to pinpoint the dimerization interface. Additionally, by probing the disulfide bond susceptibility changes via limited reduction and middle-down analysis, the structural changes of the involved domains were studied. These results highlight the critical role of sophisticated analytical methods in comprehending and addressing the complexities linked to co-formulated mAb cocktails.https://www.tandfonline.com/doi/10.1080/19420862.2025.2546074Fixed dose combinationheterodimertherapeutic antibodymonoclonal antibodynative LC-MSpost-column denaturation |
| spellingShingle | Andrew J Heindel Yang Shen Timothy N Tiambeng Yuetian Yan Shunhai Wang Ning Li CDR clipping-induced heterodimerization: identification of a novel dimerization mechanism in a co-formulated antibody cocktail via a multifaceted mass spectrometry approach mAbs Fixed dose combination heterodimer therapeutic antibody monoclonal antibody native LC-MS post-column denaturation |
| title | CDR clipping-induced heterodimerization: identification of a novel dimerization mechanism in a co-formulated antibody cocktail via a multifaceted mass spectrometry approach |
| title_full | CDR clipping-induced heterodimerization: identification of a novel dimerization mechanism in a co-formulated antibody cocktail via a multifaceted mass spectrometry approach |
| title_fullStr | CDR clipping-induced heterodimerization: identification of a novel dimerization mechanism in a co-formulated antibody cocktail via a multifaceted mass spectrometry approach |
| title_full_unstemmed | CDR clipping-induced heterodimerization: identification of a novel dimerization mechanism in a co-formulated antibody cocktail via a multifaceted mass spectrometry approach |
| title_short | CDR clipping-induced heterodimerization: identification of a novel dimerization mechanism in a co-formulated antibody cocktail via a multifaceted mass spectrometry approach |
| title_sort | cdr clipping induced heterodimerization identification of a novel dimerization mechanism in a co formulated antibody cocktail via a multifaceted mass spectrometry approach |
| topic | Fixed dose combination heterodimer therapeutic antibody monoclonal antibody native LC-MS post-column denaturation |
| url | https://www.tandfonline.com/doi/10.1080/19420862.2025.2546074 |
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