Mapping conformational changes on bispecific antigen-binding biotherapeutic by covalent labeling and mass spectrometry

Arnik Shah; Dipanwita Batabyal; Dayong Qiu; Weidong Cui; John Harrahy; Alexander R. Ivanov

doi:10.1016/j.jpha.2024.100966

Volume 14 Issue 8

Aug. 2024

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Arnik Shah, Dipanwita Batabyal, Dayong Qiu, Weidong Cui, John Harrahy, Alexander R. Ivanov. Mapping conformational changes on bispecific antigen-binding biotherapeutic by covalent labeling and mass spectrometry[J]. Journal of Pharmaceutical Analysis, 2024, 14(8): 100966. doi: 10.1016/j.jpha.2024.100966

Citation:

Arnik Shah, Dipanwita Batabyal, Dayong Qiu, Weidong Cui, John Harrahy, Alexander R. Ivanov. Mapping conformational changes on bispecific antigen-binding biotherapeutic by covalent labeling and mass spectrometry[J]. Journal of Pharmaceutical Analysis, 2024, 14(8): 100966. doi: 10.1016/j.jpha.2024.100966

Citation:

Arnik Shah, Dipanwita Batabyal, Dayong Qiu, Weidong Cui, John Harrahy, Alexander R. Ivanov. Mapping conformational changes on bispecific antigen-binding biotherapeutic by covalent labeling and mass spectrometry[J]. Journal of Pharmaceutical Analysis, 2024, 14(8): 100966. doi: 10.1016/j.jpha.2024.100966

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Mapping conformational changes on bispecific antigen-binding biotherapeutic by covalent labeling and mass spectrometry

doi: 10.1016/j.jpha.2024.100966

a. Amgen Inc., Cambridge, MA, 02141, USA;
b. Barnett Institute of Chemical and Biological Analysis, Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA, 02115, USA;
c. Amgen Inc., Thousand Oaks, CA, 91320, USA;
d. Sanofi, Waltham, MA, 02451, USA

Funds:

This research is supported by Amgen Inc., USA and the National Institutes of Health, USA (Grant Nos.: R01CA218500 (ARI) and R35GM136421 (ARI)). We would like to thank Dr. Scott Siera for providing a critical review of this manuscript and Drs. Chetan Goudar, Tiffany Thiel, and Susan Burke for their support of this project. We would also like to thank Drs. Mats Wikstroem and Palanisamy Kanakraj for providing valuable inputs and support with biophysical and binding assay, and Drs. Hao Zhang and Mengru Zhang for helpful discussions on FPOP and protein footprinting.

Received Date: Sep. 22, 2023
Accepted Date: Mar. 13, 2024
Rev Recd Date: Mar. 07, 2024
Publish Date: Mar. 16, 2024

Abstract

Abstract

Biotherapeutic's higher order structure (HOS) is a critical determinant of its functional properties and conformational relevance. Here, we evaluated two covalent labeling methods: diethylpyrocarbonate (DEPC)-labeling and fast photooxidation of proteins (FPOP), in conjunction with mass spectrometry (MS), to investigate structural modifications for the new class of immuno-oncological therapy known as bispecific antigen-binding biotherapeutics (BABB). The evaluated techniques unveiled subtle structural changes occurring at the amino acid residue level within the antigen-binding domain under both native and thermal stress conditions, which cannot be detected by conventional biophysical techniques, e.g., near-ultraviolet circular dichroism (NUV-CD). The determined variations in labeling uptake under native and stress conditions, corroborated by binding assays, shed light on the binding effect, and highlighted the potential of covalent-labeling methods to effectively monitor conformational changes that ultimately influence the product quality. Our study provides a foundation for implementing the developed techniques in elucidating the inherent structural characteristics of novel therapeutics and their conformational stability.

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