Mapping conformational changes on bispecific antigen-binding biotherapeutic by covalent labeling and mass spectrometryOA
Mapping conformational changes on bispecific antigen-binding biotherapeutic by covalent labeling and mass spectrometry
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 bis-pecific 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 tech-niques in elucidating the inherent structural characteristics of novel therapeutics and their conforma-tional stability.
Arnik Shah;Dipanwita Batabyal;Dayong Qiu;Weidong Cui;John Harrahy;Alexander R.Ivanov
Amgen Inc.,Cambridge,MA,02141,USA||Barnett Institute of Chemical and Biological Analysis,Department of Chemistry and Chemical Biology,Northeastern University,Boston,MA,02115,USAAmgen Inc.,Thousand 0aks,CA,91320,USAAmgen Inc.,Cambridge,MA,02141,USASanofi,Waltham,MA,02451,USABarnett Institute of Chemical and Biological Analysis,Department of Chemistry and Chemical Biology,Northeastern University,Boston,MA,02115,USA
Covalent labeling/footprintingLiquid chromatography-mass spectrometryFast photooxidation of proteinsDiethylpyrocarbonateHigher order structure characterization
《药物分析学报（英文）》 2024 (008)
1238-1246 / 9
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 Gou-dar,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.
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