Mapping conformational changes on bispecific antigen-binding biotherapeutic by covalent labeling and mass spectrometry
10.1016/j.jpha.2024.100966
- Author:
Shah ARNIK
1
,
2
;
Batabyal DIPANWITA
;
Qiu DAYONG
;
Cui WEIDONG
;
Harrahy JOHN
;
R.Ivanov ALEXANDER
Author Information
1. Amgen Inc.,Cambridge,MA,02141,USA
2. Barnett Institute of Chemical and Biological Analysis,Department of Chemistry and Chemical Biology,Northeastern University,Boston,MA,02115,USA
- Keywords:
Covalent labeling/footprinting;
Liquid chromatography-mass spectrometry;
Fast photooxidation of proteins;
Diethylpyrocarbonate;
Higher order structure characterization
- From:
Journal of Pharmaceutical Analysis
2024;14(8):1238-1246
- CountryChina
- Language:Chinese
-
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 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.
