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Anal Chem. 2020 Jan 7;92(1):766-773. doi: 10.1021/acs.analchem.9b03129. Epub 2019 Dec 12.

Direct Determination of Antibody Chain Pairing by Top-down and Middle-down Mass Spectrometry Using Electron Capture Dissociation and Ultraviolet Photodissociation.

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Environmental Molecular Sciences Laboratory , Pacific Northwest National Laboratory , 3335 Innovation Boulevard , Richland , Washington 99354 , United States.
e-MSion Inc. , 2121 NE Jack London Drive , Corvallis , Oregon 97330 , United States.
Linus Pauling Institute and the Department of Biochemistry and Biophysics , Oregon State University , Corvallis , Oregon 97331 , United States.
Mapp Biopharmaceutical Inc. , 6160 Lusk Boulevard #105 , San Diego , California 92121 , United States.


One challenge associated with the discovery and development of monoclonal antibody (mAb) therapeutics is the determination of heavy chain and light chain pairing. Advances in MS instrumentation and MS/MS methods have greatly enhanced capabilities for the analysis of large intact proteins yielding much more detailed and accurate proteoform characterization. Consequently, direct interrogation of intact antibodies or F(ab')2 and Fab fragments has the potential to significantly streamline therapeutic mAb discovery processes. Here, we demonstrate for the first time the ability to efficiently cleave disulfide bonds linking heavy and light chains of mAbs using electron capture dissociation (ECD) and 157 nm ultraviolet photodissociation (UVPD). The combination of intact mAb, Fab, or F(ab')2 mass, intact LC and Fd masses, and CDR3 sequence coverage enabled determination of heavy chain and light chain pairing from a single experiment and experimental condition. These results demonstrate the potential of top-down and middle-down proteomics to significantly streamline therapeutic antibody discovery.

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