Fast Comparative Structural Characterization of Intact Therapeutic Antibodies Using Hydrogen-Deuterium Exchange and Electron Transfer Dissociation.
Higher-order structural characterization plays an important role in many stages of therapeutic antibody production. Herein, we report a new top-down mass spectrometry approach for characterizing the higher-order structure of intact antibodies, by combining hydrogen/deuterium exchange (HDX), subzero temperature chromatography, and electron transfer dissociation on the Orbitrap mass spectrometer. Individual IgG domain-level deuteration information was obtained for 6 IgG domains on Herceptin (HER), which included the antigen binding sites. This is the first time that top-down HDX has been applied to an intact protein as large as 150 kDa, which has never been done before on any instrument. Ligand-binding induced structural differences in HER were determined to be located only on the variable region of the light chain. Global glycosylation profile of antibodies and HDX property of the glycoforms were also determined by accurate intact mass measurements. Although the presence of disulfide bonds prevent the current approach from being able to obtain amino acid level structural information within the disulfide-linked regions, the advantages such as minimal sample manipulation, fast workflow, very low level of back exchange, and simple data analysis, make it well-suited for fast comparative structural evaluation of intact antibodies.
†University of Victoria-Genome British Columbia Proteomics Centre, Vancouver Island Technology Park, #3101-4464 Markham St., Victoria, British Columbia V8Z 7X8, Canada.
Jingxi
J
Pan
†University of Victoria-Genome British Columbia Proteomics Centre, Vancouver Island Technology Park, #3101-4464 Markham St., Victoria, British Columbia V8Z 7X8, Canada.
Suping
S
Zhang
†University of Victoria-Genome British Columbia Proteomics Centre, Vancouver Island Technology Park, #3101-4464 Markham St., Victoria, British Columbia V8Z 7X8, Canada.
Albert
A
Chou
†University of Victoria-Genome British Columbia Proteomics Centre, Vancouver Island Technology Park, #3101-4464 Markham St., Victoria, British Columbia V8Z 7X8, Canada.
Darryl B
DB
Hardie
†University of Victoria-Genome British Columbia Proteomics Centre, Vancouver Island Technology Park, #3101-4464 Markham St., Victoria, British Columbia V8Z 7X8, Canada.
Christoph H
CH
Borchers
D041961:Deuterium Exchange Measurement; D004579:Electron Transport; D007074:Immunoglobulin G / Q000737:chemistry; D013058:Mass Spectrometry; D008958:Models, Molecular; D015394:Molecular Structure; D000068878:Trastuzumab / Q000737:chemistry*
D007074:Immunoglobulin G; D000068878:Trastuzumab