Fast Comparative Structural Characterization of Intact Therapeutic Antibodies Using Hydrogen–Deuterium Exchange and Electron Transfer Dissociation

Abstract

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.

The Role of Electron Transfer Dissociation in Modern Proteomics

“…Note, a combination of HDX and ETD also enabled analysis of the structure of antibodies in middle-down and top-down approaches discussed above. 244 , 248 , 252 Beyond HDX, ETD has been used with other surface labeling techniques, as well, 283 − 285 although HDX is the more common approach to use ETD because of the hydrogen scrambling concerns with collisional activation.…”

Comparative higher-order structure analysis of antibody biosimilars using combined bottom-up and top-down hydrogen-deuterium exchange mass spectrometry

“…Protein elution was conducted at −20°C using our recently developed subzero temperature approach [27] The UPLC system was coupled to an Orbitrap-fusion mass spectrometer (Thermo Fisher Scientific, Bremen, Germany), equipped with electron transfer dissociation (ETD) [45] The antibodies were eluted with 15-minute binary gradient from 15 to 50% B, at a flow rate of 200 μL/min. The Orbitrap spray voltage was 3500 V in the positive ion mode.…”

“…To the best of our knowledge, this kind of study has not yet been done. The HDX-MS work done so far on antibodies used either the bottom-up [39][40][41][42][43][44] or the top-down approach [27,45]. Our current work fills this gap by characterizing the higher-order structure of an originator antibody drug BEV (BEV) and two batches of its biosimilars side by side, using the two methods.…”

Tracking Higher Order Protein Structure by Hydrogen-Deuterium Exchange Mass Spectrometry

“…HDX-MS is becoming increasingly incorporated into biotherapeutic development as it provides a fast structural evaluation of intact therapeutic antibodies [58] and a structural tool for ensuring biocomparability [59]. The structural consequences due to altered glycosylation on IgG molecules has now been extensively investigated by HDX-MS [60–63].…”