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

Abstract

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.

Selectivity over coverage in de novo sequencing of IgGs

“…S1 and Table S1 †). In line with previously reported data, 27 we observed that most of the abundant fragment ions have masses between 9000 and 16 000 Da, regardless of the Trastuzumab moiety that was used.…”

“…Here, we explored the benets of electron capture dissociation in attempting to sequence antibodies. Differentiating our approach from earlier attempts, 21,27,56 we aimed deliberately for a very restricted, albeit targeted, sequence coverage, spanning just the important hypervariable CDR3 of both the light and heavy chain (LC and HC) of the mAbs. We optimized our method by rst getting rid of the sequence-wise less informative constant regions (Fc) by using the IdeS protease, to obtain F(ab 0 ) 2 fragments.…”

“…Combining ECD with supplemental collisional activation can enhance precursor fragmentation. Although this has been demonstrated by Shaw et al 27 to extend sequence coverage, collisional activation also generates additional b/y-ion series. We argue that the generation of these extra fragment ion series could be disadvantageous for straightforward and condent de novo sequencing of IgGs.…”

“…25,26 While the presence of intrachain disulde bridges complicates the detection of several variable domain regions, it enhances the detection of CDR3 fragments. 25,27 In the present work, we build upon these exciting results and further optimize ECD for the de novo sequencing of IgG CDR3s and their downstream regions, generating very clean, straightforward-to-read sequence-ladders comprised solely of cions ( Fig. 1B).…”

Higher-order structural characterisation of native proteins and complexes by top-down mass spectrometry

“…95 In comparison, UVPD with 157 nm photons (F 2 excimer laser) appears to be more effective at generating a/x ions and has specicity towards disulphide bond cleavage. 128,129 UVPD at wavelengths of 213 nm 130 and 266 nm 131 has also been reported for native TD of small proteins. In the following paragraphs, we will consider how different aspects of higher-order structure can be probed using native TD fragmentation.…”