Characterization and Comparison of Disulfide Linkages and Scrambling Patterns in Therapeutic Monoclonal Antibodies: Using LC-MS with Electron Transfer Dissociation

Abstract

Summary The disulfides in three monoclonal antibodies (mAb), the anti-HER2, anti-CD11a, and GLP-1 with IgG4-Fc fusion protein, were completely mapped by LC-MS with the combination of ETD and CID fragmentation. In addition to mapping the 4 inter- and 12 intra-chain disulfides (total 16), the identification of scrambled disulfides in degraded samples (heat-stress) was achieved. The scrambling was likely attributed to an initial breakage between the light (Cys 214) and heavy (Cys 223) chains in anti-HER2, with the same observation found in a similar therapeutic mAb, anti-CD11a. On the other hand, the fusion antibody, with no light chain but containing only 2 heavy chains, generated much less scrambling under the same heat-stressed conditions. The preferred sites of scrambling were identified, such as the intra-chain disulfide for CxxC in the heavy chain, and the C194 of the heavy chain pairing with the terminal Cys residue (C214) in the light chain. The inter-chain disulfides between the light and heavy chains were weaker than the inter-chain disulfides between the two heavy chains. The relative high abundance ions observed in ETD provided strong evidence for the linked peptide information, which was particularly useful for the identification of the scrambled disulfides. The use of SDS-PAGE helped the separation of these misfolded proteins for the determination of scrambled disulfide linkages. This methodology is useful for comparison of disulfide stability generated from different structural designs, and providing a new way to determine the scrambling patterns, which could be applied for those seeking to determine unknown disulfide linkages.

Antibody Drug Conjugates: Design and Selection of Linker, Payload and Conjugation Chemistry

“…The acquisition of mass spectra is relatively straightforward; however, it is important to remove all salts and other excipients that may interfere with protein ionization. Tandem liquid chromatography-mass spectrometry is often employed to simplify desalting and to improve resolution (7,86,87). In addition, the deglycosylation of mAb samples using enzymes such as PNGase is common practice and beneficial to simplify MS analysis.…”

Mass spectrometry-based strategies for protein disulfide bond identification

“…The exact cysteine connection can be obtained by performing MS 3 on P1-P2 and P2-P3. With the aid of SDS-PAGE separation, this approach was later used to identify the disulfide bonds of several therapeutic proteins including monoclonal antibodies as well as their scrambled forms under heat stress (Wang et al 2011). Low-energy electron-based fragmentation methods provide valuable information on disulfide-dissociated peptides; however, it requires much more effort to interpret the spectra that contain abundant peptide backbone fragments.…”

ETD and ECD Mass Spectrometry Fragmentation for the Characterization of Protein Post Translational Modifications

“…Final suggestions are the use of combined fragmentation approach and high resolution mass spectrometers (Q-TOF or Orbitrap) for the characterization of multi-and close-disulfide bonds in proteins. Wang Y et al proposed the use of ETD process for the characterization and comparison of disulfide linkages and scrambling pattens in therapeutic monoclonal antibodies [53].…”