Monoclonal antibody charge variant characterization by fully automated four-dimensional liquid chromatography-mass spectrometry

Fingerprinting trimeric SARS-CoV-2 RBD by capillary isoelectric focusing with whole-column imaging detection

“…The difference was partly related to protein glycosylation modification verified by the UPLC-MS analysis. Besides glycosylation modification, other PTMs will also result in charge heterogeneity [ 26 ]. Thus, in our next study, the variants will be further analyzed using cIEF-MS [ 27 ] to clarify the reasons for the formation of variants and explore the correlation between variants and immunogenicity.…”

3D-LC-MS with 2D Multimethod Option for Fully Automated Assessment of Multiple Attributes of Monoclonal Antibodies Directly from Cell Culture Supernatants

“…This mass difference corresponds to an F to I/L substitution in the variable part of both heavy chains. 22,28 A point mutation is at the origin of this wrong recombinant expression, and as can be concluded from the ELISA results presented in Supporting Table 13, this single mutation has huge functional consequences with a 4-times lower affinity for the target antigen IL6R. Using HDX-MS, a local structural change could be revealed as a result of this point mutation explaining the functional ELISA results and also the CEX results (Supporting Figure 4).…”

“…2 D CEX peaks 1, 2, and 4 have similar MW values to the main peak (within the mass accuracy of the instrument) pointing in the direction of deamidation or isomerization events. Harris et al 44 and Verscheure et al 28 who performed CEX fraction collection and peptide mapping in, respectively, an offline and online manner, confirmed that peaks 1 and 2 correspond to a deamidated variant and peak 4 to an isoaspartate containing variant. In 2 D CEX peak 5, a partially resolved variant with a mass increase of approximately 128 Da is observed corresponding to heavy-chain C-terminal lysine.…”

“…As such, different structural characteristics can simultaneously be assessed, a principle known as multiattribute analysis. Multidimensional liquid chromatography (mD-LC) is highly promising in that respect, and various online two- (2D), three- (3D), and four-dimensional (4D) LC-MS protein analyzers, optionally with chemical or enzymatic reactors implemented, have been described. − Analytical scientists reported on 2D-LC-MS setups for the rapid and fully automated characterization of mAbs and antibody–drug conjugates (ADCs) by combining CEX, SEC, HIC, or Protein A affinity chromatography with RPLC or SEC prior to MS or ion mobility (IM) MS analysis. − First-dimension ( 1 D) CEX, SEC, HIC, or Protein A fractions are commonly stored in loops prior to second-dimension ( 2 D) analysis. The latter is typically applied for desalting, thereby facilitating MS analysis, although in some studies, the 2 D step contributes to the overall separation.…”

“…Gstöttner et al pioneered a 4D-LC-MS method incorporating 1 D CEX, peak collection, 2 D RPLC desalting, denaturation and reduction, 3 D trypsin digestion, and 4 D RPLC-MS-based peptide mapping for the in-depth characterization of mAb charge variants . This innovation substantially reduces turnaround time, sample manipulation, loss, and artifacts and further increases information gathering over the 2D-LC-MS approach, i.e., modifications sites are highlighted and modifications resulting in zero/minor mass differences (e.g., aspartate isomerization, asparagine deamidation) are revealed. − Goyon et al reported on a variation of the above configuration for the study of ADCs by replacing 1 D CEX for 1 D SEC . Potential critical quality attributes such as aggregation, drug-to-antibody ratio (DAR), oxidation, and deamidation could be revealed in a 2 h runtime.…”