Unravelling Structural Conformers within a Therapeutic Multispecific mAb using FabALACTICA
Scientists at the University of Strasbourg and Sanofi describe the global structural characterisation of a trispecific antibody which exhibits two distinct structural isoforms when analyzed by size-exclusion chromatography (SEC). FabALACTICA proved to be a valuable tool in this study as above hinge digestion of this trispecific antibody allowed for middle-level characterisation of the isomeric Fab regions and facilitated cyclic-ion mobility (cIM) differentiation of the isomers, which was not possible in the intact molecule without prior SEC separation.
Monoclonal antibodies (mAbs) comprise the largest class in human therapeutics and are used to treat numerous diseases. Recently, advancements in antibody engineering have enabled a much larger diversity of mAb formats to be applied, ranging from small nanobodies to highly complex multispecific antibodies capable of targeting multiple antigens simultaneously. These new-generation multispecific molecules provide advantages for various therapeutic applications and may prove to be lower cost alternatives by allowing administration of single biologic therapy rather than combination therapies.
A trispecific antibody (tsAb) was recently developed to confer protection against diverse HIV strains and, during development, this tsAb exhibited an unusual two-peak profile when analyzed by SEC. Results confirmed conformational switching in one of the CDR regions. This initial study focused on structural characterization at the local level in these tsAb isomers, however, the authors in this study undertook a more global structural investigation into this tsAb. Using native SEC-MS and ion mobility-based approaches, they investigate the conformational landscape of the tsAb and aim to definitively prove the coexistence of cis/trans isomerism.
FabALACTICA was a key part of this overall strategy as it allowed characterisation at both the intact and the subunit level. This middle-level characterisation, facilitated by above hinge digestion of the tsAb and isolation of the Fab arms allowed for a more direct analysis of the specific region of isomerism. In addition, when the samples were analyzed by cIM, where there was no prior SEC separation, meaning all differentiation was a consequence of the IM dimension, the conformational isomers could be differentiated within these smaller, isolated Fab fragments which was not the case for the intact sample.
Altogether, both the native SEC-MS and IM-based characterisation methods were able to unequivocally confirm the co-existence of two tsAb conformers and that the cis/trans isomerism event occurred at a proline within a specific motif within a specific region of the tsAb. Furthermore, by performing middle-level analysis, following FabALACTICA digestion, the authors were able to generate data that was in good agreement with the intact data, but also allowed for isomeric differentiation by cIM, which was not achieved on the intact tsAb to further strengthen their findings.
The data presented in this study illustrate the benefits of combining mass spectrometry techniques, particularly when used in combination with ion mobility-based approaches, when addressing challenging issues within the biopharmaceutical industry. In addition, this study also highlights the benefits and advantages of using enzymes such as FabALACTICA to perform middle-level characterization on these classes of molecules. Digestion of molecules into smaller, more simplified subunits facilitates in-depth characterization of such complex biopharmaceuticals and in many cases, could enhance the quality of data generated by techniques such as mass spectrometry, or in fact allow the generation of data which might not be possible at the intact level depending on the complexity of the molecule.
Reference
Deslignière et al., 2022. Combination of IM-Based Approaches to Unravel the Coexistence of Two Conformers on a Therapeutic Multispecific mAb. Analytical Chemistry. https://doi.org/10.1021/acs.analchem.2c00928.
FabALACTICA – Above hinge digestion of human IgG1