Towards improved comprehension of the relationship between proteoform and conformation of therapeutic monoclonal antibodies with structural mass spectrometry – conformAbs

Monoclonal antibodies (mAbs) are considered as the most rapidly growing class of therapeutics to treat several diseases such as cancer, cardiovascular disorders, asthma, or autoimmune diseases. The therapeutic efficiency of mAbs relies mainly on their high target specificity, long circulating half-life, and the ability to induce immune cell effector response. However, in some cases the therapeutic response of mAbs is inefficient or may encounter cell tumor resistance. Alternative formats such as antibody drug conjugates (ADCs), immunocytokines (ICC), multi-specific mAbs (bi-, tri-specific), and antibody fragments (scFv or VHH), among others, have been developed aiming at increasing the therapeutic potency of mAbs, leading to a chemical diversity and heterogeneity of mAb-related proteins. The structural characterization of these therapeutic proteins is still considered as a real analytical challenge due to their intrinsically heterogeneity (tens of PTMs), the flexibility associated to the low structural stability of the hinge region, and the primary structure modifications from the different engineering processes (addition of cytotoxic molecules or immune proteins). For all these reasons, the number of 3D structures of mAbs and derived products reported in public databases is still scarce. Even though different analytical strategies have been successfully proposed in the literature to separate and determine the co-existence of different populations of therapeutic proteins (proteoforms), their impact on the global conformation of the proteins or the variation of protein gas-phase thermodynamics are often overlooked or even completely ignored. The general objective of the conformAbs project is to develop multi-level mass spectrometry (MS)-centric approaches to decipher the relationship between proteoforms and higher order structures (HOS) of a series of therapeutic mAb-related products with increasing size and complexity (nanobodies, mAbs, ADCs, and immunocitokines) through three different axes:
1) to identify and precisely localize the different proteoform modifications of different therapeutic mAb-derived products by top- and middle-down-MS (TD/MD-MS) under denaturing conditions.
2) to develop top- and middle-level ion mobility (IM)-based workflows using high resolution instrumentation, hyphenation with native chromatographic methods (ndLC) and collision induced unfolding experiments (CIU) to decipher the conformational landscape and record differences on the gas-phase stability of therapeutic proteins (HOS) under native conditions.
3) to develop a new integrative methodology combining TD/MD-MS with IM-MS based techniques under native conditions to directly unveil the influence of proteoform primary sequence on higher order structures of mAb-based proteins.
The combination of the results from ndLC-nTD/nMD-MS and ndLC-CIU will allow the correlation of the specific proteoform identification with their global conformation and precise CIU unfolding mechanism. Therefore, straight relationship between the specific location of proteoform modifications and the conformational landscape of therapeutic proteins could be stablished enabling the assessment of the structural consequences in the HOS of therapeutic proteins of a wide range of primary sequence variants. The results of this project might potentially contribute to better evaluate mAb lead candidates at an early stage, and thus avoiding delays during late-stage development, through the identification of degradation hot-spots, and providing a ranking order of mAb candidates based on stability.