Development of innovative NMR methods enabling in-depth characterization of therapeutic antibodies batches – NMR4mAbs

The aim of NMR4mAbs project is to develop innovative Nuclear Magnetic Resonance (NMR) tools to better characterize the impact of chemical modifications on structure of monoclonal Antibodies (mAbs) clinical batches. So far, 2D NMR spectra of methyl groups of therapeutic antibodies at natural abundance are used as "fingerprints" to ensure the structural compliance of mAbs batches. Although this fingerprinting approach is a powerful and effective tool for quality monitoring, it does not grant access to in-depth structural information, as it does not identify precisely the structural modifications involved on mAbs. As a result, it is challenging to explain the structural origin of any potency change after stress tests and in turn to understand precisely biotherapeutic molecules degradation pathways. A precise characterization of mAbs is limited by the inability to assign each observed NMR signal to the corresponding atom in the target mAb. To solve this bottleneck, the partners already set up new in vivo and in vitro isotopic labelling methods optimized for mAbs, enabling to initiate the first assignment of a full mAb. Based on this first assignment of a mAb, and exploiting sequence and structure similarities, our goal is to develop fast and efficient NMR-based and labelling-based methods to speed up frequencies assignment to any other mAbs. These new approaches will enable to decipher the whole structural information encoded in 2D CH3-NMR spectra, so far unexploited. While advanced assignment approaches will need to be implemented at early stages of therapeutical mAb development, the frequencies identification will be used in all the following phases of the development pipeline. In this project, we will demonstrate how previous knowledge of mAb NMR frequencies, will enable an in-depth characterization of any structural modification using natural abundance 2D CH3-NMR spectra, acquired directly on the mAb clinical batches.