and c.r.pudney@bath.ac.uk 2 Monoclonal Antibody stability can be usefully monitored using the excitation-energydependent fluorescence edge-shift Abstract Among the major challenges in the development of biopharmaceuticals are structural heterogeneity and aggregation. The development of a successful therapeutic monoclonal antibody (mAb) requires both a highly active and also stable molecule. Whilst a range of experimental (biophysical) approaches exist to track changes in stability of proteins, routine prediction of stability remains challenging. The fluorescence red edge excitation shift (REES) phenomenon is sensitive to a range of changes in protein structure. Based on recent work, we have found that quantifying the REES effect is extremely sensitive to changes in protein conformational state and dynamics. Given the extreme sensitivity, potentially this tool could provide a 'fingerprint' of the structure and stability of a protein. Such a tool would be useful in the discovery and development of biopharamceuticals and so we have explored our hypothesis with a panel of therapeutic mAbs. We demonstrate that the quantified REES data show remarkable sensitivity, being able to discern between structurally identical antibodies and showing sensitivity to unfolding and aggregation. The approach works across a broad concentration range (μg -mg/ml) and is highly consistent. We show that the approach can be applied alongside traditional characterisation testing within the context of a forced degradation study (FDS). Most importantly, we demonstrate the approach is able to predict the stability of mAbs both in the short (hours), medium (days) and long-term (months). The quantified REES data will find immediate use in the biopharmaceutical industry in quality assurance, formulation and development. The approach benefits from low technical complexity, is rapid and uses instrumentation which exists in most biochemistry laboratories without modification. Eq 1Where fi is the measured fluorescence intensity and λem is the emission wavelength. We would stress the importance of using a consistent wavelength range when reporting CSM data, as the magnitude will be dependent on the wavelength range chosen. The data are extracted by fitting the CSM versus λEx data as described in the manuscript. Data fitting and plotting was performed using OriginPro 2016 (Microcal).Antibody samples, unfolding and aggregation. Therapeutic antibodies (Figure 2A) were provided by Bath ASU and were either extensively dialysed (for urea denaturation experiments) or diluted into Tris-Cl buffered saline pH 8. All buffer components were of a spectroscopic grade. Antibody denaturation was achieved by extensive dialysis into a buffered solution of 8M urea or 0M urea as a control. Antibody aggregation was achieved through incubation at elevated temperatures and monitored by DLS as described in the manuscript.Structure-based calculations. Partial Fab region structures (X-ray crystal structures) were used for all structure-based calculations. To ensure comparabilit...