Monoclonal antibody stability can be usefully monitored using the excitation-energy-dependent 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, which would have applications in the discovery and development of biopharamceuticals. As such 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). We demonstrate the approach is able to predict the stability of mAbs both in the short (hours), medium (days) and long-term (months). The approach benefits from low technical complexity, is rapid and uses instrumentation which exists in most biochemistry laboratories without modification.

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“…[25][26][27][28] We term this quantification quantitative understanding of bimolecular edge shift, QUBES. 29 We have demonstrated this quantification of the REES effect tracks subtle changes in protein dynamics, even where the crystal structures are identical and for a broad range of mutli-Trp proteins. 23,[29][30] Briefly, REES is a fluorescence phenomenon where decreasing excitation energies are used to photo select for discrete emissive states, which manifests as inhomogeneous spectral broadening of the resultant emission spectra.…”

“…29 We have demonstrated this quantification of the REES effect tracks subtle changes in protein dynamics, even where the crystal structures are identical and for a broad range of mutli-Trp proteins. 23,[29][30] Briefly, REES is a fluorescence phenomenon where decreasing excitation energies are used to photo select for discrete emissive states, which manifests as inhomogeneous spectral broadening of the resultant emission spectra. 26, [31][32] Using our QUBES approach we are able to interpret the REES effect to show relative changes in the equilibrium of discrete conformational sub-states.…”

“…REES data was collected essentially as described previously as the matrix of excitation emission wavelengths. 23,[29][30] Fluorescence measurements were performed using a Perkin Elmer LS50B Luminescence Spectrometer (Perkin Elmer, Waltham, MA, USA) connected to a peltier heat pump (± 1 °C). 15 µM ssGDH samples were used for analysis in 100 mM pH8 HEPES.…”

“…Essentially, as the magnitude of A/R becomes larger the protein becomes more flexible; a broader range of accessible conformational substates as we have previously described. 23,[29][30] From Figure 3C, we find that the REES data suggest that, despite the small number of Hbonding changes in the active site for each substrate, there is a significant difference in the equilibrium of conformational states with each of the epimers. We find that the ternary complex with D-galactose, outside of experimental error, explores a more restricted range of conformational states, i.e.…”

“…We have previously found that, for an invariant CSM0 value, an increased A/R value is indicative of broader population of conformational states. 29 Figure 3C shows the resulting QUBES data for the ternary complex of GDH with a range of substrates versus the extracted magnitude of ∆ ‡ (Figure 2 and Table 1). Essentially, as the magnitude of A/R becomes larger the protein becomes more flexible; a broader range of accessible conformational substates as we have previously described.…”