Excitation-Energy-Dependent Molecular Beacon Detects Early Stage Neurotoxic Aβ Aggregates in the Presence of Cortical Neurons

et al. 2020

Biochemical Journal

Self Cite

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.

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Monoclonal antibody stability can be usefully monitored using the excitation-energy-dependent fluorescence edge-shift

Knight¹,

Woolley

et al. 2020

Biochemical Journal

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“…Eq 3) since it predicts a maximum magnitude of the CSM, corresponding to the fully relaxed state, (Figure 1C). Indeed, ourselves and others have observed saturation of the REES effect with non-Trp fluorophores used as molecular probes 18 or ligands, 19 and so Eq 7 is logical for the REES effect in proteins.…”

Section: Resultsmentioning

confidence: 99%

A thermodynamic model for interpreting tryptophan excitation-energy-dependent fluorescence spectra provides insight into protein conformational sampling and stability

Camacho

Winter

et al. 2021

Preprint

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It is now over thirty years since Demchenko and Ladokhin first posited the potential of the tryptophan red edge excitation shift (REES) effect to capture information on protein molecular dynamics. Whilst there have been many key efforts in the intervening years, a biophysical thermodynamic model to quantify the relationship between the REES effect and protein flexibility has been lacking. Without such a model the full potential of the REES effect cannot be realized. Here, we present a thermodynamic model of the protein REES effect that captures information on protein conformational flexibility, even with proteins containing multiple tryptophan residues. Our study incorporates exemplars at every scale, from tryptophan in solution, single tryptophan peptides to multi-tryptophan proteins, with examples including a structurally disordered peptide, de novo designed enzyme, human regulatory protein, therapeutic monoclonal antibody in active commercial development, and a mesophilic and hyperthermophilic enzyme. Combined, our model and data suggest a route forward for the experimental measurement of the protein REES effect and point to the potential for integrating bimolecular simulation with experimental data to yield novel insights.

“…We have recently demonstrated that monitoring REES for mAbs labelled with an extrinsic fluorophore can report on relatively low-n changes in oligomeric state. 36 Similarly, the REES effect has been used to infer differently folded/aggregated states of proteins using singe-Trp containing examples. 30 Based on these findings and given the sensitive discrimination of mAbs by the QUBES data shown above, we wished to explore whether differences in multi-Trp REES data are sensitive to changes in protein structure in mAbs.…”

Section: Figures 3b and 3cmentioning

confidence: 99%

“…35 Similarly, we have recently shown that extrinsic fluorophore labelling or mAbs can be used to track changes in low-n oligomer formation. 36 We term our method of quantifying REES data, QUBES (quantitative understanding of bimolecular edge shift), in order to distinguish other treatments of REES data. Based on the apparent power and potential information content of the REES phenomenon, we reason that this approach could be developed further to deliver sensitive detection of changes in a proteins dynamic profile as well as overall conformation.…”

Section: Introductionmentioning

confidence: 99%

Monoclonal antibody stability can be usefully monitored using the excitation-energy-dependent fluorescence edge-shift

Woolley

Parsons

et al. 2020

Preprint

Self Cite

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...