A study team from the University of Bath have utilized a sensitive way of protein structure stability, with instant use in the biopharmaceutical industry. The method, fluorescence red edge excitation shift (REES) exhibited high sensitivity in calling monoclonal antibody (mAb) stability in the short, medium, and long term.

REES data could always distinguish between identical monoclonal antibodies across the micro to milligram/ml range. Guarantees prompt use in quality assurance, formulation, and development processes in the biopharmaceutical sector

A significant barrier in the development of therapeutic monoclonal antibodies (mAb) is the structural heterogeneity and aggregation. Stable biomolecules are essential to the commercial viability and efficacy of mAb and other biopharmaceuticals. Methods of monitoring stability is a challenge.

<strong>REES: a Possible approach to shooting changes in a proteins profile</strong>

Several techniques have been developed to address the issue of stability; of these, fluorescence red edge excitation shift (REES) is most sensitive to a range of protein structure changes and dynamics. It harnesses the properties of tryptophan (Trp) property of excitation-emission.

Shifts at the maximum emission, along with decreasing energy or excitation are seen; this happens as reduced energy photons excite the different conformational state of their Trp-solvent system. Called the REES effect, its own typical application is in distinguishing between the folded or unfolded state of a protein

The team illustrates that direct quantification of REES data instead can disclose the dynamism of protein structures -- their conformational states, characterized since the free energy landscape (FEL) of a protein.

<span style="color: #ff6600;"><a style="color: #ff6600;" href="https://www.biorxiv.org/content/10.1101/2020.03.23.003608v1">This strategy works for multi-Trp proteins, where gaps in flexibility of molecules could be seen in structurally identical X-ray structures.</a></span>

<strong>Improving REES: measuring the data</strong>

Pudney et al.. Termed their way of measuring REES information, QUBES (quantitative understanding of bimolecular edge shift), to differentiate it from different remedies of REES data.

The team measured change in the middle of spectral mass (CSM) variance connected to the amplitude, A, of an exponential with a curvature determined by R. The plot produces a three-dimensional data stage.

Previously, Pudney et al.. Found that the measured ratio A/R varies with protein flexibility. Large A/R values reflect more flexible proteins and little A/R values reflect a more rigid protein. On the other hand, the ratio alone is insufficient to capture the complete data content. Consequently an exponential function has to be fitted to catch all info.

<span style="color: #ff6600;"><a style="color: #ff6600;" href="https://www.biorxiv.org/content/10.1101/2020.03.23.003608v1">The antibodies for which QUBES data was measured represented different classes and analyzed in buffer systems not utilized in commercial formulations so more valid comparisons could be made.</a></span>

Fluorescence measurements were conducted on therapeutic antibodies subject to unfolding and aggregation. Structure-based calculations were made with Fab regions of X-ray structures along with the rigidity employing a score termed amount value of rigid clusters, SVRC.

<strong>QUBES data are sensitive to mAb flexibility</strong>

The group expressed a parameter termed amount significance of rigid clusters (SVRC), which vary significantly despite the high structural similarity. This quantifying of this REES effect for multi-Trp proteins provides a unique'fingerprint' for distinct monoclonal antibodies, demonstrating its sensitivity.

The QUBES data is sensitive to monoclonal antibody flexibility over a variety of time and length scales -- from little internal shifts in configuration to more rapid conformational sampling moves from the hypervariable loop (HVL) regions of the Fab.

<strong>The data and sensitivity revealed by QUBES</strong>

The data of QUBES data included an ability to ascertain between native and denatured, and folded and unfolded proteins.

The sensitivity of the method was evaluated by analyzing the reaction of the antibodies to a range of chemical and physical perturbations that mimic those undergone by monoclonal antibodies during the process of fabrication.

<strong>QUBES predicts protein equilibrium</strong>

Extracted QUBES A/R data values were compared to the fractional loss of protein monomer as evaluated by DLS dynamic light scattering. The similarity between those indicates that their quantification methods may be used to predict the thermodynamic equilibrium of a sample.

<strong>A summary of the detection capability of QUBES data</strong>

The team summarized the detection capability of the QUBES data, revealing the 3D plot created may be used to forecast the nation (unfolded, destabilized, stabilized, and aggregated). Using their approach, accurate detection, separation, and quantification of protein unfolding, and early stage of soluble aggregation (as a predictor for equilibrium ) can be made.

The team stressed that the data could be simplistically interpreted with the A/R alone. Therefore, all QUBES have to be utilized to evaluate a protein's stability.

QUBES also offers the following benefits; quick (&lt;5 minutes) data acquisition facilitating large screening requirements; multi-Trp protein analysis, enabling analysis of most proteins; broad sample concentrations (μgram - mg)to minimize consumption; also, ability to monitor the stability of proteins of almost any size across a range of solvent or buffer environments.

These findings suggest a potential ways to fingerprint protein structure and stability, improving the discovery and creation of mAbs and biopharmaceuticals at large.

<strong>Story source:</strong>

Pudney, CR et al. (2020) Monoclonal antibody stability can be usefully monitored using the excitation-energy-dependent fluorescence edge-shift. DOI: 10.1101/2020.03.23.003608.