Dye Harder with UNcle: Measure Thermal Stability with SYPRO

Significance of the Topic

Thermal stability assessment is a cornerstone in the development and formulation of biologics. It enables researchers to predict shelf life, optimize buffer conditions, and identify liability hotspots in protein therapeutics. While label-free approaches preserve native conformation, extrinsic dyes such as SYPRO Orange offer a robust alternative when intrinsic fluorescence signals are insufficient.

Study Objectives and Overview

This application note examines the use of SYPRO Orange dye combined with differential scanning fluorimetry (DSF) on the UNcle stability platform. Key goals include:

• Evaluating protein melting transitions for lysozyme and a monoclonal antibody (mAb).
• Comparing thermal unfolding and aggregation profiles in different buffer systems.
• Determining the effect of protein concentration on melting temperature (Tm) measurements.

Methodology and Instrumentation

Proteins and Dye Preparation:

• Lysozyme at 2 mg/mL in PBS, pH 7.4.
• Monoclonal antibody formulated in PBS, pH 7.4 or 5 mM succinate, pH 5, at concentrations from 0.05 to 100 mg/mL.
• SYPRO Orange diluted to 20X final concentration.

DSF Protocol on UNcle:

• Sample volume: 9 µL per well in 48-well quartz cuvettes (“UNis”).
• Thermal ramp from 15–95 °C or 20–95 °C at 0.5 °C/minute.
• Excitation at 473 nm; emission spectra collected from 250–720 nm.
• Analysis of area under the curve between 510–680 nm to calculate Tm via inflection points.

Key Results and Discussion

Lysozyme Behavior:

• Single thermal unfolding transition detected at Tm = 67.2 °C, consistent with literature values.
• Spectral comparison at 37 °C vs. 75 °C revealed a dramatic fluorescence increase upon unfolding.

Monoclonal Antibody Profile:

• In succinate buffer, two unfolding transitions at 62.7 °C and 76.3 °C.
• In PBS, unfolding transitions at 66.1 °C and 75.6 °C; onset of aggregation (Tagg) observed at 75.5 °C by static light scattering (SLS).
• Post–aggregation signal decline suggests precipitation of large aggregates and dye dissociation.

Concentration Dependence:

• Tm values remained stable across 0.05–100 mg/mL (average Tm1 ≈ 63.3 °C; Tm2 ≈ 78.1 °C).
• At 100 mg/mL, lower fluorescence intensity and loss of second transition likely due to dye-protein stoichiometry or packing effects.

Instrumentation

The UNcle stability platform integrates:

• Fluorescence detection for DSF.
• Static and dynamic light scattering for aggregation and sizing.
• Precise temperature control from 15 to 95 °C.
• Sealed low-volume multi-well quartz cuvettes accommodating up to 48 samples simultaneously.

Benefits and Practical Applications

The combined SYPRO-DSF and label-free capabilities on a single instrument allow:

• Flexible method switching as project needs evolve.
• High-throughput screening of multiple formulations and constructs.
• Minimal sample consumption for early-stage developability assessments.

Future Trends and Opportunities

Ongoing enhancements may include:

• Integration with automated liquid handling for larger library screens.
• Advanced data analytics and machine learning to predict stability from raw spectral data.
• Expansion of dye chemistries to target specific hydrophobic patches or post-translational modifications.

Conclusion

SYPRO Orange–based DSF on the UNcle platform provides a rapid, reliable approach to assess protein thermal stability and aggregation. It complements label-free methods, supports a wide range of concentrations, and streamlines early formulation optimization for biologics.

Reference

1. Lavinder JJ, Hari SB, Sullivan BJ, Magliery TJ. High-throughput thermal scanning: a general, rapid dye-binding thermal shift screen for protein engineering. J Am Chem Soc 131(2009): 3794–3795.
2. Niesen FH, Berglund H, Vedadi M. The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability. Nature Protocols 2(2007): 2212–2221.