See it all with Uncle's full-spectrum analysis

Význam tématu

Protein thermal stability analysis is crucial for biologics formulation, ensuring proper folding and aggregation behavior during development and storage.
The combination of intrinsic and extrinsic fluorescence with light scattering provides orthogonal insights into protein stability.

Cíle a přehled studie / článku

This application note demonstrates Uncle, a unified platform for comprehensive protein stability profiling using full-spectrum fluorescence, static light scattering (SLS), and dynamic light scattering (DLS).
It aims to show how label-free and dye-based assays can be combined in a single thermal melt experiment to assess unfolding and aggregation of various proteins.

Použitá metodika a instrumentace

Ultraflexible multi-detection platform: Uncle integrates:

• Full-spectrum fluorescence detection (250–720 nm)
• Static light scattering (SLS)
• Dynamic light scattering (DLS)
• Temperature control from 15 to 95 °C with 0.6 °C/min ramp
• Sealed quartz cuvettes (9 µL sample, up to 48 samples)

Protein-dye experiments included:

• SYPRO Orange (400× in DMSO) with bovine IgG
• CPM (20 mg/mL in DMSO) with β-lactoglobulin A
• ANS (10 mg/mL in DMSO) with RNase A

Thermal ramps and spectral data were collected using Uncle Tm & Tagg application with Optional DLS.

Hlavní výsledky a diskuse

Intrinsic fluorescence and SLS of IgG

• Both intrinsic (300–430 nm) and SYPRO Orange fluorescence (536–650 nm) yielded similar Tm values (~70 °C), indicating minimal dye perturbation.
• SLS revealed a dye-dependent delay in aggregation onset, suggesting SYPRO Orange may shield hydrophobic patches.

Extrinsic fluorescence of RNase A with ANS

• ANS binding produced a blue-shifted emission peak at ~470 nm, enabling detection of a protein with weak intrinsic fluorescence.
• BCM analysis showed Tm increases with phosphate concentration, consistent with literature.

Probing β-lactoglobulin A with CPM

• CPM conjugation to exposed cysteine produced fluorescence increase at 400–650 nm upon heating.
• Intrinsic and CPM-based methods both determined a Tm of ~60 °C in PBS, demonstrating compatibility of dyes for lipophilic proteins.

Přínosy a praktické využití metody

The unified platform allows simultaneous measurement of multiple stability parameters in minimal sample volumes.
Combining label-free and dye-based assays enhances sensitivity for proteins lacking strong intrinsic fluorescence such as RNase A and membrane-associated targets.
High throughput (48 samples) and customizable analysis streamline developability screening in biopharmaceutical R&D.

Budoucí trendy a možnosti využití

Expanding the dye library and spectral analysis algorithms may enable characterization of multi-domain or fusion proteins.
Integration with automated sample handling to increase throughput for screening large antibody and protein libraries.
Potential to incorporate additional orthogonal techniques such as circular dichroism or mass spectrometry in a single workflow.

Závěr

Uncle multispectral fluorescence and light scattering platform provides comprehensive thermal stability profiles for a wide range of proteins. The combination of intrinsic and extrinsic fluorescence with SLS and DLS enables deeper insights into unfolding and aggregation behavior. This approach supports robust formulation design and developability assessment in biologics research.

Reference

1. Jarasch A et al. Developability assessment during the selection of novel therapeutic antibodies. J Pharm Sci. 2015;104(6):1885–98.
2. He F et al. Detection of IgG aggregation by a high throughput method based on extrinsic fluorescence. J Pharm Sci. 2010;99(6):2598–608.
3. Semisotnov G et al. Study of the molten globule intermediate state in protein folding by a hydrophobic fluorescent probe. Biopolymers. 1991;31(1):119–28.
4. Sigma-Aldrich. Product Description for 8-Anilino-1-naphthalenesulfonic acid ammonium salt.
5. Stelea S et al. Thermal unfolding of ribonuclease A in phosphate at neutral pH: deviations from the two-state model. Protein Sci. 2002;10(5):970–8.
6. Alexandrov A et al. Microscale fluorescent thermal stability assay for membrane proteins. Structure. 2008;16(3):351–9.