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Importance of the topic

In biopharmaceutical research and development, comprehensive stability profiling of proteins and viral vectors is essential for safe formulation, reliable manufacturing, and long-term storage. Conventional approaches often require multiple instruments and large volumes of sample, slowing workflows and increasing material consumption. A one-stop solution that integrates key biophysical measurements streamlines data collection, reduces sample needs, and accelerates decision-making.

Study objectives and overview

This application note introduces a unified analytical platform that combines fluorescence spectroscopy, static light scattering (SLS), and dynamic light scattering (DLS) in a single instrument. The primary aim is to demonstrate how simultaneous collection of thermal unfolding (Tm), aggregation onset (Tagg), isothermal stability, sizing, polydispersity, colloidal interaction parameters (kD, B22, G22), viscosity, and viral capsid integrity can be achieved in one automated workflow. The platform is evaluated for throughput, sensitivity, and applicability to various biologics and gene therapy vectors.

Methodology

Analyses are conducted in sealed glass capillaries using only 9 µL of sample. Up to 48 samples can be processed per run over a temperature range of 15–95 °C with programmable heating rates (0.1–10 °C/min). Measurement modes include:

• Full-spectrum fluorescence (250–720 nm detection) with 266 nm and 473 nm excitation
• Static light scattering at two wavelengths (266 nm, 473 nm) for sensitive aggregation detection
• Dynamic light scattering (660 nm laser) for hydrodynamic diameter and polydispersity

This flexible setup supports thermal ramps, isothermal holds, and customizable assay apps to track real-time stability changes and long-term degradation.

Použitá instrumentace

• Minimum sample volume: 9 µL in sealed capillaries
• Simultaneous samples per experiment: 48
• Temperature range: 15–95 °C; accuracy ±0.1 °C
• Concentration range: 0.05 mg/mL–300 mg/mL (protein dependent)
• Fluorescence: full spectral CCD detection, 250–720 nm
• SLS: dual-wavelength intensity monitoring
• DLS: avalanche photodiode detection for 0.3–1000 nm sizing

Main results and discussion

Simultaneous measurement of Tm and Tagg enables rapid identification of temperature thresholds where unfolding leads to aggregation. Adding a pre-ramp DLS read reveals the presence of preformed aggregates. Customized isothermal apps allow long-term incubation studies at fixed temperatures, detecting early aggregation trends well before visible precipitation. Colloidal interaction assays (kD, B22, G22) profile protein–protein interactions under varying excipient conditions, guiding formulation optimization. For viral vectors, SYBR Gold fluorescence detects genome release at sub-aggregation temperatures, while SLS monitors capsid aggregation.

Benefits and practical applications

• Minimal sample consumption and high throughput (48 samples in under 2 hours)
• Comprehensive data from a single run reduces experimental complexity
• Early detection of aggregation and genome leakage improves formulation design
• Versatile assay library supports proteins, dyes, and viral vectors without hardware changes

Future trends and applications

The integration of multi-mode stability data with machine learning algorithms will enable predictive formulation design and accelerated screening. Expansion of dye chemistries and assay workflows can further tailor measurements for novel biologics, nanoparticles, and other advanced therapeutics. Continuous online monitoring and higher sample multiplexing are logical next steps to support real-time process analytics in manufacturing.

Conclusion

A multi-modal instrument that unifies fluorescence, SLS, and DLS measurements significantly streamlines stability characterization of proteins and viral vectors. By reducing sample volume, automating workflows, and delivering rich data sets, this approach accelerates formulation development, ensures quality, and supports regulatory compliance in biopharmaceutical research and manufacturing.