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Significance of the topic

Reliable stability analysis of proteins and viral vectors is essential for formulation development and quality control in biopharmaceutical research and production. Traditional approaches often require multiple instruments, consume large sample volumes and extend project timelines.

Objectives and overview of the study

This work presents a one stop stability platform that integrates fluorescence, static light scattering and dynamic light scattering to assess multiple stability parameters in a single workflow. The goal is to deliver simultaneous measurements of thermal unfolding, aggregation onset, size distribution and interaction coefficients with minimal sample.

Methodology and instrumentation

The platform uses sealed capillaries with 9 microliter sample volume and can process up to 48 samples per run at temperatures from 15 to 95 C. Fluorescence is recorded across 250 to 720 nm following excitation by 266 and 473 nm lasers. Static light scattering is monitored at the same two wavelengths to detect aggregation while dynamic light scattering with a 660 nm laser diode measures hydrodynamic diameter and polydispersity.

Instrumentation used

• Sample volume 9 microliters in sealed capillaries
• Temperature control 15–95 C with 0.1 to 10 C per minute ramp
• Fluorescence detection via CCD spectrometer at full spectral range
• SLS detection of intensity changes at 266 nm and 473 nm
• DLS measurement of hydrodynamic diameter from 0.3 to 1000 nm
• Sample concentration range 0.05 to 300 mg/mL for proteins

Main results and discussion

Combined measurements enable determination of melting temperature Tm and aggregation onset temperature Tagg in a single experiment. Pre ramp DLS scans reveal existing aggregates. Isothermal studies track long term behavior. Binding assays yield second virial coefficient B22 and dissociation constant kD. Viral vector assays using DNA intercalating dyes detect capsid stability and genome release ahead of capsid disassembly.

Benefits and practical applications

• Low sample consumption accelerates screening of formulations and variants
• High throughput up to 48 samples in under two hours
• Simultaneous multi parametric output simplifies decision making
• Early detection of aggregation and binding behavior informs development
• Applicable to proteins, monoclonal antibodies, viral vectors and gene therapies

Future trends and potential applications

• Integration with predictive modeling and data analytics to forecast stability
• Extension to higher concentration formulations and complex biologics
• Automation workflows for continuous monitoring and library screening
• Development of novel fluorescent probes for specific structural insights

Conclusion

The unified platform streamlines stability assessment by combining fluorescence, SLS and DLS in a single instrument. It delivers comprehensive insights into thermal stability, aggregation, size distribution and molecular interactions with minimal sample and high throughput, supporting efficient biopharmaceutical development and quality control.