Meet Aunty – the Queen of protein stability

Significance of the topic

The thermal stability and colloidal behavior of proteins and viral vectors are critical parameters in the development, formulation, and quality control of biologic therapeutics
Aunty addresses the limitations of traditional stability assays by combining intrinsic and dye‐based fluorescence with static and dynamic light scattering in a high-throughput, low-volume format

Objectives and overview of the study

This application note demonstrates how to leverage Aunty’s integrated detection modes and temperature control to obtain key stability metrics
Key objectives include:

• Determination of melting temperatures (Tm) and aggregation onset (Tagg) via intrinsic or reporter dye fluorescence and SLS
• Particle sizing and polydispersity analysis across a broad size range (0.3–1000 nm) with DLS
• Combined thermal ramp assays integrating fluorescence, SLS, and DLS for full stability profiling
• Capsid stability assessment through genome ejection monitoring in viral vectors
• Colloidal stability screening via diffusion interaction parameter (kD), second virial coefficient (B22), and Kirkwood-Buff integral (G22)
• Isothermal stability testing under forced-degradation conditions

Methodology and used instrumentation

The core platform is the Aunty thermal stability system configured with:

• Full-spectrum fluorescence detection (LED excitation at 280 nm and 470 nm)
• Static light scattering (SLS) and dynamic light scattering (DLS) modules operating in parallel
• Automation-friendly SBS-format 96-well quartz plates (8 µL per well)
• Precise temperature control from 15 °C to 95 °C (ramps of 0.1–10 °C/min, isothermal holds)
• Reporter dyes: SYPRO Orange for protein unfolding and SYBR Gold for viral genome release

Main results and discussion

• Thermal ramp by intrinsic fluorescence and SLS of trastuzumab in PBS at 1 mg/mL and 0.025 mg/mL yielded Tm values of ~71 °C with concentration-dependent Tagg shifts (76 °C vs. 79 °C) and higher SLS signal at higher concentration
• Particle sizing of NIST polystyrene beads (20, 100, 500 nm) by DLS matched manufacturer specifications and demonstrated monodispersity (PDI ≤ 0.1)
• Combined thermal ramp assay on NISTmAb reference material produced two Tm transitions (69.7 ± 0.8 °C and 83.5 ± 1.2 °C) in agreement with DSC data, with no significant aggregation detected by SLS and minimal size increase by DLS
• Reporter dye assay using SYPRO Orange for bovine IgG (3 mg/mL) enabled Tm determination (71.5 °C) where intrinsic fluorescence was insufficient, and Tagg was determined at 69.9 °C by SLS
• Capsid stability assay of AAV9 at 5 × 10¹² vg/mL in PBS with SYBR Gold revealed genome ejection Tm at 60.6 °C and aggregation onset at 76.3 °C
• Colloidal stability screening of trastuzumab in histidine buffer with sucrose or NaCl at 25 °C and 42 °C showed positive kD and B22 values with sucrose (repulsive interactions) and negative kD with NaCl at high concentration, indicating reduced colloidal stability
• Isothermal stability of bovine IgG at 62 °C demonstrated that arginine accelerated unfolding (fluorescence shift) while inhibiting aggregation (SLS and DLS) over 14 hours

Benefits and practical applications of the method

• Rapid screening: 96 samples per minute with only 8 µL sample volume
• Multiparametric profiling: simultaneous assessment of unfolding, aggregation, size, and colloidal interactions
• Low sample consumption enables early-stage developability and formulation optimization
• Compatibility with intrinsic and extrinsic fluorescence assays for diverse analytes, including challenging proteins and viral vectors
• Automation-friendly plate format and sealed wells support long-term and isothermal studies without contamination

Future trends and potential applications

Integration of Aunty data with machine learning for predictive stability modeling
Expansion to novel excipient libraries and high-throughput formulation screening
Adoption in gene therapy and vaccine development pipelines for rapid capsid and genome stability assessments
Development of advanced reporter dyes and custom assay modules for broader biomolecule compatibility

Conclusion

Aunty represents a comprehensive, high-throughput platform for protein and viral vector stability characterization by uniting full-spectrum fluorescence with static and dynamic light scattering in a low-volume, automation-compatible format
This approach accelerates developability screening, formulation optimization, and quality control with precise thermal control and multiparametric detection

References

1. Gokarn Y, et al. Biophysical Techniques for Characterizing the Higher Order Structure and Interactions of Monoclonal Antibodies. ACS Symposium Series. 2015;1201:285–327
2. Connolly BD, et al. Weak interactions govern the viscosity of concentrated antibody solutions: High-throughput analysis using the diffusion interaction parameter. Biophysical Journal. 2012;103(1):69–78
3. Wilson W, et al. Colloidal Behavior of Proteins: Effects of the Second Virial Coefficient on Solubility, Crystallization and Aggregation of Proteins in Aqueous Solution. Current Pharmaceutical Biotechnology. 2006;6(6):427–436
4. Blanco MA, et al. Reexamining protein-protein and protein-solvent interactions from Kirkwood-Buff analysis of light scattering in multi-component solutions. Journal of Chemical Physics. 2011;134(22)
5. Kamerzell TJ, et al. Protein-excipient interactions: Mechanisms and biophysical characterization applied to protein formulation development. Advanced Drug Delivery Reviews. 2011;63(13):1118–1159
6. Ibreljic N, et al. Recombinant AAV genome size effect on viral vector production, purification, and thermostability. Molecular Therapy Methods & Clinical Development. 2024;32(1):101188